ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES ANA CATARINA FREITAS DA SILVA DE JESUS

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1 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES ANA CATARINA FREITAS DA SILVA DE JESUS JULHO 2010

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3 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES Monograph of the Master Course in Biomedical Engineering Program, Faculty of Engineering of University of Porto Ana Catarina Freitas da Silva de Jesus Graduated in Biochemistry (2000) Faculty of Science of University of Porto Graduated in Nuclear Medicine (2006) Superior School of Allied Health Sciences Polytechnic Institute of Porto Supervisor: João Manuel R. S. Tavares Assistant Professor of the Mechanical Engineering Department Faculty of Engineering of University of Porto

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5 SUMMARY The purpose of this monograph is to perform a literature search on the effect of radiation on living systems and their use as therapy to kill cancer cells. To this end, in this work I start with a description of the checkpoints of the cell cycle and apoptosis phenomena as well as the cancer cell characteristics, which are important to understand the effect of radiation on cancer cells. Then, there is a description of the biological effects of radiation and how it interacts with normal and cancer cells. Subsequently, there is a description of the radiological technique used to kill cancer cells, which will be studied in my thesis dissertation, called brachytherapy. In addition, the cell cultures and the adequate means to obtain reasonable laboratory culture of cells, without contamination, for subsequent use to study the effect of radiation on cells, are discussed. To finish this monograph it is performed a description of the basic concepts of digital image processing, highlighting the increasing importance of this technique in the image processing and analysis. ii

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7 ACKNOWLEDGEMENTS To Professor João Manuel R. S. Tavares for the support provided throughout this work, particularly for guidance, support and availability, essential for the proper and constructive development of the same. To all of those who make possible the development of this work. i

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9 CONTENTS

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11 CONTENTS CHAPTER I INTRODUCTION TO THE THEME AND REPORT ORGANIZATION Introduction Main Objectives Report Organization Major Contributions 6 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Introduction Cell Life Cycle Interphase DNA Replication Cell Division Mitosis Cytokinesis Meiosis Progression of the cell cycle Growth characteristics of malignant cells Phenotypic Alterations in Cancer Cells Immortality of Transformed Cells in Culture Decreased Requirement for Growth Factors Loss of Anchorage Dependence Loss of Cell Cycle Control and Resistance to Apoptosis Cell Cycle Regulation 29 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES iii

12 CONTENTS CDK Inhibitors Cyclins Cell Cycle Checkpoints Cell Cycle Regulatory Factors as Targets for Anticancer Agents Apoptosis Biochemical Mechanism of Apoptosis Caspases Bcl-2 Family Anoikis Resistance to Apoptosis in Cancer and Potential Targets for Therapy Summary 47 CHAPTER III CANCER CELL Introduction Cancer cell Types of cancer The uniqueness of cancer The development of tumors Genetic influence on tumors Cancer through the ages Early discovery of carcinogens The use of microscopes demonstrated changes at a cellular level Modern day research and treatment 59 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES iv

13 CONTENTS 3.5 Tissues changes in response to stimuli Metaplasia Hypertrophy and hyperplasia Dysplasia Feeding tumor growth by angiogenesis Characteristics of benign and malignant tumors Events that occur during the process of metastasis Characteristics of metastatic cells Summary 71 CHAPTER IV RADIATION EFFECT ON NORMAL AND NEOPLASTIC TISSUES Introduction Quantities and units used in radiation dosimetry Radiation measurements definitions Quantities and units Historical perspective of radiobiology Law of Bergonie and Tribendeau Ancel and Vitemberger Fractionation theory Mutagenesis Effect of oxygen Relative biologic effectiveness Reproductive failure 87 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES v

14 CONTENTS 4.4 Biologic effect of radiation Elementary phenomena Molecular damages Chromossomes irradiation Irradiation of macromolecules Dose-response relationship Linear-dose-response relationships Linear quadratic dose-response curves Dose-response curve linear quadratic Targeted theory Cell survival curves Cell Death in Mammalian Tissues Nature of Cell Populations in Tissue Cell Population Kinetics and Radiation Damage Growth Fraction and its significance Cell Kinetics in Normal Tissues and Tumors Models for Radiobiological Sensitivity of Neoplastic Tissues Hewitt Dilution Assay Lung Colony Assay System Tumor Growth and Tumor Cure Models Tumor Volume Versus Time TCD 50, Tumor Cure Radiobiological Responses of Tumors 118 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES vi

15 CONTENTS Hypoxia and Radiosensitivity in Tumor Cells Summary 122 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY Introduction Cell-Culture Laboratory Maintaining Cultures Medium The use of medium in analysis and alternatives Cytogenetic Analysis of Cell Lines The Utility of Cytogenetic Characterization Methods to Induce Cell Cycle Checkpoints Methods for Synchronizing Mammalian Cells Analysis of the Mammalian Cell Cycle by Flow Cytometry Conclusion 138 CHAPTER VI BRACHYTHERAPY Introduction Brachytherapy Sources in brachytherapy Radium Radium substitutes New sources Radiobiology of brachytherapy 148 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES vii

16 CONTENTS The four Rs of radiobiology Radiobiology of low dose-rate and fractioned irradiation Split-dose recovery from sub-lethal damages in mammalian cells Cell-cycle complication: a heterogeneous population Radiation affects cell-cycle progression itself Potentially lethal damage Dose-rate effects with human cells Time-scale of radiation action Mechanism of the dose-rate effect Dose-rate effect in human tumor cells Effect of irradiation on cell cycle progression Cell killing around an implanted radiation source Implications for clinical brachytherapy Predictive assays for radiation oncology Summary 169 CHAPTER VI I BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING Introduction Pre-processing evaluation of digital images Look-up tables Flat-field correction and background subtraction Image interpretation Digital image histogram adjustment 183 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES viii

17 CONTENTS 7.7 Spatial convolution kernels (or masks) Smoothing convolution filters (spatial averaging) Sharpening convolution filters Median filters Specialized convolution filters Unsharp mask filtering Fourier transforms Summary 195 CHAPTER VIII CONCLUSIONS AND FUTURE WORKS Final Conclusions Future Works 200 REFERENCES 201 ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES ix

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19 CHAPTER I INTRODUCTION TO THE THEME AND REPORT ORGANIZATION

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21 CHAPTER I INTRODUCTION TO THE THEME AND REPORT ORGANIZATION 1.1 INTRODUCTION The different types of radiation applied for radiobiological research is important for the determination of the biological effectiveness of ionizing photon radiation as a function of photon energy. The therapeutic dose values (few Gy per daily fraction) can be delivered in a sufficiently small irradiation duration (dose rate 1 Gy/min) to be independent from repairing processes in human cells (Zeil, 2009). Brachytherapy is a term used to describe the short distance treatment of cancer with radiation from small, encapsulated radionuclide sources. This type of treatment is made by placing sources directly into or near the volume to be treated. The dose is then delivered continuously, either over a short period of time (temporary implants) or over the lifetime of the source to a complete decay (Suntharalingam, 2002). When cells are exposed to ionizing radiation the standard physical effects between radiation and the atoms or molecules of the cells occur first and the possible biological damage to cell functions follows later. The biological effects of radiation result mainly from damage to the DNA, which is the most critical target within the cell; however, there are also other sites in the cell that, when damaged, may lead to cell death (Suntharalingam, 2002). Human tumors strongly differ in radiosensitivity and radiocurability and this is thought to stem from differences in capacity for repair of sub-lethal damage. Radiosensitivity varies along the cell cycle, S being the most resistant phase and G 2 and M the most sensitive. Therefore, cells surviving an exposure are preferentially in a stage of low sensitivity (G 1 ), i.e. synchronized in a resistant cell cycle phase. They progress thereafter together into S and then to the more sensitive G 2 and M phases. A new irradiation exposure at this time will have a larger biological effect (more cell kill) (Mazeron, 2005). Brachytherapy is used to treat patients with cancer cells and the irradiated cells will be studied by me in my dissertation thesis, as the continued work from this monograph. One of the most widely used steps in the process of obtaining information from images is image segmentation: dividing the input image into regions that hopefully ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 3

22 CHAPTER I INTRODUCTION TO THE THEME AND REPORT ORGANIZATION correspond to structural units in the scene or distinguish objects of interest (Russ, 1998). 1.2 MAIN OBJECTIVES Since the 1980s, radiation oncologists and biologists have recognized the need for additional assays on an individual patient basis that would select the most advantageous treatment approach. Though, it s important to have in mind that the cellular radiation sensitivity of the tumor may differ among individuals, even for tumors of the same histological type. If the radiosensitivity of the individual's tumor were precisely known, perhaps total radiation doses could be adjusted before the end of therapy to maximize tumor response (Joslin, 2001). The main objective of this monograph is to emphasize the importance and application of brachytherapy in the cure of cancer patients. To do this, it s performed a description of the theory important to understand the underlying biochemical events upon irradiation of the cells. These concepts include the knowledge of the cancer cell, regulation of cell cycle and apoptosis and the biological effects of radiation. This theoretic knowledge is important to proceed with to my dissertation thesis which consists in the image processing and analysis of the electron microscopic cell images of cancer irradiated cells with the brachytherapy radiation technique. 1.3 REPORT ORGANIZATION It was intended to organize this document in a self-directed and self-regulating approach to improve the access to various topics structured in eight chapters. So, it will be described very succinctly what is treated in each remaining chapter: ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 4

23 CHAPTER I INTRODUCTION TO THE THEME AND REPORT ORGANIZATION Chapter II Cell cycle regulation and apoptosis In this chapter takes place a description of key concepts related to the cell cycle checkpoints, to the behavior of the malignant cells and to the cellular death mechanisms among other information related to the normal and malignant cells. Chapter III Cancer cell This chapter focuses the characteristics of the cancer cells in comparison with normal cells, as well as the stages that the normal cell passes to become a cancer cell. Chapter IV Radiation and biological effects in cancer cells In this chapter it is presented a description of the irradiated carcinogenesis as well as the cell death mechanisms. It is also described important issues regarding the cellular behavior upon irradiation. Chapter V Cell culture and flow cytometry In this fifth chapter it is performed an approach of some important issues regarding the safety manipulation and maintenance of cells when performing cell culture techniques. It is also described the methods to induce cell cycle checkpoints and the flow cytometry technique. Chapter VI Brachytherapy In this chapter a description of one of the radiation technique to kill cancer cells is made. In addition it is mentioned the types of sources used in this radiation technique as well as the biological events occurring in the cancer cells upon irradiation. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 5

24 CHAPTER I INTRODUCTION TO THE THEME AND REPORT ORGANIZATION Chapter VII Image Processing and Analysis In this chapter it is performed a description of the basic concepts of the image processing. This chapter is important to emphasize the image processing and segmentation that will be performed in my thesis to extract information of the irradiated cancer cell images. This analysis will be performed using the MATLAB image processing toolbox. Chapter VIII Final Conclusions and Future Works In the last chapter it is presented the final conclusions of the work performed, as well as the future perspectives regarding the execution of the correspondent thesis. 1.4 MAJOR CONTRIBUTIONS This work consists in exposing the theory about cell cycle regulation and checkpoints that help to understand the behavior of cells when they are irradiated with the radiation technique named brachytherapy. This information will be helpful to study the electron microscopy images of breast cancer cells submitted to brachytherapy for the thesis work. In addition a description of the image processing and analysis is made, which is very important to understand the steps that need to the performed to be able to extract useful information of images. It is also important to highlight the importance of this tool as a technical aid and complement to the extraction of information on biological and biochemical events. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 6

25 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS

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27 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS INTRODUCTION The development of knowledge about the biochemistry and cell biology of cancer comes from a number of disciplines. Some of this knowledge has come from research initiated a century or more ago. There has been a flow of information about genetics into a knowledge base about cancer, starting with Gregor Mendel and the discovery of the principle of inherited traits and leading through Theodor Boveri s work on the chromosomal mode of heredity and chromosomal damage in malignant cells to Avery s discovery of DNA as the hereditary principle, Watson and Crick s determination of the structure of DNA, the human genome project, DNA microarrays, and proteomics. Not only has this information provided a clearer understanding of the carcinogenic process, it has also provided better diagnostic approaches and new therapeutic targets for anticancer therapies (Ruddon, 2007). Cancer cells contain many alterations, which accumulate as tumors develop. Over the last 25 years, considerable information has been gathered on the regulation of cell growth and proliferation leading to the identification of the proto-oncogenes and the tumor suppressor genes. The proto-oncogenes encode proteins, which are important in the control of cell proliferation, differentiation, cell cycle control and apoptosis. Mutations in these genes act dominantly and lead to a gain in function. In contrast the tumor suppressor genes inhibit cell proliferation by arresting progression through the cell cycle and block differentiation. They are recessive at the level of the cell although they show a dominant mode of inheritance. In addition, other genes are also important in the development of tumors. Mutations leading to increase genomic instability suggest defects in mismatch and excision repair pathways. Genes involved in DNA repair, when mutated, also predispose the patient to developing cancer (Macdonald, 2005). A crucial decision in every proliferating cell is the decision to continue with a further round of cell division or to exit the cell cycle and return to the stationary phase. Similarly quiescent cells must make the decision, whether to remain in the stationary phase (G 0 ) or to enter into the cell cycle. Entry into the cycle occurs in response to mitogenic signals and exit in response to withdrawal of these signals. To ensure that ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 9

28 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS DNA replication is complete and that any damaged DNA is repaired, cells must pass through specific checkpoints. Tumor cells undergo uncontrolled proliferation either due to mutations in the signal transduction pathways or because of mutations in the regulatory mechanism of the cell cycle (Macdonald, 2005). In this chapter, it is provided a detailed description of the cell cycle, its progression and the cellular events involved in transforming normal cells into malignant cells. For this purpose, the chapter starts with the explanation of the cell cycle followed by the description of the progression of the cell cycle, the growth characteristics of the malignant cells and the cell cycle regulation. After this, the chapter focuses the importance of the apoptosis phenomena and ends referring the resistance to apoptosis in cancer cells and potential targets for therapy. 2.2 CELL LIFE CYCLE The cell life cycle includes the changes a cell undergoes from the time it is formed until it divides to produce two new cells. The life cycle of a cell has two stages, an interphase and a cell division stage, Figure 2.1 (Seelev, 2004). Figure 2.1 Cell cycle (from (Seeley, 2004)) Interphase Interphase is the phase between cell divisions. Ninety percent or more of the life cycle of a typical cell is spent in interphase and, during this time the cell carries out ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 10

29 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS the metabolic activities necessary for life and performs its specialized functions such as secreting digestive enzymes. In addition, the cell prepares to divide which includes an increase in cell size; because many cell components double in quantity, and a replication of the cell s DNA. Consequently, the centrioles within the centrosome are also duplicated, when the cell divides, each new cell receives the organelles and DNA necessary for continued functioning. Interphase can be divided into three subphases, called G 1, S, and G 2. During G 1 (the first gap phase) and G 2 (the second gap phase), the cell carries out routine metabolic activities. During the S phase (the synthesis phase), the DNA is replicated (new DNA is synthesized) (Seelev, 2004). Many cells in the human body do not divide for days, months, or even years. These resting cells exit and enter the cell cycle that is called the G 0 phase, in which they remain, unless, stimulated to divide (Seelev, 2004) DNA Replication DNA replication is the process by which two new strands of DNA are made, using the two existing strands as templates. During interphase, DNA and its associated proteins appear as dispersed chromatin threads within the nucleus. When DNA replication begins, the two strands of each DNA molecule separate from each other for some distance, Figure 2.2. Then, each strand functions as a template, or pattern, for the production of a new strand of DNA, which is formed as new nucleotides pair with the existing nucleotides of each strand of the separated DNA molecule. The production of the new nucleotide strands is catalyzed by DNA polymerase, which adds new nucleotides at the 3` end of the growing strands. One strand, called the leading strand, is formed as a continuous strand, whereas the other strand, called the lagging strand, is formed in short segments going in the opposite direction. The short segments are then spliced by DNA ligase. As a result of DNA replication, two identical DNA molecules are produced, each of them having one strand of nucleotides derived from the original DNA molecule and one newly synthesized strand (Seelev, 2004). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 11

30 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure 2.2 Replication of DNA (from (Seelev, 2004)) Cell Division New cells necessary for growth and tissue repair are produced by cell division. A parent cell divides to form two daughter cells, each of which has the same amount and type of DNA as the parent cell. Because DNA determines cell structure and function, the daughter cells have identical structure and perform the same functions as the parent cell. Cell division involves two major events: the division of the nucleus to form two new nuclei, and the division of the cytoplasm to form two new cells. Each of the new cells contains one of the newly formed nuclei. The division of the nucleus occurs by mitosis, and the division of the cytoplasm is called cytokinesis (Seelev, 2004) Mitosis Mitosis is the division of the nucleus into two nuclei, each of which has the same amount and type of DNA as the original nucleus. The DNA, which was dispersed as chromatin in interphase, condenses in mitosis to form chromosomes. All human somatic cells, which include all cells except the sex cells, contain 46 chromosomes, which are referred to as a diploid number of chromosomes. Sex cells have half the number of chromosomes as somatic cells (Seelev, 2004). The 46 chromosomes in somatic cells are organized into 23 pairs of chromosomes. Twenty-two of these pairs are called autosomes. Each member of an ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 12

31 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS autosomal pair of chromosomes looks structurally alike, and together they are called a homologous pair of chromosomes. One member of each autosomal pair is derived from the person s father, and the other is derived from the mother. The remaining pair of chromosomes is the sex chromosomes. In females, the sex chromosomes look alike, and each is called an X chromosome. In males, the sex chromosomes do not look similar. One chromosome is an X chromosome, and the other is smaller and is called a Y chromosome. One X chromosome of a female is derived from her mother and the other is derived from her father. The X chromosome of a male is derived from his mother and the Y chromosome is derived from his father (Seelev, 2004). Mitosis is divided into four phases: prophase, metaphase, anaphase, and telophase. Although each phase represents major events, mitosis is a continuous process, and no discrete jumps occur from one phase to another. Learning the characteristics associated with each phase is helpful, but a more important concept is how each daughter cell obtains the same number and type of chromosomes as the parent cell. The major events of mitosis are summarized in Figure 2.3 (Seelev, 2004). Figure 2.3 Mitosis. (1) Interphase; (2) Prophase; (3) Metaphase; (4) Anaphase; (5) Telophase; (6) Interphase, Cytokinesis (from (Seelev, 2004)) ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 13

32 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Cytokinesis Cytokinesis is the division of the cytoplasm of the cell to produce two new cells (Figure 2.3). Cytokinesis begins in anaphase continues through telophase and ends in the following interphase. The first sign of cytokinesis is the formation of a cleavage furrow, or puckering of the plasma membrane, which forms midway between the centrioles. A contractile ring composed primarily of actin filaments pulls the plasma membrane inward, dividing the cell into two halves. Cytokinesis is complete when the membranes of the two halves separate at the cleavage furrow to form two separate cells (Seelev, 2004) Meiosis All cells of the body are formed by mitosis, except sex cells that are formed by meiosis. In meiosis the nucleus undergoes two divisions resulting in four nuclei, each containing half as many chromosomes as the parent cell. The daughter cells that are produced by cytokinesis differentiate into gametes, or sex cells. The gametes are reproductive cells sperm cells in males and oocytes (egg cells) in females. Each gamete not only has half the number of chromosomes found in a somatic cell but also has one chromosome from each of the homologous pairs verified in the parent cell. The complement of chromosomes in a gamete is referred to as a haploid number. Oocytes contain one autosomal chromosome from each of the 22 homologous pairs and an X chromosome. Sperm cells have 22 autosomal chromosomes and either an X or Y chromosome. During fertilization, when a sperm cell fuses with an oocyte, the normal number of 46 chromosomes in 23 pairs is reestablished. The sex of the baby is determined by the sperm cell that fertilizes the oocyte. The sex is male if a Y chromosome is carried by the sperm cell that fertilizes the oocyte and female if the sperm cell carries an X chromosome (Seelev, 2004). The first division during meiosis is divided into four phases: prophase I, metaphase I, anaphase I, and telophase I, Figure 2.4. As in prophase of mitosis, the nuclear envelope degenerates, spindle fibers form, and the already duplicated chromosomes become visible. Each chromosome consists of two chromatids joined by a centromere. In prophase I, however, the four chromatids of a homologous pair of ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 14

33 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS chromosomes join together, or synapse, to form a tetrad. In metaphase I the tetrads align at the equatorial plane and in anaphase I each pair of homologous chromosomes separate and move toward opposite poles of the cell (Seelev, 2004). For each pair of homologous chromosomes, one daughter cell receives one member of the pair, and the other daughter cell receives the other member. Thus each daughter cell has 23 chromosomes, each of which is composed of two chromatids. Telophase I with cytokinesis is similar to telophase of mitosis and two daughter cells are produced. Interkinesis is the phase between the formation of the daughter cells and the second meiotic division. No duplication of DNA occurs during this phase. The second division of meiosis also has four phases: prophase II, metaphase II, anaphase II, and telophase II. These stages occur much as they do in mitosis, except that 23 chromosomes are present instead of 46 (Seelev, 2004). The chromosomes align at the equatorial plane in metaphase II, and their chromatids split apart in anaphase II. The chromatids then are called chromosomes, and each new cell receives 23 chromosomes. In addition to reducing the number of chromosomes in a cell from 46 to 23, meiosis is also responsible for genetic diversity for two reasons: A random distribution of the chromosomes is received from each parent. One member of each homologous pair of chromosomes was derived from the person s father and the other member from the person s mother. The homologous chromosomes align randomly during metaphase I when they split apart, each daughter cell receives some of the father s and some of the mother s chromosomes. The number of chromosomes each daughter cell receives from each parent is determined by chance; However, when tetrads are formed, some of the chromatids may break apart, and part of one chromatid from one homologous pair may be exchanged for part of another chromatid from the other homologous pair, Figure 2.5. This exchange is called crossing-over; as a result, chromatids with different DNA content are formed, Figure 2.5. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 15

34 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS With random assortment of homologous chromosomes and crossing-over, the possible number of gametes with different genetic makeup is practically unlimited. When the distinct gametes of two individuals unite, it is virtually certain that the resulting genetic makeup never has occurred before and never will occur again. The genetic makeup of each new human being is unique (Seelev, 2004). Figure 2.4 Meiosis (from (Seelev, 2004)) ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 16

35 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure 2.5 Crossing-over (from (Seelev, 2004)) PROGRESSION OF THE CELL CYCLE The cell cycle is controlled by a complex pattern of synthesis and degradation of regulators together with careful control of their spatial organization in specific subcellular compartments. In addition, checkpoint controls can modulate the progression of the cycle in response to adverse conditions such as DNA damage. Cells either enter G 1 from G 0 in response to mitogenic stimulation or follow on from cytokinesis if actively proliferating (i.e. from M to G 1 ). Removal of mitogens allows them to return to G 0. The critical point between mitogen dependence and independence is the restriction point or R which occurs during G 1. It is here that cells reach the point of no return and are committed to a round of replication (Macdonald, 2005), Figure 2.6. Figure 2.6 Restriction point, R (from (Griffiths, 1999)) ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 17

36 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Synthesis of the D-type cyclins begins at the G 0 /G 1 transition and continues so long as growth factor stimulation persists. This mitogen stimulation of cyclin D is in part dependent on RAS activation, a role which is highlighted by the ability of anti-ras antibodies to block the progression of the cell cycle if added to cells prior to mitogen stimulation. The availability of cyclin D activates CDK4 and 6 and these complexes then drive the cell from early G 1 through R to late G 1 ; largely by regulation of RB which exists in a phosphorylated state at the start of G 1 complexed to a large number of proteins. Cyclin D-CDK4/6 activation begins phosphorylation of Rb during early G 1. This initial phosphorylation leads to release of histone deacetylase activity from the complex alleviating transcriptional repression. The E2F transcription factor remains bound to Rb at this stage but can still transcribe some genes including cyclin E. Therefore, levels of cyclin E increase and lead to activation of CDK2, which can then complete phosphorylation of Rb. Consequently, complete phosphorylation of Rb results in the release of E2F to activate genes required to drive cells through the G 1 /S transition (Macdonald, 2005), Figure 2.7. Figure 2.7 Regulation of the G1 to S transition (from (Griffiths, 1999)) The CKIs also play a role in control of cell cycle progression at this stage and in response to antimitogenic signals, oppose the activity of the CDKs and cause cell cycle arrest. INK4 inhibitors bind to CDK4/6 to prevent cyclin D binding and CIP/KIP inhibitors similarly inhibit the kinase activity of cyclin ECDK2, Figure 2.8. CIP/KIP inhibitors also interact with cyclin D-CDK4/6 complexes during G 1, but rather than blocking cell cycle progression, this interaction is required for the complete function of ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 18

37 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS the complex and allows G 1 progression. This interaction sequesters CIP/KIP, preventing its inhibition of cyclin E-CDK2 and thereby facilitating its full activation to contribute to G 1 progression. In the presence of an antimitogenic signal, levels of cyclin D-CDK4/6 are reduced, CIP/KIP is released, which can then interact with and inhibit CDK2 to cause cell cycle arrest (Macdonald, 2005). Cells which have suffered DNA damage are prevented from entering S phase and are blocked at G 1. This process is dependent on the tumor suppressor gene p53 and p21. Activation of p53 by DNA damage results in increased p21 levels which can then inactivate cyclin E-CDK2 to prevent phosphorylation of Rb and inhibit the release of E2F to promote transcription of genes involved in DNA synthesis, Figure 2.8. This causes the cell cycle to arrest in G 1. Clearly, loss or mutation of p53 will lead to loss of this checkpoint control and cells will be able to enter S phase with damaged DNA. After cells have entered S phase, cyclin E is rapidly degraded and CDK2 is released. In S phase, a further set of cyclins and CDKs, cyclin A-CDK2, are required for continued DNA replication. Two A-type cyclins have been identified to date: cyclin A1 is expressed during meiosis and in early cleavage embryos whereas cyclin A2 is present in all proliferating cells. Cyclin A2 is also induced by E2F and is expressed from S phase through G 2 and M until prometaphase when it is degraded by ubiquitin-dependent proteolysis (Macdonald, 2005). Cyclin A2 binds to two different CDKs. Initially, during S phase, it is found complexed to CDK2 following its release from cyclin E and subsequently in G 2 and M it is found complexed to CDC2 (also known as CDK1). Cyclin A2 has a role in both transcriptional regulation and DNA replication and its nuclear localization is crucial to its function. Cyclin A regulates the E2F transcription factor and in S phase, when E2F directed transcription is no longer required, cyclin A directs its phosphorylation by CDK2 leading to its degradation. This down-regulation by cyclin A2 is required for orderly S phase progression and in its absence apoptosis occurs. Recently, cyclin A as well as cyclin E have been shown to be regulators of centrosome replication and are able to do so because of their ability to shuttle between nucleus and cytoplasm, Figure 2.9 (Macdonald, 2005). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 19

38 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure 2.8 Cell cycle arrest at G 1 /S, mediated by cdk inhibitors (from (Shapiro, 1999)) Figure 2.9 Dynamics of the DNA synthesome (from (Frouin, 2003)) ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 20

39 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS The final phase of the cycle is M phase that comprises mitosis and cytokinesis. The purpose of mitosis is to segregate sister chromatids into two daughter cells so that each cell receives a complete set of chromosomes, a process that requires the assembly of the mitotic spindle. Mitosis is split into a number of stages that includes prophase, prometaphase, metaphase, anaphase and telophase (Macdonald, 2005). Cytokinesis, the process of cytoplasmic cleavage, follows the end of mitosis and its regulation is closely linked to mitotic progression. Mitosis involves the last of cyclin/cdks, cyclin B1 and CDC2 as well as additional mitotic kinases. These include members of the Polo family (PLK1), the aurora family (aurora A, B and C) and the NIMA family (NEK2) plus kinases implicated at the mitotic checkpoints (BUB1), mitotic exit and cytokinesis (Macdonald, 2005). Entry into the final phase of the cell cycle, mitosis, is signaled by the activation of the cyclin B1-CDC2 complex also known as the M phase promoting factor or MPF. This complex accumulates during S and G 2, but is kept in the inactive state by phosphorylation of tyrosine 15 and threonine 14 residues on CDC2 by two kinases, WEE1 and MYT1. WEE1 is nuclear and phosphorylates tyrosine 15, whereas MYT1 is cytoplasmic and phosphorylates threonine 14. At the end of G 2, the CDC25 phosphatase is stimulated to dephosphorylate these residues thereby activating CDC2. These enzymes are all controlled by DNA structure checkpoints which delay the onset of mitosis if DNA is damaged. Regulation of cyclin B1-CDC2 is also regulated by localization of specific subcellular compartments. It is initially localized to the cytoplasm during G 2, but is translocated to the nucleus at the beginning of mitosis. A second cyclin B, cyclin B2, also exists in mammalian cells and is localized to the Golgi and endoplasmic reticulum where it may play a role in disassembly of the Golgi apparatus at mitosis (Macdonald, 2005). A further checkpoint exists at the end of G 2 which checks that DNA is not damaged before entry into M. Once more p21 activation by p53 can arrest the cell cycle as at the end of G 1. In addition, the CHK1 kinase can phosphorylate CDC25 to create a binding site for the protein, a process which inactivates CDC25, thereby preventing dephosphorylation of CDC2 and halting the cell cycle, Figure 2.10 (Macdonald, 2005). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 21

40 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Tumor cells can enter mitosis with damaged DNA, suggesting a defect in the G 2 /M checkpoint. Tumor cell lines have been shown to activate the cyclin B-CDC2 complex irrespective of the state of the DNA. Activation of cyclin B1-CDC2 leads to phosphorylation of numerous substrates including the nuclear lamins, microtubulebinding proteins, condensins and Golgi matrix components that are all needed for nuclear envelope breakdown, centrosome separation, spindle assembly, chromosome condensation and Golgi fragmentation respectively. During prophase, the centrosomes structures which organize the microtubules and which were duplicated during G 2 separate to define the poles of the future spindle apparatus, a process regulated by several kinases including the NIMA family member NEK2, as well as aurora A. At the same time centrosomes begin nucleating the microtubules which make up the mitotic spindle (Macdonald, 2005). Chromatin condensation also occurs accompanied by extensive histone phosphorylation to produce well defined chromosomes. Nuclear envelope breakdown occurs shortly after centrosome separation. The nuclear envelope is normally stabilized by a structure known as the nuclear lamin which is composed of lamin intermediate filament proteins. This envelope is broken down as a result of hyperphosphorylation of lamins by cyclin B-CDC2 (Macdonald, 2005). During prometaphase, the microtubules are captured by kinetochores, the structure which binds to the centromere of the chromosome. Paired sister chromatids interact with the microtubules emanating from opposite poles resulting in a stable bipolar attachment. Chromosomes then sit on the metaphase plate where they oscillate during metaphase. Once all bipolar attachments are complete anaphase is triggered. This is characterized by simultaneous separation of all sister chromatids. Each chromosome must be aligned in the center of the bipolar spindle such that its two sister chromatids are attached to opposite poles. If this is correct, the anaphasepromoting complex (APC) together with CDC20 is activated to control degradation of proteins such as securin. This in turn activates the separin protease which cleaves the cohesion molecules between the sister chromatids allowing them to separate. At this stage, there is one final checkpoint, the spindle assembly checkpoint, at the metaphase to anaphase transition, which checks the correct assembly of the mitotic ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 22

41 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS apparatus and the alignment of chromosomes on the metaphase plate. The gatekeeper at this checkpoint is the APC complex. Unaligned kinetochores are recognized and associate with the MAD2 and BUB proteins which can prevent activation of APC and cell arrest at metaphase preventing exit from mitosis. In tumor cell abnormalities of spindle formation are found, suggesting that checkpoint control is lost (Macdonald, 2005). Mitotic exit requires that sister chromatids have separated to opposite poles. During telophase, nuclear envelopes can begin to form around the daughter chromosomes and chromatin decondensation occurs. The spindle is also disassembled and cytokinesis is completed. The control of these processes requires destruction of both the cyclins and other kinases such as NIMA and aurora family members by ubiquitin dependent proteolysis mediated by APC. Daughter cells can now re-enter the cell cycle (Macdonald, 2005). Figure 2.10 Cell cycle regulation of cyclin dependent kinase (Cdk1) Cyclin-B (CycB) complex (from (Novák, 2010)) ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 23

42 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS GROWTH CHARACTERISTICS OF MALIGNANT CELLS Cancer can be characterized as a disease of genetic instability, altered cellular behavior and altered cell extracellular matrix interactions. These alterations lead to dysregulated cell proliferation, and ultimately to invasion and metastasis. There are interactions between the genes involved in these steps. For example, the genes associated with loss of control of cell proliferation may also be involved in genetic instability (rapidly proliferating cells have less time to repair DNA damage) and tumor vascularization that leads to dysregulated proliferation of cells, which in turn eats up more oxygen, creates hypoxia, and turns on HIF-1 and additional angiogenesis. Similarly, genes involved in tumor cell invasion may also be involved in loss of growth control (invasive cells have acquired the skills to survive in hostile new environments) and evasion of apoptosis (less cell death even in the face of a normal rate of cell proliferation produces more cells). The molecular genetic alterations of cancer cells lead to cells that can generate their own growth-promoting signals are less sensitive to cell cycle checkpoint controls, evade apoptosis, and thus have almost limitless replication potential. This redundancy makes design of effective signal transduction-targeted chemotherapeutic drugs that target a single pathway very difficult indeed (Ruddon, 2007). Cancer cells can also subvert the environment in which they proliferate. Alterations in both cell cell and cell extracellular matrix interactions also occur, leading to creation of a cancer-facilitating environment. For example, a common alteration in epithelial carcinomas is alteration of E-cadherin expression, which is a cell cell adhesion molecule found on all epithelial cells. Cancer cells exhibit remarkable plasticity and have the ability to mimic some of the characteristics of other cell types as they progress and became less well differentiated. For example, cancer cells may assume some of the structure and function of vascular cells. As cancer cells metastasize, they may eventually take on a new phenotype such that the tissues of origin may become unclear so-called cancers of unknown primary site (Ruddon, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 24

43 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Phenotypic Alterations in Cancer Cells Treatment of animals or cells in culture with carcinogenic agents is a means of studying discrete biochemical events that lead to malignant transformation, Figure However, studies of cell transformation in vitro have many pitfalls. These tissue culture artifacts include overgrowth of cells not characteristic of the original population of cultured cells (e.g., overgrowth of fibroblasts in cultures that were originally primarily epithelial cells), selection for a small population of variant cells with continued passage in vitro, or appearance of cells with an abnormal chromosomal number or structure (karyotype). Such changes in the characteristics of cultured cell populations can lead to spontaneous transformation that mimics some of the changes seen in populations of cultured cells treated with oncogenic agents. Thus, it is often difficult to sort out the critical malignant events from the noncritical ones (Ruddon, 2007). Figure 2.11 Cellular response (from (Gil, 2006)) Although closer to the carcinogenic process in humans, malignant transformation induced in vivo by treatment of susceptible experimental animals with carcinogenic chemicals or oncogenic viruses or by irradiation, is even more difficult because it is hard to discriminate toxic from malignant events and to determine what role a myriad of factors, such as the nutritional state of the animal, hormone levels, or endogenous infections with microorganisms or parasites, might have on the in vivo carcinogenic events. Moreover, tissues in vivo are a mixture of cell types, and it is ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 25

44 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS difficult to determine in which cells the critical transformation events are occurring and what role the microenvironment of the tissue plays. Thus, most studies designed to identify discrete biochemical events occurring in cells during malignant transformation have been done with cultured cells, since clones of relatively homogeneous cell populations can be studied and the cellular environment defined and manipulated. The ultimate criterion that establishes whether cells have been transformed, however, is their ability to form a tumor in an appropriate host animal. The generation of immortalized normal cell lines of a given differentiated phenotype from human embryonic stem cells, has enhanced the ability to study cells of a normal genotype from a single source. Such cell lines may also be generated by transfection of the telomerase gene into cells to maintain chromosomal length (Ruddon, 2007). Over the past 60 years, much scientific effort has gone into research aimed at identifying the phenotypic characteristics of in vitro transformed cells that correlate with the growth of a cancer in vivo. This research has tremendously increased our knowledge of the biochemistry of cancer cells. However, many of the biochemical characteristics initially thought to be closely associated with the malignant phenotype of cells in culture has subsequently been found to be dissociable from the ability of those cells to produce tumors in animals. Furthermore, individual cells of malignant tumors growing in animals or in humans exhibit marked biochemical heterogeneity, as reflected in their cell surface composition, enzyme levels, immunogenicity, response to anticancer drugs, and so on. This has made it extremely difficult to identify the essential changes that produce the malignant phenotype (Ruddon, 2007) Immortality of Transformed Cells in Culture Most normal diploid mammalian cells have a limited life expectancy in culture. For example, normal human fibroblast lines may live for 50 to 60 population doublings (the Hayflick index ), but then viability begins to decrease rapidly, unless they transform spontaneously or are transformed by oncogenic agents. However, malignant cells, once they become established in culture, will generally live for an indefinite number of population doublings, provided the right nutrients and growth factors (Ruddon, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 26

45 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS It is not clear what limits the life expectancy of normal diploid cells in culture, but it may be related to the continual shortening of chromosomal telomeres each time cells divide. Transformed cells are known to have elevated levels of telomerase that maintain telomere length. Transformed cells that become established in culture also frequently undergo karyotypic changes, usually marked by an increase in chromosomes (polyploidy), with continual passage. This suggests that cells with increased amounts of certain growth-promoting genes are generated and/or selected during continual passage in culture. The more undifferentiated cells from cancers of animals or patients also often have an atypical karyology, thus the same selection process may be going on in vivo with progression over time of malignancy from a lower to a higher grade (Ruddon, 2007) Decreased Requirement for Growth Factors Other properties that distinguish transformed cells from their non transformed counterparts are decreased density-dependent inhibition of proliferation and the requirement for growth factors for replication in culture. Cells transformed by oncogenic viruses have lower serum growth requirements than do normal cells. Cancer cells may also produce their own growth factors that may be secreted and activate proliferation in neighboring cells (paracrine effect) or, if the same malignant cell type has both the receptor for a growth factor and the means to produce the factor, selfstimulation of cell proliferation (autocrine effect) may occur. One example of such an autocrine loop is the production of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR1 by diffuse large cell lymphoma. Co-expression of TNF-α and its receptor are negative prognostic indicators of survival, suggesting that autocrine loops can be powerful stimuli for tumor aggressiveness and thus potentially important diagnostic and therapeutic targets Loss of Anchorage Dependence Most freshly isolated normal animal cells and cells from cultures of normal diploid cells do not grow well when they are suspended in fluid or a semisolid agar gel. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 27

46 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS However, if these cells contact with a suitable surface they attach, spread, and proliferate. This type of growth is called anchorage-dependent growth. Many cell lines derived from tumors and cells transformed by oncogenic agents are able to proliferate in suspension cultures or in a semi solid medium (methylcellulose or agarose) without attachment to a surface. This is called anchorage-independent growth and this property of transformed cells has been used to develop clones of malignant cells. This technique has been widely used to compare the growth properties of normal and malignant cells. Another advantage that has been derived from the ability of malignant cells to grow in soft agar (agarose), is the ability to grow cancer cells derived from human tumors to test their sensitivity to chemotherapeutic agents and to screen for potential new anticancer drugs (Ruddon, 2007) Loss of Cell Cycle Control and Resistance to Apoptosis Normal cells respond to a variety of suboptimal growth conditions by entering a quiescent phase in the cell division cycle, the G 0 state. There appears to be a decision point in the G 1 phase of the cell cycle, at which time the cell must make a commitment to continue into the S phase, the DNA synthesis step, or to stop in G 1 and wait until conditions are more optimal for cell replication to occur. If this waiting period is prolonged, the cells are said to be in a G 0 phase. Once cells make a commitment to divide, they must continue through S, G 2, and M to return to G 1. If the cells are blocked in S, G 2, or M for any length of time, they die. The events that regulate the cell cycle are called cell cycle checkpoints (Ruddon, 2007). The loss of cell cycle check point control by cancer cells may contribute to their increased susceptibility to anticancer drugs. Normal cells have mechanisms to protect themselves from exposure to growth-limiting conditions or toxic agents by calling on these check point control mechanisms. Cancer cells, by contrast, can continue through these checkpoints into cell cycle phases that make them more susceptible to the cytotoxic effects of drugs or irradiation. For example, if normal cells accrue DNA damage due to ultraviolet (UV) or X-irradiation, they arrest in G 1 so that the damaged DNA can be repaired prior to DNA replication. Another check point in the G 2 phase allows repair of chromosome breaks before chromosomes are segregated at mitosis, ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 28

47 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure Cancer cells, which exhibit poor or absent check point controls, proceed to replicate the damaged DNA, thus accounting for persisting and accumulating mutations (Ruddon, 2007) CELL CYCLE REGULATION Cyclin-dependent protein kinases (CDKs), of which CDC2 is only one, are crucial regulators of the timing and coordination of eukaryotic cell cycle events. Transient activation of members of this family of serine/threonine kinases occurs at specific cell cycle phases (Ruddon, 2007). Figure Major pathways where Plks may play a role in intra-s-phase checkpoint in mammalian systems (from (Suqing, 2005)) In budding yeast G 1 cyclins encoded by the CLN genes, interact with and are necessary for the activation of, the CDC2 kinase (also called p34cdc2), driving the cell cycle through a regulatory point called START (because it is regulated by the cdc2 or start gene) and committing cells to enter S phase. START is analogous to the G 1 restriction point in mammalian cells. The CDKs work by forming active heterodimeric complexes following binding to cyclins, their regulatory subunits. CDK2, 4, and 6, and ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 29

48 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS possibly CDK3 cooperate to push cells through G 1 into S phase. CDK4 and CDK6 form complexes with cyclins D1, D2, and D3, and these complexes are involved in completion of G 1. Cyclin D dependent kinases accumulate in response to mitogenic signals and this leads to phosphorylation of the Rb protein. This process is completed by the cyclin E1- and E2-CDK2 complexes. Once cells enter S phase, cyclin E is degraded and A1 and A2 cyclins get involved by forming a complex with CDK2. There are a number of regulators of CDK activities; where they act in the cell cycle is depicted in Figure 2.13 (Ruddon, 2007). Figure Restriction point control and the G1-S transition (from (Ruddon, 2007)) CDK Inhibitors The inhibitors of CDKs include the Cip/Kip and INK4 family of polypeptides. The Cip/Kip family includes p21cip1, p27kip1, and p57kip2. The actions of these proteins are complex. Although the Cip/Kip proteins can inhibit CDK2, they are also involved in the sequestration of cyclin D-dependent kinases that facilitates cyclin E-CDK2 activation necessary for G 1 /S transition (Ruddon, 2007). The INK4 proteins target the CDK4 and CDK6 kinases, sequester them into binary CDKINK4 complexes, and liberate bound Cip/Kip proteins. This indirectly inhibits cyclin E CDK and promotes cell cycle arrest. The INK4-directed arrest of the cell cycle in G 1 keeps Rb in a hypophosphorylated state and represses the expression of S-phase genes. Four INK4 proteins have been identified: p16ink4a, p15ink4b, p18ink4c, and ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 30

49 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS p19ink4d. INKA4a loss of function occurs in a variety of cancers including pancreatic and small cell lung carcinomas and glioblastomas. INK4a fulfills the criteria of a tumor suppressor and appears to be the INK4 family member with the most active role in this regard. The INK4a gene encodes another tumor suppressor protein called ARF (p14arf). Mice with a disrupted ARF gene have a high propensity to develop tumors, including sarcomas, lymphomas, carcinomas, and CNS tumors. These animals frequently die at less than 15 months of age. ARF and p53 act in the same pathway to insure growth arrest and apoptosis in response to abnormal mitogenic signals such as myc-induced carcinogenesis, Figure 2.14 (Ruddon, 2007). Figure Regulation of the Rho pathway and the cytoskeleton by cyclin-dependent kinase (CDK) inhibitors (from (Besson, 2004)) Cyclins The originally discovered cyclins, cyclin A and B, identified in sea urchins, act at different phases of the cell cycle. Cyclin A is first detected near the G 1 /S transition and ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 31

50 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS cyclin B is first synthesized during S phase and accumulates in complexes with p34cdc2 as cells approach the G 2 -to-m transition. Cyclin B is then abruptly degraded during mitosis. Thus, cyclins A and B regulate S and M phase, but do not appear to play a role in G 1 control points such as the restriction point (R point), which is the point where key factors have accumulated to commit cells to enter S phase (Ruddon, 2007). Three more recently discovered mammalian cyclins, C, D1, and E, are the cyclins that regulate the key G 1 and G 1 /S transition points. Unlike cyclins A and B, cyclins C, D1, and E are synthesized during the G1 phase in mammalian cells. Cyclin C levels change only slightly during the cell cycle but peak in early G 1. Cyclin E peaks at the G 1 S transition, suggesting that it controls entry into S. Three distinct cyclin D forms, D1, 2, and 3, have been discovered and are differentially expressed in different mouse cell lineages. These D cyclins all have human counterparts and cyclin D levels are growth factor dependent in mammalian cells: when resting cells are stimulated by growth factors, D-type cyclin levels rise earlier than cyclin E levels, implying that they act earlier in G 1 than E cyclins. Cyclin D levels drop rapidly when growth factors are removed from the medium of cultured cells. All of these cyclins (C, D, and E) form complexes with, and regulate the activity of various CDKs and these complexes control the various G 1, G 1 S, and G 2 M transition points, Figure 2.15 (Ruddon, 2007). Interestingly, negative growth regulators also interact with the cyclin-cdk system. For example, TGF-b1, which inhibits proliferation of epithelial cells by interfering with G 1 -S transition, reduced the stable assembly of cyclin E-CDK2 complexes in mink lung epithelial cells, and prevented the activation of CDK2 kinase activity and the phosphorylation of Rb. This was one of the first pieces of data suggesting that the mammalian G 1 cyclin-dependent kinases are targets for negative regulators of the cell cycle (Ruddon, 2007) Cell Cycle Checkpoints The role of various CDKs, cyclins, and other gene products in regulating checkpoints at G 1 to S, G 2 to M, and mitotic spindle segregation have been described in detail previously. Alterations of one or more of these checkpoint controls occur in most, if not all, human cancers at some stage in their progression to invasive cancer. A ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 32

51 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS key player in the G 1 S checkpoint system is the retinoblastoma gene Rb (Ruddon, 2007). Figure Cell-cycle regulation (from (Charles, 2004)) Phosphorylation of the Rb protein by cyclin D dependent kinase releases Rb from the transcriptional regulator E2F and activates E2F function. Inactivation of Rb by genetic alterations occurs in retinoblastoma and is also observed in other human cancers, for example, small cell lung carcinomas and osteogenic sarcomas (Ruddon, 2007). The p53 gene product is an important cell cycle checkpoint regulator at both the G 1 S and G 2 M checkpoints but does not appear to be important at the mitotic spindle checkpoint because gene knockout of p53 does not alter mitosis. The p53 tumor suppressor gene is the most frequently mutated gene in human cancer, indicating its important role in conservation of normal cell cycle progression. One of p53 s essential roles is to arrest cells in G 1 after genotoxic damage, to allow for DNA repair prior to DNA replication and cell division. In response to massive DNA damage, p53 triggers the apoptotic cell death pathway. Data from short-term cell-killing assays, using normal and minimally transformed cells, have led to the conclusion that mutated p53 protein confers resistance to genotoxic agents (Ruddon, 2007). The spindle assembly checkpoint machinery involves genes called bub (budding uninhibited by benomyl) and mad (mitotic arrest deficient). There are three bub genes and three mad genes involved in the formation of this checkpoint complex. A protein ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 33

52 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS kinase called Mps1 also functions in this checkpoint function. The chromosomal instability, leading to aneuploidy in many human cancers, appears to be due to defective control of the spindle assembly checkpoint. Mutant alleles of the human bub1 gene have been observed in colorectal tumors displaying aneuploidy. Mutations in these spindle checkpoint genes may also result in increased sensitivity to drugs that affect microtubule function because drug-treated cancer cells do not undergo mitotic arrest and go on to die (Ruddon, 2007). Maintaining the integrity of the genome is a crucial task of the cell cycle checkpoints. Two checkpoint kinases, called Chk1 and Chk2 (also called Cds1), are involved in checkpoint controls that affect a number of genes involved in maintenance of genome integrity. Chk1 and Chk2 are activated by DNA damage and initiate a number of cellular defense mechanisms that modulate DNA repair pathways and slow down the cell division cycle to allow time for repair. If DNA is not successfully mended, the damaged cells usually undergo cell death via apoptosis. This process prevents the defective genome from extending its paternity into daughter cells (Ruddon, 2007). Upstream elements activating the checkpoint signaling pathways such as those turned on by irradiation or agents causing DNA double strand breaks include the ATM kinase, a member of the phosphatidylinositol 3-kinase (PI3K) family, which activates Chk2 and its relative ATR kinase that activates Chk1. There is also cross talk between ATM and ATR that mediates these responses. Chk1 and Chk2 phosphorylate CDC25A and C, which inactivate them. In its dephosporylated state CDC25A activates the CDK2- cyclin E complex that promotes progression through S phase. It should be noted that this is an example of dephosphorylation rather than phosphorylation activating a key biological function. This is in contrast to most signal transduction pathways, where the phosphorylated state of a protein (often a kinase) is the active state and the dephosphorylated state is the inactive one. In addition, Chk1 renders CDC25A unstable, which also diminishes its activity. CDC25A also binds to and activates CDK1- cyclin B, which facilitates entry into mitosis. G 2 arrest induced by DNA damage induces CDC25A degradation and, in contrast, G 2 arrest is lost when CDC25A is overexpressed. A number of proteins are now known to act as mediators of checkpoint responses by impinging on the Chk1 and 2 pathways. These include the BRCT domain containing ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 34

53 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS proteins 53BP1, BRCA1, and MDC1.These proteins are involved in activation of Chk1 and Chk2 by acting through protein protein interactions that modulate the activity of these checkpoint kinases. In general, these modulators are thought to be tumor suppressors (Ruddon, 2007). Chk1 and 2 have overlapping roles in cell cycle regulation, but different roles during development. Chk1 but not Chk2 is essential for mammalian development, as evidenced by the early embryonic lethality of Chk1 knockout mice. Chk2-deficient mice are viable and fertile and do not have a tumor-prone phenotype unless exposed to carcinogens, and this effect is more evident later in life. As illustrated in Figure 2.16, there are interactions between the Chk kinases and the p53 pathway. Chk2 phosphorylates threonine-18 or serine-20 on p53, which attenuates p53 s interaction with its inhibitor MDM2, thus contributing to p53 stabilization and activation. However, Chk2 and p53 only have partially overlapping roles in checkpoint regulation because not all DNA-damaging events activate both pathways, Figure 2.16 (Ruddon, 2007) Cell Cycle Regulatory Factors as Targets for Anticancer Agents The commonly observed defects in cell cycle regulatory pathways in cancer cells distinguish them from normal cells and provide potential targets for therapeutic agents. One approach is to inhibit cell cycle checkpoints in combination with DNAdamaging drugs or irradiation. The rationale for this is that normal cells have a full complement of checkpoint controls, whereas tumor cells are defective in one or more of these and thus are more subject to undergoing apoptosis in response to excessive DNA damage. This has been accomplished by combining ATM/ATR inhibitors such as caffeine or Chk1 inhibitors in combination with DNA-damaging drugs. So far this approach has not been demonstrated clinically, and indeed is somewhat counter intuitive, since p53 mutant tumor cells are more resistant to many chemotherapeutic drugs. p53 is a key player in causing cell death in drug treated, DNA-damaged cells (one exception to that is the microtubule inhibitor paclitaxel), and active, unmutated p53 is needed for this response (Ruddon, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 35

54 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure Simplified scheme of cell-cycle checkpoint pathways induced in response to DNA damage (here DSBs), with highlighted tumor suppressors shown in red and proto-oncogenes shown in green (from (Kastan, 2004)) Another approach is to target the cyclin dependent kinases directly. Alteration of the G 1 S checkpoint occurs in many human cancers. Cyclin D1 gene amplification occurs in a subset of breast, esophageal, bladder, lung, and squamous cell carcinomas. Cyclins D2 and D3 are overexpressed in some colorectal carcinomas. In addition, the cyclin D associated kinases CDK4 and CDK6 are over expressed or mutated in some ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 36

55 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS cancers. Mutations or deletions in the CDK4 and CDK6 inhibitor INK4 have been observed in familial melanomas, and in biliary tract, esophageal, pancreatic, head and neck, non small cell lung, and ovarian carcinomas. Inactivating mutations of CDK4 inhibitory modulators p15, p16, and p18 have been observed in a wide variety of human cancers. Cyclin E is also amplified and overexpressed in some breast and colon carcinomas and leukemias (Ruddon, 2007). Human cancers have a variety of mutations in cell cycle regulatory genes. This includes overexpression of D1 and E1 cyclins and CDKs (mainly CDK4 and CDK6) as noted above. Loss of CDK inhibitory functions (mainly INK4a and 4b and Kip1) also occurs, as does loss of Rb, one of the first tumor suppressor genes identified. Loss of Kip1 function and overexpression of cyclin E1 occur frequently and are associated with poor prognosis in breast and ovarian cancers (Ruddon, 2007). The mitogen-stimulated proliferation of cells is mediated via a retinoblastoma (Rb) pathway that involves phosphorylation of Rb, its dissociation form and activation of the E2F family of transcription factors, and subsequent turn-on of genes involved in G 1 S transition and DNA synthesis. Disruption of this pathway by overexpression of cyclin D1, loss of the INK4 inhibitor p16, mutation of CDK4 to a p16-resistant form, or loss or mutation of Rb is frequently seen in cancer cells. The activation of CDK inhibitory factors such as p16ink4 or p27kip1 and inhibition of cyclin dependent kinases are, therefore, potential ways to interdict the overactive cell proliferation pathways in cancer cells. Thus, inhibition of cyclins D1 and E and CDKs, especially CDK4 and CDK6, could be targets for inhibiting growth of cancers. As more knowledge of the complicated steps in cell cycle regulation is gained, more potential targets become available (Ruddon, 2007) APOPTOSIS Apoptosis (sometimes called programmed cell death) is a cell suicide mechanism that enables multicellular organisms to regulate cell number in tissues and to eliminate unneeded or aging cells as an organism develops. The biochemistry of apoptosis has been well studied in recent years, and the mechanisms are now reasonably well understood (Ruddon, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 37

56 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS The apoptosis pathway involves a series of positive and negative regulators of proteases called caspases, which cleave substrates, such as poly (ADP-ribose) polymerase, actin and lamin. In addition, apoptosis is accompanied by the intranucleosomal degradation of chromosomal DNA, producing the typical DNA ladder seen for chromatin isolated from cells undergoing apoptosis. The endonuclease responsible for this effect is called caspase-activated DNase, or CAD (Ruddon, 2007). A number of death receptors have also been identified, they are cell surface receptors that transmit apoptotic signals initiated by death ligands, Figure The death receptors sense signals that tell the cell that it is in an uncompromising environment and needs to die. These receptors can activate the death caspases within seconds of ligand binding and induce apoptosis within hours. Death receptors belong to the tumor necrosis factor (TNF) receptor gene superfamily and have the typical cystine rich extracellular domains and an additional cytoplasmic sequence termed the death domain (Ruddon, 2007). Figure Apoptosis signaling through death receptors (from (Frederik, 2002)) ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 38

57 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS The best-characterized death receptors are CD95 (also called Fas or Apo1) and TNF receptor TNFR1 (also called p55 or CD120a). The importance of the apoptotic pathway in cancer progression is seen when there are mutations that alter the ability of the cell to undergo apoptosis and allow transformed cells to keep proliferating rather than die. Such genetic alterations include the translocation of the bcl-2 gene in lymphomas that prevents apoptosis and promotes resistance to cytotoxic drugs. Other genes involved as players on the apoptosis stage include c-myc, p53, c-fos, and the gene for interleukin-1b-converting enzyme (ICE). Various oncogene products can suppress apoptosis, like the adenovirus protein E1b, ras, and n-abl (Ruddon, 2007). Mitochondria plays a pivotal role in the events of apoptosis by at least three mechanisms: 1) Release of proteins, e.g., cytochrome c, that triggers activation of caspases; 2) Alteration of cellular redox potential; 3) Production and release of reactive oxygen species after mitochondrial membrane damage. Another mitochondrial link to apoptosis is implied by the fact that Bcl-2, the anti-apoptotic factor, is a mitochondrial membrane protein that appears to regulate mitochondrial ion channels and proton pumps, Figure 2.18 (Ruddon, 2007) Biochemical Mechanism of Apoptosis Multicellular organisms, from the lowest to the highest species, must have a way to get rid of excess cells or cells that are damaged in order for the organism to survive. Apoptosis is the mechanism that they use to do this. It is the way that the organism controls cell numbers and tissue size and protects itself from rogue cells. A simplified version of the apoptotic pathways can be visualized in Figure 2.19 (Ruddon, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 39

58 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure Apoptosis signaling through mitochondria (from (Frederik, 2002)) The death receptor mediated pathway is turned on by members of the death receptor superfamily of receptors including Fas receptor (CD95) and TNF receptor 1, which are activated by Fas ligand and TNF, respectively. Interaction of these ligands with their receptors induces receptor clustering, binding of the receptor clusters to Fas-associated death domain protein (FADD), and activation of caspase-8, Figure This activation step is regulated by c-flip. Caspase-8, in turn, activates caspase-3 and other executioner caspases, which induce a number of apoptotic substrates. The DNA damage induced pathway invokes a mitochondrial-mediated cell death pathway that involves pro-apoptotic factors like Bax (blocked by the anti-apoptotic protein Bcl- 2). This results in cytochrome c release from the mitochondria and triggering of downstream effects facilitating caspase-3 activation, which is where the two pathways intersect. There are both positive and negative regulators that also interact on these pathways (Ruddon, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 40

59 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure The two main apoptotic signaling pathways (from (Frederik, 2002)) Figure Illustration of the main TNF receptor signaling pathways (from (Dash, 2003)) ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 41

60 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Caspases Caspases are a family of cysteine proteases that are activated specifically in apoptotic cells. This family of proteases is highly conserved through evolution all the way from hydra and nematodes up to humans. Over 12 caspases have been identified and although most of them appear to function during apoptosis, the function of all of them is not yet clear. The caspases are called cysteine-proteases because they have a cysteine in the active site that cleaves substrates after asparagines in a sequence of asp-x, with the four amino acids amino-terminal to the cleavage site determining a caspase s substrate specificity (Ruddon, 2007). The importance of the caspases in apoptosis is demonstrated by the inhibitory effects of mutation or drugs that inhibit their activity. Caspases can either inactivate a protein substrate by cleaving it into an inactive form or activate a protein by cleaving a pro-enzyme negative regulatory domain. In addition, caspases themselves are synthesized as pro-enzymes and are activated by cleavage at asp-x sites. Thus, they can be activated by other caspases, producing elements of the caspase cascade shown in Figure Figure 2.21 Caspase activation (from (Dash, 2003)) ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 42

61 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Also, as illustrated in Figure 2.21, caspases are activated in a number of steps by proteolytic cleavage by an upstream caspase or by protein protein interactions, such as that seen for the activation of caspase-8 and the interaction of cytochrome c and Apaf-1 in the activation of caspase-9. A number of important substrates of caspases have been identified, including the caspase-activated DNase (CAD), noted above, which is the nuclease responsible for the DNA ladder of cells undergoing apoptosis. Activation of CAD is mediated by caspase-3 cleavage of the CAD-inhibitory subunit. Caspase-mediated cleavage of other specific substrates has been shown to be responsible for other typical changes seen in apoptotic cells, such as the cleavage of nuclear lamins required for nuclear shrinkage and budding, loss of overall cell shape by cleavage of cytoskeleton proteins, and cleavage of PAK2, a member of the p21- activated kinase family, that mediates the blebbing seen in dying cells Bcl-2 Family Mammalian Bcl-2 was first identified as anti-apoptotic protein in lymphomas cells. It turned out to be a homolog of an anti-apoptotic protein called Ced-9 described in C. elegans and protects from cell death by binding to the pro-apoptotic factor Ced-4. Similarly, in mammalian cells, Bcl-2 binds to a number of pro-apoptotic factors such as Bax, Figure One concept is that pro- and anti- apoptotic members of the Bcl-2 family of proteins form heterodimers, which can be looked on as reservoirs of plus and minus apoptotic factors waiting for the appropriate signals to be released (Ruddon, 2007) Anoikis Anoikis is a form of apoptosis that occurs in normal cells that lose their adhesion to the substrate or extracellular matrix (ECM) on which they are growing. Adherence to a matrix is crucial for the survival of epithelial, endothelial, and muscle cells. Prevention of their adhesion usually results in rapid cell death, which occurs via apoptosis. Thus, anoikis is a specialized form of apoptosis caused by prevention of cell adhesion (Ruddon, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 43

62 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure 2.22 Apoptotic pathways. Two major pathways lead to apoptosis: the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling (from (Zhang, 2005)) The term anoikis means homelessness in Greek and although the observation of this phenomen occurs only with cultured cells, it is likely to occur also in vivo because it is known that cell-cell and cell-ecm interactions are crucial to cell proliferation, organ development, and maintenance of a differentiated state. This may be a way that a multicellular organism protects itself from free-floating or wandering cells (such as occurs in tumor metastasis). The basic rule for epithelial and endothelial cells appears to be attach or die. Interestedly, cells that normally circulate in the body such as hematopoietic cells do not undergo anoikis (Ruddon, 2007). Cell attachment is mediated by integrins, and ECM integrin interactions transduce intracellular signaling pathways that activate genes involved in cell ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 44

63 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS proliferation and differentiation. Although the cell death pathways induced by disruption of these cell attachment processes are not clearly worked out, cell detachment induced anoikis does result in activation of caspases-8 and -3 and is inhibited by Bcl-2 and Bcl-XL, indicating some similarities to the typical apoptosis mechanisms. In addition, integrin-ecm interaction activates focal adhesion kinase (FAK) and attachment-mediated activation of PI3-kinase. Both of these steps protect cells from anoikis, whereas inhibition of the PI3-kinase pathway induces anoikis (Ruddon, 2007). Disruption of cell-matrix interactions also turns on the JNK /p38 pathway, a stress-activated protein kinase. The mitogen-activated kinase system may also be involved, since caspase mediated cleavage of MEKK-1 occurs in cells undergoing anoikis. As stated earlier, one of the hallmarks of malignantly transformed cells growing in culture is their ability to grow in an anchorage independent manner, whereas normal cells do not. Thus, cancer cells may develop resistance to anoikis. This may be a way that metastatic cancer cells can survive in the bloodstream until they seed out in a metastatic site (Ruddon, 2007) RESISTANCE TO APOPTOSIS IN CANCER AND POTENTIAL TARGETS FOR THERAPY It would be a mistake to portray apoptosis as only a mechanism to kill cells damaged by some exogenous insult such as DNA-damaging toxins, drugs, or irradiation. Apoptosis is, in fact, a usual mechanism used by all multicellular organisms to facilitate normal development, selection of differentiated cells that the organism needs, and control of tissue size. For example, studies of nematodes (C. elegans), fruit flies, and mice indicate that apoptotic-mediated mechanisms similar to those described here are intrinsic and required for normal development. Dysfunction of these pathway results in developmental abnormalities and disease states (Ruddon, 2007). In the human, development of the immune system is perhaps the best example of the role for apoptosis in normal development. In the immune system, apoptosis is a fundamental process that regulates T- and B-cell proliferation and survival and is used to eliminate immune cells that would potentially recognize and destroy host tissues ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 45

64 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS ( anti-self ). Mechanisms involving Apo-1/FAS (CD95)-mediated signaling of the caspase cascade are employed in lymphocytic cell selection. In the case of T lymphocytes, pre-t cells are produced in the bone marrow and circulate to the thymus where they differentiate and rearrange their T-cell receptors (TCRs). Those cells that fail to rearrange appropriately their TCR genes, and thus cannot respond to self major histocompatibility complex (MHC) peptide complexes, die by neglect, Figure Those T cells that pass the TCR selection tests mature and leave the thymus to become the adult peripheral T-cell pool. The mature T-cell pool thus passes through a number of selection steps to ensure self-mhc restriction and self-tolerance. Apoptosis also is used to delete mature peripheral T cells that are insufficiently stimulated by positive growth signals, and this is a mechanism to downregulate, or terminate, an immune response (Ruddon, 2007). B lymphocytes undergo selection and maturation in the bone marrow and germinal centers of the spleen and other secondary lymphoid organs. Those with low antigen affinity or those autoreactive are eliminated by apoptosis. Those that pass this test mature into memory B cells and long-lived plasma cells. The ability of lymphoid progeny cells to avoid apoptosis may lead to lymphatic leukemias or lymphomas. In addition, cancers develop multiple mechanisms to evade destruction by the immune system such as a decreased expression of MHC molecules on cancer cell surfaces and production of immunosuppressive cytokines. Several cell proliferations promoting events take place in cancer cells as they evolve over time into growth dysregulated, invasive, metastatic cell types. These events include activation of proliferationpromoting oncogenes such as ras and myc, overexpression of cell cycle regulatory factors such as cyclin D, increased telomerase to overcome cell senescence, and increased angiogenesis to enhance blood supply to tumor tissue (Ruddon, 2007). The cancer-related alterations in the apoptotic pathway provide a number of cancer chemotherapeutic targets. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 46

65 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS Figure The role of apoptosis in the development and function of T lymphocytes. Major pro-apoptotic and antiapoptotic signals/molecules (from (Zhang, 2005)) SUMMARY At the end of this chapter is possible to conclude that many of the controls that govern the transition between quiescence and active cell cycling in mammals operate in G 1 phase. Loss of R point control appears to be a common, possibly even universal step in tumor development, and a number of genetic lesions that can contribute to this deregulation have been identified. Loss of survival proteins can also contribute to apoptosis. The antiapoptotic gene, BCL2, has been shown to be repressed by p53 and, therefore, contributes to apoptosis by blocking survival signals mediated by BCL2. The choice as to whether a cell undergoes apoptosis or cell cycle arrest and DNA repair depends on a number of factors. Some may be independent of p53 such as extracellular survival factors, the existence of oncogenic alterations and the availability of additional transcription factors. However, the extent of DNA damage may also contribute to the choice by affecting the level of activity of p53 induced. Activation of apoptosis has been associated with higher levels of p53 than those required for cell cycle arrest which may reflect a lower affinity of cell cycle arrest target gene promoters for p53. In addition, the type of cell may affect the response to p53. Importantly, it is vital to identify why transformed cells die in response to p53, whereas normal cells undergo cell cycle ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 47

66 CHAPTER II CELL CYCLE REGULATION AND APOPTOSIS arrest and DNA repair as this may be of great potential for the development of cancer therapies (Macdonald, 2005). This loss of cell cycle check point control by cancer cells may contribute to their increased susceptibility to anticancer drugs. Normal cells have mechanisms to protect themselves from exposure to growth-limiting conditions or toxic agents by calling on these check point control mechanisms. Cancer cells, by contrast, can continue through these checkpoints into cell cycle phases that make them more susceptible to the cytotoxic effects of drugs or irradiation (Ruddon, 2007). Apoptosis occurs in most, if not all, solid cancers. Ischemia, infiltration of cytotoxic lymphocytes, and release of TNF may all play a role in this and it would be therapeutically advantageous to tip the balance in favor of apoptosis over mitosis in tumors, if that could be done. Clearly, a number of anticancer drugs induce apoptosis in cancer cells but the problem is that they usually do this in normal proliferating cells as well. Therefore, the goal should be to manipulate selectively the genes involved in inducing apoptosis in tumor cells, although understanding how those genes work may go a long way to achieving this goal. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 48

67 CHAPTER III CANCER CELL

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69 CHAPTER III CANCER CELL INTRODUCTION Cancer is an abnormal growth of cells caused by multiple changes in gene expression leading to dysregulated balance of cell proliferation and cell death and, ultimately evolving into a population of cells that can invade tissues and metastasize to distant sites, causing significant morbidity and, if untreated, death of the host (Ruddon, 2007). Cancer is a group of diseases of higher multicellular organisms. It is characterized by alterations in the expression of multiple genes, leading to dysregulation of the normal cellular program for cell division and cell differentiation. This results in an imbalance of cell replication and cell death that favors growth of a tumor cell population (Ruddon, 2007). The characteristics that delineate a malignant cancer from a benign tumor are the abilities to invade locally, to spread to regional lymph nodes, and to metastasize to distant organs in the body (Ruddon, 2007). Cancer cells contain many alterations which accumulate as tumors develop. Over the last 25 years, considerable information has been gathered on the regulation of cell growth and proliferation leading to the identification of the proto-oncogenes and the tumor suppressor genes. The proto-oncogenes encode proteins which are important in the control of cell proliferation, differentiation, cell cycle control and apoptosis (MacDonald, 2005). Mutations in these genes act dominantly and lead to a gain in function. In contrast the tumor suppressor genes inhibit cell proliferation by arresting progression through the cell cycle and block differentiation. They are recessive at the level of the cell although they show a dominant mode of inheritance. In addition, other genes are also important in the development of tumors. Mutations leading to increased genomic instability suggest defects in mismatch and excision repair pathways. Genes involved in DNA repair, when mutated, also predispose the patient to developing cancer, as described in chapter 3 (MacDonald, 2005). In this chapter is held a description of the tumor cell; the types of cancers; ongoing research and treatments; tissue changes upon stimuli; tumor angiogenesis; benign and malignant cell characteristics and the process of metastasis. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 51

70 CHAPTER III CANCER CELL The description of the tumor cell is important in this work since then, in my thesis, I will focus on studies of cancer cells. This chapter and the chapters three and four complement each other, describing the steps for the formation of a tumor cell, the changes in the cell cycle and finally the role of radiation in killing/give rise to cancer cells. 3.2 CANCER CELL In normal cell growth there is a finely controlled balance between growthpromoting and growth-restraining signals such that proliferation occurs only when required. The balance is tilted when increased cell numbers are required, for example during wound healing and during normal tissue turnover. Differentiation of cells during this process occurs in an ordered manner and proliferation ceases when no longer required. In tumor cells this process is disrupted, continued cell proliferation occurs and loss of differentiation may be found (MacDonald, 2005). In addition the normal process of programmed cell death may no longer operate and cancers arise from a single cell which has undergone mutation. Gene mutations give the cell increased growth advantages compared to others and allow them to escape normal controls on proliferation. The initial mutation will cause cells to divide to produce a genetically homogeneous clone. In turn, additional mutations occurs which further enhance the cells growth potential. These mutations give rise to subclones within the tumor each with differing properties so that most tumors are heterogeneous (MacDonald, 2005). Tumors can be divided into two main groups, benign or malignant. Benign tumors are rarely life threatening, grow within a well-defined capsule which limits their size and maintain the characteristics of the cell of origin and are thus usually well differentiated. Malignant tumors invade surrounding tissues and spread to different areas of the body to generate further growths or metastases. It is this process which is often the most life threatening. Different clones within a tumor will have differing abilities to metastasize, a property which is genetically determined. The process of ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 52

71 CHAPTER III CANCER CELL metastasis is likely to involve several different steps and only a few clones within a tumor will have all of these properties (MacDonald, 2005). Tumor cells show a number of features which differentiate them from normal cells: (1) They are no longer as dependent on growth factors as normal cells either because they are capable of secreting their own growth factors to stimulate their own proliferation, a process termed autocrine stimulation, or because growth factor receptors on the surface are altered in such a way that binding of growth factors is no longer necessary to stimulate proliferation; (2) normal cells require contact with the surface in the extracellular environment to be able to grow whereas tumor cells are anchorage independent; (3) normal cells respond to the presence of other cells, and in culture will form a monolayer due to contact inhibition, whereas tumor cells lack this and often grow over or under each other; (4) tumor cells are less adhesive than normal cells; (5) normal cells stop proliferating once they reach a certain density but tumor cells continue to proliferate (MacDonald, 2005). In the most basic sense, cancer is the abnormal, uncontrolled growth of previously normal cells. The transformation of a cell results from alterations to its DNA that accumulates over time. The change in the genetic information causes a cell to no longer carry out its functions properly. A primary characteristic of cancer cells is their ability to rapidly divide, and the resulting accumulation of cancer cells is termed a tumor. As the tumor grows and if it does not invade the surrounding tissues, it is referred to as being benign (Figure 3.1a). If, however, the tumor has spread to nearby or distant tissues then it is classified as malignant (Figure 3.1b) (Almeida, 2010). Metastasis is the breaking free of cancer cells from the original primary tumor and their migration to either local or distant locations in the body where they will divide and form secondary tumors (Almeida, 2010). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 53

72 CHAPTER III CANCER CELL Types of cancer Figure 3.1 Benign vs. malignant cancers. (a) Benign tumor (b) Malignant tumor (from Almeida, 2010). Cancer is not a single disease; there are over 100 identified types, all with different causes and symptoms. To distinguish one form from another the cancers are named according to the part of the body in which they originate. Some tumors are identified to reflect the type of tissues they arise from, with the suffix -oma, meaning tumor, added on. For example, myelos- is a Greek term for marrow. Thus, myeloma is a tumor of the bone marrow, whereas hepatoma is liver cancer (hepato- = liver), and melanoma is a cancer of melanocytes, cells found primarily in the skin that produce the pigment melanin (Almeida, 2010). The four major types of cancer are carcinomas, sarcomas, leukemias, and lymphomas. Approximately 90% of human cancers are carcinomas, which arise in the skin or epithelium (outer lining of cells) of the internal organs, glands, and body cavities. Tissues that commonly give rise to carcinomas are breast, colorectal, lung, prostate, and skin (Almeida, 2010). In my thesis I will study, in term of image processing, adenocarcinomas of the prostate and breast before and after irradiation of the cells (Figure 3.2). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 54

73 CHAPTER III CANCER CELL Figure 3.2 Adenocarcinoma of the prostate (from IPO Porto, radiobiology department). Sarcomas are less common than carcinomas and involve the transformation of cells in connective tissue such as cartilage, bone, muscle, or fat. There are a variety of sarcoma subtypes and they can develop in any part of the body, but most often arise in the arms or legs. Liposarcoma is a malignant tumor of fat tissue (lipo- = fat) whereas a sarcoma that originates in the bone is called osteosarcoma (osteo- = bone). Certain forms of cancer do not form solid tumors. For example, leukemias are cancers of the bone marrow, which leads to the overproduction and early release of immature leukocytes (white blood cells). Lymphomas are cancers of the lymphatic system. This system, which is a component of the body s immune defense, consisting of lymph, lymph vessels, and lymph nodes, serves as a filtering system for the blood and tissues (Almeida, 2010) The uniqueness of cancer While there are certain commonalities shared by cancers of a particular type, each may be unique to a single individual. This is because of different cellular mutations that are possible, and can depend on whether the disease is detected at an ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 55

74 CHAPTER III CANCER CELL early or advanced stage. As a result, two women diagnosed with breast cancer may or may not receive the same treatment (Almeida, 2010). The impact of the disease on the individual, as well as the final outcome of the disease, is unique in every case. Still, several types of cancers can have a similar set of symptoms, which may be shared with several other conditions, making screening, detection, and diagnosis a complex problem. A tumor can impact the function of the tissue in which it resides or those in the surrounding areas. Tumors provide no useful function themselves and may be considered parasites, with every step of their advance being at the expense of healthy tissue. While most types of cancers form tumors, many do not form discrete masses. As previously stated, leukemia is a cancer of the blood that does not produce a tumor, but rather rapidly produces abnormal blood cells in the bone marrow at the expense of normal blood cells (Almeida, 2010) The development of tumors All tumors begin with mutations (changes) that accumulate in the DNA (genetic information) of a single cell causing it and its offspring to function abnormally. DNA alterations can be sporadic or inherited. Sporadic mutations occur spontaneously during the lifespan of a cell for a number of reasons: a consequence of a mistake made when a cell copies its DNA prior to dividing, the incorrect repair of a damaged DNA molecule, or chemical modification of the DNA, each of which interferes with expression of the genetic information. Inherited mutations are present in the DNA contributed by the sperm and/or egg at the moment of conception. To date, 90 95% of diagnosed cancers appear to be sporadic in nature and thus have no heredity basis. Whether the mutations that result in a cancer are sporadic or inherited, certain genes are altered that negatively affect the function of the cells (Almeida, 2010) Genetic influence on tumors A link between a particular genetic mutation and one or more types of cancers is made by analyzing and comparing the DNA of malignant tissue samples obtained ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 56

75 CHAPTER III CANCER CELL from patients and members of families with a high incidence of a particular cancer and comparing it to the DNA from healthy individuals. For example, a study could be conducted in which the DNA isolated from tumor cells obtained from liver cancer patients is analyzed and determined to possess certain versions of genes whereas different versions of those same genes are present in the DNA of liver cells of healthy persons. An association could then be drawn between the bad versions of those genes and liver cancer (Almeida, 2010). This type of analysis has been crucial in identifying certain versions of genes associated with a predisposition for the development of particular forms of cancer. For example, studies have demonstrated that there is an elevated risk of breast or ovarian cancer associated with certain versions of the BRCA1 and/or BRCA2 genes. Another example is retinoblastoma, a rare tumor of the eye typically found in infants and young children, which is associated with alterations within the Rb gene (Almeida, 2010). 3.3 CANCER THROUGH THE AGES Although not specifically identified as such, cancer has been known for many centuries. In fact, there is evidence of tumors in the bones of five thousand year old mummies from Egypt and Peru. The disease itself was not very common, nor explored or understood, because in ancient times fatal infectious diseases resulted in shorter lifespan. Given that the vast majority of cancers are sporadic, there was less opportunity for the accumulation of the mutations necessary to transform normal cells into cancerous ones (Almeida, 2010). The word cancer was first introduced by Hippocrates ( BC), the Greek physician and father of medicine. He coined the term carcinoma, from the Greek word karcinos, meaning crab, when describing tumors. This is because tumors often have a central cell mass with extensions radiating outward that mimic the shape of the shellfish (Figure 3.3) (Almeida, 2010). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 57

76 CHAPTER III CANCER CELL Figure 3.3 Cancer cell (from Almeida, 2010) Early discovery of carcinogens Also published in 1761 was a paper by John Hill, an English physician. In it he made the first causal link between substances in the environment and cancer when he described a relationship between tobacco snuff and nasal cancer. This brought about the awareness of carcinogens (chemical agents that have been demonstrated to cause cancer). In 1775, the English surgeon Sir Percivall Pott observed and noted a high rate of scrotal cancer among chimney sweepers. He postulated that it was caused by longterm exposure to the chemicals in the soot-soaked ropes worn as harnesses. His research led to studies that associated particular occupations with an increased risk of developing specific forms of cancer the forerunner to the field of public health and cancer (Almeida, 2010) The use of microscopes demonstrated changes at a cellular level The development of improved microscopes in the late nineteenth century allowed for more thorough examinations of cells and their activities than was previously possible. It was realized that cancer cells were different in both appearance and behavior from normal cells within the same tissue or organ (Figure 3.4). Early twentieth century accomplishments in the development of cell culture, new and improved diagnostic techniques, the discovery of chemical carcinogens, and the use of ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 58

77 CHAPTER III CANCER CELL chemotherapy (powerful anticancer drugs) all had significant impacts upon the understanding and treatment of cancer (Almeida, 2010). Figure 3.4 (a) Note the abundance of the thin, sheet-like extensions from the cell bodies of the healthy cells. (b) Note the rounded appearance of the cancer cells (from Almeida, 2010). 3.4 MODERN DAY RESEARCH AND TREATMENT The radioactive element radium, isolated by Marie and Pierre Curie in 1898, was found to be effective in the treatment of tumors in While both healthy and cancerous cells are susceptible to the damage caused by X-rays, cancer cells are inherently less able to repair the damage and recover. Once safe dosage levels were determined, radiation therapy became a standard form of treatment for many cancers (Almeida, 2010). Tumor formation, growth, and metastasis reflect that the regulation of the cell cycle is critical in maintaining the structural and functional integrity of all tissues. The inability to control passage of a cell through each of the cycle checkpoints can result in unwanted growth within a tissue. The growth not only can disrupt the function of that tissue but also of those nearby. The situation becomes much more serious if cells break free from the tumor and travel to other tissues where they may take up residence, multiply, and create additional problems (Almeida, 2010). 3.5 TISSUES CHANGES IN RESPONSE TO STIMULI Our cells experience many different types of chemical and physical stimuli on an almost constant basis. For example, our cells are exposed to both beneficial and ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 59

78 CHAPTER III CANCER CELL harmful chemicals in the air, food, and water we take into our bodies, hormones surging through our bloodstreams relaying messages to the cells to which they bind, and stresses and strains are applied when we move heavy objects. The type and strength of the stimuli cells receive or are subjected to affect the structural and functional changes they will undergo. The cellular changes that occur in response to stimuli are an indication of both the susceptibility to signals and the adaptability that cells exhibit in response to changes in their environment (Almeida, 2010). It is logical to expect that if a certain stimulus causes a cell to change in a particular way, then the cell should revert back to its original condition upon removal of the stimulus. The transformation that cells in a tumor have undergone is often the result of changes brought about by certain stimuli. A unique aspect of tumor cells is that the cellular changes remain even after the stimulus that led to their transformation is no longer present (Almeida, 2010) Metaplasia Epithelial cells that line certain portions of the respiratory tract are known to undergo changes in appearance and function when exposed to noxious chemicals in the air. The pathway of air through the respiratory tract begins in the nose or back of the throat and travels down through the trachea or windpipe, the tube that leads from the back of the throat to the lungs (Figure 3.5). The base of the trachea branches to form two bronchi, one going to each of the lungs, which then progressively branch into many smaller tubes called bronchioles that spread to all areas of the lungs. At the ends of the bronchioles are tiny balloon-like sacs called alveoli where gas exchange between the area and bloodstream occurs. The surface layer of the trachea, bronchi and some of the bronchioles consists of pseudostratified columnar epithelial cells (Figure 3.6) (Almeida, 2010). These cells are somewhat rectangular in shape and aligned side by side in a single layer. The term pseudostratified comes from the fact that there appears to be more than one layer of cells present (pseudo- = false; stratified = layered) because the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 60

79 CHAPTER III CANCER CELL position of the nuclei in adjacent cells alternates from being centrally located to being at the bottom of the cell(almeida, 2010). Figure 3.5 Respiratory system (from Almeida, 2010). Figure 3.6 Pseudostratified columnar epithelium (from Almeida, 2010). The respiratory epithelium serves a protective function. Among the columnar cells are specialized goblet cells that secrete thick, sticky mucus that coats the epithelium. The sticky mucus traps particulate matter in the air, such as dust and microorganisms, preventing it from getting deeper into the lungs. The exposed surface of a columnar epithelial cell possesses cilia, short hair like structures that beat back ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 61

80 CHAPTER III CANCER CELL and forth to sweep the mucus and anything trapped in it upward to the back of the throat. The material brought up by the so-called ciliary escalator can be either swallowed and destroyed in the highly acidic environment of the stomach or expectorated (spat out) (Almeida, 2010). Smoke is a mixture of many different types of chemicals, liquids, and solids and is an irritant of the respiratory epithelium. Over time, smoke paralyzes the cilia of the respiratory epithelium, allowing mucus to build up in the airways and material to travel deeper into the lungs. Also observed in tobacco smokers is the replacement of pseudostratified columnar epithelium with stratified squamous epithelium (Figure 3.6). The multiple layers of flattened cells in this form of epithelium protect underlying tissues against abrasion. The cells in the outer layers are regularly sloughed off and replaced by the replication of the cells in the lower layers. Stratified squamous epithelium is normally present in the outer layer of skin and the inner lining of the digestive tract, but not in the respiratory tract (Almeida, 2010). The previously described condition is often exhibited in the airways of smokers. It is an example of metaplasia, which is the change of mature and differentiated cells from one normal cell type to another normal cell type. It is important to note that what is abnormal about metaplastic tissue when observed under the microscope is not the presence of abnormal cells, but rather the presence of a type of cell that is normally found in other types of tissue. The stratified squamous epithelium that may be present along the airway of a smoker can have a normal appearance (Almeida, 2010). The problem is that it should not be present in that location. Metaplasia is not a normal process that cells undergo; there must be an inciting stimulus that triggers the structural and functional changes that occur. A typical characteristic of metaplasia is that it is reversible when the signal that initiated the changes is no longer present, healthy cells should revert back to their original form. A concern arises when the inciting stimulus that resulted in the metaplastic changes is no longer present, yet the cells do not revert back to their normal structure and function. In some cases, the permanent alterations are the result of genetic mutations that have negatively ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 62

81 CHAPTER III CANCER CELL affected oncogenes and tumor suppressor genes. These types of cellular mutations certainly enhance the likelihood that cells will become cancerous (Almeida, 2010) Hypertrophy and hyperplasia Metaplasia is not a form of growth, which means either an increase in individual cell size or an increase in the number of cells. The purpose of the majority of new growth that occurs between conception and adulthood is to form the variety of differentiated body tissues. In adults, tissues are mature in their size, structure, and function, and the primary role of cell division is the replacement of those cells that have either died of old age, are lost due to abrasion (occurs to outer layer of skin and inner lining of the digestive tract), or are damaged beyond repair (Almeida, 2010). There are, however, times when growth does occur in adults. For example, the goal of resistance or weight training is the growth of muscle tissue. A muscle is composed of individual muscle cells known as muscle fibers, and each fiber contains bundles of particular proteins that are responsible for contraction and relaxation. The lifting of heavy weights damages the protein bundles. During the recovery or repair period, the cells destroy and replace the damaged proteins. The cells, in an attempt to be stronger and prevent similar damage from occurring again, increase the number of protein bundles. This form of growth, which is due to an increase in size but not number, is known as hypertrophy (Figure 3.7a) (Almeida, 2010). Similar to metaplasia, hypertrophic growth occurs in response to an inciting stimulus and is reversed when that stimulus is no longer present. When resistance training is stopped, there will be a loss in muscle size and tone since there is no longer a need to maintain the greater number of protein bundles. Another example of growth that occurs in adulthood is the increase in breast size that occurs during pregnancy. This form of growth is known as hyperplasia and is the result of an increase in the number of cells in a tissue (Figure 3.7b). The combinations and levels of hormones produced during pregnancy act as an inciting signal for the cells of the breast s mammary tissue to progress through the cell cycle and divide. This growth results in the development of mammary glands and ducts that produce milk to nourish the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 63

82 CHAPTER III CANCER CELL newborn child. A mother will continue to lactate (produce milk) for as long as the child breast feeds (Almeida, 2010). When a woman stops breast feeding, there is an absence of the inciting stimuli (i.e., pregnancy hormones and infant suckling) that led to the development, activity, and maintenance of the mammary tissues. The result is that the cells formed in response to the stimuli now undergo apoptosis, effectively reverting the tissue to its original state. Hyperplastic growth can also occur in the absence of proper stimuli. This occurs when there is a loss of regulation at the checkpoints of the cell cycle. The most common cause for a loss of cell cycle regulation is an accumulation of gain-of-function mutations within proto-oncogenes, converting them into oncogenes, and/or loss-offunction mutations in tumor suppressor genes. Mutations in caretaker tumor suppressor genes may cause structural and functional changes that do not allow cells to interact with one another in an organized fashion (Almeida, 2010) Dysplasia A Pap test is often part of a woman s routine gynecological exam. The test entails obtaining cells from the inner surface of the cervix and the lower portion of the uterus, followed by their examination under a microscope. Less than 5% of Pap tests display dysplasia, a disorganized arrangement of cells, which is typically reported as mild, moderate, or severe (Figure 3.7c). Mild cases often clear up on their own and are typically followed up with repeat Pap tests every 3 6 months. Moderate and severe cases require the use of treatment methods to remove the abnormal cells. The stimuli that result in cervical dysplasia, which is most common in women between 25 and 35 years of age, are unknown, although women who are infected with the human papilloma virus have an increased risk of exhibiting the condition (Almeida, 2010). Dysplasia is an indication that the cells are not functioning properly, and is considered a pre-cancerous condition. The risk associated with dysplasia is the potential for the cells to progress to a state of neoplasia. A neoplastic growth has, in addition to a disorganized arrangement of cells, a larger than normal number of cells capable of dividing (Figure 3.7d) (Almeida, 2010). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 64

83 CHAPTER III CANCER CELL Figure 3.7 Types of tissue growth (from Almeida, 2010). A neoplasm is also known as a cancer or tumor because its growth is the result of disruptions to the normal regulation of the cell cycle resulting in uncontrolled progression through the cell cycle and cell division (Almeida, 2010). 3.6 FEEDING TUMOR GROWTH BY ANGIOGENESIS The formation of new blood vessels, a process known as angiogenesis (angio = blood and lymph vessel; genesis = production), first occurs during embryonic development and continues until early adulthood. Expansion of a blood supply is the result of the division and proliferation of the cells of the blood vessels currently in a tissue. Therefore, it is a form of growth. As mentioned previously, growth of new tissue is not a regular occurrence in adults but typically occurs only during the repair of injured tissue (Almeida, 2010). Similar to the way that the cell cycle is regulated by balancing a set of opposing signals from the activities of protooncogenic and tumor suppressor proteins, ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 65

84 CHAPTER III CANCER CELL angiogenesis is under the control of competing signals from many activator and inhibitor molecules. To date, there are more than two dozen proteins and small molecules that have been identified as angiogenic activators and inhibitors. In adults, the concentration of angiogenic inhibitors is higher than that of activators, thus restricting angiogenesis. A shift in the balance so that the concentration of the activators is higher than that of the inhibitors will have an opposite effect and result in the formation of new blood vessels (Almeida, 2010). Capillaries, the smallest blood vessels, are abundant in tissues to ensure that the nearby cells are provided with a continuous supply of essential nutrients and a way to remove the metabolic waste products. New tissue growth without a concomitant expansion of the blood supply is limited to 1 2 mm 3 in size, which is approximately the size of the head of a pin. Tumors are able to exceed that growth limit by stimulating angiogenesis. In an attempt to support growth, tumor cells may secrete the potent angiogenic activator vascular endothelial growth factor (VEGF; vascular = pertaining to vessels) (Figure 3.8a). This protein diffuses to the endothelial cells of a nearby blood vessel. The binding of VEGF to the appropriate receptors in the outer membranes of endothelial cells initiates a signal transduction cascade within the cells that results in changes in gene expression and cell function. For example, the expression of protooncogenes is enhanced while that of tumor suppressor genes is inhibited so that the cells will progress through the cell cycle and divide (Almeida, 2010). As the endothelial cells divide, they will form a bud that protrudes from the blood vessel wall into the surrounding tissue (Figure 3.8b). As the number of endothelial cells increases, the bud elongates and the endothelial cells produce matrix metalloproteinases (MMPs). MMPs are enzymes that breakdown the extracellular matrix proteins to enable the growing blood vessel to migrate between the tissue cells toward the cancer cells. Once established, a tumor s blood supply will grow along with the tumor, nourishing it and removing its wastes (Figure 3.8c). Angiostatin and endostatin are two angiogenic inhibitors that have been used in extensive animal studies and human trials. Tests conducted with mice have indicated that the treatment of tumors with angiogenic inhibitors are effective at inhibiting their growth and can limit the number of secondary tumors that may form (Almeida, 2010). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 66

85 CHAPTER III CANCER CELL Figure 3.8 Tumor angiogenesis. (a) Cancer cells secrete vascular endothelial growth factor (VEGF), an angiogenic activator, which binds to VEGF receptors on endothelial cells of a capillary causing a change in gene expression within the endothelial cells of the capillary. In response to VEGF signaling an endothelial cell will divide and secrete matrix metalloproteinases (MMPs). (b) Many rounds of endothelial cell division produce a bud off of a capillary that grows and forms additional branches. (c) Endothelial cell growth toward a tumor supports its growth (from Almeida, 2010). 3.7 CHARACTERISTICS OF BENIGN AND MALIGNANT TUMORS Neoplasms are classified into two broad categories, benign and malignant. The classification of a tumor is most often done by a pathologist, a physician who specializes in interpreting and diagnosing changes in bodily fluids and tissues that occur in response to disease. The assessment of a neoplastic growth is based on a biopsy (bio- = life; -opsy = look or appearance), a macro- and microscopic examination of either a portion of or an entire tumor that has been surgically removed. Microscopic ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 67

86 CHAPTER III CANCER CELL analysis provides a number of distinguishing features that are key to differentiating between benign and malignant tissue (Almeida, 2010). A benign tumor is noncancerous and classified as in situ, or contained solely within the tissue in which it originated; the abnormal cells have not spread to surrounding tissues or other areas of the body. In fact, there is typically a well-defined border between a benign neoplastic growth and normal tissue. Benign neoplastic growths are usually slow growing and although they are generally not life-threatening, they can become dangerous based on their location and whether or not their growth disrupts or interferes with normal healthy tissue functions. Their self-contained nature is an added benefit that often allows the entire tumor to be surgically removed, unless it is in an inoperable position, such as within an organ rather than on the surface or adjacent to major blood vessels or the spinal cord (Almeida, 2010). A concern with benign growths is that they can progress into the far more serious malignant or cancerous neoplasms. Principal among the distinguishing features of malignant tumors is that they are not contained solely within the tissue in which they originally developed. This means that a portion of the tumor has grown into one or more of the surrounding tissues or has spread to a distant location in the body. Metastasis, the process by which malignant cells travel from the original (primary) tumor to other (secondary) sites in the body, is often accomplished through the use of either the circulatory or lymphatic systems (Figure 3.9) (Almeida, 2010). Normal tissues consist of differentiated cells performing specific functions. Malignant tissue typically exhibits anaplasia, the presence of undifferentiated cells that bear no resemblance to the cells normally found in that location. The presence of undifferentiated cells is a reflection of what is normally present during embryonic and fetal development when tissues are going through their formative stages. Since undifferentiated cells are involved in tissue formation they divide frequently. As a result, malignant tissues often exhibit a high mitotic index, the ratio between the number of cells undergoing mitosis and the total number of cells within the field of view. This accounts for the faster growth rate of malignant tumors. Malignant tumors are considered life-threatening because of the rapid growth and production of ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 68

87 CHAPTER III CANCER CELL undifferentiated cells that are invasive and disruptive to the structure and function of surrounding tissues. In addition, the anaplastic nature of malignant cells is an important factor in the likelihood that they will metastasize and wreak similar havoc on other locations in the body. Surgery alone is not a sufficient form of treatment for malignant tumors because of the possibility that some of the cells have spread to locations throughout the body. Chemotherapy, the use of toxic drugs, is a more systemic form of treatment used to target the destruction of undetectable metastatic cancer cells in an attempt to prevent the growth and formation of new tumors (Almeida, 2010). Figure 3.9 Metastasis. (a) Neoplastic cells grow, (b) produce proteases that breakdown the basement membrane, and then invade the surrounding tissue. (c) Malignant cells can gain access to the circulatory or lymphatic system, and then (d) exit and take up residence elsewhere in the body. (from Almeida, 2010). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 69

88 CHAPTER III CANCER CELL 3.8 EVENTS THAT OCCUR DURING THE PROCESS OF METASTASIS The structural and functional changes that occur to the cells within a tumor can be a consequence of external growth factor signals and/or mutations to DNA. It is common for tumors, particularly those that are malignant, to exhibit tumor progression the cells mutate independently of one another as they grow, thereby generating a collection of genetically different subpopulations. The genetic differences between cells result in their unique growth and metastatic potentials (Figure 3.10). It is only the more potent cells that are likely to have the ability to invade the surrounding tissues, gain access to the circulatory or lymphatic systems, travel to and invade a tissue in a new location in the body, proliferate, stimulate angiogenesis, and form a secondary tumor (Almeida, 2010). Figure 3.10 Tumor progression (from Almeida, 2010) Characteristics of metastatic cells Approximately 90% of all human cancers are carcinomas, which mean that epithelial cells have undergone a neoplastic transformation. All epithelial tissue is attached to a basement membrane, a upporting layer of extracellular material composed of a variety of glycoproteins (proteins with sugars bound to them) and carbohydrates (Figure 3.11). The membrane provides a defining boundary between the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 70

89 CHAPTER III CANCER CELL epithelium and an underlying layer of connective tissue. In order for cells within a carcinoma to invade surrounding tissue, they must be able to maneuver between other cells of that tissue and among the extracellular matrix, as well as degrade the basement membrane (Figure 3.9). These functions are the result of specific cellular changes that occur during the malignant transformation and tumor progression processes. Certain cells develop the ability to secrete proteases, the enzymes that destroy the proteins involved in cell-to-cell and cell-to-extracellular matrix connections as well as those of the basement membrane (Almeida, 2010). Figure 3.11 Basement membrane of epithelium (from Almeida, 2010). Associated with the fixed location most cells in the body have within tissues is an absolute requirement for anchorage dependence they adhere themselves to neighboring cells and the extracellular matrix. Red and white blood cells are the exception to the rule since they circulate freely in the blood stream. The ability to migrate through tissues depends on having a reduced need to be anchored. Malignant cells that encounter and gain entrance to capillaries and lymphatic vessels by migrating between their outer layer of endothelial cells can be carried to secondary tissue sites (Figure 3.9d) (Almeida, 2010). 3.9 SUMMARY At the end of this chapter is possible to conclude that a neoplasm can be either malignant, able to spread and become worse, or benign, not inclined to spread and not likely to become worse. Although benign tumors are usually less dangerous than ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 71

90 CHAPTER III CANCER CELL malignant tumors, they can cause problems. As a benign tumor enlarges, it can compress surrounding tissues and impair their functions. In some cases (e.g., brain tumors), the result can be death (Seeley, 2004). Malignant tumors can spread by local growth and expansion or by metastasis, which results from tumor cells separating from the main neoplasm and being carried by the lymphatic or circulatory system to a new site, where a second neoplasm forms. Malignant neoplasms lack the normal growth control that is exhibited by most other adult tissues, and in many ways they resemble embryonic tissue. Rapid growth is one characteristic of embryonic tissue, but as the tissue begins to reach its adult size and function, it slows or stops growing completely. This cessation of growth is controlled at the individual cell level (Seeley, 2004). Cancer results when a cell or group of cells, for some reason, breaks away from that control. This breaking loose involves the genetic machinery and can be induced by viruses, environmental toxins, and other causes. The illness associated with cancer usually occurs as the tumor invades and destroys the healthy surrounding tissues, eliminating their functions (Seeley, 2004). Tumors tend to acquire more aggressive characteristics as they develop, and in 1957 Foulds pointed out that tumor progression occurred in a stepwise fashion, each step determined by the activation, mutation or loss of specific genes. Over the next two decades biochemical and cytogenetic studies demonstrated the sequential appearance of subpopulations of cells within a tumor, attributable, in part at least, to changes in the genes themselves (MacDonald, 2005). The evidence suggests that, in the majority of cases, cancers arise from a single cell which has acquired some heritable form of growth advantage. This initiation step is believed to be caused frequently by some form of genotoxic agent such as radiation or a chemical carcinogen. The cells at this stage, although altered at the DNA level, are phenotypically normal. Further mutational events involving genes responsible for control of cell growth lead to the emergence of clones with additional properties associated with tumor cell progression. Finally, additional changes allow the outgrowth of clones with metastatic potential. Each of these successive events is likely to make the cell more unstable so that the risk of subsequent changes increases. Animal models of carcinogenesis, primarily based on models of skin cancer development in ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 72

91 CHAPTER III CANCER CELL mice, have enabled these steps to be divided into initiation events, promotion, malignant transformation and metastasis (MacDonald, 2005). Although it is clear that multiple changes are necessary for tumor development, it is not clear whether the order in which the changes occur is critical. Evidence suggests, however, that it is the accumulation of events that is important rather than the order in which they occur (MacDonald, 2005). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 73

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93 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELLS

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95 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL INTRODUCTION When cells are exposed to ionizing radiation the standard physical effects between radiation and the atoms or molecules of the cells occur first and the possible biological damage to cell functions follows later. The biological effects of radiation result mainly from damage to the DNA, which is the most critical target within the cell; however, there are also other sites in the cell that, when damaged, may lead to cell death (Suntharalingam, 2002). Many aspects of the response of tissue systems are strongly affected by the state of the cell in its cycle, for example, the state of oxygenation of the cell. The supply of metabolic substrates and the removal of metabolic products also play a role in modifying the response of tissue systems. The most significant aspect of the radiosensitivity of a tissue or organ system centers on the state of reproductive activity and, this proliferative state varies widely among the tissues of any mammalian species. At one extreme are the tissues of the central nervous system, some of which rarely, if ever, undergo division during the organism's adult life, and for which loss of clonogenic ability is an irrelevant end point. At the other extreme are the blood forming organs, which are proliferating at a rate approaching that of an exponentially growing, in vitro culture (Alpen, 1998). This chapter focuses on the most relevant aspects of radiation and provides a detailed description of the effects of radiation on normal and neoplastic tissues. The main objectives covered in this chapter include: knowledge about radiation dosimetry; description of some important milestones in radiobiology, the types of cell death in mammalian cells and undertake a relative exhaustive description of the radiation effects in the environment. After this item, it is performed a description of the nature of cell population in tissues and of the cell population kinetics and radiation damage. Subsequently, the chapter focuses on the cell kinetics in normal and tumor tissues, on the models for radiobiology sensitivity of neoplastic tissues and the tumor growth and cure models. Finally, it ends with a description of the radiobiological responses, hypoxia and radiosensitivity of the tumor cell. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 76

96 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL 4.2 QUANTITIES AND UNITS USED IN RADIATION DOSIMETRY The physical interactions of the various types of ionizing radiation with living matter are the first stage of a series of events that lead to biological changes, whose manifestations may occur over time, until many years after irradiation occurred. The radiation gives energy to the medium, thus inducing physical, chemical and biological processes that will lead to the changes mentioned previously. That part of biology that studies the chain of phenomena, from physical interaction to the external consequences, it is called Radiobiology. Given its complexity, not yet known in detail, many of the physic-chemical triggered the constituent molecules of living cells after irradiation (Dendy, 2000). The disproportion between the kinetic energy and its biological consequences emphasizes this complexity. Indeed, if the energy transferred to the body of an animal, subject to deadly radiation, was transformed into heat it would only raise the body temperature of a few thousandths of a degree. However, the kinetic energy that is transferred to the cells upon irradiation with ionizing radiation, though small, has major implications as it is released at the molecular level (Dendy, 2000). Ionizing radiation can then be defined as any type of radiation capable of removing an orbital electron of an atom or may carry electrons to higher energy levels (outer orbital), causing their activation or arousal. Radiation can be divided into: a) Particulate radiation (corpuscular) (Dendy, 2000): i. Alpha particles (α) - is a particle equivalent to a helium nucleus 2 He 4 (2p + 2n) and has two positive charges. Due to its high density of ionization, the energy of the α-particle is rapidly transferred to the medium, which makes its power of penetration rather limited (approximately 5 cm in air or about 100 mm in soft tissue). ii. Beta particles (β) - is a more common process among the light nuclei, which have excess of neutrons or protons in relation to the corresponding stable structure, Figure 4.1. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 77

97 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Depending on their energy, a β-particle can go through 10 to 100 cm in air and 1 to 2 cm in biological tissue. b) Radiation of electromagnetic waves: are high intranuclear energies transmitted in the form of wave motion, generated by radioactive isotopes. This emission is for the release of excess energy from the nucleus core and/or is produced by special equipment such as x-ray machines or linear accelerators. These waves have neither mass nor electric charge and can be divided into (Dendy, 2000): i. X-rays - are produced when fast-moving electrons collide with a metal object. The kinetic energy of the electron is transformed into electromagnetic energy. It is important to remember that the origin of this radiation is extranuclear; that is, is formed in the electronic layer of the atom. The function of the X-ray machine is to provide a sufficient flow intensity of electrons in a controlled manner, for the production of an X-ray beam with the quality and quantity desired. ii. Gamma (γ) radiation - are bundles of energy, of nuclear origin, transmitted in the form of wave motion, and with great power of penetration, Figure 4.2. This emission is intended to release excess energy of an unstable atomic nucleus. Figure 4.1 Particulate radiation emission (from Jefferson, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 78

98 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Figure 4.2 Penetration power of the main forms of radioactivity (from Suntharalingam, 2002). When a beam of ionizing radiation passes through the matter, there are three types of important physical information: 1. Their spectral energy distribution; 2. The intensity of the flow of particles; 3. The amount of energy that is released per unit mass in the area of irradiated material(yadunath, 2010). The action of ionizing radiation in air can be used to evaluate the last two physical information s, although the measurement of radiation is complex given the large number of units involved (Pisco, 2003) RADIATION MEASUREMENTS DEFINITIONS i. Directly Ionizing Particles Directly ionizing particles are charged particles that have sufficient kinetic energy to produce ionization by collision. This energy certainly must be greater than the minimum electron binding energy in the medium in which the interaction takes place (Alpen, 1998). ii. Indirectly Ionizing Particles Indirectly ionizing particles are uncharged particles that can produce ionizing particles through kinetic interaction with the medium or that can initiate a nuclear ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 79

99 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL transformation. For example, neutrons can interact with the medium to produce high kinetic energy protons or, atomic nuclei through collisions or through the release of secondary directly ionizing particles after nuclear interactions between the neutron and a target atom (Alpen, 1998). iii. Gamma Rays and X-Rays Gamma rays and X-rays are electromagnetic radiations, that is, photons, of high enough energy to produce ionization. Gamma rays are identical to X-rays in their physical properties, but, by convention, it has become practice to call ionizing photons produced in "machines" X-rays, whereas ionizing photons from radioactive sources are called gamma rays (Alpen, 1998) QUANTITIES AND UNITS Usually the exposure is a term related with the radiation source and is used to express the intensity of radiation, from a beam of X-rays or γ rays, measuring the ability of ionizing radiation in ionizing the air. The exposure is defined as the total charge released per unit mass of air when all electrons released by interactions with photons are completely stopped in air. The display units come in coulombs per kilogram [C/kg] in the international system (SI) or roentgens [R] with 1R = 2.58 x10-4 C/kg. The exposure of a beam of X-rays or γ rays varies inversely with the square of the distance from the source (Pisco, 2003). The Kerma (Kinetics Energy Released in the Medium) is the kinetic energy released in the medium, being defined as the kinetic energy transferred from neutral particles (photons and neutrons) to charged particles (electrons and protons) when radiation interacts with matter. The Kerma is specified in units of joules per kilogram [J/kg]. Additionally, in air or water Kerma can replace roentgen as a measure of exposure (Pisco, 2003). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 80

100 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Absorbed dose (D) translate the amount of radiation energy (E) absorbed per unit mass (M) of the absorbing medium:, being specified in gray (G) in the SI system and in rad (radiation absorbed dose). One gray equals 1 J of energy deposited per kilogram and 1 rad equals 100 ergs of energy deposited per kg: 1 Gy = 100 rad, 1 rad = 10 mgy. The absorbed dose is independent of the radiation source, being dependent of the absorbing medium, which is placed in the radiation field, so both the absorbing medium and the location should be specified (Pisco, 2003). The factor-f is a conversion factor between exposure and absorbed dose, determined by the relationship between the absorbed dose (D) and exposure (X): where f is the conversion factor from roentgen to rad (Pisco, 2003). The linear energy transfer (LET) is the energy absorbed by the medium per unit of the traversed distance [kev/mm]. The high LET radiation is more effective in producing biological damage than low LET radiation. When considering the biological effect of radiation, the total amount of energy absorbed (dose) and the effectiveness of radiation in causing biological damage (LET) should be considered parameters (Pisco, 2003). The dose equivalent (H) quantifies the biological damage resulting from the deposition of ionizing radiation in tissues and is mainly used in radiation protection. It is defined as the absorbed dose (D) multiplied by the quality factor (QF) of the radiation. The quality factor depends on the LET value: for sources with low LET (electrons, beta particles, X-rays and γ rays) QF=1, for sources with high LET (protons, neutrons and α particles) QF can reach the value 20. The dose equivalent is expressed in sievert (Sv) in the SI system and in rems: 1 Sv=100 rem and 1 rem= 10 msv (Pisco, 2003). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 81

101 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL 4.3 HISTORICAL PERSPECTIVE OF RADIOBIOLOGY Three incidents triggered the beginning of radiobiology: Wilhelm Conrad Roentgen's discovery of X-rays in 1895; Henri Becquerel's observance of rays being given off by a uranium-containing substance in 1896 (Marie Curie subsequently would call this radioactivity); the discovery of radium by Pierre and Marie Curie in 1898 (Forshier, 2008). Early radiobiology observations included skin erythema (radiation induced skin reddening), epilation (radiation induced hair loss), and anemia. Because of unshielded fluoroscopic apparatus, radiologists had to have fingers amputated, and compared with other doctors, had superior incidence of leukemia (Forshier, 2008). The first United States X-rays fatality occurred in Clarence Daly, an assistant of Thomas Edison, had collaborated with him in producing the fluoroscope and fluorescent screens. In working long days, Daly was subjected to doses above modern lifetime limits. In Edison s day, shielding was seldom used for personnel or x- ray tubes (Forshier, 2008). The early observations of Becquerel, the Curies, and early radiologists sparked much research into the effects of radiation exposure on biological processes. Beginning in the early 1900s through the 1950s and 1960s, many theories were developed to define and explain these effects (Forshier, 2008) Law of Bergonie and Tribendeau In 1906 two Frenchmen, J. Bergonie and L. Tribendeau, exposed rodent testicles to X-rays, and observed the effect of radiation. These researchers selected the testicles since this organ contains both mature cells (spermatozoa), which execute the organ s principal function and immature cells (spermatogonia and spermatocytes), whose only purpose is t evolve into mature, functional cells. Not only do these cells functions differ, but their rate of mitosis also differs. The spermatogonia (immature) cells divide frequently, whereas the spermatozoa (mature) cells do not divide. After exposing the testicles to radiation, Bergonie and Tribendeau noticed that the immature cells were injured at doses lower than mature cells. Supported by these ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 82

102 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL founds, they proposed a law describing the radiation sensitivity for all body cells. Their law maintains that actively mitotic and undifferentiated cells are most susceptible to damage from ionizing radiation (Forshier, 2008). The law of Bergonie and Tribondeau states that: 1. Steam cells are more radiosensitive than mature cells. The more mature a cell is, the more radioresistant. 2. Younger tissues and organs are more radiosensitive than older tissues and organs. 3. The higher the metabolic activity of a cell, the more radiosensitive it is. 4. The greater the proliferation and growth rate for tissues, the greater the radiosensitivity. This law informs us that compared to a child or mature adult, the fetus is most radiosensitive (Forshier, 2008) Ancel and Vitemberger In 1925 the law of Bergonie and Tribondeau was modified by P. Ancel and P. Wittenberg. These researchers suggested that the intrinsic susceptibility of damage by any cell by ionizing radiation is the same, but that the timing of manifestation of radiation-produced damage varies according to the types of cells. In experiments on mammals, they determined that there are two factors, which affect the appearance of radiation damage to the cell (Forshier, 2008): 1. The amount of biological stress the cell receives. 2. Pre- and post-irradiation conditions that the cell is exposed to. Ancel and Vitemberger theorized that the most significant biological stress on the cell is the need to division. In their terms, a given dose of radiation will cause the same degree of damage to all cells (the innate susceptibility is comparable for all cells) but only if and when a cell divides will damage be demonstrated (Forshier, 2008). Although Ancel and Vitemberge communicate radiosensitivity differently than Bergonie and Tribondeau, they do agree with them by placing a significant emphasis on the amount of mitotic activity involved (Forshier, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 83

103 Cell survival CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL In the 1920s researchers learned that the process of ionization in tissues was the cause of biologic results. The two mechanisms recognized were, Figure 4.3: Direct ionization along charged particles tracks caused direct effects (original ionization occurs directly on the targeted molecule). The formation of free radicals caused indirect effects (original ionization occurs with water, and transfers ionization to target molecule). Figure 4.3 Radiation path with low and high LET (from Yadunath, 2010) Fractionation Theory The 20s and 30s brought the fractionation theory from France. Ram testicles were exposed to large doses of ionizing radiation. Even though the rams could be sterilized with one large dose, this quantity of radiation also caused the skin next to the ram s scrotum to have a reaction. However, it was found, that if the large dose was fractioned (smaller doses spread out over a period of time, Figure 4.4), the animals would still become sterile, but with considerably less damage to their skin (Forshier, 2008). Neutrons X-rays Dose (Gy) Figure 4.4 Effect of fractionation (from Cherry, 2006). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 84

104 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Mutagenesis In 1927, H. Muller discovered that ionizing radiation produced mutations through his experiments with fruit flies. His finding is termed mutagenesis. This researcher found that the radiation-induced mutations were the same as those produced by nature. Irradiating the fruit flies did not create any unusual effects, but the frequency of mutations was intensified. This implies that the effects of ionizing were not unique to radiation, that is, they could have been caused by things other than radiation (Forshier, 2008) Effect of Oxygen The oxygen effect was the subject of experimentation during the 1940s and 1950s. Oxygen is a radiosensitizer because it increases the cell-killing effects of a given dose of radiation. This occurs as a result of the increased production of free radicals when ionizing radiation is delivered in the presence of oxygen (Forshier, 2008). The oxygen effect is known as Oxygen Enhancement Ratio (OER) and numerically defined as (Forshier, 2008): It is necessary the presence of oxygen in order to form free radicals during ionization of water. Without free radicals, hydrogen peroxide is not formed, and thus cell damage is reduced (Forshier, 2008). The OER is dependent on LET, being more pronounced for low LET radiation and less effective for high LET radiation. Because of the physical differences between high and low LET radiations the quantity of damage done by high LET radiation would be beyond repair. Thus, having oxygen present would not intensify the response to radiation the same magnitude, as would be the case with the low LET radiation, (Forshier, 2008), Figure 4.5. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 85

105 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Figure Oxygen effect of the LET (from Forshier, 2008) Relative Biologic Effectiveness The relative effect of LET is quantitatively described by the relative biologic effectiveness (RBE). RBE is a comparison of a dose of test radiation to a dose of 250 kev X-ray which produces the same biologic response, being expressed as follows (Forshier, 2008): The RBE measures the biological effectiveness of radiation having different LET values. Factors which influence RBE include radiation type, cell or tissue kind, physiologic conditions, biologic result being examined, and the radiation dose rate. In comparing LET and RBE, as LET increases, RBE increases also, Figure 6. Accordingly, low LET radiations have a low RBE, and high LET radiation have a high RBE (Forshier, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 86

106 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Figure RBE versus LET (from Forshier, 2008) Reproductive Failure In 1956, Puck and Marcus exposed human uterine cervix cells to varying doses of radiation. Thus, experimentally determined reproductive failure by counting the number of colonies formed by these irradiated cells (Forshier, 2008). As scientists began to research the effects of radiation exposure had on biological processes, there occurred a need to measure the levels of radiation causing specific effects. Units of measurement were developed to quantify radiation levels and thus track the effects of exposure to varying the levels of exposure (Forshier, 2008). 4.4 BIOLOGIC EFFECTS OF RADIATION Ionizing radiation transferring energy to biologic systems causes, in several successive stages, biological consequences. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 87

107 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Elementary phenomena Physic interactions - these interactions vary according to the nature of radiation. Photons (X-rays or gamma rays) put in motion, during collisions with atoms of the medium, electrons to which they transfer whole or part of its energy in the form of kinetic energy. This kinetic energy is expended in the course of interactions with electrons belonging to atoms of the medium, and is subjected to the electric field of the incident electron (excitation and ionization) and these interactions "consume" an energy that was subsequently transferred, through ionizing radiation, to the medium. This phase is very brief (Pedroso Lima, 2003). The proportion of these modified atoms is minimal; however, they are grouped along the path of electrons, at varying distances. Although the amount of energy transferred is low, its concentration along these trajectories into bundles of energy whose value is relatively high (10 to 100 ev) gives a great efficiency. The other charged particles (alpha particles, protons set in motion when the interactions of neutrons with the medium) cause the same excitations and ionizations along its own path but at much shorter distances (the beam energy has the same value but is closer) (Pedroso Lima, 2003). Radiochemical phenomena in a second phase, equally brief, the ionization of an atom within a molecule leads, in general, to her collapse and the fragments formed, called radicals. These radicals are chemically very "active" since they are able to react with other molecules initiating various chemical reactions. The effect is direct when the ionization directly affects the molecules damaging them, or indirect when the injury is caused by free radicals formed during the breakdown of water molecules - radiolysis - which constitute the bulk of biologic systems, Figure 4.7. The final product of the water radiolysis is the formation of an ion pair, H + and OH -, and two free radicals H* and OH*. These chemical species are highly reactive radicals that play an important role and constitute the starting point of many molecular changes. Half of the molecular injuries are due to direct effect and the other half to indirect effect. When the distance between ionizations is short, these radicals react with each other and their concentration along the trajectories increases the effectiveness of these ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 88

108 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL reactions. Therefore, for the same amount of energy absorbed the number of damaged molecules is larger (Pedroso Lima, 2003). Radicais livres OH*, H* Água H 2 O Iões HOH +, HOH - Iões OH -, H - Figure 4.7 Radiolysis of water molecules (Forshier, 2008). The human body is composed of 80% water so the irradiation of water is involved in most interactions involving radiation Molecular Damages All biological molecules can be altered but the consequences vary according to the importance of the injured molecules. The molecules of deoxyribonucleic acid or DNA are those where the damage is more serious, since each has a specific role. Indeed, each cell contains information that will allow, according to a preconceived plan, the appropriate development and reaction to external events. The genetic material, or hereditary material, consists of DNA molecules that are the backbone of information. Damage to DNA molecules is the key mechanism of ionizing radiation action (Suntharalingam, 2002). Deoxyribonucleic acid or DNA - The structure is the same in all living species. The elementary constituent of DNA molecule is the nucleotide, which is formed by a phosphate group, a sugar (desoxirribose) and one base. A DNA molecule consists of two long strands or fibers of millions of nucleotides that form as a ladder whose bars would be the sequence of alternating sugars and phosphate groups, and the lanes ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 89

109 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL would be two bases joined together. This string wraps around its axis (Suntharalingam, 2002), Figure 4.8. Figure Deoxyribonucleic acid molecule (DNA) (from Seeley, 2004). There are four different types of bases: adenine (A), cytosine (C), guanine (G) and thymidine (T), that are always available to form these dishes, paired as follows: adenine with thymine and guanine with cytosine, forming these four pairs possible: AT, TA, GC and GC. The order of bases in one of the molecule chains determines, unambiguously, the order of bases on the other chain (from Seeley, 2004). The orders in which the bases follow one other constitute one code, and a sequence of three bases (triplets) determines the amino acid that is present in the encoded protein. The set of "triplets" that encode a protein constitutes a gene. Thus, a gene consists of a sequence of several thousand of nucleotides coding for a specific protein that is synthesized from the information contained in this gene. This information is transmitted to the cytoplasm by a messenger RNA (from Seeley, 2004). Besides the coding genes, other DNA sequences constitute regulatory systems that, for example, activate ('operators' genes) or repress ('repressive' genes) the expression of a gene and, consequently, the synthesis of the protein encoded by this gene. These regulatory mechanisms, not yet fully understood, and to which are certainly devoted numerous DNA sequences, definitely explain the disproportion ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 90

110 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL between the number of genes identified and the total of DNA mass (from Seeley, 2004). When radiation interacts with the cell, the ionization and excitation may occur in the macromolecules (for example, DNA) or in the medium they are (for example, water). Depending on the site of interaction, the effect is called direct or indirect (Suntharalingam, 2002). The direct interaction occurs when a first ionization reaches a macromolecule (for example, DNA, RNA, proteins or enzymes). If the macromolecule is ionized it is considered abnormal or mutated (Suntharalingam, 2002). The indirect interaction occurs if the initial ionization takes place at a distance not critical of the macromolecule and, then takes place the transfer of ionization energy to the molecule (Suntharalingam, 2002) Chromosomes Irradiation In multicellular species the DNA molecules are the heart of chromosomes, which are essential constituents of the cell nucleus. Each species is characterized by the number and shape of chromosomes. Human cells, for example, have 46 chromosomes grouped in 23 pairs of 2 chromosomes apparently identical (size, shape, etc.), one from the mother and one from the father. One of these 23 pairs is unique, the sex chromosomes. In women, the two chromosomes called X are similar; in men, they look different: one, called X, is similar to the woman and the other called Y, is much smaller (Forshier, 2008). Each chromosome consists of a single molecule of DNA coiled about itself and closely tied to protein molecules, Figure 4.9. The length of a chromosome is about 0.1 μm, but if the DNA molecule was stretched it would have a length of approximately 4 cm that is times longer. Its width is 2 nm (Forshier, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 91

111 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Figure 4.9 DNA Compaction (from Seeley, 2004). At the time of cell division, chromosomes can be observed microscopically. It is then possible to count them and identify them by size, shape and after stained, by structure. In this phase, it is feasible to study chromosomal abnormalities (Forshier, 2008). When the chromosomes are irradiated, the radiation interaction can be direct or indirect. The result of any of the interactions is a mutation. The mutation causes a visible chromosomal change, Figure 4.10, and represents critical lesions in DNA (Forshier, 2008). Figure 4.11 depicts the effects of a single mutation caused by an irradiation in the G1 phase of the cell cycle. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 92

112 D. One break in two chromossomes Dicentrics C. One break in two chromossomes Translocation B. Two break in one chromossome A. One break in one chromossome CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Quebra Recombinação Replicação Separação Anafásica Figure Chromossome Aberrations (from Forshier, 2008). Replication in S and pass through the G 2 phase Irradiation in G 1 phase Causes chromatid breaks Visualization in M phase Figure Simple Mutation in G1 phase (from Forshier, 2008). Radiochemical effects on DNA and chromosomes - the main damage caused by ionizing radiation are: Modifications of bases: adenine, cytosine, guanine and specially thymidine. A pair of bases may be absent or replaced by ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 93

113 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL another. The modification of the order or nature of the bases causes an alteration of the information carried by the gene (point mutation). Changes in DNA conformation: a rupture in one of the two chains (these lesions are easily repairable - Figure 4.12) or rupture of the two chains (these injuries are difficult to repair) Endonuclease Polimerase Χ Ligase Figure 4.12 Schematic of the repair mechanism of excision-resynthesis (from Forshier, 2008). Other intersection injuries (cross links) form links, for example, between two DNA strands, DNA-DNA bonds, or between one nucleic acid and protein: DNA-binding protein. Several remodeling of chromosome structure: a single or multiple rupture can cause the loss of a fragment - deletion - if it occurs in S phase of the cell cycle takes place the replication of the deletion and, in metaphase the abnormal chromosome looks like the normal chromosome despite lacking information in the terminal region; the setting of this fragment on another chromosome is called translocation. When two chromosomes exchange pieces thus speaks of reciprocal translocation. This fragment can then re-weld abnormally on the same chromosome (inversion). If in G1 phase of the cell cycle occurs two mutations in the same chromosome, the two ends can ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 94

114 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL 'weld' and form a 'ring' chromosome; chromossomes can weld again in a more complex way, forming dicentric chromosomes, etc. The quality of the adhesion ability of damaged chromosomes is a determining factor in the joining of the chromatid, Figure 4.13 (Forshier, 2008). Ring Dicentric Irradiation in G 1 phase Causes chromatid breaks Bind during S phase Visualization in M phase Figure Chromosomal aberrations of multiple mutations (from Forshier, 2008). The morphological study of chromosomes in a cell is of enormous practical interest, since the number of abnormalities is dose dependent and can assess their importance from relatively low values (0.25 Gy). Chromosomal aberrations may make it impossible the balance of genetic material between two daughter cells and, lead to cell death at the time of cell division or non-viability of the two daughter cells (Forshier, 2008). Cellular constituents other than DNA can suffer injuries caused by ionizing radiation, for example, fatty acids that make up cell membranes, proteins such as enzymes, involved in all stages of cellular life. Although, if the points of impact of ionizing radiation are numerous, the biological effect resulting primarily from lesions in the DNA molecules (Forshier, 2008). Molecular DNA repair there are many chemical or physical agents that can damage DNA and so life would not be possible without repair. The total length of DNA ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 95

115 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL contained in the cells of the body (2m in length per cell) is about 60 million kilometers. Per day is born 200 billion cells, the length of DNA synthesized is 400 million kilometers a day. These long and narrow molecules are fragile and therefore the thermal agitation and chemical reactions harm it constantly. Consequently, becomes, necessary systems to repair the damage, particularly, due to external factors such as ultraviolet radiation, chemicals, etc. If the injuries were permanent, the impact of a single photon at the level of a molecule would result in an irreversible alteration of a gene, and the smallest radiation harm. Thanks to the final repair the damage is much less than the damage we would get if were added all the molecular lesions (Forshier, 2008). When the injuries are related to one of the two chains, restoration is usually full; however, if the two chains simultaneously suffer injury, repair mechanisms are more complex and can result in a repair deficient, that is, has an error (mutation) whose consequences can lead to cell death or start their cancer (Forshier, 2008). Biological consequences of irradiation - At the cell level the effects are multiple. Irreversible DNA injuries can result: a mutation, that is, a final modification of the property inherited from the cell; loss of viability, that is, the inability to divide and give rise to normal daughter cells, which can express themselves since the first cell division or during the first five divisions (delayed death). The proportion of surviving cells, i.e., is, those which retained the ability to divide many times, it decreases with the dose. Besides depending on the dose, this ratio also depends on the nature of radiation and dose rate, as well as suffering from the influence of the environment of cells (for example, the decrease of oxygen content increases radiation resistance) (Forshier, 2008) Irradiation of Macromolecules The occurrence of molecular derangements or injuries may be classified either effects on macromolecules or effects on water. Irradiating macromolecules gives very different results when compared to the irradiation of water, Figure If ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 96

116 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL macromolecules are exposed to ionizing radiation in vitro (outside the body or cell), a significant dose of radiation is needed to produce a measurable effect. Irradiating macromolecules in vivo (inside the living cell) shows that when cells are in their natural conditions, they are much more radiosensitive (Forshier, 2008). Figure 4.14 Macromolecules mutations (from Forshier, 2008). The three primary effects of irradiating macromolecules in vitro include mainchain scission, cross-linking and point lesions. Main chain scission - occurs when the thread or backbone of the long-chain molecule is broken. This results in the long-chain molecule being reduced to numerous smaller molecules, which can still be macromolecular in nature. Not only the size of the macromolecule is reduced, but its viscosity (thickness) is also reduced (Forshier, 2008). Cross-linking - certain macromolecules have spurlike extensions off the main chain. Others develop these spurs after being irradiated. After being irradiated, these spurs can as if they had a sticky material on their ends. This stickiness causes the macromolecule to connect to another macromolecule, or to another section of the same molecule. This is termed cross-linking. Viscosity is increased by radiationproduced molecular cross-linking (Forshier, 2008). Point lesions - Irradiating macromolecules may result in disturbance of single chemical bonds, which create molecular lesions or point lesions. Point lesions may ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 97

117 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL cause slight molecular changes, which in turn cause the cell to function incorrectly (Forshier, 2008). At low doses of radiation, point lesions are regarded to be the cellular radiation damage that is responsible for late radiation effects, which are observed at the whole-body level (Forshier, 2008). Irradiating macromolecules may result in either death of the cell or late effects. Throughout the cell cycle proteins are constantly being created, and occur in greater number than nucleic acids. Abundant copies of unique protein molecules always exist in the cell. These factors allow protein to be more radioresistant than the nucleic acids. In addition, numerous copies of m-rna and t-rna exist in the cell, although they are not as plentiful as the protein molecules. Conversely, DNA molecules, having their distinctive base arrangements, are not so frequent. Because of this, DNA molecule is considered the most radioresistant macromolecule. RNA radiosensitivity is midway between that of DNA and protein macromolecules (Forshier, 2008). There can be visible chromossome abnormalities or cytogenetic damage if the radiation damage to the DNA is intense enough. DNA can be injured without producing visible chromosomal aberrations. Even though this damage is reversible, it can lead to death of the cell, and ultimately destroy tissues and organs (Forshier, 2008). Metabolic activity can also be affected by DNA damage. The primary characteristic of radiation-induced malignancies is the uncontrolled reproduction of cells. If germ cells receive DNA damage, the response may be detected in future offspring (Forshier, 2008). Figures 4.15 A-D, illustrate DNA aberrations that are reversible types of damage. They may involve the sequence of bases being changed, thus changing the triplet code of codons. This is considered a genetic mutation at the molecular level (Forshier, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 98

118 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Damage type shown in Figure 4.15-E also involves the change of or loss of a base. This type of damage destroys the triplet code as well, and may not be reversible; this is considered a genetic mutation (Forshier, 2008). These molecular genetic mutations are termed point mutations, and are common with low LET radiation. Point mutations may be either of minor or major significance to the cell. A key effect of these point mutations would be the genetic code being incorrectly transferred to daughter cells (Forshier, 2008). A base deletion B base substitution C Hydrogen bond disruption or or D Single strand E Double strand (not repairable) or Low LET (x-ray) High LET (α particle) Figure 4.15 DNA aberrations (from Forshier, 2008) Dose-response relationship The dose-response relationships, also referred to as dose-response curves, are graphical correlations between the observed effects (response) from radiation and dose of radiation received, Figure 4.16 (Forshier, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 99

119 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Dose-response curves differ in two ways (Forshier, 2008): They are either linear or non-linear; They are either threshold or nonthreshold. Figure Dose-response Relationship (from Forshier, 2008). Linear means that an observed response is directly proportional to the dose. On the other hand, nonlinear means that an observed response is not directly proportional to the dose. Additionally, threshold assumes that there is a radiation level reached below which there would be no effects observed, and nonthreshold assumes that any radiation dose produces an effect. Diagnostic radiology is primarily concerned with linear, nonthreshold dose-response relationships (Forshier, 2008) Linear-Dose-Response Relationships Since dose-response relationship A and B intersect the dose (x) axis at either zero or on the y-axis, they are considered linear, nonthreshold, Figure All linear dose-response relationships exhibit an effect regardless of the dose. This is demonstrated by relationship A. Even at zero doses, A exhibits a measurable response (RA). This RA is termed the ambient or natural response. Dose-response relationships C and D intercept the dose axis (x) at a dose value greater than zero. Thus, C and D are considered linear, threshold. At doses below the respective C and D values, o response would be anticipated (Forshier, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 100

120 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Linear Quadratic Dose-Response Curves In 1980, the Committee on the Biological Effects of Ionizing Radiation (BEIR Committee) concluded that the effects of low doses of low LET radiation follow a linear, quadratic dose-response relationship, Figure At low doses, the curve is linear and at high doses, the curve becomes curvilinear and is no threshold (Forshier, 2008). The portion of the curve where increases in dose shows no or light increase in the effect is named as the toe. The shoulder is considered the area of the curve in which a leveling off occurs, again demonstrating no or little increase off or flattened (Forshier, 2008). In 1990, with 10 additional years of human data, the BEIR committee revised its radiation risk estimates and adopted the linear, nonthreshold dose-response relationship as most relevant (Forshier, 2008). Current radiation dose-response curve, there is a nonlinear relationship between dose and effect, meaning that the effect is not directly proportional to the dose (Forshier, 2008). Figure 4.17 Linear quadratic dose-response curve (from Forshier, 2008) Dose-response curve linear quadratic The sigmoid dose-response curve s applied predominantly to the high dose effects observed in radiotherapy, Figure Sigmoid means S-shaped. There is usually a threshold below which no observable effects occur. With a sigmoid dose- ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 101

121 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL response curve, there is a nonlinear relationship between dose and effect, meaning that the effect is not directly proportional to dose (Forshier, 2008). Figure 4.18 Sigmoid dose-response curve (from Forshier, 2008) Targeted Theory As cells contain a profusion of molecules, radiation damage to these molecules is not likely to result in significant cell injury because additional molecules are present to assist in cell survival. However, there are molecules that are not in abundance that are considered necessary for the cell survival. Irradiating these could have serious consequences, because there may not be others available to maintain cell survival. This idea of a sensitive critical molecule is the foundation for the targeted theory. According to the targeted theory, there will be cell death only if cell s targeted molecules is inactivated. It is theorized that DNA is the critical molecular target (Forshier, 2008). The target is regarded to be the area of the cell that contains the target molecule. Because radiation interaction with cells is random, target interactions also occur randomly. The radiation shows no favoritism toward the targeted molecules (Forshier, 2008). When a target is irradiated, this is considered a hit. Both direct and indirect effects cause hits, Figure Direct versus indirect hits are not distinguishable. With low LET radiation in an anoxic condition, chances for a hit on the targeted molecule are low because of the large distances between ionizing events (Forshier, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 102

122 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL In an aerobic state with low LET radiation, the indirect effect is intensified, as more free radicals are formed, and the volume of action surrounding each interaction enlarged. This increases the likelihood of a hit (Forshier, 2008). Using high LET radiation, ionization distances are so close together that there is a high probability that a direct hit will take place, probably even higher than for the low LET, indirect effect (Forshier, 2008). Adding oxygen to high LET radiation will probably not result in additional hits, as the high LET has already produced the maximum number of hits possible (Forshier, 2008) Cell Survival Curves Cellular sensitivity studies began in the middle 1950s with Puck and Marcus. They performed in vitro studies using HeLa cells. Their initial study was on failure of reproduction in which they exposed HeLa cells to differing radiation doses and then totaled the number of colonies formed (Forshier, 2008). Figure 4.19 Targeted theory (from Forshier, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 103

123 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL This information may be illustrated graphically by plotting the radiation doses on a linear scale on the x-axis, and plotting the fraction of surviving cells on a logarithmic scale on the y-axis. This graphical representation of the relationship between the dose and surviving cells is a survival curve (Forshier, 2008). It was stated previously that radiation interaction is random in nature. Therefore, it must be determined how many hits are necessary to cause cell death. This may be demonstrated using a cell survival curve (Forshier, 2008). The model most used is the linear-quadratic model, whereby there are two components responsible for cell death: a dose-proportional, which corresponds to the initial portion of the curve and represents the cell death caused by lethal damage, and another component proportional to the square of the dose, related to the steeper region of the curve and is linked to the deaths caused by lethal damage, potentially lethal damage, and especially the accumulation of sub-lethal damage (Suntharalingam, 2002). In simple cells such as bacteria, if there are additional hits to the same cell, these hits do not matter. In complex cells such as human cells, it is theorized that in order to cause cell death, more than one hit is required (Forshier, 2008). The graphs of simple versus complex cells are very different, Figure Graph A represents a survival curve for simple cells, represented by a straight line. Graph B represents a survival curve for complex cells, represented by a line which displays a shouldered area where effects are not apparent until some targets have received enough multiple hits to be killed. The targeted theory can be used to explain this shoulder section of the curve (Forshier, 2008). The shoulder of the cell survival curve shows that some damage must accrue before there can be cell death. The accumulated damage is called sub-lethal damage. The wider the shoulder, the more sub-lethal damage the cell can endure. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 104

124 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Figure 4.20 Simple versus complex cell survival curves (from Forshier, 2008). 4.5 CELL DEATH IN MAMMALIAN TISSUES The clonogenic potential is the essential element for the maintenance of a cell line, either in vitro or in organized tissues, although there are other important issues in the maintenance associated with complex tissue systems. Normal senescence of cells is one of these important issues and the other is the removal of cells that are in the wrong place at the wrong time. Examples of this would be the metastatic arrival of tumor cells transported from a primary tumor elsewhere or the resolution of inflammatory processes (Alpen, 1998). It is possible to define at least two different types of cell death that go beyond the end point of clonogenic potential and involve the actual disappearance of the cell: necrosis and apoptosis (Alpen, 1998). Necrosis is characterized by a tendency for cells to swell and ultimately to lyse, which allows the cell's contents to flow into the extracellular space, this is usually accompanied by an inflammatory response. In the case of neoplasms, necrosis is most often seen in rapidly growing tumors, where the tumor mass outgrows its blood supply and regions of the tumor become undernourished in oxygen and energy sources. In this case inflammation is not a characteristic of the necrotic process (Alpen, 1998). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 105

125 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Apoptosis involves shrinkage of the nucleus and cytoplasm, followed by fragmentation and phagocytosis of these fragments by neighboring cells or macrophages. The contents of the cell do not usually leak into extracellular space, so there is no inflammation. Since there is no inflammation accompanying apoptosis, the process is histologically quite inconspicuous (Alpen, 1998). Figure Structural changes of cells undergoing necrosis or apoptosis (from Goodlett, 2001). The concept of apoptosis as a mechanism for the control of cell population numbers and cell senescence has been around for several decades, but the mechanisms of apoptosis have received extensive research attention only in the nineties. This interest in apoptosis was engendered by the discovery that tumor suppressor genes and oncogenes were central control agents for the process. The principal focus of these studies has been the role of the p53 tumor suppressor gene, already described in chapter II. The p53 gene is a transcriptional activator that may include activation of genes that regulate genomic stability, cell cycle progression, and cellular response to DNA damage. The synthesis of the p53 product is known to be responsible for the induction of apoptosis in many cell lines in which this gene is present in unmutated form. The mutational absence of this gene is often accompanied by the inability of a cell line to initiate apoptosis. For radiation pathology, the important finding is that even small amounts of DNA damage in G 1 cells cause ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 106

126 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL synthesis of the p53 product and ultimate apoptosis of the cells. It is pertinent for radiation pathology that cells of the lymphoid system generate high concentrations of p53 gene product after cell damage. This is particularly true for low doses of ionizing radiation. Clearly, the generation of the p53 product is not sufficient for the onset of apoptosis, but it is certainly necessary (Alpen, 1998). Another significant gene involved in apoptosis is the bcl-2 gene (described in chapter II). This gene encodes a protein that blocks physiological cell death (apoptosis) in many mammalian cell types, including neurons, myeloid cells, and lymphocytes. This gene is able to prevent cell death after the action of many noxious agents (Alpen, 1998). The role of apoptosis as a mechanism for cell death following ionizing radiation exposure remains unclear at this time, particularly the relative importance of the agonistic role of p53 and the antagonistic role of bcl-2. However, it must be important, as that the detection of small nicks and errors in the DNA of G 1 cells is crucial to the recovery of irradiated tissues and the reduction of genomic misinformation (Alpen, 1998). 4.6 NATURE OF CELL POPULATIONS IN TISSUE One of the earlier systematic overviews of the nature of cell population kinetics in normal and malignant tissues was that of Gilbert, Their classification of the various kinetic systems found in mammalian (and, incidentally, in other organisms) organs and tissues is shown in Figure 4.22 (Alpen, 1998). Figure Classification of cell kinetic types in the system of Gilbert, 1965 (from (Alpen, 1998)). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 107

127 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL From Figure 4.22, the definitions of each of the systems are the following (the double arrows in classifications D, E, and F, are meant to signify the mitotic division of one of the cells of the compartment, giving rise to two daughter cells): A. Simple transit population. Fully functional cells are added to the compartment while a population of either aging or randomly destroyed cells disappear from the pool. There are many examples of functional end cells that are in this category. Examples are spermatozoa, which are constantly being replaced, as well as red cells or other end cells of the blood. B. Decaying population. The cell numbers decrease with time without replacement. The population of oocytes in the mammalian female is often quoted as an example, if not the only example. Populations of this classification are rare in mammalian systems, but not in insects. C. Closed, static population. There is neither decrease nor increase in cell numbers during life. It is unlikely that such a population truly exists. The differentiated neurons of the central nervous system are quoted as an example of a static population, but there is probably a decline in cell numbers even in this population. D. Dividing, transit population. In addition to the transiting cells, division of the cells within the compartment occurs that leads to a larger number leaving than entering. It is assumed in this model that the number of cells in the compartment remains more or less static. The differentiating and proliferating blood cell types (for example, the proerythroblast of the bone marrow) that follow the stem cell are examples of this type of population. E. Stem cell population. A self-sustaining population, that relies on selfmaintenance for its continued existence. All the progeny of this type of cell line depend upon the continued existence of the stem cell pool. Every self-maintaining, dividing cell population must have such a precursor pool. Examples are the stem cells responsible for sustained spermatogenesis or hematopoiesis. F. Closed, dividing population. Such a population is best represented by neoplastic growth. No cells enter or leave the compartment in the early stages of tumor growth. In the long run, neoplastic growth is probably best represented as a stem cell population, since as the tumor enlarges, there is cell death, suppression of growth by metabolic and other nutrient shortages, and a highly variable rate of ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 108

128 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL division. The epithelial cells responsible for cell renewal in the lens of the eye are another example of this type of population (Alpen, 1998). 4.7 CELL POPULATION KINETICS AND RADIATION DAMAGE It should be almost self-evident that the kinetic types represented by D, E, and F of Figure 4.22 will be most vulnerable to radiation damage. It has been established that for clonogenic death of the cell the principal target of ionizing radiation is the genome, and the genome is certainly at its most vulnerable to radiation damage during G 2 and mitosis (M), when replication has been completed. The principal outcome of disturbances to the dynamic replicative activity of the genome is altered clonogenic ability. That is indeed the case, and the most critically sensitive of these systems would be the stem-cell-type tissue (E), which depends for its continuing function on its own continued clonogenic potential, since there is no precursor compartment to replace deficiencies (Alpen, 1998). The ultimate functional viability of a tissue that is dependent on stem cell activity will be determined by whether, after radiation exposure, there are adequate numbers of surviving and still clonogenic stem cells to repopulate the compartment and finally to produce functionally competent progeny. The most resistant tissues are those that require neither input of cells from a prior compartment nor division within the compartment. The closed static model is such a case, and in the case of the central nervous system, its high degree of radioresistance can be attributed to its lack of need for cell replication and replacement (Alpen, 1998) Growth Fraction and its significance The concept of growth fraction as a descriptive parameter for the kinetics of proliferating tissue appears to have been first proposed by Mendelsohn (1962) as the result of his observations that all cells in a growing tumor are not in the active process of proliferation as determined by the cellular incorporation of radioactive labels of DNA synthesis. Lajtha (1963), based on his own studies as well as those of others, proposed the concept of the G 0 phase of the cell cycle, a state of the cell in which the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 109

129 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL cell was not engaged in active proliferation, but in which the cell could reenter the proliferative state. The G 0 cell was visualized as a cell that has been removed from the actively dividing population by regulatory activities rather than as a result of metabolic deprivation. Subsequently, it became apparent that cells also could be removed from active division in a reversible manner by deprivation of oxygen, glucose, or other metabolites (Hlatky et al., 1988). Restoration of the lacking nutrient led to reentry of the cell into active proliferation (Alpen, 1998). Figure 4.23 Cell cycle phases (from (Goldwein, 2006)). The growth fraction is defined as the fraction of the total cellular population that is clonogenically competent and is actually in the active process of DNA replication and cell division. The growth fraction may be estimated by any one of several techniques, most of which depend on incorporation of a radioactively labeled DNA precursor into those cells that are actively dividing. One of the simpler methods for determination of the growth fraction is the exposure of a growing culture of cells, in vitro or in vivo, to an appropriate radioactive label for the synthesis of DNA. A typical and frequently used label is 3 H-thymidine. The cells are exposed to the radioactive label in the medium or by injection into the intact animal for at least the full length of a cell cycle (and usually for half again as long). Under these conditions, all cells that synthesize DNA, thus indicating their passage through the S period of the cell cycle, are labeled and can be identified by autoradiography. The percentage of cells that is labeled constitutes the growth fraction, since every cell in cycle will have passed ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 110

130 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL through the S period at least once during exposure to the radioactive label (Alpen, 1998). The radiobiological significance of the growth fraction was unclear until the appearance of new data in the late 1980s. In 1980, Dethlefsen indicated that the role of quiescent cells in radiobiological response was not satisfactorily delineated. Recent studies indicate that cells that are out of cycle are capable of a more significant amount of repair of potentially lethal damage, simply because there is more time before the cell is called on to replicate its DNA. It is possible, but by no means proved, that the concentration of enzymes necessary for repair of DNA damage may be depleted in the noncycling cell, but, in spite of this, the additional time allows effective repair to proceed with the lower concentration of repair enzymes (Alpen, 1998). 4.8 CELL KINETICS IN NORMAL TISSUES AND TUMORS Both normal and neoplastic tissues have a cellular kinetic pattern that follows the accepted model of a G 1 -S-G 2 -M cycle, and, indeed, the cell cycle parameters are not very different for tumors as compared to other growing tissues. The total cycle time and the time devoted to DNA synthesis in the S period are very much alike for both tissue types. However, there are significant differences in some of the characteristics of the kinetic pattern as the tumor reaches a size where vascularization is required for continued tumor growth. The orderly vascularization of normal tissues that originates in embryonic life and that is maintained throughout the existence of normal, nonpathological function assures that the supply of oxygen and nutrients is adequate for survival of cells. Most, if not all, tumors, on the other hand, originate as nonvascularized aggregations of cells and develop a vascular supply sometime after the origination of tumor growth. The development of vascular supply in a tumor depends on the activities of angiogenic factors that occur in normal tissues. The newly developing vascular supply is, at best, chaotic and disorganized (Alpen, 1998). Some parts of the tumor tissue will be so far from the source of oxygen and nutrients that cell survival will be impossible, Figure Other parts of the tumor will have nutrient and oxygen supplies that are adequate only for survival of cells without replication. The lack of oxygen and glucose can lead to a decrease in the growth ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 111

131 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL fraction, and probably to cell death and necrosis. Several nutrients and metabolic products, including oxygen, glucose, and lactic acid, play an important role in the determination of quiescent and proliferating cells in tumors (Alpen, 1998). One important difference between normal tissues and tumor tissues is the determinant of the fraction of quiescent cells in the organ or tumor. Because of the orderly vascular architecture of normal tissue, the movement of cells from the proliferating to the quiescent compartment is probably not the result of nutrient lack, but, rather, the result of the activity of normal soluble growth factors and naturally occurring inhibitors that regulate the growth and development of the tissue (Alpen, 1998). 4.9 MODELS FOR RADIOBIOLOGICAL SENSITIVITY OF NEOPLASTIC TISSUES The earliest attempts to assay the sensitivity of organized tissue systems were directed at establishing the radiosensitivity of tumor tissues. This was partly because these tissues offered opportunities for analysis that were not available for normal tissues. The possibility for syngeneic transplantation of the cell lines from host to recipient animal was the most important characteristic of these in vivo tissue systems. Figure Role of hypoxia in tumour angiogenesis (from Carmeliet, 2000). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 112

132 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL After irradiation of the tumor in the host in which it was growing, it was possible to transplant the tumor cells to an unirradiated recipient animal and to observe the growth response of the irradiated tumor cells. There was also strong interest in understanding tumor biology arising from the treatment of cancer by radiotherapy. It was important to establish the role of oxygen in the sensitivity of cancer cells, as well as the importance of the fraction of G 0 cells and repair or repopulation in these tissues. The overall goal was practical: to maximize the effectiveness of radiotherapy for cancer control in patients, while reducing damage to normal tissues in the radiation field (Alpen, 1998) Hewitt Dilution Assay Probably the first in vivo assay for mammalian tissues was that developed by Hewitt and Wilson (1959) with a syngeneic mouse tumor system. At that time a number of tumor cell lines that were grown in the peritoneal cavity of mice had been developed. The cells from these ascites tumors could be harvested or allowed to continue to grow in the peritoneal cavity of the host, which would cause the death of the animal. It occurred to Hewitt and Wilson that this end point - death of the host animal could be used to measure the clonogenic potential of the tumor cells after irradiation. Figure 4.25 shows the essentials of a Hewitt assay for a single dose point at 10 Gy (Alpen, 1998). Figure Typical data set for a Hewitt dilution assay (from Alpen, 1998). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 113

133 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Cells harvested from the mouse ascites tumor P388 and unirradiated cells were collected from the donor and a series of dilutions was prepared from a stock suspension of the tumor cells. A typical microbiological-type binary dilution was carried out to produce cell suspensions with low concentrations of cells that will allow the recipient animal to be injected with cell numbers that are correct for killing about half of the animals. For the tumor line used, the usual cell dose required to kill half of the animals is about two to three cells. A small number of animals (5-10) are injected with the same cell dose and the survival is followed. The same procedure is used for several additional cell doses. The resulting data on percent survival at each of the cell doses are plotted as shown in Figure 4.25, and the LD 50 (lethal dose for 50% of the animals) is determined by graphical or analytical means. The procedure is repeated, but with the cell suspension prepared from animals that were irradiated before cell collection. Animals are irradiated at several doses and injections proceed as just described for each dose. The LD 50 values can be used to construct a survival curve. Figure 4.25 shows an example for only one radiation dose on the right panel and for unirradiated cells on the left panel, with the calculated surviving fraction. The surviving fraction is estimated for each of the other doses, and a survival curve of surviving fraction against dose is plotted in the usual way (Alpen, 1998). The Hewitt assay has been the tool used for a number of significant studies of tumor cell sensitivity to radiation. Figure 4.26 is a very good example of such studies. Andrews and Berry (1962) developed survival curves for three mouse tumors, two leukemias, and a sarcoma. Some of the data were Berry's own previously unpublished observations and some were provided by Hewitt. The clonogenic survival curves were developed for both anoxic and oxic conditions. All three cell lines could be plotted on the same curve for oxic cells or for anoxic cells as appropriate, and the line produced was a good fit for the appropriate condition of oxygenation. The oxygen enhancement ratio (OER) for these cells was about 2.4, which is not far from the 2.8 or so for cell lines that are irradiated in vitro and analyzed for clonogenic survival in vitro. The D o for the cells irradiated under oxic conditions was about 150 cgy, and the extrapolation number was about 3-4 for this set of data (Alpen, 1998). A significant shortcoming of the dilution assay system is that donor cells that are grown in ascites fluid are usually irradiated when the cell number in the peritoneal ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 114

134 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL cavity is very large. Under these conditions, it is not always clear that the cells are fully oxygenated at the time of irradiation. If that is indeed the case, there is the possibility of significant anoxic protection of the cells and, subsequently, there is an overestimation of the resistance of the cells to the irradiation. The data reported in the Berry study do not seem to be affected by such hypoxia. The D o (oxic) is about 150 cgy, a number quite consistent with that found for many cell systems in vitro. The OER of 2.4 or so is, again, not very different from the seen for in vitro systems. We must conclude, at least for the cell lines reported in this study, that adequate oxygenation probably existed at the time of irradiation (Alpen, 1998). Another shortcoming of the Hewitt method is that the irradiated tumor cells must be capable of expressing clonogenic potential while growing in the ascites medium. For example, most leukemias grow readily in this environment, and usually require an inoculum of only 1-3 cells to cause the death of 50% of the recipient animals. For the Berry data just described, the sarcoma cells required an inoculum of more than 80 cells to kill 50% of the recipients. In many cases no cell growth is seen and no assay is possible. To avoid this shortcoming, other assays have been developed (Alpen, 1998). Figure The survival curve obtained by Berry (1964) via the Hewitt assay method for two mouse leukemias and a sarcoma (from Alpen, 1998). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 115

135 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Lung Colony Assay System A modification to the Hewitt assay was developed by Hill and Bush (1969) to measure clonogenic survival of cells derived from solid tumors. In principle, the assay measures the clonogenic survival of tumor cells by determining their ability to form colonies in the lung of recipient syngeneic mice. The cells from a tumor, irradiated either in vivo or, after dissection and cell dissociation, in vitro, are injected into a recipient mouse, and after days the animals are killed, the lungs are dissected, and the number of tumor colonies in the lung is counted. Hill and Bush were able to demonstrate a linear relationship between cell number injected and the number of colonies formed in the lung. A very large enhancement of the number of colonies in the lung was found if, along with the experimentally irradiated cells, a large number of heavily irradiated, nonclonogenic cells were injected. Typically, such a procedure produced a fold increase in the number of colonies formed from the clonogenic survivors. Hill and Bush were not able to establish the mechanism of this enhancement, but it was not due to an immune response on the part of the recipient. Very consistent survival curves were obtained, and, for the KHT transplantable sarcoma, the D o was 134 cgy, with an extrapolation number of about 9.5. Again, these data were found to be quite consistent with the values found for the same tumor with the Hewitt assay. Such an agreement not only validates the lung colony assay, it also demonstrates that there was little protection from radiation damage due to partial hypoxia for the KHT cells irradiated as solid tumors and tested by the dilution assay (Alpen, 1998). A significant limitation of the lung colony assay is that cells must be injected into syngeneic recipient mice, that is, inbred mouse lines of the same genotype as that from which the tumor is derived (Alpen, 1998) TUMOR GROWTH AND TUMOR CURE MODELS Since there is a very limited set of models for examining the clonogenic potential of tumor cells, much of the radiation biology of tumors has been developed using a set of tools that was developed for general use in tumor biology. Therefore, ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 116

136 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL some of these tools have been more valuable than others for radiation effect studies because of the inherent inability to effect precise quantitation Tumor Volume versus Time A widely used and relatively powerful tool in tumor radiobiology is the tumor growth curve after implantation of an inoculum of cells, usually in the flank region of recipient syngeneic mice or rats. The simplest application of the growth curve for implanted tumors is the analysis on the increase rate of the tumor volume. For analysis of the radiation effect we can measure the time for the tumor to reach a preselected volume. The measurements of tumor volume are at best imprecise. The volume is usually determined from a caliper measurement of two or more diameters of the growing tumor and calculation of the volume from the average diameter (Alpen, 1998). After the tumor has been irradiated, the time course of volume change is as shown in Figure There may be a slowing of growth for a brief time, followed by a period of decreasing tumor volume. This decrease is due to lack of replacement of the normal cell loss from tumors, associated with local necrosis, nutrient lack, or other causes unrelated to the radiation exposure. It is not due to the interphase death of cells as the result of irradiation. As the surviving clonogenic cells repopulate the tumor, regrowth will be observed; the surviving clonogenic cells will ultimately produce progeny exceeding the cell-loss factor (Alpen, 1998). Figure Tumor volume versus time (from Alpen, 1998). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 117

137 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL The criterion for measurement of the radiation dependent response is the time for the cell volume to again reach the value observed at the time of irradiation. This time is shown in Figure 4.27, and it is measured, as shown, as the time from irradiation until the tumor volume achieves the value existing at the time irradiation occurred. This time value is called the growth delay. The important limitation of the growth delay model for testing the radiobiological response of tumors is that a significant number of transplantable tumors does not show any decrease in the volume of tumor after irradiation (Alpen, 1998). Presumably, this failure to decrease in volume is the result of a small cell-loss fraction in the growing tumor. When irradiation takes place, clonogenic activity is reduced until repopulation from competent clonogenic cells occurs. During the period before regrowth commences as the result of repopulation, the normally small cell-loss fraction of the tumor does not lead to reduction in tumor volume. In these cases it is necessary to revert to the simpler measure of tumor volume versus time and the use of the time to reach a preset volume. Alternatively, differences in this time for control and irradiated tumors may be taken as the end point (Alpen, 1998) TCD 50, Tumor Cure Another end point that is widely used in tumor biology is the dose required to "cure" an implanted tumor. For this model, a large number of implanted tumors are irradiated with graded doses at the same time period after implantation of the tumor inoculum. The end point is the fraction of animals that has received a given dose in which the growth of the tumor is controlled. This local control index can be plotted for each of the doses, and the dose required to control tumor growth in 50% of the animals is estimated by a variety of statistical techniques. This value is usually called the 50% tumor cure dose -TCD 50 (Alpen, 1998) RADIOBIOLOGICAL RESPONSES OF TUMORS Using a number of end points, including dilution assay, lung colony assay, primary cell cultures, and tissue derived in vitro cultures, it has been possible to define ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 118

138 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL rather clearly the radiobiological responsiveness of various tumor lines, both animal and human. With only a few important exceptions, the various tumor cell lines in wide and long term experimental use have been found to have clonogenic survival characteristics that are generally stable and for which the relevant survival parameters are not very variable, considering the range of cell types and tissues from which these transformed and immortal cell lines have been derived (Alpen, 1998). Rather different findings have been reported for the survival curve parameters of freshly derived culture systems grown from naturally occurring malignant tumors. Extensive efforts have been devoted to characterization of the radiosensitivity of cell lines from human tumors. The best fit to the data for a large number of human cell lines, both nontransformed fibroblasts and tumors, is the linear-quadratic (LQ) model. The radiosensitivity of the various cell lines can be divided into three groups with a very good correlation with the known responsiveness of the tumors to radiotherapy: lymphomata, known to be highly curable, were the most radiosensitive of the derived cell lines, and melanomata revealed to be the most resistant for tumor curability and the most radioresistant in the survival of the cell lines in culture (Alpen, 1998). It is important to realize that the immediate responsiveness of a tumor to radiation, as determined by reduction in the tumor volume, does not necessarily predict the curability of the tumor with high efficiency. The degree of responsiveness will be determined by many of the cell kinetic parameters of the tumor system. A high cell-loss factor and a high growth factor associated with a small fraction of cells out of cycle and associated with inherent cellular radiosensitivity, will assure a high degree of responsiveness of the tumor, as measured by volume changes. Curability, on the other hand, will depend in a complex way on the ability of the few remaining clonogenic cells to repopulate the tumor after irradiation is over (Alpen, 1998) HYPOXIA AND RADIOSENSITIVITY IN TUMOR CELLS Under circumstances where severe anoxia can occur in tissues or cellular preparations, one should expect to see significant protection from the effects of ionizing radiation. It is expected to find conditions of moderate to severe anoxia in growing tumors in vivo. For cells grown in suspension, careful attention to culture ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 119

139 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL conditions usually will prevent the development of such anoxic conditions with concomitant radioprotection. For the tissue assay systems, such as the Hewitt dilution assay and others, there is clearly a protective effect of oxygen lack under the correct conditions. Figure 4.26 shows such radioprotection for cells deliberately made anoxic by killing the host animal or by allowing the cell number for cells growing in the peritoneal cavity to reach very high levels. Figure 4.28 demonstrates methods by which the fraction of hypoxic cells in a mixture with fully oxygenated cells can be detected and measured quantitatively. The radioresistant "tail" for the dashed line survival curve shown in Figure 4.28 (10% anoxic cells) is a common observation for cells from tumors and indicates the presence of a mixed population of cells, part of which have a radioresistance relative to the remainder of the population. This resistant fraction may be due to hypoxia and the radioprotection that this state affords (Alpen, 1998). Figure Survival curve for the irradiation of a cell suspension containing a fraction of hypoxic cells (from Alpen, 1998). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 120

140 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL The well known work of Thomlinson and Gray (1955) laid the foundations for our understanding of hypoxia as well as reoxygenation in tumors during growth and regrowth. Figure 4.29 (from Thomlinson, 1967) illustrates the processes proposed by this author. The very young tumor is well oxygenated, since it is so small that no cells are beyond the effective diffusion distance of oxygen from nearby capillaries. As the tumor continues to grow, portions of the tumor volume may be beyond easy access to diffusing oxygen. The tumor must depend for its supply of oxygen on the development of newly formed vessels that arise from the adjacent normal tissue and penetrate the tumor volume. This neovascularization of the tumor is not as well organized as the blood supply in normal tissues, and the expanding volume of tumor will contain regions in which oxygen is inadequate for the maintenance of metabolism, and some fraction of the cells will be anoxic. Figure 4.29 illustrates that the fraction of anoxic cells in the growing tumor may rise to several percent and in some tumor types, to as much as 10%. According to the model of Thomlinson, when the tumor is irradiated (position R1 in the figure) the more radiosensitive, fully oxygenated cells are killed, and the remaining hypoxic cells are in an environment of dead and dying cells with lesser demand for metabolic oxygen (Alpen, 1998). Figure Development of hypoxia and reoxygenation in an irradiated tumor (from Alpen, 1998). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 121

141 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Shrinking of the tumor volume and lowered oxygen demand allow for reoxygenation of the hypoxic cells, which is indicated by a rapid fall to near zero for the anoxic fraction. After this period of reoxygenation, tumor regrowth commences and the complete cycle is repeated. The significance of the reoxygenation phase in fractionated radiotherapy of human tumors is undergoing careful reexamination, partly because treatment modalities designed to optimize the kill of anoxic cells (high linear energy transfer (LET) radiation, radiation under hyperbaric oxygen conditions, and so on) have not been particularly successful. According to Figure 4.29, the optimum time for a second irradiation of a fractionated scheme would be at point H in the curve, when the population of hypoxic clonogenic cells is at a minimum. Recent data suggest that the reoxygenation phenomenon actually occurs very soon after irradiation, and indeed may take place while the irradiation is in progress (Alpen, 1998) SUMMARY Human tumors strongly differ in radiosensitivity and radiocurability and this is thought to stem from differences in capacity for repair of sub-lethal damage. Radiosensitivity varies along the cell cycle, S being the most resistant phase and G 2 and M the most sensitive. Therefore, cells surviving an exposure are preferentially in a stage of low sensitivity (G 1 ), i.e. synchronized in a resistant cell cycle phase. They progress thereafter together into S and then to the more sensitive G 2 and M phases. A new irradiation exposure at this time will have a larger biological effect (more cell kill). However, while this synchronization effect has explained some experimental results, redistribution has never been shown to play a measurable role in the clinic of radiotherapy (Mazeron, 2005). Cells surviving an irradiation keep proliferating, increasing the number of clonogenic cells, i.e. the number that must eventually be sterilized to eradicate cancer. An inappropriate development of intratumoral vasculature leads to a large proportion of poorly oxygenated cells and the proportion of hypoxic cells increases with the tumor size (Mazeron, 2005). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 122

142 CHAPTER IV RADIATION AND BIOLOGICAL EFFECTS IN CANCER CELL Acutely hypoxic cells are far more radioresistant than well oxygenated cells. Hypoxic cells usually survive irradiation, but they progressively (re)oxygenate due to the better supply of oxygen available after well oxygenated cells have died. This restores radiosensitivity in the tumor by several mechanisms, but re-oxygenation occurring at long intervals is probably due to tumor shrinkage leading to a reduction of the intercapillar distance (Mazeron, 2005). The effects of ionizing radiation, even at low doses, are potentially capable of causing serious and lasting biological damage. The potentially harmful effects of ionizing radiation must be recognized and understood. It is important that radiologists should have a good appreciation of the risks associated with the examinations they carry out. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 123

143 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY

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145 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY 5.1 INTRODUCTION Cell culture is an invaluable tool for researchers in numerous fields. It facilitates the analysis of biological properties and processes that are not readily accessible at the level of the intact organism. Successful maintenance of cells in culture, whether primary or immortalized, requires knowledge and practice of a few essential techniques (Helgason, 2005). The use of cells in analytical chemistry, engineering, and biology requires a dedicated space for cell culture and maintenance. The proper handling of cells and tissues requires a level of diligence and constant education, to mitigate health and safety risks. Cell culture requires a system of mutual separation of sample and scientist to avoid contamination of either. Each time a culture flask and the dish is opened is, in essence, an opportunity for a single bacterium or fungal cell to ruin an experiment. Likewise, every time cell cultures or tissues are handled, there is a risk to the scientist. It is therefore needed to understand the protective countermeasures required to handle cells properly (Pappas, 2010). This chapter presents the importance of the laboratory conditions in the manipulation and maintenance of cell culture. Subsequently, it is explained the cytogenetic analysis of cell line and I performed a description of the methods to induce cell cycle checkpoints. In the end of the chapter, it is presented a description of the methods for synchronizing mammalian cells and the analysis of the mammalian cell cycle by flow cytometry CELL-CULTURE LABORATORY Setting up a laboratory (or space within an existing lab) for cell culture is not a daunting task, but requires some planning and strict adherence to regulations. Most universities, research institutes, and hospitals have a safety committee (some committees specialize in biosafety) that is in place in part to help a research establish a cell lab. While the government guidelines typically set the standard for safety rules, the research institution may have additional guidelines to follow. Therefore, the safety ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 126

146 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY committee is therefore indispensable in the planning and setting up of a cell lab, as well as in the subsequent (and often frequent) safety inspections. The main issues when setting up and maintaining a culture lab are safety, sterility, and contamination. All three of these issues are linked by the common safe practices and proper use of equipment, and all three require that individuals working in the lab are properly educated (Pappas, 2010). Working in the lab requires universal precautions, assuming that all cell cultures and related materials may contain hazardous pathogens. This assumption maintains a more vigilant attitude, and reduces the risk of accidental exposure to a real pathogen. Moreover, the possibility that cultures can be cross-contaminated requires additional albeit similar precautions. In short, careful procedures will result in productive research in a safe environment for cells and individuals. For those new to cells and cell culture, this chapter will not only serve as an introduction to the tools required for a cell lab, but will also detail some of the practical aspects to setting up a culture facility. For those with cell culture experience, the discussion of analytical equipment should prove useful (Pappas, 2010). 5.3 MAINTAINING CULTURES The proper maintenance of cells includes homeostasis during culture, cell storage and the correct preparation of cells for analysis. The latter case is of the most importance, as often analysis and homeostasis are incongruent. Buffers must be changed, different media used, and the cells, at times, are exposed to drastically diverdse conditions for analysis. In some cases, the change in conditions can affect the outcome of the experiment negatively. In other instances, the conditions suitable for cell analysis are fatal to the cell (e.g., electron microscopy). There are many works available on the culture of almost every cell type imaginable (Pappas, 2010). When culturing primary or immortal cells for analysis, sterility and crosscontamination must also be monitored at all times. A few bacteria in a sample can wreak havoc in a short time, rendering any analytical data useless. The crosscontamination of cultures is at best a nightmare, as extensive genetic testing is required to purify cell populations and yield accurate data. Considering the cost of ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 127

147 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY cells, reagents, instrumentation, and lab upkeep, at least as much thought should be placed on the maintenance of cell cultures for appropriate analysis. The type of environment the cell encounters can directly affect the outcome of an analytical experiment: cell-growth conditions, analysis buffers and reagents can affect the cell phenotype, cell signaling, and a host of other parameters. By careful maintenance of primary and immortal cells, accurate and reproducible cell analyses can be conducted (Pappas, 2010) Medium More than any other reagent in a cell-analysis laboratory, a steady supply of culture medium and the choice of correct medium type is essential for cell analysis. There are, in general, two classes of medium one can consider for cell analysis. First, medium that is used to maintain a culture in between experiments, and second, medium used in the analysis itself. Often these two can be one in the same, although in some cases a modified medium or supplemented buffer is needed during the analysis or processing phase (Pappas, 2010). There are many types of medium available and the supplements that can be added to them expand the palette of options even further. Table 4.1 lists some medium types that are common to cellular analysis, by cell type. The table is not inclusive, but serves to highlight the differences in medium types, and that some medium formulations are applicable to many cell lines. In most cases, the medium in Table 4.1 is used during the culture (maintenance) phase, and a different buffer or medium may be used during the analysis itself (Pappas, 2010). Medium can be classified as basic or complete, depending on whether or not serum is included, respectively. Basic medium has many of the components required for cell metabolism. Basic media, such as DMEM and RPMI 1640 (see Table 4.1), contain salts (partly from buffer action), amino acids, vitamins (such as biotin, folic acid, B-12, etc.), and molecules involved in energy production (glucose, pyruvate). Basic medium also often contains other buffers (such as HEPES) and a colorimetric acid base indicator, such as phenol red. The latter serves as a quick visual inspection ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 128

148 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY of the age of the medium in culture. As cells consume nutrients and produce waste, the culture medium acidifies, resulting in a shift in color for the ph indicator. The formulations of most culture media are available and should be examined for potential interference in the analysis. For example, staining using Annexin-V-based apoptosis probes requires relatively high Ca 2+ concentrations and at the same time, the presence of phenol red in the medium will interfere with fluorescence measurements of fluorescein, green fluorescent protein (GFP), and other fluorophores with similar emission spectra. Fluorescence from phenol red itself makes sensitive fluorescence measurements nearly impossible (Pappas, 2010). Table 1 Medium types common to cell analysis (from (Pappas, 2010)) Medium Serum Additives Cell lines RPMI % FBS Antibacterial-Antifungal Jurkat, HuT 78, RPMI 8226, CCRF-CEM, U937, HL-60 Dulbecco`s modified Antibacterial-Antifungal, NIH 3T3, RBL-1, HT-29, 10% FBS Eagle Medium (DMEM) L-Glutamine HeLa Antibacterial-Antifungal, Clavcomb`s Medium 10% FBS Norepunephrine, L- HL-1 Glutamine Cell Mab 0-10% FBS Varies Designed for monoclonal antibody production Leibovitz`s L-15 Hemolymph Bag neuronal cells Eagle`s Minimum Essential Medium 0-10% FBS L-Glutamine F % FBS L-Glutamine Designed for primary cells Iscove`s Modified DMEM 0-10% FBS L-Glutamine HuT 78 T Cells FBS = Fetal Bovine Serum Medium is, in essence, a man-made attempt to mimic the life support found in vivo. It is, therefore, lacking in many essential compounds for cell growth. Many cell ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 129

149 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY lines can function in basic medium without additional materials, but for the most routine culture and analysis, serum must be added to form the complete medium (Pappas, 2010). Serum is typically derived from animal sources, the most common being fetal bovine serum (FBS). FBS and other sera contain growth factors such as epidermal growth factor (EGF), some interleukins, and transferrin. Furthermore, present are adhesion-promoting proteins and peptides, for example, fibronectin and laminin and other components including insulin and various minerals. FBS and other animal-based sera are by far the most common supplements used for culture maintenance (Pappas, 2010). Being derived from animal sources, serum is inherently difficult to use from a quality-control perspective and since it is derived from different animal types this can affect experiment outcome. For example, the use of FBS instead of native rat serum was shown to affect the outcome of rat leukocyte immunological response. In addition to species variability, serum varies from lot to lot, as well as by country of origin, so if cell products are to be analyzed over long time periods (months of experimentation) it is best to purchase a large quantity of serum from one particular lot. Given the high cost of medium, this may not always be practical since serum cost increases as the level of quality control improves. The more consistent and well characterized the medium, the higher the cost (Pappas, 2010). Another negative aspect of dealing with serum is that the serum, or animal of origin, is subject to contamination, just like any other primary derived material. Certain viruses, bacteria, and mycoplasma have been shown to be transmitted via serum. There are several replacement sera that can be substituted for FBS. For example, the FetalClone series and Bovine Growth Serum, both from HyClone, are non-fetal animal sera supplemented with various growth factors, minerals, and other compounds. Since they are not derived from fetal animals, there is less variability between lots (especially for the added compounds). None of the alternative sera offers much relief as far as cost is concerned, but the increase in quality control is a major improvement (Pappas, 2010). Some cells readily grow in serum-free medium; most, however, must be acclimated to a serum-free environment. This requirement is especially true if the cell ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 130

150 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY line in question is already being cultured in serum-enriched medium (typically 10% v/v). It is possible to reduce serum content in medium; in some cases, it is advisable to do so, because reducing the amount of serum added can reduce costs, as serum is the most expensive component of the complete medium. Reducing serum also lowers the total protein content of the medium, facilitating collection of cell products, and minimizing sources of contamination. For cells growing in serum-enriched medium, a method of systematically reducing medium can be implemented (Pappas, 2010). One must first consider the growth of cells in culture, before discussion of how to achieve serum reduction can initiate, Figure 4.1. Cell growth in culture whether the cells are adherent or suspended is characterized by several stages. The lag phase, during which minimal or no cell division occurs, is a brief period after inoculation. The lag phase occurs as cells adjust to a new cell-culture environment, and as adherent cells begin the process of reattaching to the culture substrate. The lag phase is followed by the log or exponential phase. This is the major phase of cell division. The doubling time, an indicator of cell growth, is determined during this period (Pappas, 2010). Figure Cell growth in culture (from (Pappas, 2010)). The time for the cell population to double, Figure 5.1, can be determined at any point during the log phase, although it is most accurate at the center of that phase. After the log phase, the culture reaches the stationary phase (Pappas, 2010). High cell density, contact inhibition, and consumption of nutrients signal a slowing of the cell cycle, and the cell concentration remains constant. Cell crowding, ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 131

151 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY depletion of nutrients and accumulation of waste eventually causes a sharp drop in cell concentration, called the death phase. This latter phase can be confirmed by microscopy, where the presence of a large number of dead cells, cell debris, and acidified medium (if an indicator is present) can be observed (Pappas, 2010). The glucose content of basic medium varies and is sometimes supplemented with additional glucose. The high glucose content of many medium types is intended to stimulate growth of the culture. However, some cell lines change phenotypic properties in high or low glucose. When culturing for conditions close to those encountered in vivo, the glucose concentration should be adjusted to reflecting the physiological value as much as possible. Like serum reduction, the impact of changes in glucose concentration can be monitored using the culture doubling time (Pappas, 2010). When formulating complete medium, care must be taken to preserve sterility of the final mixture. If all components are sterile to begin with, then aseptic handling in the biosafety cabinet will prevent contamination of the complete medium. If any of the reagents are not sterile at the onset, then filtration can be employed to remove contaminating organisms The use of medium in analysis and alternatives Medium is primarily used to maintain cultures and samples before analysis. The medium can also be used during the analysis; in other instances, components of the medium may produce artifacts or otherwise interfere. The presence of several components of medium can interfere with fluorescence measurements. Phenol red, one of the most common ph indicators added to medium, has a broad absorption band that interferes with most green fluorescence. Phenol red is also weakly fluorescent, creating an additional problem for green-emitting fluorophores. If the cell homeostasis is not required, then any buffer devoid of phenol red will work for fluorescence. On the other hand, if the cells are to be kept alive for long periods, then phenol-red-free medium is available from most medium manufacturers. In addition to the weakly fluorescent properties of phenol red, other compounds present at ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 132

152 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY relatively high concentrations can interfere with fluorescence detection. Riboflavin is also weakly fluorescent, but the relatively large volume of the medium contributes to an unacceptable background signal. Proteins such as albumin, one of the major components of serum, also contribute strongly to autofluorescence of medium. The exact medium used for culture depends on the cell type, the culture conditions, and the desired end result. For analysis, a similar selection process must be undertaken. The final medium or buffer used for analysis must be of low background, minimal interference, and when possible capable of sustaining cell viability and function for the experiment duration (Pappas, 2010). 5.4 CYTOGENETIC ANALYSIS OF CELL LINES The Utility of Cytogenetic Characterization Countless cell lines have been established more than 1000 from human hematopoietic tumors alone and the novelty and utility of each new example should be proven prior to publication. For several reasons, karyotypic analysis has become a core element for characterizing cell lines, mainly because of the unique key cytogenetics provides for classifying cancer cells. Recurrent chromosome changes provide a portal to underlying mutations at the DNA level in cancer, and cell lines are rich territory for mining them. Cancer changes might reflect developmentally programmed patterns of gene expression and responsiveness within diverse cell lineages. Dysregulation of certain genes facilitates evasion of existing antineoplastic controls, including those mediated by cell cycle checkpoints or apoptosis. The tendency of cells to produce neoplastic mutations via chromosomal mechanisms, principally translocations, duplications, and deletions, renders these changes microscopically visible, facilitating cancer diagnosis by chromosome analysis. Arguably, of all neoplastic changes, those affecting chromosomal structures combine the greatest informational content with the least likelihood of reversal. This is particularly true of the primary cytogenetic changes that play key roles in neoplastic transformation and upon the presence of which the neoplastic phenotype and cell proliferation ultimately depend. Nevertheless, the usefulness of karyotype analysis for ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 133

153 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY the characterization of cell lines lies principally among those derived from tumors with stronger associations with specific chromosome rearrangements (i.e., hematopoietic, mesenchymal, and neuronal, rather than epithelial tumors) (Helgason, 2005). Cytogenetic methods facilitate observations performed at the single-cell level, thus allowing detection of intercellular differences. Accordingly, a second virtue of cytogenetic data lies in the detection of distinct subclones and the monitoring of stability therein. Except for doublings in their modal chromosome number from 2n to 4n tetraploidization, cell lines appear to be rather more stable than is commonly supposed. Indeed, chromosomal rearrangement in cells of the immune system could reach peak intensity in vivo during the various phases of lymphocyte development in vivo. A further application of cytogenetic data is to minimize the risk of using false or misidentified cell lines. At least 18% of new human tumor cell lines have been crosscontaminated by older, mainly classic, cell lines, which tend to be widely circulated. This problem, first publicized over 30 years ago but neglected of late, poses an insidious threat to research using cell lines (Helgason, 2005). In the event of cross-contamination with cells of other species, cytogenetic analysis provides a ready means of detection. Although modal chromosome numbers were formerly used to identify cell lines, their virtue as descriptors has declined along with the remorseless increase in the numbers of different cell lines in circulation. Thus, species identification necessarily rests on the ability to distinguish the chromosome banding patterns of diverse species. Fortunately, cells of the most prolific mammalian species represented in cell lines (primate, rodent, simian, as well as those of domestic animals) are distinguishable by experienced operators (Helgason, 2005). 5.5 METHODS TO INDUCE CELL CYCLE CHECKPOINTS The way cells respond to radiation or chemical exposure that damages deoxyribonucleic acid (DNA) is important because induced lesions left unrepaired, or those that are misrepaired, can lead to mutation, cancer, or lethality. Prokaryotic and eukaryotic cells have evolved mechanisms that repair damaged DNA directly, such as nucleotide excision repair, base excision repair, homology-based recombinational repair, or nonhomologous end joining, which promote survival and reduce potential ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 134

154 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY deleterious effects. However, at least eukaryotic cells also have cell cycle checkpoints capable of sensing DNA damage or blocks in DNA replication, signaling the cell cycle machinery, and causing transient delays in progression at specific phases of the cell cycle. These delays are thought to provide cells with extra time for mending DNA lesions before entry into critical phases of the cell cycle, such as S or M, events that could be lethal with damaged DNA (Lieberman, 2004). The precise mechanisms by which checkpoints function is under intensive investigation and details of the molecular events involved are being pursued vigorously. This owes not only to the complexity and the intellectually and technically challenging aspects of the process but also to the relevance of these pathways to the stabilization of the genome and carcinogenesis. Nevertheless, it is clear that checkpoint mechanisms are very sensitive and can be induced by the presence of relatively small amounts of DNA damage. For example, in the yeast Saccharomyces cerevisiae, as little as a single double-strand break in DNA can cause a delay in cell cycle progression. One important aspect of studying cell cycle checkpoint mechanisms is an understanding of how to induce the process (Lieberman, 2004). The application of radiations, such as gamma rays and ultraviolet (UV) light, are capable of causing DNA damage, and thus leading to the induction of cell cycle checkpoints. Certain chemicals or the use of temperature- sensitive mutants to disrupt DNA replication, are also used routinely to induce checkpoints. Gamma rays cause primarily single- and double-strand breaks in DNA but can infrequently induce nitrogenous base damage as well. In contrast, UV light (i.e., 254 nm) causes a preponderance of bulky lesions, such as pyrimidine dimers, although single-base damage and strand breaks are a smaller part of the array of lesions that can be produced. Regulation of cell cycle checkpoints induced by ionizing radiation versus UV light is mediated by overlapping but not identical genetic elements (Lieberman, 2004). 5.6 METHODS FOR SYNCHRONIZING MAMMALIAN CELLS When studying cell cycle checkpoints, it is often very useful to have large numbers of cells that are synchronized in various stages of the cell cycle. A variety of methods have been developed to obtain synchronous (or partially synchronous) cells, ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 135

155 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY all of which have some drawbacks. Many cell types that attach to plastic culture dishes round up in mitosis and can then be dislodged by agitation. This mitotic shake-off method is useful for cells synchronized in metaphase, which on plating into culture dishes move into G 1 phase in a synchronous manner. A drawback to the mitotic shakeoff method is that only a low percentage (2 4%) of cells are in mitosis at any given time, so the yield is very small. Also, cells rapidly become asynchronous as they progress through G 1 phase, so the synchronization in S phase and especially G 2 phase is not very good. The first limitation can be overcome by plating multiple T150 flasks with cells, using roller bottles, or blocking cells in mitosis by inhibitors such as Colcemid or nocodazole (Lieberman, 2004). Mitotic cells that are collected can be held on ice for an hour or so while multiple collections are done to obtain larger numbers of cells. To obtain more highly synchronous populations of cells in S phase, the mitotic shake-off procedure can be combined with the use of deoxyribonucleic acid (DNA) synthesis inhibitors, such as hydroxyurea (HU) or aphidicolin (APH), to block cells at the G 1 /S border (but probably past the G 1 checkpoint). APH inhibits DNA polymerase α, whereas HU inhibits the enzyme ribonucleotide reductase, though it may operate by other mechanisms also. On release from the block, cells move in a highly synchronized fashion through S phase and into G 2 phase. In terms of the number of synchronized cells, this method has the same limitation as discussed above, because the starting cell population derives from the mitotic shake-off procedure. In addition, the block of cells with drugs can cause unbalanced cell growth, so one cannot necessarily conclude that all biochemical processes are also synchronized (Lieberman, 2004). Large numbers of synchronous cells can be obtained using centrifugal elutriation, Figure 5.2. This method requires the use of a special rotor in a large floor centrifuge and separates cells into the cell cycle based on cell size. Cells may be obtained in early or late G 1 phase, or primarily in S phase. However, the cell populations are not highly synchronous in S phase but instead have significant populations of G 1 - and G 2 -phase cells included. Nevertheless, it is possible to synchronize very large numbers of cells using this method, and biochemical processes are not perturbed (Lieberman, 2004). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 136

156 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY Figure Centrifugal elutriation (from (Wahl, 2001)). Another method that results in highly synchronous populations is based on labeling cells with a viable dye for DNA (Hoechst 33342). Cells stained with this dye can then be sorted by cell cycle phase. Sorted G 1 cells will be distributed throughout G 1, cells in S phase can be sorted into a small window in S phase and thus will be highly synchronized, but only a small number of cells can be obtained. G 2 phase cells will be contaminated with late S phase cells. Furthermore, some cell types do not stain well with Hoechst 33342, so sufficiently good DNA histograms cannot be obtained Hoechst (Lieberman, 2004). 5.7 ANALYSIS OF THE MAMMALIAN CELL CYCLE BY FLOW CYTOMETRY One of the most common uses of flow cytometry is to analyze the cell cycle of mammalian cells. Flow cytometry can measure the deoxyribonucleic acid (DNA) content of individual cells at a rate of several thousand cells per second and thus conveniently reveals the distribution of cells through the cell cycle (Lieberman, 2004). The DNA-content distribution of a typical exponentially growing cell population is composed of two peaks (cells in G 1 /G 0 and G 2 /M phases) and a valley of cells in S phase, Figure 5.3. G 2 /M-phase cells have twice the amount of DNA as G 1 /G 0 -phase cells, and S-phase cells contain varying amounts of DNA between that found in G 1 and ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 137

157 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY G 2 cells. Most flow-cytometric methods of cell cycle analysis cannot distinguish between G 1 and G 0 cells or G 2 and M cells, so they are grouped together as G 1 /G 0 and G 2 /M. However, there are flow cytometric methods that can distinguish four or even all five cell cycle subpopulations: G 0, G 1, S, G 2, and M. Furthermore, each subpopulation can be quantified. Obviously, flow cytometry with these unique features is irreplaceable for monitoring the cell cycle status and its regulation (Lieberman, 2004). Figure A typical cell cycle distribution of DNA content (from (Cooper,2004)). Cell cycle checkpoint genes are key elements in cell cycle regulation. Checkpoint gene mutation can lead to defects in one or more cell cycle checkpoint controls, which can then result in cell death or cancer. Many of the cell cycle checkpoint genes are tumor suppressors, such as p53, ataxia-telangiectasia mutant (ATM), ataxia-telangiectasia and Rad3 (ATR), and BRCA1 (Lieberman, 2004). In mammalian cells, the cell cycle checkpoint controls that can be analyzed by flow cytometry are G 1 arrest, suppression of DNA replication, and ATM dependent as well as independent G 2 arrest. Exposure to a genotoxic agent can activate some or all the checkpoints (Lieberman, 2004). 5.8 CONCLUSION Effective in vitro maintenance and growth of animal cells requires culture conditions similar to those found in vivo with respect to temperature, oxygen and ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 138

158 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY carbon dioxide concentrations, ph, osmolality, and nutrients. Within normal tissue in vivo, animal cells receive nutrients through blood circulation. For growth in vitro, animal cells require an equivalent supply of a complex combination of nutrients. For this reason, the first attempts in animal cell culture were based on the use of biological fluids such as plasma, lymph and serum, as well as on extracts from embryonic-derived tissue (Castilho, 2008). Medium composition is one of the most important factors in the culture of animal cells. Its function is to provide appropriate ph and osmolality for cell survival and multiplication, as well as to supply all chemical substances required by the cells that they are unable to synthesize themselves. Some of these substances can be provided by a culture medium consisting of low molecular weight compounds, known as basal media. However, most basal media fail to promote successful cell growth by themselves and require supplementation with more complex and chemically undefined additives such as blood serum (Castilho, 2008). Some cultivation processes are based on operational strategies that allow cells to remain viable, but in a nonproliferative state, so as to prolong the productive phase and to increase the productivity of the process. By these strategies cell proliferation may be controlled by adding chemical additives that arrest the cell cycle, usually in the G 1 phase, increasing specific productivity. However, concomitantly undesirable effects such as cytotoxicity may be observed, which result in a decrease in cell viability and in the impossibility of maintaining the culture in a nonproliferative state for long periods of time. Deprivation of specific nutrients and growth factors can also stop cell proliferation, but in this case cell viability decreases and programmed cell death apoptosis is activated. Currently, much research on the biochemical control of cell cultures based on preventing the cell death mechanisms, to avoid cell death instead of inhibiting cell growth, is being carried out with the aim of prolonging the productive period of a cell culture process (Castilho, 2008). Any process, industrial or laboratory-based, presents a series of important variables that represent its state. In the case of cell culture, there are the variables related to the environment to which the cells are exposed, such as temperature, ph, dissolved oxygen, nutrients in the culture medium, and metabolite concentrations, as ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 139

159 CHAPTER V CELL CULTURE AND FLOW CYTOMETRY well as those related to the cell itself, such as concentration, average size, or the profile of intracellular enzyme activities (Castilho, 2008). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 140

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161 CHAPTER VI BRACHYTHERAPY

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163 CHAPTER VI BRACHYTHERAPY 6.1 INTRODUCTION Brachytherapy was for many years in a state of decline, principally because of the radiation hazards to users and those associated with the management of patients. The introduction of afterloading machines in the 1960s provided the means to control the movement and position of individual radioactive sources and greatly reduced the radiation exposure to staff. As a result, brachytherapy underwent a renaissance and provided the necessary stimulus to promote the development of afterloading brachytherapy techniques. These developments have been further supported by the availability of nuclides, particularly cobalt-60, cesium-137, and iridium-192 and, more recently, radioactive seeds of iodine-125 and palladium-105. In parallel with the technological advances in afterloading machines, there have been major developments in imaging techniques and computerized planning (Joslin, 2001). Cancer management generally has undergone major advances since the 1960s and brachytherapy has played an increasingly important role. The optimal management of cancer patients requires expert teams who specialize in certain cancer sites within which brachytherapy may have a specific place. Much of this work is now being provided on an outpatient or day-care basis and prolonged hospital stay is proving to be unnecessary (Joslin, 2001). This chapter starts with a brief explanation of the brachytherapy fundaments to further understand the mechanisms used by this technique to kill the cancer cells. So, it will be made a description of the sources used in brachytherapy followed by an approach of the radiobiology of brachytherapy. At the end of the chapter a description is made about the dose-rate effect in human cells and a brief come up about predictive assays for radiation oncology. The present chapter is central in this thesis project since it is with this technique that the cancer cells will be killed. The changes that occur in the cells will be analyzed by image processing and analysis techniques. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 143

164 CHAPTER VI BRACHYTHERAPY 6.2 BRACHYTHERAPY The different types of radiation applied for radiobiological research has one important issue: there the determination of the biological effectiveness of ionizing photon radiation as a function of photon energy represents a major scientific objective. Very intense, low-energetic, quasi-monochromatic, and energy tunable ( kev) channeling radiation (CR) is generated by channeling of relativistic electrons in diamond crystals (Zeil, 2009). Usually radiobiological studies are performed on conventional high-voltage X- ray tubes or medical acceleration facilities. Both sources deliver broad polychromatic bremsstrahlung with a high photon flux. Thus, therapeutic dose values (few Gy per daily fraction) can be delivered in a sufficiently small irradiation duration (dose rate 1 Gy/min) to be independent from repairing processes in human cells. Due to the high reproducibility of beam parameters of conventional radiation sources, a large number of samples can be irradiated in stable conditions in order to cope with the biological diversity. Considering the dosimetry a standardized radiation field is used. All changes in the radiation geometry resulting in differences of beam absorption, scattering or dose build up effect are taken into consideration by applying tabled correction factors. In practical irradiation experiments, cell samples are irradiated at a vertical beam and the delivered dose is controlled by presetting certain irradiation duration (Zeil, 2009). Brachytherapy (sometimes referred to as curietherapy or endocurie therapy) is a term used to describe the short distance treatment of cancer with radiation from small, encapsulated radionuclide sources. This type of treatment is made by placing sources directly into or near the volume to be treated. The dose is then delivered continuously, either over a short period of time (temporary implants) or over the lifetime of the source to a complete decay (permanent implants). Most common brachytherapy sources emit photons; however, in a few specialized situations β or neutron emitting sources are used. There are two main types of brachytherapy treatment (Suntharalingam, 2002): Intracavitary, in which the sources are placed in body cavities close to the tumor volume; Interstitial, in which the sources are implanted within the tumor volume. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 144

165 CHAPTER VI BRACHYTHERAPY The biological effects of radiotherapy depend on dose distribution, treated volume, dose rate, fractionation and treatment duration. However, these various factors are of different importance in determining the outcome of external beam radiotherapy or of brachytherapy (Suntharalingam, 2002). In brachytherapy, the dose is prescribed to an isodose encircling a small targeted volume with a very heterogeneous dose distribution. It is minimal at distance of the radioactive sources, but much higher doses and dose rates are delivered in their immediate vicinity (Suntharalingam, 2002). Therefore, the average dose given to the targeted volume is always higher than the prescribed dose, prescribed at the periphery of the target. This is an important point to notice as the treatment report contains information regarding only the dose and dose rate at the reference isodose (Suntharalingam, 2002). Another distinct feature of brachytherapy is that the doses within an implant are higher than the tolerance dose levels accepted in external beam irradiation, yet they are well tolerated because of the volume-effect relationship (very small volumes can tolerate very high dose levels) (Suntharalingam, 2002). Finally, time-dose factors differ widely between external beam radiotherapy and brachytherapy. In external beam radiotherapy, the total dose is delivered in small, daily fractions of a few seconds or minutes, allowing for full repair between exposures. The treatment is protracted over several weeks. In contrast, in brachytherapy the dose is delivered continuously, and treatments tend to be short (several hours to several days). However, there is a variety of schedules depending on the type of equipment used (Suntharalingam, 2002). According to International Comission on Radiation Units & Measurements (ICRU) report 38, treatment dose rates fall into three categories (Mazeron, 2005): Low Dose Rate (LDR) brachytherapy ranges between 0.4 and 2 Gy/h. On the other hand, in routine clinical practice, LDR brachytherapy is usually delivered at dose rates between 0.3 and 1 Gy/h. This is compatible with conventional manual or automatic afterloading techniques. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 145

166 CHAPTER VI BRACHYTHERAPY Medium Dose Rate (MDR) brachytherapy ranges between 2 and 12 Gy/h. MDR can also be delivered by manual or automatic afterloading, although the latter is far more frequent. High Dose Rate (HDR) brachytherapy delivers the dose at 12 Gy/h or more, and only automatic afterloading can be used because of the high source activity. A new category is pulsed dose rate (PDR) brachytherapy, which delivers the dose in a large number of small fractions with short intervals, allowing only for incomplete repair, aiming at achieving a radiobiological effect similar to low dose rate over the same treatment time, typically a few days. Finally, permanent implants deliver a high total dose (for example, 150 Gy) at a very low dose rate, over several months (Mazeron, 2005). 6.3 SOURCES IN BRACHYTHERAPY RADIUM Radium was discovered by Marie Curie in Within 3 years of this discovery, the first patients were treated with radium implanted into their tumors (Joslin, 2001). In the UK, St Bartholomew's Hospital received its first radium for clinical use in Early clinical experience with these sources led to radiation necrosis, and it became clear that this was due, in part, to the intense beta-ray dose from the radium. It was not until 1920 that successful filtration of the beta-rays was achieved (Joslin, 2001). Radium was then used extensively throughout the world. Physicists in the major clinical centers developed dosimetry systems for interstitial and intracavity brachytherapy. However, in general, radium has been replaced by other radionuclides because, although it has a long half-life, it has several disadvantages (Joslin, 2001): Radium and several of its descendant products, including radon, are alpha emitters. Radon is a noble gas which is soluble in tissue. This gas could escape through a hairline crack - not easily detected by a visual check - in the radium capsule. If an implanted radium source were to be ruptured within the patient's ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 146

167 CHAPTER VI BRACHYTHERAPY body, radium and its daughter products may become deposited more or less permanently in the bone. There is also the possibility of damage by incineration or mechanical means - when the sources are lost, or while they are being processed, with the subsequent release of toxic radioactivity to the environment. The gamma radiation from a radium source is of higher energy than is necessary for brachytherapy. Radiation protection for these sources requires large thicknesses of lead, which can cause problems when it comes to: o transporting sources in heavy containers using very weighty protective screens around the patient; o the need for a heavy rectal shield in applicators used for gynecological treatment. The practical maximum activity concentration (the specific activity) of radium salt is low (approximately 50 MBq mm -3 of active volume). Therephore, sources of higher activity are bulky and u suitable for afterloading systems RADIUM SUBSTITUTES This was the phrase used to describe the first set of new (artificial) radionuclides which were found useful for brachytherapy from about 1950 onwards, though it is only very recently that most radiotherapy centers have stopped using radium. It was found that there were very few radionuclides with the appropriate properties of the ideal brachytherapy source. These properties are as follows (Joslin, 2001): Photon energy should be low to medium ( MeV) to minimize radiation protection problems (with the proviso that low-energy radionuclides should not be used near bone because of the enhanced dose to bone at these energies). For permanent stock, a long half-life is desirable such that the radioactive decay within the practical lifetime of the source and its container (typically 10 years) is small. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 147

168 CHAPTER VI BRACHYTHERAPY For permanent implantation, a fairly short half-life is essential in order to minimize the time over which special precautions, towards relatives of a radioactive patient and members of the public, need to be in place. The nuclide should be available at high specific activity. There should be no gaseous disintegration product. The nuclide should be available in a form which does not powder or otherwise disperse if the source is damaged or incinerated. The first sources to be used as alternatives to radium were cobalt-60, gold-198, cesium-137 and iridium-192. These are all described briefly below. The most commonly used sources at this time are cesium-137 and iridium-192, both of which are used in after-loading systems. Iridium-192 has the possibility of high specific activity, which allows it to be used as a high dose-rate (HDR) source (Joslin, 2001) NEW SOURCES The newer sources are not known as radium substitutes, mainly because they have very different properties from radium, namely very much higher specific activity (for example, the HDR iridium-192 source) and very different energy. The only new source that has been accepted into routine clinical use in certain centers throughout the world is iodine-125. Palladium-103 is also now available as a standard commercial source (Joslin, 2001). The other sources that are still at the research stage of development, to find out whether they can be of use clinically, are samarium-145, americium-241, and ytterbium-169 (Joslin, 2001). 6.4 RADIOBIOLOGY OF BRACHYTHERAPY The biological damage inflicted by irradiation of human cells with ionizing radiation can be divided into three consecutive steps (Mazeron, 2005): A very short initial physical phase (about s), during which photons interact with orbital electrons, raising them to higher energy levels inside the atoms ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 148

169 CHAPTER VI BRACHYTHERAPY (excitation), or ejecting some of them from the atoms (ionization). This is the energy deposition phase. A chemical phase, again very short (about 10-3 s), during which ionized and excited atoms interact, leading either directly or indirectly effects through the formation of free radicals to the breakage of chemical bonds. Free radicals are highly reactive and can induce chemical changes in biologically important molecules like DNA. Single-strand or double-strand break in DNA appears to be the basic damage leading to biological effects. A biological phase, much longer (seconds to years), during which the cells react to the inflicted chemical damage. Specific repair enzymes can successfully repair the vast majority of lesions in DNA. However, few lesions however may not be repaired and may consequently lead to cell death. Cell death is not immediate and usually occurs during the next cell division (apoptosis is a minor process in most human cells). On the other hand, death due to a lethal lesion may be delayed for a limited number of mitotic divisions (up to 5 or 6). Because the stem cells are the only cells which divide in normal tissues, the earliest effect observed is a deficit in stem cells. Later, the loss of stem cells will lead to a deficit in differentiated cells causing the observed clinical reactions. The early reactions are seen during the first days or weeks after irradiation (for example, diarrhea or acute mucositis). They are temporary because the cell deficit is compensated for by the repopulation of stem cells and subsequently of differentiated cells. Late reactions due to damage to the late-reacting tissues, for instance blood vessel damage, fibrosis, telangiectasia, etc., may be seen after months or years. Damage to these late reacting normal tissues is poorly repaired and is responsible for most severe complications of radiotherapy. Tolerance of these tissues is the limiting factor for radiation therapy THE FOUR RS OF RADIOBIOLOGY A number of biological processes take place during irradiation and modify the radiation response. These processes are often described as the four Rs of radiobiology. Each follows a specific time pattern (Mazeron, 2005): ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 149

170 CHAPTER VI BRACHYTHERAPY Repair of DNA damage - it is often referred as repair of sub-lethal damage. Experimental and clinical studies have shown that human tumors strongly differ in radiosensitivity and radiocurability. This is thought to stem from differences in capacity for repair of sub-lethal damage. Similar differences are seen between normal tissues, the haemopoetic system being more sensitive than the kidney. Reassortment or redistribution - the cell cycle is divided in four consecutive stages: G 1, S, G 2 and M. G 1 is a gap of apparent inactivity after a mitosis (M), before DNA synthesis (S-phase) resumes in view of the following cell division. G 2 is a second gap of apparent inactivity between S phase and M, Figure 6.1. Radiosensitivity varies along the cell cycle, S being the most resistant phase and G 2 and M the most sensitive. Therefore, cells surviving an exposure are preferentially in a stage of low sensitivity (G 1 ), i.e. synchronized in a resistant cell cycle phase. They progress thereafter together into S and then to the more sensitive G 2 and M phases. A new irradiation exposure at this time will have a larger biological effect (more cells killed). However, while this synchronization effect has explained some experimental results, redistribution has never been shown to play a measurable role in the clinic of radiotherapy. Repopulation - cells surviving an irradiation keep proliferating. This increases the number of clonogenic cells, i.e. the number that must eventually be sterilized to eradicate cancer. Consequently, repopulation has a detrimental effect as far as cancer control is concerned. Stem cells do also proliferate in normal tissues, which has in this case a protective effect (it helps the tissue to recover from radiation damage and it adds to DNA repair in cells). Reoxygenation - because of an inappropriate development of intratumoral vasculature, every tumor of clinically detectable size contains a large proportion of poorly oxygenated cells. In addition, the proportion of hypoxic cells increases with the tumor size. Acutely hypoxic cells are far more radioresistant than well oxygenated cells. This is expressed by the oxygen enhancement ratio (OER), i.e. the ratio between radiation doses required in hypoxia and air to produce the same biological effect. Hypoxic cells usually survive irradiation, but they progressively (re)oxygenate due to the better ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 150

171 CHAPTER VI BRACHYTHERAPY supply of oxygen available after well oxygenated cells have died, Figure 6.2. This restores radiosensitivity in the tumor by several mechanisms, but reoxygenation occurring at long intervals is probably due to tumor shrinkage leading to a reduction of the intercapillar distance. Figure The cell cycle (from Murray, 1993). Figure Re-oxygenation due to tumor shrinkage (from Mazeron, 2005) RADIOBIOLOGY OF LOW DOSE-RATE AND FRACTIONED IRRADIATION For exposure to sparsely ionizing radiations such as X-rays or gamma-rays, the degree of a biological effect produced can depend as much on the dose rate as on the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 151

172 CHAPTER VI BRACHYTHERAPY total dose received. The importance of dose rate and dose fractionation effects has been recognized for more than 70 years (Joslin, 2001). Studies of Regaud and his collaborators were perhaps the first to show the potential therapeutic advantages of dose fractionation in the treatment of patients with cancer by radiation. Since that time, the evolution of treatment regimes involving dose time variations have increasingly improved cancer radiotherapy and the evolution continues even today. In cancer radiotherapy, the dose rate and dose fractionation are not the only important factors, but also in connection with the mutagenic and oncogenic hazards of radiation exposure (Joslin, 2001). Normally, reducing the dose rate decreases the biological effectiveness, that is, decreasing the dose rate generally increases the dose necessary to yield the same level of effect. A number of factors can contribute to the dose rate or dose fractionation effect, depending on the conditions and cell or tissue system involved. For example, in a tissue or tumor exposed over a period of weeks or months, cells may migrate into or out of the radiation field, or the oxygenation status may change to alter the intrinsic radiosensitivity of the cells during the course of treatment (Joslin, 2001) SPLIT-DOSE RECOVERY FROM SUB-LETHAL DAMAGE IN MAMMALIAN CELLS For ionizing radiation damage in mammalian cells, the first direct demonstration of a cellular repair process affecting cell killing that could explain dose rate and dose fractionation effects seen in mammalian tissues or tumors was provided by Elkind and Sutton. These researchers reasoned that because the shouldered survival curves for mammalian cells exposed to X-rays or gamma-rays indicate the involvement of a damage accumulation process in cell killing, then cells surviving a dose beyond the shoulder region of the curve (survivals below about 10%) would contain sub-lethal damage capable of interacting with further damage to become lethal. Elkind and Sutton questioned whether this sub-lethal damage might remain in surviving cells, in which case their dose response at some later time would not be 'shouldered'. Alternatively, if the sub-lethal damage were repaired, the cells would be expected to respond as if they had never been irradiated, i.e., the surviving cells would display the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 152

173 CHAPTER VI BRACHYTHERAPY same shouldered survival curve for subsequent irradiation. The latter was found to be the case, as is illustrated in Figure 6.3 from their early work (Joslin, 2001). The curve indicated by filled circles in Figure 6.3 illustrates a dose-response curve for irradiations requiring only a few minutes each - high dose rate (HDR) or 'acute' exposures - over a range of doses from 0 (zero) to about 12.5 Gy. Curves starting at a dose of 5.05 Gy illustrate dose-response curves for cells surviving a first dose of 5.05 Gy followed by various additional doses given either immediately after the first dose (filled circles) or 18 h following the first dose (open circles). During the time interval between the first and second doses, the surviving cells 'restored themselves to good (original) condition. They had repaired this so-called sub-lethal damage so they again had to accumulate damage for cell killing (Joslin, 2001). This sub-lethal damage repair (SLDR) is a repair process operationally defined in terms of the observations demonstrating the phenomenon, i.e., the increase in the fraction of cells surviving. It says nothing about what is being damaged and repaired (Joslin, 2001). Figure 6.3 Initial survival curve (closed circles) and fractionation curve (open circles) for clone A cultured Chinese hamster cells (from Joslin, 2001). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 153

174 CHAPTER VI BRACHYTHERAPY CELL-CYCLE COMPLICATION: A HETEROGENEOUS POPULATION In the early 1960s, Terasima and Tolmach first showed with synchronized cultures of HeLa cells that cellular responses varied greatly throughout the cell cycle. During mitosis cells become very loosely attached to the surface of the culture vessel and these were collected by a 'shakeoff method', leaving the interphase cells behind in the flask. Appropriate numbers of mitotic cell populations were inoculated into dishes. After various periods of incubation, different sets of the synchronously progressing cells were irradiated when they were (for the most part) at a particular stage of the cycle. When the dose was the same for all cultures, but the time after mitotic shake-off was varied, the proportion surviving to form colonies varied. Parallel cultures were flash labeled with tritiated thymidine ( 3 H TdR) to monitor the synchronous progression of cells into and out of S phase. For irradiation of mitotic cells survival was low, indicating a high sensitivity for this cell cycle phase. As cells progressed into mid-g 1 (2-6 h), the cells were more resistant. At around the G 1 /S border and in early S phase cells were again more sensitive, and as cells progressed toward late S phase and early G 2 the cells again became more resistant. Because there is some variation from one cell to the next in the cell cycle transit times, particularly through G 1, there is an increasing decay in synchrony and therefore the resolution of experimental data on cycle-dependent radiosensitivity with time. Nevertheless, there is clearly a large variation in the radiation response of cells through the cell cycle. Other cells have shown similar cellcycle-dependent variations in radiosensitivity, although the peak of resistance in G 1 is not well resolved experimentally in cells with very short G 1 transit times. The sensitivity of cells in different parts of G 2 is difficult to determine by the synchronization procedure described above, because of synchrony decay during the passage of the starting population of mitotic cells through their first G1 and S phase, and because G 2 transit times are relatively short (about 1-2 h). However, a modification of the technique allows a much greater resolution for studying G 2 sensitivity. This is sometimes called 'retroactive synchronization': cells are first irradiated and then, as a function of time, cells arriving in mitosis are harvested by mitotic shake-off and plated for survival (Joslin, 2001). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 154

175 CHAPTER VI BRACHYTHERAPY RADIATION AFFECTS CELL-CYCLE PROGRESSION ITSELF Radiation effects on cell cycle progression are yet another factor that influences dose rate effects. Ionizing radiation reduces the mitotic index within a short time after exposure (mitotic delay). This delay has been studied extensively in more recent times, and the timing for the reduction in mitotic index and subsequent recovery clearly indicates the delay is reversible and occurs sometime during G 2. The production of this effect is very radiosensitive (Joslin, 2001). Appreciable proportions of the cells are delayed by doses of the order of tens of cgy. The G 2 delay increases with dose and frequently corresponds to about 1-3 hgy 1 depending on the particular cells and on the stage in the cycle when the cells are irradiated. Most of the extensive work on cell cycle progression delays in cultured mammalian cells was carried out in the 20-year period between about 1965 and 1985 using 'transformed' or tumorigenic cell lines. Delays in G 1 or S phase were relatively minor and, in many cases, undetectable in the 0-5 Gy dose range. As it turned out, the generalization or extrapolation of the results to normal or untransformed cells was unwarranted. Some investigators during this period, even as early as 1968, reported appreciable delays in the progression of 'non-transformed' cells from G 1 into S phase or in the transition from the non-cycling G 0 to the cycling state after low dose or low dose-rate (LDR) irradiation (Joslin, 2001). In a split-dose experiment, the first dose kills a fraction of the cells, but this fraction is different in all portions of the cell cycle. Survival for cells in the most sensitive phases will be much lower and, in resistant phases much higher than the average. Thus, after the first dose the population of cells surviving will not be distributed around the cell cycle as it normally is, but will be highly enriched in cells from more radioresistant phases. It is these surviving cells that determine the further reduction in survival measured by the second dose. If the first dose is of sufficient magnitude to bring the survival down to, say, 10% or less, then these surviving cells will still contain sub-lethal damage capable of interacting with an additional dose. Thus, if the additional dose were given immediately after the first, the survival reduction would effectively continue down along the single dose survival curve. With a time delay, however, three things happen (Joslin, 2001): ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 155

176 CHAPTER VI BRACHYTHERAPY First, the sub-lethal damage begins to repair, and the half-time for this process is relatively fast being 0.5-2h depending on the system. The effect of this repair process on the surviving cells is to make them more resistant to a second dose, so the proportion surviving will increase with an increasing time interval between the first and second doses. This process is 90% or more complete within about 2-4 h. Second, the cells surviving the first dose which were already in the more resistant phases of the cycle begin to progress and, at least for the first few hours; this progression can only be toward a more sensitive state. For initially log phase populations it is no longer surprising then that with increasing time, between about 3 to 6 or 7 h after the first dose, the survival after the second dose actually decreases. The first dose also produces a mitotic and division delay, so the increase in number of surviving colonies with increasing time before the second dose is not due to an actual increase in numbers of surviving cells from cell division, at least for the first few hours. For example, after a first dose of 5 Gy, there would be essentially no cell division for some 5-10 h, depending on the cells. Third, after the mitotic delay, cell division would resume, so instead of having only one viable cell per surviving colony, as would be the case immediately after the first dose, some, and eventually all, would have two or more viable cells, both of which would have to be killed to prevent colony formation at that locus. Especially appropriate for cell culture applications are 'normal' or 'nontransformed' cells, which form so-called contact-inhibited monolayers. In such monolayers, the cells enter a non-cycling G0 state, where they are no longer a heterogeneous population with respect to the radiosensitivity of subpopulations and, of course, where cell cycle progression and cell division during treatment do not complicate the picture. One additional issue that does arise with the use of contactinhibited monolayer systems as well as organized tissues in vivo is that another, perhaps related, repair process known as 'potentially lethal damage' repair (PLDR), also plays an important role (Joslin, 2001). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 156

177 CHAPTER VI BRACHYTHERAPY POTENTIALLY LETHAL DAMAGE When contact-inhibited monolayers of non-transformed cells are irradiated for a cell survival experiment, the flasks must, of course, be sub-cultured and plated at a low enough density to allow surviving cells to form colonies for the surviving fraction to be assessed. As it turns out, the proportion of irradiated cells surviving a single acute dose in such cultures depends greatly on whether the cells are sub-cultured, diluted, and plated for the colony forming assay immediately after irradiation, or the sub-culture is delayed for some hours, in which case the survival is much higher. The interpretation of this phenomenon is that because damage is lethal in some cells under one set of circumstances (e.g., immediate subculture) but is not under another set (e.g., delayed subculture), such damage must be considered not as 'inevitably lethal' but only 'potentially lethal', depending on the circumstances (Joslin, 2001). Another factor for the study of cellular radiation responses relevant to normal tissue effects, is that virtually no normal tissue contains cells existing in the abundant nutrient conditions of in vitro culture and which are proliferating with growth fractions near 1.0 and doubling times of h. Perhaps intestinal crypt stem cells come as close to this unusual situation as any in vivo. The non-cycling contact-inhibited state for normal cells in culture may fail to simulate all conditions in vivo, but the conditions are perhaps a little closer in general to those in most cell renewal tissues, and much closer with respect to the cell cycling status (Joslin, 2001). 6.5 DOSE-RATE EFFECTS WITH HUMAN CELLS The term 'dose-rate effect' refers to the change in sensitivity or tissue response when the dose rate of irradiation is modified. Dose-rate effects are common in mammalian cell systems, including human tumors and normal tissues. The response of these tissues is complex, depending in part on the radiosensitivity of the stem cells (or 'clonogenic' cells) of the tissue, but also on the modifying effects of cell proliferation and such physiological parameters as oxygenation and growth factors (Joslin, 2001). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 157

178 CHAPTER VI BRACHYTHERAPY TIME-SCALE OF RADIATION ACTION Time-scale of biological effects of ionizing radiation is illustrated in Figure 6.4. It is the operation of some of the processes represented in this chart that gives rise to dose-rate effects. Immediately after exposure, free-radical processes take place leading to damage of many constituents of the cell. Because of its vital nature and the relative uniqueness of its genetic message, DNA is the most important of these damaged molecules (Joslin, 2001). Under physiological conditions the rapid free-radical reactions are complete within around 1 ms, during the subsequent few minutes enzymatic processes begin to operate on the damaged molecules. Some of these act to repair the damage; others leave the molecules in a changed but stable form and this is described as 'misrepair.' Within a few hours these enzymatic processes will be complete (Joslin, 2001). Repair of radiation damage to DNA is highly effective in most cell types: a 1 Gy dose will induce upwards of 1000 DNA strand breaks in every irradiated cell. Roughly half of the cells will survive this dose, so strand-break rejoining must be a remarkably error-free process. Most strand breaks are to one strand only of the double helix, but a small proportion can be recognized as affecting both DNA strands (double-strand breaks - dsb). There is evidence that these are much more serious for the viability of the cell. Even so, the great majority of dsb are also successfully repaired, and of particular importance are dsb that arise from clusters of ionizations at the end of the tracks of secondary electrons: these can involve severe damage to the DNA molecule (so-called 'multiply damaged sites') and, it may be that these events have a relatively low probability of successful repair and a correspondingly high likelihood of leading to cell death or mutation (Joslin, 2001). At longer intervals after irradiation cell proliferation will take place within tissues, leading to the replacement of radiation-damaged cells. In tumors this may lead to recurrence or to a reduced likelihood of success as a result of subsequent treatment (Joslin, 2001). In normal tissues, proliferation may prevent tissue breakdown and the observed early effects of irradiation will then be minimal. However, if the level of cell killing is greater and of such a severity that it cannot be counteracted by proliferation, ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 158

179 CHAPTER VI BRACHYTHERAPY then serious tissue damage may appear. At even longer time intervals after irradiation (months to years), the very long-term effects will become apparent, including tissue failure, formation of new tumors and mutational effects in germ cells (Joslin, 2001). Figure Time-scale of the effects of radiation exposure on biological systems (from Joslin, 2001) MECHANISMS OF THE DOSE-RATE EFFECT Observed dose-rate effects derive from the operation of the processes just described. Usually, clinical external-beam treatments are given within a few minutes. These brief exposures are long enough for the initial chemical effects of irradiation to be complete, but are too short for the subsequent enzymatic and proliferation processes to take place. As radiation dose rate is lowered, the irradiation time for a given dose, increases and it becomes possible for such processes to take place during radiation exposure. These will modify the extent of damage and thus lead to a doserate effect (Joslin, 2001). Four main processes lead in this way to the dose-rate effect. They are the '4Rs of radiobiology': repair, redistribution, repopulation, and reoxygenation, as described before. Among these repair is the fastest, the time required to repair half the induced damage is about 1 h. This means that as soon as the duration of exposure becomes a significant fraction of an hour some repair will take place during irradiation. At the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 159

180 CHAPTER VI BRACHYTHERAPY other extreme, repopulation is a much slower process: repopulation requires cell multiplication and human cells cannot divide in less than about a day. Therefore, repopulation will only have a significant effect when the exposure time is a day or more. Redistribution and reoxygenation probably have a speed that is intermediate between these two processes. Figure 6.5 illustrates the range of dose rates over which each of these processes might be expected to influence radiation action. For dose rates in excess of a few gray per minute none of the processes will take place significantly during irradiation and there will be no dose-rate effect due to them (Joslin, 2001). At much higher dose rates than illustrated a further process, the consumption of oxygen by radiochemical reactions leading to partial hypoxia, may have an effect. At dose rates around 1 Gymin -1, sometimes used for 'high dose rate' or 'acute' irradiations, there may be a small amount of repair during irradiation and such treatments will be slightly less effective than if given at a higher dose rate (Joslin, 2001). Figure Range of dose rates over which repair, reassortment, and repopulation may influence radiation effects (from Joslin, 2001). The curves drawn in Figure 6.5 to represent the effects of repopulation or reassortment are diagrammatic. Repopulation is a much slower process than repair and, only when the exposure time becomes a significant proportion of a cell cycle time (perhaps 1-4 days in human tumor and normal tissue cells) will it have a significant ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 160

181 CHAPTER VI BRACHYTHERAPY effect during the period of irradiation. Reassortment (otherwise known as redistribution) refers to the effects that derive from the movement of surviving cells through the cell cycle after a first dose or increment of dose radiation (Joslin, 2001). These effects may modify the response of a tissue or cell system to subsequent irradiation and, occur over a dose rate range that is somewhere intermediate between those of repair and repopulation. The comparative effects of repair and repopulation are further illustrated in Figure 6.6. This figure shows actual calculations for a typical human cell line, based on a repair half-time of 0.85 h and an α/β ratio of 3.7 Gy (Joslin, 2001). Curves of Figure 6.6 are drawn for four different cell population doubling times and the calculations show the radiation doses (i.e., ED 50 values) for a survival of For these parameter values, there is no effect of proliferation at dose rates above 1 cgymin -1, but as dose rate is lowered to 0.01 cgymin -1 dramatic effects are predicted, depending on the cell population doubling time. The implication for brachytherapy is that above 1 cgymin -1 repopulation effects can be ignored, but below this dose rate they can, under some circumstances, predominate over effects due to incomplete repair (Joslin, 2001). Figure In human cell systems proliferation probably affects radiation response for dose rates below about 1Gyh -1 (from Joslin, 2001) DOSE-RATE EFFECTS IN HUMAN TUMOR CELLS Pioneering experimental studies of the dose-rate effect were made in a number of publications by Hall, Bedford and Mitchell ( Dose rate: its effect on the survival of ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 161

182 CHAPTER VI BRACHYTHERAPY HeLa cells irradiated with gamma rays, Radiat Res 1964; 22: ). The experiments were performed on a variety of cell lines, mainly derived from experimental animals but also including the long established HeLa cell line (derived from a human cervix carcinoma). They showed that the dose rate effect mainly appeared over the range of dose rates from 1 Gymin -1 down to 0.1 cgymin -1. There was considerable variation in the magnitude of the dose-rate effect (i.e., the relative radiosensitivities at high and low dose rates). Steel et al. analyzed these data and showed that derived values for the half-time for repair of radiation damage ranged widely: from below 0.1 h to above than 1 h (Joslin, 2001). Studies on human tumor cell lines taken from a variety of tumor types were reported by Steel et al ( The dose-rate effect in human tumor cells, Radiother Oncol 1987; 9: ). Most of the cell lines were newly established. In some cases the cells were taken directly from human tumors that had first been grown as xenografts in immune-deficient mice; other studies were made on cell lines established in tissue culture. They were irradiated with cobalt-60 gamma-radiation at dose rates ranging from 1 to 150 cgymin -1 at body temperature and under conditions of controlled oxygenation. Cell survival was measured using a colony assay, either in soft agar or in monolayer, depending on the growth characteristics of the cell line. Data on four cell lines are shown in Figure 6.7, covering the range of responses seen in a larger group of human tumor cell lines. Figure 6.7a shows results at high dose rate. The data are fitted by a linear quadratic equation; there is a well-defined initial slope to the data, which are clearly consistent with a continuously bending relationship. The range of sensitivities is considerable (Joslin, 2001). The doses required for a survival of 0.01 range from 3.6 Gy in the HX142 neuroblastoma to 10.9 Gy in the RT112 bladder carcinoma (i.e., by a factor of 3). In the initial dose region the factor is greater. Figure 6.7b shows the results for the same cell lines at the low dose rate of 1.6 cgy min '. The curves have fanned-out and become straight or almost so on the semi-logarithmic plot. It can be seen that at low dose rate the lines seem to extrapolate the initial slopes of the high dose-rate curves (Joslin, 2001). The range of sensitivities among the cell lines is now larger: by a factor of approximately 10. The data shown in Figure 6.7 indicate the range of sensitivities seen ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 162

183 CHAPTER VI BRACHYTHERAPY among tumors of different histological types. Less information is available about the range of sensitivities among tumors of the same type, from diverse patients. Kelland and Steel ( Differences in radiation response among human cervix carcinoma cell lines, Radiother. Oncol., 13,225-32) studied five cell lines newly established from human cervical carcinomas. They found that at high dose rate the dose to produce a surviving fraction of 0.01 ranged from 5 to 10.5 Gy. The dose-rate sparing factors (the dose at 1.6 cgymin -1 compared with the dose at 150 cgymin -1 ) ranged from 1.1 to 1.6. This showed that among tumors of the same type there were considerable radiobiological differences that could be clinically significant. There may be a number of causes of failure in brachytherapy and these include the inherent insensitivity of the tumor cells to radiation. A so-far insufficiently explored aspect of brachytherapy is the attempt to develop predictive tests of radiosensitivity in order to identify patients most at risk of recurrence. The data in Figure 6.7 clearly indicate that such tests should be made at low dose rate, where the differences among cell lines are greatest (Joslin, 2001). Figure Cell survival curves for four human tumor cell lines irradiated at (a) 150 cgy min-1 or (b) 7.6 cgy min -1 HX142, neuroblastoma; HX58, pancreas carcinoma; HX156, cervix carcinoma; RT112, bladder carcinoma (from Joslin, 2001) EFFECT OF IRRADIATION ON CELL CYCLE PROGRESSION Irradiation at high dose rate blocks cell entry into mitosis. The cell cycle may be interrupted at a number of so called 'check-points', and the biochemical processes ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 163

184 CHAPTER VI BRACHYTHERAPY involved in these arrests are the subject of intense laboratory research at the present time. At high dose rate, there are two reasons why proliferation effects during irradiation are unimportant: irradiation times are too short, and the cells are subject to mitotic delay and therefore inhibited from proliferating. As dose rate is reduced, both these factors become less severe and cell cycling takes place during irradiation, thus counteracting the effect of irradiation (Joslin, 2001). Skladowski et al. ( Cell-cycle progression during continuous irradiation of a human bladder carcinoma cell line Radiother. Oncol., 28,219-27) concluded that cellcycle effects in tumor cells are unlikely to be of any great significance, in relation to the cell-killing effect at different distances from an implanted radiation source. Overall treatment times in brachytherapy tend to be short compared with external-beam treatment and proliferation effects are correspondingly of less significance (Joslin, 2001) CELL KILLING AROUND AN IMPLANTED RADIATION SOURCE The non-uniformity of radiation field around an implanted source has important radiobiological consequences. Close to the source, the dose rate is high and the amount of cell killing will be close to that indicated by the acute-radiation survival curve. As the distance from the source is increased, two changes take place: cells will be less sensitive to lower dose rates, and within a given period of implantation the accumulated dose will also be less. These two factors lead to a very rapid change of cell killing with distance from the source. This is illustrated in Figure 6.8 for the case of a point radioactive source. A source strength was chosen that gives 75 Gy in 6 days at a range of 2 cm. Three different tumor-cell sensitivities were assumed, as shown in the upper panel. It is the low dose-rate sensitivities that matter for this calculation. For spherical shells containing 109 clonogenic cells at different distances from the source, it was possible to calculate the surviving fraction from 6 days irradiation, the absolute number of surviving clonogenic cells, and thus the probability that all cells in the shell would be killed. The results are shown in the lower panel. For cells of any given level of radiosensitivity there will be cliff-like change from high to low local cure probability, ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 164

185 CHAPTER VI BRACHYTHERAPY taking place over a radial distance of a few millimeters. Note that the order of the lines in the upper and lower panels of this figure is reversed: very sensitive tumor cells (lines A) can be cured out to a greater radius than less sensitive cells (B) or very radioresistant cells (C). The steepness of the tumor control curves derives in part from the underlying assumed Poisson relationship between the average number of surviving cells per shell and the control probability. As is the case with tumor control by external-beam irradiation, in reality, there will be factors that make the tumor control curves less shallow: heterogeneity, for instance (Joslin, 2001). Within tissues (tumor or normal) that are close to the source, the level of cell killing will be so high that cells of any radiosensitivity will be killed. Further out, the effects will be so low that even the most radiosensitive cells will survive. Between these extremes there is a critical zone in which differential cell killing will occur. In this critical region the radiation dose rate will be low. For this reason, one would argue that the low dose-rate survival curves as shown in Figures 6.5 and 6.6 are more clinically realistic than the high dose-rate curves, certainly for brachytherapy. Figure 6.9 contrasts this situation with external beam radiotherapy, where the aim is to deliver a uniform radiation dose across the tumor. Only in a narrow zone around an implanted source (where the surviving fraction changes from, say, to 10-6 ) will radiobiological considerations be of interest or importance in relation to tumor control. The same principle will apply to normal tissue damage: serious damage to normal structures depends on making sure that they are outside the corresponding 'cliff' (Joslin, 2001). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 165

186 CHAPTER VI BRACHYTHERAPY Figure The likelihood of cure varies steeply with distance from a point radiation source. The radius at which failure occurs depends upon the steepness of the survival curve at low dose rate (upper panel) (from Joslin, 2001). Figure Variation of cell kill around a point source of radiation (from Joslin, 2001). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 166

187 CHAPTER VI BRACHYTHERAPY IMPLICATIONS FOR CLINICAL BRACHYTHERAPY The radiobiology of low dose-rate irradiation is now fairly well understood. Although data are not available on a wide range of human tumors, the data that one have do indicate the range of responses that are seen for human cells in tissue culture. It is likely that these will be realistic for effects on well-oxygenated cells in the patient. Much less is known about the effects of low dose-rate irradiation on hypoxic cells in vivo. These are, of course, less sensitive to high dose-rate irradiation. The work of Ling et al. ( The variation of OER with dose rate, Int. J. Radial. Oncol. Biol. Phys.,11, ) showed that the sparing effect of low dose-rate irradiation as a function of oxygen concentration was complex. Lowering the dose rate initially had more effect on the oxic cells than on the hypoxic cells. Further lowering of dose rate had consequently more effect on the hypoxic cells. Although for such reasons there is much that still needs to be understood about the tumor effects of brachytherapy, some simple conclusions can be drawn: 1. In the dose-rate range from a few Gymin -1 down to a few cgymn -1, repair of radiation damage is the main modifying process on radiosensitivity. The effects are large, leading to a change in the isoeffective radiation dose by a factor of 2 or more. Below 1 cgymhr -1, cell proliferation will play an increasingly strong role in making tumors or normal tissues less sensitive to radiation damage. 2. There is evidence for a dose-rate effect in the region of 1 Gymin -1. If, in external-beam radiotherapy, a change of machine or of source-skin distance leads to a substantial lowering of dose rate, then a dose rate correction should be considered. 3. The biological effect of irradiation changes rapidly at dose rates around 10 cgymin -1. This may mean that greater precision in dosimetry and dose prescription is required in high dose-rate brachytherapy than when a low dose rate is used. 4. Tumor cells of different origins show very different response to low dose-rate irradiation. Theoretical calculations suggest that as one move out from an implanted radiation source the local tumor control probability will change rapidly, i.e., there will be sudden failure to eradicate all clonogenic tumor cells. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 167

188 CHAPTER VI BRACHYTHERAPY The prediction that the range at which this occurs will depend strongly on the low dose-rate radiosensitivity of the tumor cells could be clinically important. There is a strong case for predictive testing of tumors that are to be treated with curative intent by brachytherapy in order to predict those that require a greater or lesser range of dose distribution (Joslin, 2001). 6.6 PREDICTIVE ASSAYS FOR RADIATION ONCOLOGY Since the 1980s, radiation oncologists and biologists have recognized the need for additional assays on an individual patient basis that would select the most advantageous treatment approach. Hence, it should emphasize assays for individual patients for several reasons (Joslin, 2001). First, the cellular radiation sensitivity of the tumor may differ among individuals, even for tumors of the same histological type. If the radiosensitivity of the individual's tumor were precisely known, perhaps total radiation doses could be adjusted before the end of therapy to maximize tumor response. Alternatively, the option of using radiation sensitizers for 'radioresistant' tumors would have a more rational basis (Joslin, 2001). Second, normal-tissue radiation sensitivity may differ among individuals. This is an important point because the total radiation dose that can be delivered to a patient's tumor is often limited by normal tissue tolerance. Stated differently, frequently radiation oncologists are compelled to treat a patient's tumor with radiation doses that are dictated not by tumor sensitivity but by normal-tissue tolerance, which in many instances results in inadequate dose to the tumor. If one assumes there is a Gaussian distribution of normal-tissue radiosensitivities among humans, then the most sensitive individuals in the population may well dictate radiation tumor doses utilized in the clinic. Because the radiation tumor control dose response curve is quite steep for many tumors, modest increases in the total radiation dose delivered would be expected greatly to enhance tumor control. If it were determined that the patient's normal-tissue radiation response were toward the 'radioresistant' edge of the Gaussian distribution, consideration could be given to ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 168

189 CHAPTER VI BRACHYTHERAPY administering higher radiation doses. Alternatively, if the patient's normal-tissue radiation response was toward the 'radiosensitive' edge of the Gaussian distribution, the use of radioprotectors could be considered. Unfortunately, selective normal-tissue radioprotectors have yet to be identified (Joslin, 2001). Third, biological, environmental, and physiological factors of tumors may differ among individuals. Factors such as tumor ph, hypoxia, blood flow, and growth of the tumor in terms of cell-cycle parameters and potential tumor doubling times (Tpot) can influence the overall radiation responsiveness of the tumor. If these factors were known prior to therapy, the use of hypoxic cell radiosensitizers or, in the case of Tpot values, alteration of fractionation/time schedules could be considered (Joslin, 2001). Numerous predictive assays have been developed over the past two decades to address many of the points cited above and several have been evaluated in a clinical setting (Joslin, 2001). 6.7 SUMMARY Brachytherapy is an important radiation technique in the treatment of malignant disease that allows conformal treatment without heavy technological involvement. However, since it generally involves invasive procedures (interstitial brachytherapy), except for special instances in which intracavitary techniques may be employed, brachytherapy is relegated to second place behind external beam radiotherapy in the treatment of malignant disease (Suntharalingam, 2002). A typical radiation oncology department will treat about 80% of its patients with the various external beam techniques and about 10 20% of its patients with brachytherapy. The basic principles of brachytherapy have not changed much during the past 100 years of radiotherapy; however, the advent of remote afterloading brachytherapy has made brachytherapy much more efficient for the patient and safer for staff from the radiation protection point of view. In terms of physics human resource needs, a brachytherapy patient requires considerably more involvement than an average external beam patient (Suntharalingam, 2002). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 169

190 CHAPTER VI BRACHYTHERAPY Nearly every malignant disease in the human body has been treated with brachytherapy; however, gynaecological cancer treatments provide the greatest success and permanent prostate implants are becoming increasingly common. (Suntharalingam, 2002) There are also various sites for which brachytherapy has proven a complete failure. The newest application of brachytherapy is intravascular (also referred to as endovascular) brachytherapy, used for the prevention of restenosis in arteries following coronary arterial angioplasty (Suntharalingam, 2002). This radiation technique was used to kill the prostate and breast cancer cells that will be studied by me in my dissertation thesis. ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 170

191 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING

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193 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING INTRODUCTION Digital image processing is an area characterized by the need for extensive experimental work to establish the viability of proposed solutions to a given problem. An important characteristic underlying the design of an image processing system is the significant level of testing and experimentation that normally is required before arriving at an acceptable solution. This characteristic implies that the ability to formulate approaches and quickly prototype candidate solutions generally plays a major role in reducing the cost and time required to arrive at a viable system implementation (González, 2004). MATLAB is a high-performance language for technical computing. It integrates computation, visualization, and programming in an easy-to-use environment where problems and solutions are expressed in familiar mathematical notation. Typical uses include the following: Math and computation; Algorithm development; Data acquisition; Modeling, simulation and prototyping; Data analysis, exploration and visualization; Scientific and engineering graphics; Application development, including graphical user interface building. MATLAB is an interactive system whose basic data element is an array that does not require dimensioning. This allows formulating solutions to many technical computing problems, especially that involving matrix representation, in a fraction of the time it would take to write a program in a scalar non-interactive language such as C or Fortran (González, 2004). The name MATLAB stands for matrix laboratory and was written originally to provide easy access to matrix software developed by the LINPACK (Linear System Package) and EISPACK (Eigen System Package) projects (González, 2004). The Image Processing Toolbox is a collection of MATLAB functions (called M- functions or M-files) has extended the capability of the MATLAB environment for the solution of digital image processing problems (González, 2004). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 173

194 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING In this chapter, it is performed a description of the basic concepts of digital image processing to provide background information of what is performed with the cells images that I will study in my dissertation thesis. The cell images will be processed using the image processing program MATLAB. 7.2 PRE-PROCESSING EVALUATION OF DIGITAL IMAGES After digital images have been captured, and prior to initiating processing algorithm applications, each image should be evaluated with regard to its general characteristics, including noise, blur, background intensity variations, brightness and contrast, and the general pixel value distribution (histogram profile). Attention should be given to shadowed regions to determine how much detail is present, as well as bright features (or highlights) and areas of intermediate pixel intensity (Davidson, 2007). Each image-editing program has a statistics or status window that enables the user to translate the mouse cursor over the image and obtain information about specific pixel values at any location in the image. For example, the Photoshop Info Palette provides continuously updated pixel information, including x and y coordinates, RGB (red, green, and blue) color values, CMYK (cyan, magenta, yellow, black) conversion percentages, and the height and width of a marquee selection within the image. Preference options in the palette display include selecting alternative colorspace models for information readout. Among the models available in Photoshop are grayscale, HSB (hue, saturation, and brightness), web color (the 216 colors that overlap in the Windows and Macintosh 8-bit or 256 color display palettes), actual color, opacity, and Lab color (device-independent color space) (Davidson, 2007). By evaluating the intensities (grayscale and color) and histogram positions of various image features, the black and white set points for stretching and sliding of the entire histogram for contrast adjustments can be determined. The image should also be checked for clipping, which is manifested by the appearance of saturated white or underexposed black regions in the image. In general, clipping should be avoided, both during image acquisition, and while the image is being processed. Images that have been adversely affected by background intensity variations should be corrected by flat- ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 174

195 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING field techniques or background subtraction prior to applying histogram manipulations (Davidson, 2007). 7.3 LOOK-UP TABLES Several of the fundamental digital image processing algorithms commonly employed in optical microscopy function through a technique known as single-image pixel point operations, which perform manipulations on sequential individual pixels rather than large arrays. The general equation utilized to describe single-image pixel point processes for an entire image array is given by the relationship: where I(x,y) represents the input image pixel at coordinate location (x,y), O(x,y) is the output image pixel having the same coordinates, and M is a linear mapping function. In general, the mapping function is an equation that converts the brightness value of the input pixel to another value in the output pixel. Because some of the mapping functions utilized in image processing can be quite complex, performing these operations on a large image, pixel-by-pixel, can be extremely time-consuming and wasteful of computer resources. An alternative technique used to map large images is known as a look-up table (LUT), which stores an intensity transformation function (mapping function) designed so that its output gray-level values are a selected transformation of the corresponding input values (Davidson, 2007). (a) (b) (c) Figure 7.1 Inversion and threshold map look-up table operation (from Davidson, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 175

196 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING When quantized to 8 bits (256 gray levels) each pixel has a brightness value that ranges between 0 (black) and 255 (white), to yield a total of 256 possible output values. A look-up table utilizes a 256-element array of computer memory, which is preloaded with a set of integer values defining the look-up table mapping function. Thus, when a single-pixel process must be applied to an image using a look-up table, the integer gray value for each input pixel is utilized as an address specifying a single element in the 256-element array. The memory content of that element (also an integer between 0 and 255) overrides the brightness value (gray level) of the input pixel and becomes the output gray value for the pixel. For example, if a look-up table is configured to return a value of 0 for input values between 0 and 127 and to return a value of 1 for input values between 128 and 255, then the overall point process will result in binary output images that have only two sets of pixels (0 and 1). Alternatively, to invert contrast in an image, a look-up table can return inverse values of 0 for 255, 1 for 254, 2 for 253, and so forth. Look-up tables have a significant amount of versatility and can be utilized to produce a wide variety of manipulations on digital images (Davidson, 2007). Image transformations that involve look-up tables can be implemented by either one of two mechanisms: at the input so that the original image data are transformed, or at the output so that the transformed image is displayed but the original image remains unmodified. A permanent transformation of the original input image may be necessary to correct for known defects in detector properties (for example, nonlinear gain characteristics) or to transform the data to a new coordinate system (from linear to logarithmic or exponential). When only the output image should be modified, the image transformation is performed just before the digital image is converted back to analog form by the digital-to-analog converter for display on a computer monitor. In some cases, the results of the transformation specified by the output look-up table(s) are displayed visually on the monitor, but the original image data are not altered (Davidson, 2007). Look-up tables are not restricted to linear or monotonic functions and a variety of nonlinear look-up tables are utilized in signal processing to correct for camera response characteristics or to emphasize a narrow region of gray levels. A good example of the utility of a nonlinear look-up table is the correction of recorded images ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 176

197 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING that have been inadvertently captured with an incorrect camera gamma adjustment. In addition, monochrome or color images can also be converted to generate negatives for photography. Other applications include pseudocoloring and sigmoidal look-up tables that emphasize a selected range of gray values targeted to enhance desired features or to adjust the amount of image contrast (Davidson, 2007). Presented in Figure 7.1 are look-up table mapping functions for image contrast inversion using both a 256-element memory pre-loaded register and a table map (Figure 7.1(a)), and a thresholding operation using only a table map (Figure 7.1(b)). The input pixel gray level is utilized to specify the address of the look-up table element whose content provides the gray level of the output pixel in the memory register (Figure 7.1(a)). The square look-up table map presents an alternative method of calculating output pixel values based on those of the input pixel. To use the map, first determine the input pixel gray-level value, and then extend a vertical line from the input value to the mapping function. A horizontal line is then drawn from the intersection of the vertical line and the mapping function to produce the output pixel gray level on the vertical axis of the map (Figure 7.1(b) and 7.1(c)). In the case of the thresholding operation (Figure 7.1(c)), all pixels having an input value below 100 are mapped to black (0), while other input pixel intensities are unaltered (Davidson, 2007). 7.4 FLAT-FIELD CORRECTION AND BACKGROUND SUBTRACTION A digital image acquired from a microscope, camera, or other optical device is often described as a raw image prior to processing and adjustment of critical pixel values (see Figure 7.2). In many cases, the raw image is suitable for use in target applications (printing, web display, reports, etc.), but such an image usually exhibits a significant level of noise and other artifacts arising from the optical and capture system, such as distortions from lens aberrations, detector irregularities (pixel nonuniformity and fixed-pattern noise), dust, scratches, and uneven illumination. In addition, improper bias signal adjustment can increase pixel values beyond their true photometric values, a condition that leads to significant errors in measuring the amplitudes of specific image features. Errors in the raw image are manifested as dark shadows, excessively bright highlights, specks, mottles, and intensity gradients that ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 177

198 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING alter the true pixel values. In general, these errors are particularly evident in digital images having bright, uniform backgrounds, which are produced by a variety of common microscope illumination modes, including brightfield, oblique, phase contrast, and differential interference contrast (DIC). Fluorescence images having medium gray or bright backgrounds, though relatively rare, may suffer from similar errors (Davidson, 2007). Figure 7.2 Flat-field correction of a digital image (from Davidson, 2007). Applying flat-field correction techniques to raw digital images can often ensure photometric accuracy and remove common image defects to restore the fidelity of features and achieve a visual balance. These correction steps should be undertaken before measuring light amplitudes or obtaining other quantitative information from pixel intensity values, although the corrections are not necessary in order to display or print an image. Flat-field and background subtraction techniques usually require collection of additional image frames under conditions similar to those employed to capture the primary raw specimen image (Davidson, 2007). Most of the flat-field correction schemes utilize two supplemental image frames, in addition to the raw image, to calculate final image parameters (Figure 7.2). A flat-field reference frame can be obtained by removing the specimen and capturing the featureless view field at the same focus level as the raw image frame. Flat-field reference frames should display the same brightness level as the raw image and take ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 178

199 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING advantage of the full dynamic range of the camera system to minimize noise in the corrected image. If both the raw image and flat-field reference frame have low signal amplitudes and contain a significant amount of noise, the corrected image will also be dark and noisy. In order to compensate for noise and low intensity, flat-field reference frames can be exposed for longer periods than those used for capturing raw images. Several averaged frames (3-20) can be added together to create a master flat-field reference frame with a very low noise level (Davidson, 2007). In addition to a flat-field reference frame, a dark reference frame is collected, which effectively records the output level of each pixel when the image sensor is exposed to a dark scene, absent microscope illumination. The dark frame contains the pixel bias offset level and noise acquired from electronic and thermal sources that contaminate the raw image. Offset pixel values derive from the positive voltage applied to the image sensor in order to digitize analog intensity information from each photodiode. Electronic noise originates from camera readout and related sources, and thermal noise is generated by kinetic vibration of silicon atoms in the collection wells and substrate of semiconductor-based sensors. Collectively, these noise sources are referred to as dark noise, and are a common artifact in digital image sensors, which can contribute up to 20 percent of apparent pixel amplitudes. In order to ensure photometric accuracy, these sources must be subtracted from the flat-field reference frame and raw image. Dark frames are generated by integrating the image sensor output for the same period as the raw image, but without opening the camera shutter. Master dark frames can be prepared by averaging several individual dark frames together to increase signal intensity (Davidson, 2007). Once the necessary frames have been collected, flat-field correction is a relatively simple operation that involves several sequential functions. First, the master dark frame is subtracted from both the raw image and flat-field reference frames, followed by the division of the resulting values (Figure 3). In effect, the raw frame is divided by the flat-field frame after the dark frame has been subtracted from each frame and the quotient is multiplied by the mean pixel value in order to maintain consistency between the raw and corrected image intensities. Individual pixels in the corrected image are constrained to have a gray level value between 0 and 255, as a precaution against sign inversion in cases where the dark reference frame pixel value ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 179

200 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING exceeds that of the raw image. The flat-field correction illustrated in Figure 3 shows a plot of intensity profile across a selected region of the image versus pixel number for the raw, flat-field, and dark frames, as well as that for the corrected image (Davidson, 2007). Background subtraction is a technique that results in localized alterations of each pixel value in the raw image, depending upon the intensity of a corresponding pixel at the same coordinate location in the background image. As a result, non uniformities in detector sensitivity or illumination (including mottle, dirt, scratches, and intensity gradients) can be compensated by storing a background image of an empty microscope field as a reference image. Video-enhanced contrast (VEC) microscopy is critically dependent on background subtraction for removal of both stray light and artifacts from highly magnified images of specimens having poor contrast. In this case, the background image is obtained by defocusing or displacing the specimen from the field of view. The resulting background image is stored and continuously subtracted from the raw image, producing a dramatic improvement in contrast. This technique is also useful for temporal comparisons to display changes or motion between view fields (Davidson, 2007). (a) (b) Figure 7.3 Surface function background subtraction technique (from Davidson, 2007). When it is not feasible to capture a background image in the microscope, a surrogate image can be created artificially by fitting a surface function to the background of the captured specimen image (see Figure 7.3). This artificial background image can then be subtracted from the specimen image. By selecting a number of points in the image that are located in the background, a list of brightness values at ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 180

201 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING various positions is obtained. The resulting information can then be utilized to obtain a least squares fit of a surface function that approximates the background. In Figure 7.3, eight adjustable control points are used to obtain a least squares fit of the background image with a surface function B(x, y) of the form: where c(0)... c(5) are the least squares solutions, and (x, y) represents the coordinates of a pixel in the fitted background image. The specimen presented in Figure 7.3 is a young starfish captured digitally with an optical microscope configured to operate in oblique illumination. The control points should be chosen so that they are evenly distributed across the image, and the brightness level at each control point should be representative of the background intensity. Placing many points within a small region of the image while very few or none are distributed into surrounding regions will result in a poorly constructed background image. In general, background subtraction is utilized as an initial step in improving image quality, although (in practice) additional image enhancement techniques must often be applied to the subtraction image in order to obtain a useful result (Davidson, 2007). Images modified by flat-field correction appear similar to those obtained with background subtraction, but performing the operation by division (flat-field correction) is preferred because the technique yields images that are photometrically more accurate. The primary reason for this difference is that images result from light amplitude values derived by a multiplicative process that combines the luminous flux and exposure time. After application of flat-field correction techniques (but not necessarily background subtraction algorithms), the relative amplitudes of specimen features will be photometrically accurate. As an added benefit, flat-field correction removes a majority of the optical defects that are present in the raw image (Davidson, 2007). 7.5 IMAGE INTEGRATION Because a digital image is composed of a matrix of integers, operations such as the summation or integration of images can readily be conducted at high speed. If the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 181

202 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING original images were digitized with 8-bit resolution, the storage region, or digital frame memory, which holds the accumulated images, must have sufficient capacity to accommodate a sum that exceeds 8 bits. If it is assumed that a few pixels in an 8-bit digital image have the maximum gray-level value of 255, then summation of 30 frames would result in a local pixel gray-level value of 7650 and require a storage register with 13-bit capacity. To sum 256 frames, the storage capacity must equal 65,536 gray levels, or 16 bits, to accommodate the brightest pixels (Davidson, 2007). Although modern computer monitors are capable of displaying images having more than 256 gray levels, the limited response of the human eye (35-50 gray levels) suggests that 16-bit digital images should be scaled to match the limitations of the display and human visual ability. When the useful information of the image resides only in a subregion of the 16-bit stored image, only this portion should be displayed. This is a beneficial approach when displaying images captured by a slow-scan CCD camera of a view field with a large intrascene range of intensities. The process involves searching through the 16-bit image for the visually meaningful portion (Davidson, 2007). When images obtained with a video-rate analog or CCD camera are summed into a 16-bit frame memory, display of a meaningful 8-bit image is usually accomplished by dividing the stored sum by a constant. For example, a 96-frame summation of a view field can be divided by 96, 64, 32, or 24. Division by 32 is equivalent to a threefold increase in gain and results in utilization of the full 255 graylevel range. However, division by 24 is equivalent to a fourfold gain increase and results in image saturation and loss of information (Davidson, 2007). Image integration using digital image processing techniques often enables visualization of a faint object that is barely detectable above the camera noise. Integration may be of particular value in low-light-level imaging when the brightness of the image cannot be increased by additional image intensification. However, it is important to realize that, from signal-to-noise considerations, integration directly on the sensor is always preferable to integration in the processing software. Each image integration step in the software introduces analog-to-digital noise as well as camera readout noise (Davidson, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 182

203 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING 7.6 DIGITAL IMAGE HISTOGRAM ADJUSTMENT A majority of the digital images captured in an optical device, such as a camera or microscope, require adjustments to either the look-up table or the image histogram to optimize brightness, contrast, and general image visibility. Histograms of digital images provide a graphical representation of image contrast and brightness characteristics, and are useful in evaluating contrast deficiencies such as low or high contrast, and inadequate dynamic range. An image histogram is a graphical plot displaying input pixel values on the x-axis (referred to as a bin) versus the number (or relative number) of pixels for any given bin value on the y axis. Each bin in a grayscale histogram depicts a subgroup of pixels in the image, sorted by gray level. The numeric range of input values, or bins, on the x-axis usually corresponds to the bit depth of the captured image (0-255 for 8-bit images, for 10-bit images, and for 12-bit images). Mathematical operations may be performed on the histogram itself to alter the relative distribution of bins at any gray level. Manipulation of the histogram can correct poor contrast and brightness to dramatically improve the quality of digital images (Davidson, 2007). Histogram stretching involves modifying the brightness (intensity) values of pixels in the image according to a mapping function that specifies an output pixel brightness value for each input pixel brightness value (see Figure 7.4). For a grayscale digital image, this process is straightforward. For an RGB color space digital image, histogram stretching can be accomplished by converting the image to a hue, saturation, intensity (HSI) color space representation of the image and applying the brightness mapping operation to the intensity information alone. The following mapping function is often utilized to compute pixel brightness values: In the above equation, the intensity range is assumed to lie between 0.0 and 1.0, with 0.0 representing black and 1.0 representing white. The variable B represents the intensity value corresponding to the black level, while the intensity value ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 183

204 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING corresponding to the white level is represented by the variable W. In some instances, it is desirable to apply a nonlinear mapping function to a digital image in order to selectively modify portions of the image (Davidson, 2007). Histogram equalization (also referred to as histogram leveling) is a related technique, which results in the reassignment of pixel gray-level values so that the entire range of gray levels is utilized and the number of counts per bin remains constant. The process yields a flat image histogram with a horizontal profile that is devoid of peaks. Pixel values are reassigned to ensure that each gray level contains an equal number of pixels while retaining the rank order of pixel values in the original image. Equalization is often utilized to enhance contrast in images with extremely low contrast where a majority of the pixels have nearly the same value, and which do not respond well to conventional histogram stretching algorithms. The technique is effective in treating featureless dark, and flat-field frames, and to rescue images with low-amplitude gradients. In contrast, histogram stretching spaces gray-level values to cover the entire range evenly. The auto-enhance or automatic levels (contrast) features of many image processing software packages utilize one of these histogrambased transformations of the image (Davidson, 2007). Figure 7.4 Contrast enhancement by histogram stretching (from Davidson, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 184

205 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING Digital image histograms can be displayed in several motifs that differ from the conventional linear x and y plots of pixel number versus gray level value. Logarithmic histograms chart the input pixel value on the x-axis versus the number of pixels having that value on the y-axis, using a log scale. These histograms are useful to examine pixel values that comprise a minority of the image, but exhibit a strong response to histogram stretching. Another commonly employed variation, the integrated or cumulative histogram, plots input pixel values on the x-axis and the cumulative number of all pixels having a value of x, and lower, on the y-axis. Cumulative histograms are often utilized to adjust contrast and brightness for images gathered in phase contrast, DIC, and bright field illumination modes, which tend to have light backgrounds (Davidson, 2007). In some cases, images have regions of very high intensity, manifested by large peaks near the histogram 255 gray level, where the video signal is saturated and all pixels have been rendered at the maximum gray value. This situation is termed graylevel clipping and usually indicates that a certain degree of detail has been lost in the digital image because some regions of the original image that might have different intensities have each been assigned to the same gray value. Clipping of the histogram may be acceptable in some circumstances if detail is lost only from unimportant parts of the image. Such a situation might occur, for example, if the system has been adjusted to maximize the contrast of stained histological slides under brightfield illumination, with the clipping occurring only in bright background regions where there is no cellular structure (Davidson, 2007). 7.7 SPATIAL CONVOLUTION KERNELS (OR MASKS) Some of the most powerful image processing tools utilize multipixel operations, in which the integer value of each output pixel is altered by contributions from a number of adjoining input pixel values. These operations are classically referred to as spatial convolutions and involve multiplication of a selected set of pixels from the original image with a corresponding array of pixels in the form of a convolution kernel or convolution mask. Convolutions are mathematical transformations of pixels, carried out in a manner that differs from simple addition, multiplication, or division, as ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 185

206 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING illustrated in Figure 7.5 for a simple sharpening convolution kernel mask (Davidson, 2007). In the simplest form, a two-dimensional convolution operation on a digital image utilizes a box convolution kernel. Convolution kernels typically feature an odd number of rows and columns in the form of a square, with a 3 x 3 pixel mask (convolution kernel) being the most common form, but 5 x 5 and 7 x 7 kernels are also frequently employed. The convolution operation is performed individually on each pixel of the original input image, and involves three sequential operations, which are presented in Figure 7.5. The operation begins when the convolution kernel is overlaid on the original image in such a manner that the center pixel of the mask is matched with the single pixel location to be convolved from the input image. This pixel is referred to as the target pixel (Davidson, 2007). Figure 7.5 The convolution operation sequence (from Davidson, 2007). Next, each pixel integer value in the original (often termed the source) image is multiplied by the corresponding value in the overlying mask (Figure 7.5). These products are summed and the grayscale value of the target pixel in the destination image is replaced by the sum of all the products, ending the operation. The convolution kernel is then translocated to the next pixel in the source image, which ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 186

207 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING becomes the target pixel in the destination image, until every pixel in the original image has been targeted by the kernel (Davidson, 2007). Convolution kernels may contain all positive, or positive and negative values, and thus can result in negative totals, or results that exceed the maximum 255 limit that a pixel can hold. Appropriate divisor and offset values are needed to correct this. The smoothing convolution kernel illustrated in Figure 7.6(a) has a value of unity for each cell in the matrix, with a divisor value of 9 and an offset of zero. Kernel matrices for 8-bit grayscale images are often constrained with divisors and offsets that are chosen so that all processed values following the convolution fall between 0 and 255. Many of the popular software packages have user-specified convolution kernels designed to fine-tune the type of information that is extracted for a particular application (Davidson, 2007). Convolution kernels are useful for a wide variety of digital image processing operations, including smoothing of noisy images (spatial averaging) and sharpening images by edge enhancement utilizing Laplacian, sharpening, or gradient filters (in the form of a convolution kernel). In addition to convolution operations, local contrast can be adjusted through the application of maximum, minimum, or median filters that rank the pixels within each local neighborhood. Furthermore, the use of a Fourier transform to convert images from the spatial to the frequency domain makes possible another class of filtering operations. The total number of algorithms developed for image processing is enormous, but several operations enjoy widespread application among many of the popular image processing software packages (Davidson, 2007). 7.8 SMOOTHING CONVOLUTION FILTERS (SPATIAL AVERAGING) Specialized convolution kernels, often termed smoothing filters, are often used for reducing random noise in digital images. A typical smoothing convolution filter is illustrated in Figure 7.6(a), and is essentially a matrix having an integer value of 1 for each row and column. When an image is convolved with this type of kernel, the gray value of each pixel is replaced by the average intensity of its eight nearest neighbors and itself. Random noise in digital images is manifested by spurious pixels having unusually high or low intensity values. If the gray value of any pixel overlaid by the ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 187

208 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING convolution kernel is dramatically different than that of its neighbors, the averaging effect of the filter will tend to reduce the effect of the noise by distributing it among all of the neighboring pixels (Davidson, 2007). Figure 7.6 Smoothing and sharpening convolution kernels (from Davidson, 2007). The nine integers in each smoothing kernel illustrated in Figure 7.6 add to a value of 1 when summed and divided by the number of values in the matrix. These kernels are designed so that the convolution operation will produce an output image having an average brightness that is equal to that of the input images (however, in some cases, this may be only approximate). In general, the sum of terms in most convolution kernels will add to a value between zero and one in order to avoid creating an output image having gray values that exceed the dynamic range of the digital-to-analog converter utilized to display the image (Davidson, 2007). Smoothing convolution kernels act as low-pass filters to suppress the contribution of high spatial frequencies in the image. The term spatial frequency is analogous to the concept of frequency with respect to time (temporal frequency), and describes how rapidly a signal changes with respect to position in the image. A low spatial frequency might exhibit only a few cycles across the width of an image, while a high spatial frequency often displays numerous cycles in the same linear dimensions. An excellent example is the minute orderly arrays of miniature pores and striate exhibited by diatom frustules, which alternate between very high and low intensities over very short distances. A low spatial frequency might exhibit only a few cycles across the width of an image (manifested as widely spaced stripes, for example), ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 188

209 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING whereas a high spatial frequency undergoes numerous cycles across the lateral dimensions of an image. The highest spatial frequency that can be displayed in a digital image has a period equal to the width of two pixels (Davidson, 2007). The type of random noise typically observed in digital images has a high spatial frequency that can be effectively removed by applying a smoothing convolution kernel to the image, pixel by pixel. However, other "real" image features that are desirable, such as object boundaries and fine structural details, may also have high spatial frequencies that can unfortunately be suppressed by the smoothing filter. Consequently, application of a smoothing convolution kernel will often have the undesirable effect of blurring an input image. Furthermore, the larger the kernel (5 x 5, 7 x 7, and 9 x 9), the more severe this blurring effect will be (Figure 8). For most applications, the size and form of the smoothing kernel must be carefully chosen to optimize the tradeoff between noise reduction and image degradation. A Gaussian filter is a smoothing filter based on a convolution kernel that is a Gaussian function, and provides the least amount of spatial blurring for any desired amount of random noise reduction. Smoothing filters are good tools for making simple cosmetic improvements to grainy images that have a low signal-to-noise ratio, but these filters can also undesirably reduce the image resolution as a consequence (Davidson, 2007). 7.9 SHARPENING CONVOLUTION FILTERS In direct contrast to the action of smoothing convolution filters, sharpening filters are designed to enhance the higher spatial frequencies in a digital image, while simultaneously suppressing lower frequencies. A typical 3 x 3 convolution mask and its effect on a digital image captured with an optical microscope is illustrated in Figure 7(c). In addition to enhancing specimen boundaries and fine details, sharpening filters also have the effect of removing slowly varying background shading. Thus, these filters can sometimes be utilized to correct for shading distortion in an image without having to resort to background subtraction algorithms. Unfortunately, sharpening convolution filters have the undesirable effect of enhancing random noise in digital images (Davidson, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 189

210 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING Figure 7.7 Kernel size effects on smoothing convolution operations (from Davidson, 2007). The kernel size can be adjusted to optimize the effects of sharpening filters and to fine-tune the masks to operate on a specific range of spatial frequencies. A typical 3 x 3 mask (see Figures 7.5 and 7.6) has the greatest effect on image features that vary over the spacing interval of a single pixel. Doubling or tripling the size of the kernel will target lower spatial frequencies that extend over two or more pixels (Davidson, 2007) MEDIAN FILTERS Median filters are primarily designed to remove image noise, but are also very effective at eliminating faulty pixels (having unusually high or low brightness values) and reducing the deterioration caused by fine scratches. These filters are often more effective at removing noise than smoothing (low pass) convolution kernels. Median kernels are applied in a manner that is different from standard smoothing or sharpening kernels. Although the median filter operates in a local neighborhood that is translated from pixel to pixel, there is no convolution matrix applied. At each successive pixel location, the pixels under scrutiny are ordered in rank according to their intensity magnitude. A median value is then determined for all of the pixels covered by the neighborhood, and that value is assigned to the central pixel location in the output image (Davidson, 2007). Median filters are useful for removing random intensity spikes that often occur in digital images captured in the microscope. Pixels contributing to the spike are replaced with the median value of the local neighborhood pixels, which produces a more uniform appearance in the processed image. Background regions that contain infrequent intensity spikes are rendered in a uniform manner by the median filter. In ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 190

211 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING addition, because the median filter preserves edges, fine specimen detail, and boundaries, it is often employed for processing images having high contrast (Davidson, 2007) SPECIALIZED CONVOLUTION FILTERS Derivative filters provide a quantitative measurement for the rate of change in pixel brightness information present in a digital image. When a derivative filter is applied to a digital image, the resulting data concerning brightness fluctuation rates can be used to enhance contrast, detect edges and boundaries, and to measure feature orientation. One of the most important derivative filters is the Sobel filter, which combines two orthogonal derivatives (produced by 3 x 3 kernel convolutions) to calculate the vector gradient of brightness. These convolutions are very useful for edge enhancement of digital images captured in the microscope. Edges are usually one of the most important features in a microscopic structure, and can often be utilized for measurements after appropriate enhancement algorithms have been applied (Davidson, 2007). Laplacian filters (often termed operators) are employed to calculate the second derivative of intensity with respect to position and are useful for determining whether a pixel resides on the dark or light side of an edge. The Laplacian enhancement operation generates sharp peaks at the edges, and any brightness slope, regardless of whether it is positive or negative, is accentuated, bestowing an omnidirectional quality to this filter. It is interesting to note that in the human visual system, the eye-brain network applies a Laplacian-style enhancement to every object in the viewfield. Human vision can be simulated by applying a Laplacian-enhanced image to the original image, using a dual-image point process, to produce a modified image that appears much sharper and more pleasing (Davidson, 2007). An important issue that arises within the convolution process methodology centers on the fact that the convolution kernel will extend beyond the borders of the image when it is applied to border pixels. One technique commonly utilized to remedy this problem, referred to as centered, zero boundary superposition, is simply to ignore the problematic pixels and to perform the convolution operation only on those pixels ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 191

212 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING that are located at a sufficient distance from the borders. This method has the disadvantage of producing an output image that is smaller than the input image. A second technique, called centered, zero padded superposition, involves padding the missing pixels with zeroes. Yet a third technique regards the image as a single element in a tiled array of identical images, so that the missing pixels are taken from the opposite side of the image. This method is called centered, reflected boundary superposition and has the advantage of allowing for the use of modulo arithmetic in the calculation of pixel addresses to eliminate the need for considering border pixels as a special case. Each of these techniques is useful for specific image-processing applications. The zero padded and reflected boundary methods are commonly applied to image enhancement filtering techniques, while the zero boundary method is often utilized in edge detection and in the computation of spatial derivatives (Davidson, 2007) UNSHARP MASK FILTERING Unsharp mask algorithms operate by subtraction of a blurred image from the original image, followed by adjustment of gray level values in the difference image. This operation enables preservation of high-frequency detail while allowing shading correction and background suppression. The popular technique is an excellent vehicle to enhance fine specimen detail and sharpen edges that are not clearly defined in the original image. The first step in an unsharp mask process is to produce a slight blur (by passage through a Gaussian low-pass filter) and a reduction in amplitude of the original image, which is then subtracted from the unmodified original to produce a sharpened image. Regions in the image that have uniform amplitude are rendered in a medium gray brightness level, whereas regions with larger slopes (edges and boundaries) appear as lighter or darker gradients (Davidson, 2007). In general, unsharp mask filters operate by subtracting appropriately weighted segments of the unsharp mask (the blurred original) from the original image. Such a subtraction operation enhances high-frequency spatial detail at the expense (attenuation) of low-frequency spatial information in the image. This effect occurs because high-frequency spatial detail removed from the unsharp mask by the Gaussian ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 192

213 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING filter is not subtracted from the original image. In addition, low-frequency spatial detail that is passed by the Gaussian filter (to the unsharp mask) is almost entirely subtracted from the original image. Increasing the size of the Gaussian filter allows the smoothing operation to remove larger size detail, so that those details are retained in the difference image (Davidson, 2007). One of the primary advantages of the unsharp mask filter over other sharpening filters is the flexibility of control, because a majority of the other filters do not provide any user-adjustable parameters. Like other sharpening filters, the unsharp mask filter enhances edges and fine detail in a digital image. Because sharpening filters also suppress low frequency detail, these filters can be used to correct shading distortion throughout an image that is commonly manifested in the form of slowly varying background intensities. Unfortunately, sharpening filters also have the undesirable side effect of increasing noise in the filtered image. For this reason, the unsharp mask filter should be used conservatively, and a reasonable balance should always be sought between the enhancement of detail and the propagation of noise (Davidson, 2007) FOURIER TRANSFORMS The Fourier transform is based on the theorem that any harmonic function can be represented by a series of sine and cosine functions, differing only in frequency, amplitude, and phase. These transforms display the frequency and amplitude relationship between the harmonic components of the original functions from which they were derived. The Fourier transform converts a function that varies in space to another function that varies with frequency. It should also be noted that the highest spatial frequencies of the original function are found the farthest away from the origin in the Fourier transform (Davidson, 2007). ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 193

214 CHAPTER VII BASIC CONCEPTS OF DIGITAL IMAGE PROCESSING Figure 7.8 Fourier transform filtering (from Davidson, 2007). Spatial filtering involving Fourier techniques can be utilized to manipulate images through deletion of high or low spatial-frequency information from an image by designing a Fourier filter that is nontransmitting at the appropriate frequency. This technique is especially useful for removing harmonic noise from an image such as the herringbone or sawtooth patterns often apparent in video images (see Figure 7.8). Because the added noise is harmonic, it will be found in localized discrete regions of the Fourier transform. When these local peaks are removed from the transform with the appropriate filter, the re-formed image is essentially unaltered except that the offending pattern is absent. Similar filtering techniques can also be applied to remove sine wave, moiré, halftone, and interference patterns, as well as noise from video signals, CCDs, power supplies, and electromagnetic induction (Davidson, 2007). Illustrated in Figure 7.8(a) is a video image of a diatom frustule imaged in darkfield illumination with a superimposed sawtooth interference pattern. Adjacent to the diatom image (Figure 7.8(b)) is the Fourier transform power spectrum for the image, which contains the spatial frequency information. After applying several filters (Figure 7.8(d)) and re-forming the image, the sawtooth pattern has been effectively eliminated (Figure 7.8(c)), leaving only the image of the frustules (Davidson, 2007). The decision as to whether to utilize Fourier filtering or convolution kernel masks depends on the application being considered. The Fourier transform is an ANALYSIS OF STRUCTURES IN BIOMEDICAL IMAGES 194

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