34 Apoptosis Programmed cell death is vital to the health and development of multicellular organisms.

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1 Principles of Biology contents 34 Apoptosis Programmed cell death is vital to the health and development of multicellular organisms. Apoptosis is the reason we have separate fingers and toes. During embryonic development, the cells between the fingers and toes go through apoptosis, or programmed cell death. Picture Partners/Science Source. Topics Covered in this Module Programmed Cell Death Major Objectives of this Module Explain the adaptive value of programmed cell death. Give examples of intrinsic and extrinsic triggers for apoptosis. Describe how apoptosis proceeds. page 180 of pages left in this module of biology / /1

2 Principles of Biology contents 34 Apoptosis What is the difference between a duck's foot and a chicken's foot? A duck's foot is webbed and the chicken's foot is not the difference is due to apoptosis. The spaces between the toes of a chicken's foot (and the fingers and toes of other vertebrates) form during early development because the cells that exist between the digits die. Apoptosis is a form of programmed cell death. This type of cell death is the result of a cellular "self destruct" mechanism that is initiated when the cell responds to specific signals, leading to the activation of cell destroying enzymes within the cell. Ducks have webbed feet because the cells between their toes do not undergo apoptosis. A protein called bone morphogenetic protein 4 (BMP4) is the signal for the excess cells around the toes to undergo apoptosis. Both chicken and duck embryos express BMP4 in the space between the toes. However, duck embryos also express the protein Gremlin between their toes, whereas chicken embryos do not. Gremlin binds to BMP4, inhibiting its apoptosis inducing action, ensuring that ducks have webbed feet. Programmed Cell Death Apoptosis is critically important during development. But apoptosis also continues to be essential throughout the life of an organism. By some estimates, a million cells die in the human body every second. Many of these cells are parts of tissues and organs deep within the body. Yet these dead and dying cells do not induce inflammatory responses or other triggers that would damage the surrounding healthy tissue. This is due, at least in part, to apoptosis. Apoptosis also serves as a means of removing abnormal cells. It is important that the process of cell death be tightly controlled since clearly problems will arise if there is too little or too much cell death. Understanding apoptosis could improve cancer treatment because some tumor cells manage to escape the apoptotic process. Why do cells need to self destruct? Programmed cell death is adaptive for an organism for the following reasons: A cell may no longer be needed by an organism. For instance the webbing between the toes of a chicken, and the tail of a tadpole undergoing metamorphosis into a frog, are cells that are no longer needed. Programmed cell death is how animals regulate the size and structure of the developing nervous system. As cells age they may be exposed to more pathogens or have sustained damage or injury. New cells will take their place, but the old cells must be removed. A cell may be killed to play a non living structural role. An example of this would be the death of the skin cells that become the keratinized top layer of skin of an animal or the water conducting xylem cells of a plant that consist of strong structural cell walls only. In plants, pathogen infected cells "seal themselves off" from the surrounding tissue and then undergo apoptosis, as a means of preventing the spread of the pathogen to the rest of the plant. This is called the hypersensitive response. The term apoptosis was coined to distinguish it from necrosis, which is cell death due to external factors such as toxins, lack of oxygen, lack of nutrients, or some pathological reason. In contrast with apoptosis, necrosis does not involve gene expression or the activation of a controlled, predictable cellular process. Necrosis is the catastrophic loss of cellular homeostasis. A necrotic cell loses membrane integrity and organization, resulting in cellular rupture and death. Cells undergoing apoptosis appear very different from cells undergoing necrosis. As apoptosis begins in animals, the cell detaches itself from the surrounding cells. The chromatin aggregates and components of the nucleus and cytosol condense. The DNA is hydrolyzed by DNases, and parts of the nucleus and cytoplasm partition off into small membrane bound vesicles called apoptotic bodies. The cell membrane fragments by forming blebs, which are membranous lobes. Apoptotic cells and cell fragments are typically taken up by neighboring cells or phagocytes. However, there is no activation of the immune response. Observation of many kinds of multicellular animals has revealed a type of apoptosis with a common morphological appearance, suggesting a common programmed cell death pathway. The hypothesis that the mechanism of programmed cell death was evolutionarily conserved is supported by recent molecular evidence of homologous elements of the apoptosis pathway in animals as different as nematodes, insects and humans. Single celled fungi (yeasts) have several molecular mechanisms of apoptosis that are homologous to those in animals, suggesting that these pathways arose many millions of years ago, before animals evolved as a distinct lineage. Apoptosis signals can arise from many factors. Since the concept of apoptosis was firmly established in 1972, researchers have identified hundreds of genes whose products control and conduct apoptosis. There are two main pathways that lead to the apoptosis of a cell: one is through death receptors on the cell surface (extrinsic pathway), and the other is through the mitochondria (intrinsic pathway) (Figure 1). of biology / /1 1/6

3 Figure 1: Apoptosis can be initiated by two pathways. Apoptosis can be activated through death receptors on the cell surface (also known as the extrinsic pathway) or through mitochondria (also known as the intrinsic pathway). The intrinsic pathway is activated in response to a variety of internal damage signals such as UV radiation damage or the improper folding of proteins Nature Education All rights reserved. Figure Detail The extrinsic pathway is controlled by the signaling molecules that bind to the cell surface death receptors (DRs). The intrinsic pathway is controlled by the Bcl 2 family of proteins and involves the disruption of mitochondrial membranes in response to internal signals of damage. Both pathways eventually activate an enzyme called an initiator caspase. Caspases (cysteine proteases that cleave after an aspartate) are proteolytic enzymes that carry out the cell death response. The initiator caspase activates one or more executioner caspases. Executioner caspases cleave death substrates, which eventually results in apoptosis (see Figure 1). Points of crossover of the two pathways allows for crosstalk between elements of each pathway. For instance, after apoptosis induction through death receptors, the cleavage of one particular Bcl 2 protein (BID) by caspase 8 can activate the intrinsic mitochondrial pathway, amplifying the apoptotic response (see Figure 1). How does apoptosis proceed? Apoptosis starts with a condition that signals the cell to "commit suicide." Such triggers may be a decline in the local concentration of a growth factor, damage to DNA or the cytoskeleton, or infection. These signals, acting through the intrinsic or extrinsic pathways, activate the caspase cascade. The executioner caspases target various cellular proteins, leading to the characteristic morphological events of apoptosis. The cell detaches itself from the surrounding cells. The chromatin aggregates and components of the nucleus and cytosol condense (Figure 2). The DNA is hydrolyzed by DNases, which digest the DNA in regions unprotected by the nucleosomes. Evidence of DNA hydrolysis is seen as a ladder of evenly spaced fragments in gel electrophoresis analysis of DNA extracted from cells at this stage. Parts of the nucleus and cytoplasm partition off into small membranebound vesicles called apoptotic bodies. The cell membrane fragments by forming membranous lobes called blebs. Apoptotic cells and cell fragments are typically taken up by neighboring cells or phagocytes. of biology / /1 2/6

4 Figure 2: How does apoptosis look compared to necrosis? Images of DNA labeled cells in culture showing: a) normal control cells (live cells show green nuclei); b) cells undergoing necrosis after treatment with a toxin (dead cells show non condensed orange nuclei); and c) cells undergoing apoptosis; the white arrowheads point to cells with nuclei that display chromatin aggregation in the early stages of apoptosis, while the yellow arrowheads point to dead cells that display the chromatin clumping typical of apoptosis Nature Publishing Group Bezabeh, T. et al. Detection of drug induced apoptosis and necrosis in human cervical carcinoma cells using 1H NMR spectroscopy. Cell Death and Differentiation 8, (2001) doi: /sj.cdd Used with permission. Figure Detail Apoptotic cells are removed by phagocytosis by either neighboring cells or phagocytic cells like macrophages. Cells undergoing necrosis are engulfed by macrophages, which triggers the inflammatory response of innate immunity. Why does phagocytosis of apoptotic cells by macrophages not result in an immune response? The apoptotic cells attract macrophages with "eat me" signals that are different from the signals that characterize necrotic cells. After phagocytosis of apoptotic cells, instead of up regulating the immune response, these macrophages appear to "calm" the immune response. This calming is likely accomplished by suppression of chemical signals that promote inflammation and cell killing and regulation of immune system activation by inducing changes in how the immune system signals are presented to the rest of the immune system (Figure 3). of biology / /1 3/6

5 Figure 3: Phagocytic clearance of apoptotic cells. Apoptotic cells express proteins that serve as "eat me" flags that promote their phagocytosis by macrophages. Besides serving as waste disposal, this process has acquired new meaning as an immune system modulating process Nature Education All rights reserved. Figure Detail There is a delicate balance of cell death and cell division required for maintaining health. Excessive apoptosis contributes to many pathological conditions, including Alzheimer's disease and Huntington's disease. Deficient apoptosis is linked to many pathological conditions, such as autoimmune diseases and cancer. Therapeutic approaches that affect the apoptotic pathway are currently very active areas of research. Future perspectives. Ongoing work in apoptosis signaling is an avenue for cancer therapies. One of the many proteins that play a role in the development of cancer, the p53 protein is a key player in the cell's response to cellular stresses, such as DNA damage. Following DNA damage, p53 regulates the expression and activity of genes to mediate DNA repair, cell cycle arrest, senescence or apoptosis (Figure 4). Loss of p53 activity is often found in cancerous cells. This enables them to escape apoptosis. Figure 4: The p53 pathway and its role in apoptosis and cancer. The p53 protein is a tumor suppressor protein that regulates the cell cycle and is involved in preventing cancer. Following cellular stress, p53 mediates apoptosis and senescence, which lead to cancer cell clearance. As an adjunct treatment for cancer, reactivation of p53 in cancer cells could help clear cancer cells Nature Publishing Group Vazquez, A., et al. The genetics of the p53 pathway, apoptosis and cancer therapy. Nature Reviews Drug Discovery 7, (2008) doi: /nrd2656. Used with permission. Traditional cancer therapies work by inducing cellular stress, and p53 helps to remove the cancer cells. Enhancement or reactivation of p53 activity in cancer cells has the potential to improve the response to cancer therapies. Other approaches in cancer therapy are the development of pro apoptotic therapies. One promising approach under study is pro apoptotic receptor agonists (PARAs) that target the extrinsic apoptosis pathway. BIOSKILL Interpret a Signaling Pathway Problems with apoptosis underlie many human diseases, such as stroke, heart failure, Alzheimer's disease, Parkinson's disease, cancer and the development of autoimmune diseases. Consequently, ways to enhance or interrupt the pathways identified in apoptosis are actively being sought by researchers. Some of the biochemical targets that are being investigated include Bcl 2, FLIP (a caspase inhibitor) and the caspases. One of the challenges is how to induce apoptosis in one target cell type without affecting non target cells or organs. Figure 5 shows just a small fraction of what is known about apoptotic pathways with potential therapeutic approaches for each drug target (for which potential drug leads are being pursued) highlighted in blue. Ligands, such as the cytokine tumor necrosis factor (TNF), bind to pro apoptotic "death" receptors on the surface of the target cell, inducing the formation of a death inducing signaling complex. Caspase 8/10 is activated, and the intrinsic pathway is also activated via the Bcl 2 family protein BID. Caspase 8/10 is negatively regulated by FLIP, which is of biology / /1 4/6

6 structurally similar to a caspase but lacks enzymatic activity. On the left of Figure 5, a caspase activated DNase (CAD) moves into the nucleus and fragments the cell's DNA in a characteristic pattern defined by the spacing of the nucleosomes. The caspase 8/10 and the Bcl 2 pathways also come together at this point via caspase 9. Figure 5: Apoptosis pathways and drug targets. A small fraction of what is known about the apoptosis pathway is shown here, potential targets for drugs are highlighted in light green Nature Education All rights reserved. Figure Detail Test Yourself Many cancers (for example, lymphoma a white blood cell cancer) over express BCL 2, a protein that inhibits apoptosis. Think of an approach that may lead to an effective therapy. Submit BIOSKILL What are other mechanisms of programmed cell death? Autophagy is another form of programmed cell death in which cells break down their own cytoplasm and organelles. The breakdown products are metabolized within the cell as a source of nutrients during times of limited resources. It is likely that future research will provide more opportunities for understanding these different forms of cell death in both animals and plants. Test Yourself How might researchers distinguish between autophagy and apoptosis? of biology / /1 5/6

7 Submit IN THIS MODULE Programmed Cell Death Summary Test Your Knowledge WHY DOES THIS TOPIC MATTER? Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting edge research lead to prevention and treatment strategies that could make cancer obsolete? PRIMARY LITERATURE Identifying genes linked to cystic fibrosis Genome wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2. Classic paper: X rays reveal the structure of myoglobin (1958) A three dimensional model of the myoglobin molecule obtained by X ray analysis. page 181 of pages left in this module of biology / /1 6/6

8 1/20/2015 Summary of Apoptosis Principles of Biology from Nature Education Principles of Biology contents 34 Apoptosis Summary OBJECTIVE Explain the adaptive value of programmed cell death. Apoptosis is a form of programmed cell death that is used to remove cells that are no longer needed in development or to remove cells that may be damaged or irregular in some way. Defects in apoptosis play a role in many diseases. Studies on the molecular mechanisms of apoptosis regulation have provided several potential targets for future therapies. OBJECTIVE Give examples of intrinsic and extrinsic triggers for apoptosis. Apoptosis can be triggered along either the intrinsic or extrinsic pathway. There are points of convergence between the two pathways. An example of an intrinsic signal is damaged DNA, and an example of an extrinsic signal would be a ligand for the death receptors, such as tumor necrosis factor. Signals acting through the intrinsic or extrinsic pathways activate the caspase cascade. The executioner caspases process various cellular proteins, leading to the characteristic morphological events of apoptosis. OBJECTIVE Describe how apoptosis proceeds. As apoptosis begins in animals, the cell detaches itself from the surrounding cells. The chromatin aggregates, and components of the nucleus and cytosol condense. The DNA is hydrolyzed by DNases, and parts of the nucleus and cytoplasm partition off into small membrane bound vesicles called apoptotic bodies. The cell membrane fragments by forming blebs, which are membranous lobes. Apoptotic cells and cell fragments are typically taken up by neighboring cells or phagocytes. However there is no activation of the immune response. Key Terms apoptosis Programmed cell destruction or death. autophagy Process of breaking down and recycling malfunctioning or worn out cell parts. blebs Membranous lobes created as the cell membrane breaks up in apoptosis. caspase A type of proteolytic enzyme that is involved in initiating and executing programmed cell death. DNA hydrolysis Process by which DNA is broken into small fragments. hypersensitive response Infected cells are sealed off and die so as to protect the rest of the plant. necrosis Cell death caused by toxins, hypoxia, lack of nutrients, or some pathological reason. IN THIS MODULE Programmed Cell Death Summary Test Your Knowledge WHY DOES THIS TOPIC MATTER? Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting edge research lead to prevention and treatment strategies that could make cancer obsolete? PRIMARY LITERATURE Identifying genes linked to cystic fibrosis Genome wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2. Classic paper: X rays reveal the structure of myoglobin (1958) A three dimensional model of the myoglobin molecule obtained by X ray analysis. of biology / /2 1/2

9 1/20/2015 Summary of Apoptosis Principles of Biology from Nature Education page 182 of pages left in this module of biology / /2 2/2

10 Principles of Biology contents 34 Apoptosis IN THIS MODULE Test Your Knowledge 1. Which of the following is NOT an adaptive advantage of apoptosis? A cell may no longer be needed by an organism. A cell may be killed to play a nonliving structural role. to protect the rest of the organism from pathogens activation of the immune response to fight pathogens All answers are correct. Programmed Cell Death Summary Test Your Knowledge WHY DOES THIS TOPIC MATTER? Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting edge research lead to prevention and treatment strategies that could make cancer obsolete? Which of the following scenarios would most likely involve apoptosis? destruction of muscle cells in the muscles (formerly used to move a caterpillar's body) during its metamorphosis to a butterfly killing of skin cells by contact with an extremely hot surface the breakdown of a cell's internal structures (mitochondria, membrane vesicles, etc.) during times of limited resources phagocytosis and killing of an invading bacterium by a macrophage All answers are correct. Which of the following is/are an example of an intrinsic trigger for apoptosis? damaged DNA mitochondrial damage a ligand binding to the death receptor All answers are correct. damaged DNA and mitochondrial damage only PRIMARY LITERATURE Identifying genes linked to cystic fibrosis Genome wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2. Classic paper: X rays reveal the structure of myoglobin (1958) A three dimensional model of the myoglobin molecule obtained by X ray analysis. 4. Which of the following is NOT a step in apoptosis in animal cells? The cytosol leaks out of the cell. The cell membrane forms blebs. The cell detaches itself from the surrounding cells. Caspases digest their target proteins. The DNA is hydrolyzed by DNAses. 5. Which step(s) occur in the apoptosis of both plant and animal cells? Macrophages engulf the cell and trigger an immune response. The DNA is hydrolyzed by DNAses. The cytosol leaks out of the cell. The cell membrane forms blebs. All answers are correct. Submit page 183 of of biology / /3 1/1