Programmed Cell Death (Apoptosis) Apoptosis: from a Greek word meaning "falling off," as leaves from a tree Molecular Biology of Cancer 1 1

What purposes does this massive cell death serve? 2 Molecular Biology of Cancer What purposes does this massive cell death serve? In some cases, the answers are clear Mouse paws, for example, are sculpted by cell death during embryonic development Figure 17-35. Sculpting the digits in the developing mouse paw by apoptosis. (A) The paw in this mouse embryo has been stained with a dye that specifically labels cells that have undergone apoptosis. The apoptotic cells appear as bright green dots between the developing digits. (B) This interdigital cell death eliminates the tissue between the developing digits, as seen one day later, when few, if any, apoptotic cells can be seen. (From W. Wood et al., Development 127:5245 5252, 2000. © The Company of Biologists.) 2

In many other cases, cell death helps regulate cell numbers. 3 Molecular Biology of Cancer In other cases, cells die when the structure they form is no longer needed When a tadpole changes into a frog, the cells in the tail die, and the tail, which is not needed in the frog, disappears In many other cases, cell death helps regulate cell numbers. In the developing nervous system, for example, cell death adjusts the number of nerve cells to match the number of target cells that require innervation. Figure 17-36. Apoptosis during the metamorphosis of a tadpole into a frog. As a tadpole changes into a frog, the cells in the tadpole tail are induced to undergo apoptosis; as a consequence, the tail is lost. All the changes that occur during metamorphosis, including the induction of apoptosis in the tail, are stimulated by an increase in thyroid hormone in the blood. 3

In adult tissues, cell death exactly balances cell division 4 Molecular Biology of Cancer In adult tissues, cell death exactly balances cell division If part of the liver is removed in an adult rat, liver cell proliferation increases to make up the loss. Conversely, if the rat liver cell division is stimulated by phenobarbital treatmnt and then the phenobarbital treatment is stopped liver enlargement occur first apoptosis in the liver greatly increases until the liver has returned to its original size Thus, the liver is kept at a constant size through the regulation of both the cell death rate and the cell birth rate. 4

5 Molecular Biology of Cancer Cell death Cell necrosis: acute injury typically causes the cells to swell and burst. They spill their contents all over their neighbors causing a potentially damaging inflammatory response. Apoptosis: causes cells to die neatly, without damaging its neighbors. The cell shrinks and condenses The cytoskeleton collapses the nuclear envelope disassembles the nuclear DNA breaks up into fragments Most importantly, the cell surface is altered, displaying properties that cause the dying cell to be rapidly phagocytosed, either by a neighboring cell or by a macrophage before any leakage of its contents occurs This not only avoids the damaging consequences of cell necrosis but also allows the organic components of the dead cell to be recycled by the cell that ingests it. 5

Apoptosis is mediated by an intracellular proteolytic cascade 6 Molecular Biology of Cancer Apoptosis is mediated by an intracellular proteolytic cascade Caspases: a family of proteases that have a cysteine at their active site and cleave their target proteins at specific aspartic acids. Caspases are synthesized in the cell as inactive precursors, or procaspases, which are usually activated by cleavage at aspartic acids by other caspases Figure 17-38. The caspase cascade involved in apoptosis. (A) Each suicide protease is made as an inactive proenzyme (procaspase), which is usually activated by proteolytic cleavage by another member of the caspase family. As indicated, two of the cleaved fragments associate to form the active site of the caspase. The active enzyme is thought to be a tetramer of two of these units (not shown). 6

another cleaves a protein that normally holds a DNAse inactive 7 Molecular Biology of Cancer Once activated, caspases cleave, and thereby activate, other procaspases, resulting in an amplifying proteolytic cascade Figure 17-38. The caspase cascade involved in apoptosis. (B) Each activated caspase molecule can cleave many procaspase molecules, thereby activating them, and these can then activate even more procaspase molecules. In this way, an initial activation of a small number of procaspase molecules (called initiator caspases) can lead, via an amplifying chain reaction (a cascade), to the explosive activation of a large number of procaspase molecules. Some of the activated caspases (called effector caspases) then cleave a number of key proteins in the cell, including specific cytosolic proteins and nuclear lamins, leading to the controlled death of the cell Some cleave the nuclear lamins, causing the irreversible breakdown of the nuclear lamina another cleaves a protein that normally holds a DNAse inactive freeing the DNAse to cut up the DNA in the cell nucleus 7

How are procaspases activated to initiate the caspase cascade? 8 Molecular Biology of Cancer How are procaspases activated to initiate the caspase cascade? Adaptor protein Initiator procaspases aggregate Initiator procaspases A general principle is that the activation is triggered by adaptor proteins They bring multiple copies of specific procaspases, known as initiator procaspases, close together in a complex or aggregate. In other cases Conformational change In some Small amount of protease activity they cleave each other Active caspases Active caspases procaspases 8

9 Molecular Biology of Cancer Apoptosis activation from outside the cell by the activation of cell surface death receptors For example: Fas ligand on the surface of killer lymphocytes and the death receptor protein Fas on the surface of the target cell Some stressed or damaged cells kill themselves by producing both the Fas ligand and the Fas protein Figure 17-39. Induction of apoptosis by either extracellular or intracellular stimuli. (A) Extracellular activation. A killer lymphocyte carrying the Fas ligand binds and activates Fas proteins on the surface of the target cell. Adaptor proteins bind to the intracellular region of aggregated Fas proteins, causing the aggregation of procaspase-8 molecules. These then cleave one another to initiate the caspase cascade. 9

Apoptosis activation from inside the damaged or stressed cells 10 Molecular Biology of Cancer Apoptosis activation from inside the damaged or stressed cells Mitochondria are induced to release the electron carrier protein cytochrome c into the cytosol It binds and activates an adaptor protein called Apaf-1 10

11 Molecular Biology of Cancer Figure 17-39. Induction of apoptosis by either extracellular or intracellular stimuli. (B) Intracellular activation. Mitochondria release cytochrome c, which binds to and causes the aggregation of the adaptor protein Apaf-1. Apaf-1 binds and aggregates procaspase-9 molecules, which leads to the cleavage of these molecules and the triggering of a caspase cascade. Other proteins that contribute to apoptosis are also released from the mitochondrial intermembrane space (not shown). p53 usually can activate the transcription of genes (the Bcl-2 family) whose proteins promote the release of cytochrome c from mitochondria. 11

12 Molecular Biology of Cancer The Bcl-2 family of intracellular proteins helps regulate the activation of procaspases. Bcl-2 Bcl-XL Bad Bax Bid Bak Some members of this family, like Bcl-2 itself or Bcl-X L inhibit apoptosis at least partly by blocking the release of cytochrome c from mitochondria. Other members of the Bcl-2 family are not death inhibitors, but instead promote procaspase activation and cell death. Some of these apoptosis promoters, such as Bad, function by binding to and inactivating the death-inhibiting members of the family, whereas others, like Bax and Bak, stimulate the release of cytochrome c from mitochondria. If the genes encoding Bax and Bak are both inactivated, cells are remarkably resistant to most apoptosis-inducing stimuli, indicating the crucial importance of these proteins in apoptosis induction. Bax and Bak are themselves activated by other apoptosis-promoting members of the Bcl-2 family such as Bid. 12