For a complete list of defined terms, see the Glossary. Transformation the process by which a cell acquires characteristics of a tumor cell. LESSON 3.2 WORKBOOK How do normal cells become cancer cells? The key factor that determines whether a normal cell will become a tumor is the kind of mutations it acquires. Most mutations do not affect cell function, and those that do usually lead to cell death. However, a minority of mutations can both affect cell function and allow the cell to live. Of those mutations, only a minority leads to cancer. In this lesson, we will explore how cells acquire random mutations and how carcinogens increase the chance that very rare cancer-causing mutations will accumulate. DNA mutations and tumor formation In Unit 2, we learned how critical it is for cells in tissues to work together as a community, and for tissue communities to work together to regulate organ function. Tumor formation occurs when cells lose their ability to participate as members of a tissue community, and this may eventually disrupt organ function too. The key change in cellular behavior that precipitates this loss of function occurs when cells accumulate mutations in their DNA that modify critical proteins. This change in a normal cell s DNA so that the cell forms a tumor is called transformation. DNA acquires random mutations for a variety of reasons, but the most common include: DNA polymerase errors the DNA polymerase enzyme duplicates DNA during S phase as the cell prepares for mitosis. DNA polymerase makes a mistake once in every 10 million bases it copies. Mistakes during mitosis for mitosis to occur cleanly chromosomes must be divided equally between the daughter cells. This does not always occur. Effects of environmental agents environmental agents called mutagens can modify the chemical structure of the DNA bases themselves, or promote errors DNA polymerase and/or mitosis. Most mutagens are also carcinogens, but not every carcinogen is a mutagen. 1. Transformation describes which of the following types of cell? A cell that has acquired mutations A cell acquiring spreading traits A cell that can replicate indefinitely A cell entering the blood stream 88
Mutagen any chemical or agent that is capable of mutating DNA sequence. Somatic cell any cell that forms the body of an organism that is not a germ cell. Germline mutation any detectable mutation or variation of DNA present within germ cells that is inherited by offspring of that individual. BRCA1 a tumor suppressor gene involved in DNA repair, whose mutated form is associated with breast and ovarian cancer as well as other cancers. BRCA2 another tumor suppressor gene involved in DNA repair, whose mutated form is associated with breast and ovarian cancer as well as other cancers. As we know, there are two types of cell in the body: Germ cells (eggs and sperm) and somatic cells. Germ cells contain one copy of the genome on 23 chromosomes, while somatic cells contain two copies of the genome on 23 pairs of chromosomes. When germ cells fuse they form a zygote with two copies the genome on 23 pairs of chromosomes. Whatever mutations the germ cells have acquired will therefore be inherited by the zygote, so that each somatic cell in the offspring will also Figure 1: Pictures of normal epithelial cells of the mammary have the mutation. Inheritable duct (left) compared to transformed epithelial cells of the mutations like this are called mammary duct (right). Normal cells are generally more structured and ordered within the tissue compared to transformed germline mutations. If the germline mutations have the cells. potential to cause the cell to transform into a tumor they can genetically predispose individuals that have them to develop cancer. One example of a germline mutation that plays a critical role in predisposition to a number of different cancers occurs to the tumor suppressor protein BRCA1 that is involved in DNA repair Mutated forms of BRCA1, which stands for BReast CAncer susceptibility protein, have been conclusively linked to predisposition to developing breast cancer. When mutations to BRCA1 are seen together with mutations to another tumor suppressor protein, BRCA2 the likelihood of developing breast cancer by the age of 70 increases to 50-65% while the likelihood of developing ovarian cancer increased to 35-46%. Mutations to BRCA2 alone increase the likelihood of developing breast cancer to 40-57% and ovarian cancer to 13-23%. Mutations in BRCA1/2 are also risk factors for colon, prostate, and pancreatic cancer. Unlike germline mutations that are found in eggs and sperm and therefore inherited by every zygote produced when the germ cells fuse, somatic mutations are found in somatic, not germ cells. As a result they will only affect the individual who acquired the mutation, but will not be inherited by their offspring. Somatic mutations also only affect the cell that acquired that mutation, and no other cell in the body. Somatic mutations that lead to tumor formation and hence cancer are therefore found only in those tumor/ cancer cells and not throughout the body. 2. Which of the following can lead to cell transformation? (Circle all correct.) DNA polymerase errors. Mistakes of mitosis. Exposure to mutagens. Exposure to carcinogens. 3. Which of the following is true of BRCA1? (Circle all correct.) It is a tumor suppressor gene. It is mostly active in breast and ovarian cancers. Mutations in the gene cannot be passed to offspring. Mutations in the gene increase cancer risk. 89
Somatic mutation a change in DNA sequence of a cell that is not inherited by the offspring. Neutral mutations mutations that do not affect the ability of a cell to function or survive. These include any mutation in a noncoding sequence, or synonymous DNA mutations that do not affect protein sequence. Synonymous mutations mutations within a protein coding sequence that do not affect the amino acid sequence. Nonsynonymous mutations mutations within a protein coding sequence that alter the amino acid sequence. Types of DNA mutations Mutations occurring during DNA replication and mitosis Mutations caused by the errors that occur during DNA replication or mitosis are limited to cells that are actively dividing. Most cells that are terminally differentiated are no longer dividing, and are therefore protected from these kinds of errors. However, stem cells and progenitor cells that are still dividing are capable of developing mutations each time DNA is replicate Normally, DNA polymerase is very accurate and the DNA repair proteins are vigilant to identify mistakes of DNA replication. As a result, the normal mutation rate is only approximately 175 mutations total per duplication of the genome. Most often, cells that accumulate mutations will die, because they prevent the cell from being a productive member of the cell community. Most of the remaining mutations will have no observable effect on the cell. These neutral mutations typically occur in sequences that do not encode proteins, or, if they do occur in coding sequences do not alter the protein sequence. These so-called synonymous mutations are possible because each amino acid has more than one codon. Most mutations a cell acquires will be neutral: Only 2% of the genome encodes Figure 2: A gene fusion is formed when the ends of chromosome 9 and chromosome 22 recombine, forming a longer chromosome 9 and shorter chromosome 22. This forms the bcr-abl gene fusion, which is a hyperactive form of two proto-oncogenes. proteins, so the chances of a mutation changing amino acid sequence (so-called nonsynonymous mutations) and affecting cell behavior are very low. Moreover, the types of acceptable errors in DNA replication is very limite Hence development of mutations In a cell is a very slow and rare process. However, if a random mutation compromises cell cycle control mechanisms (e.g. DNA repair proteins, Rb, or p53), then the cell will begin to divide rapidly, thereby increasing the number of mutations that the cell can accumulate. In fact, the mutations accumulated in tumors can increase by as much as 100,000 mutations per cell compared to normal cells. Errors in mitosis are also relatively rare in normal cells, but these also will increase if cell cycle control mechanisms are damage These errors of mitosis may lead to chromosomes inappropriately mixing together, a process called chromosome recombination. When chromosomes recombine, they may 4. Which of the following is the MAIN difference between germline and somatic mutations? Germline mutations cannot affect gene expression. Germline mutations do not cause transformation. Somatic mutations cannot be inherite Somatic mutations cannot alter sequence of tumor suppressor genes. 5. True or False: Most mutations that occur in a cell cause cell death. True. False. 90
Chromosome recombination the process by which portions of different chromosomes are mixed together. Gene fusion a mutant gene that is formed by two genes that were previously separate were joined through chromosome recombination. Point mutation a change in a single nucleotide of a DNA sequence. Single nucleotide polymorphism (SNP) a type of mutation where only a single nucleotide is altered either inserted, deleted, or substitute create new types of genes by merging portions of two genes together into one gene. A notable example of this process, called a gene fusion, involves chromosomes 9 and chromosome 22 swapping portions of their chromosomes, producing a fusion of two proto-oncogenes bcr and abl. This fusion (bcr-abl) is responsible for causing a specific type of leukemia, as well as being involved in the development of many other types of cancer. Mutations caused by agents in the environment While mutations caused by DNA replication and mitosis are limited to dividing cells, environmental mutagens can cause mutations in any cells that are exposed to them (i.e. both dividing and terminally differentiated cells). While both replicating and non-replicating (terminally differentiated) cells are susceptible, replicating cells are particularly vulnerable since environmental mutagens may also promote errors of DNA replication and mitosis. Most exposed surfaces of our body are composed of terminally differentiated epithelial cells; if these cells Figure 3: Ultimate carcinogens can chemically bind to DNA bases. This affects how DNA polymerase detects the sequence, and leads to mutation of DNA. are mutated by mutagens, they are easily killed and replaced by the epithelial progenitor and stem cells buried just beneath them deeper in the tissue. Stem and progenitor cells are harder to replace if they are mutated, so mutagens that can penetrate deeper into the tissue can have more profound effects in causing cancer. Perhaps the most common chemical mutations are stimulated by the reactive oxygen species (ROS) that are produced when oxygen is metabolized in cells. When DNA bases such as guanidine (G) are exposed to ROS they undergo a chemical reaction called oxidation, which produces 8-oxo-guanine. DNA repair proteins incorrectly identify this base as a thymidine nucleotide and convert the G to a T. This type of mutation in one nucleotide is called a point mutation. Another name for a point mutation is a single nucleotide polymorphism (SNP). SNPs are any single base mutation such as when a single base is added, removed, or substituted in a DNA sequence. UV radiation causes SNPs by chemically linking thymidines together. DNA repair proteins then replace these thymidines with an adenine nucleotide. Carcinogens, such as those found in tobacco, can bind DNA and cause damage (see Figure 3). Modification of DNA sequences by carcinogens leads to errors in DNA repair, which may make the cell more susceptible to more mutations, particularly if that mutation occurs in a DNA repair protein. 6. If random mutations are rare, why do cancer cells typically have thousands of mutations? Cancer cells are exposed to more carcinogens. Epithelial cells have less efficient DNA repair proteins. Most mutations are neutral mutations. One key mutation leads to accumulation of many mutations. 7. How does carcinogen exposure lead to DNA mutations? (Circle all correct.) DNA repair proteins cannot repair chemically altered nucleotides. DNA polymerase cannot recognize chemically altered nucleotides. Carcinogens chemically modify nucleotides. Carcinogens bind to DNA polymerase. 91
Effects of DNA mutations on gene function In many cases, the types of mutations a cell must acquire to become transformed are very specific and very rare. The random DNA mutations due to carcinogens or errors of DNA replication or mitosis are rarely preserved in a cell, and the affected cells are usually killed through apoptosis Firstly, the mutations must only occur in a subset of genes and must be just the right types of mutations. In previous lessons we have discussed how mutations of proto-oncogenes to form oncogenes and of tumor suppressor are necessary for cellular transformation to occur. Furthermore, they must be just the right type of mutations i.e. the mutations must hyperactivate proto-oncogenes and inactivate the tumor suppressor genes. Mutations that inactivate proto-oncogenes or hyperactivate tumor suppressor genes will not lead to cancer, and will most likely lead to cell death. However, if the cell has acquired just the right combination of mutations in tumor suppressor genes and/ or proto-oncogenes, it may be able to avoid death by apoptosis. Accumulation of DNA mutations that cause a normal cell to become a tumor is just the first step of many physiological changes a transformed tumor cell has to make on its path to becoming a cancer cell. One relevant DNA mutation is not enough to cause cell transformation, rather it has been estimated that a minimum of 3-6 key DNA mutations are necessary. It is important to realize that while all these mutations are required, cells do not all acquire them in the same order, and differences between the order in which key mutations are acquired can lead to key differences between individual tumors and cancers, such as how quickly they can spread from the primary site to different tissues. 8. Which of the following mistakes are caused by carcinogens? (Circle all correct.) Gene fusions. Germline mutations. Point mutations. Somatic mutations. 9. Which of the following is a type of mutation that will promote transformation? (Circle all correct.) Hyperactivation of protooncogene. Inactivation of proto-oncogene. Hyperactivation of tumor suppressor gene. Inactivation of tumor suppressor gene. 92
STUDENT RESPONSES Describe 2-3 types of DNA mutations and explain why most DNA mutations result in death of the cell. Remember to identify your sources 93
TERMS TERM DEFINITION BRCA1 A tumor suppressor gene involved in DNA repair, whose mutated form is associated with breast and ovarian cancer as well as other cancers. BRCA2 Another tumor suppressor gene involved in DNA repair, whose mutated form is associated with breast and ovarian cancer as well as other cancers. DEFINITIONS OF TERMS Chromosome recombination The process by which portions of chromosomes are mixed together forming variants of sequence within each chromosome. For a complete list of defined terms, see the Glossary. Gene fusion Germline mutation A mutant gene that is formed by two genes that were previously separate were joined through chromosome recombination. Any detectable mutation or variation of DNA present within germ cells that is inherited by offspring of that individual. Mutagen Any chemical or agent that is capable of mutating DNA sequence. Neutral mutations Mutations that do not affect the ability of a cell to function or survive. Nonsynonymous mutations Mutations within a protein coding sequence that alter the amino acid sequence. Point mutation A change in a single nucleotide of a DNA sequence. Single nucleotide polymorphism (SNP) A type of mutation where only a single nucleotide is altered either inserted, deleted, or substitute Somatic cell Any cell that forms the body of an organism that is not a germ cell. Somatic mutation A change in DNA sequence of a cell that is not inherited by the offspring. Synonymous mutations Mutations within a protein coding sequence that do not affect the amino acid sequence. Transformation The process by which a cell acquires characteristic of a cancer cell. 94