Cell Cycle Regulation/Apoptosis

Cell Cycle Regulation
Apoptosis

One of the reasons that people are so interested in the regulation of mammalian cell division is that it’s widely believed that cancer is a malfunction of the normal controls on cell division. We’ll talk about this in more detail later, but I want briefly mention the kinds of proteins that are defective in cancer cells. One kind we’ve already discussed--the so-called oncogenes that encode proteins in cell signaling pathways. It’s thought that mutations in these proteins cause cancer by permanently activating some signaling protein that normally is tightly regulated; that is, a protein that normally exists in both active and inactive states is mutated into the permanently active conformation.. One example of this is the Ras oncogene, which can mutate so that it does not hydrolyze GTP, and thus constantly activates the MAP kinase pathway. Many other proteins in this pathway have been found to be defective in cancer cells.
However, as you might guess from our discussion of yeast and Far1, there are also proteins in mammalian cells that are inhibitors of cell division. If these are mutated so that they become inactive, then a brake on division is removed and the cell can enter the cell cycle. Such a protein was found to be mutated in people who developed retinoblastoma, a tumor of the eye. This disease has a hereditary form that runs in families, and so it was possible to use this clue and the techniques of molecular genetics to identify the gene and its protein product. The Rb gene has been called a tumor suppressor, because in its normal, active form it prevents cell division. It does this by binding to a regulatory protein, called E2F, that is necessary for transcription of some of the genes required for cell division (see Fig. 13-31 in Lodish et al.). When growth factors are present, the Rb protein is phosphorylated, by a cyclin/CDK complex, and the phosphorylated form is inactive. Thus it unbinds from the transcription factor, allow transcription to proceed, and producing the proteins necessary for cell division. So in normal cells the Rb protein and probably several others like it, are part of the mechanism that allows the cell to divide when appropriate growth factors are present, but that prevent division otherwise. When the Rb gene is mutated, it apparently can no longer bind to and inhibit the regulatory factor, even when it is not phosphorylated, so the transcription of cell division genes proceeds unimpeded, and the cell divides constantly, the mark of cancer.
There are in fact a number of places during the cell cycle at which the cell must "take stock" of itself, and assess whether it is prepared, in a sense, to proceed to the next stage. These are referred to as "checkpoints", and they occur at the end of G1 before the transition to S phase, at the end of S before transition to G2, in G2 before the start of mitosis (M), and again in M. We've already discussed what is known about the last of these checkpoints, which is mediated by chromosomal alignment on the mitotic spindle, involving Bub and Mad proteins. Until all chromosomes align, the APC is inactive, and the cell doesn't enter anaphase. A second checkpoint in S phase is sensitive to chromosome replication. Failure to completely reproduce the DNA somehow leads to a arrest of the cell cycle in S phase. The other two checkpoints in G1 and G2 screen for major chromosomal damage of the type produced by ultraviolet or X-ray radiation. In both places a crucial component of the cell's self-examination machinery is a protein called p53, because it has a molecular weight of about 53 kD. A variety of experiments, initially done by Bert Vogelstein and collaborators at Johns Hopkins, originally identified p53 because it was often mutated in cancerous cells. Subsequently, it has been shown that p53 is a crucial inhibitor of the cell cycle in DNA-damaged cells. As shown in Fig. 13-35, cells with wild-type p53 that are damaged by gamma irradiation (similar to X-rays) arrest in G1 or G2; essentially none are found in S (or M). Cells with two copies of a mutant p53 gene, however, continue to move through G1 into S (and through G2 into M). It appears that p53 is a transcription factor, that activates transcription of a number of genes that are involved both in inhibiting the cell cycle and in induction of cell death, or apoptosis. One of these proteins is called p21^cip (which means that it has a molecular weight of 21 kD (but there are lots of proteins with that MW), and that it is a cyclin inhibitor protein. P53 is known to be a very unstable protein that is constantly degraded by proteases, and thus is normally at low levels in the cell. In a way not yet understood, chromosomal damage diminishes the rate of degradation of p53, allowing it to accumulate. It then stimulates the transcription of p21^cip, which acts as an inhibitor of all the human cyclin/cdk complexes, thus preventing movement of the cell out of G1 or G2. The interest in p53 is generated by the observation that in over half of all human cancers, both p53 genes are mutated; this implies that this protein is necessary to eliminate defective cells and prevent them from dividing out of control.
I want to switch gears slightly now from cell division (or cell reproduction) to cell death, though I hope to convince you that these are in effect two sides of the same coin. Cells die in two ways--if they are injured or diseased (e.g., infected) they undergo a messy process of death called necrosis, which usually involves disruption of the cells, spillage of their contents, and then a complex reaction called inflammation which brings phagocytic cells to an area, causes redness and swelling, etc. For a long time people thought that all cells died this way as a result of injury or other problem, but about 1970 it was suggested that some cells can self destruct in a controlled way without inducing an inflammation. There's now plenty of evidence that this is in fact a common and normal feature of all multicellular organisms, which is called programmed cell death, naturally occurring cell death (to distinguish it from artificially induced cell death), or formally, apoptosis. Cells can die in this way either because they are deprived of something they need, because are induced to do so by an extracellular signal, or because something is seriously wrong with them. Most importantly, the cell itself is an active participant in its own destruction, which involves activation of a variety of protein and nucleic acid degrading enzymes, which cause degradation of essential proteins, fragmentation of DNA, and a series of internal morphological changes that lead to disintegration of the cell and quiet gobbling up of the resultant fragments by phagocytic cells, without inflammation. (See Fig. 23-45).
A great deal of information about the molecular basis of apoptosis has been learned from studies on the roundworm, Caenorhabditis elegans, that has only about 1000 cells, a couple of hundred of which die off by apoptosis during development. Mutants that had excessive or reduced cell death were isolated and characterized, allowing identification of a group of enzymes, called caspases (which is a contraction of cysteine-dependent, aspartate specific enzyme, because they cleave other proteins at aspartate residue, using an active site that contains cysteine) that are involved in apoptosis. People who were studying cancer cells also identified a group of proteins that were often mutant in cancers and that were important internal regulators of cell death.
A summary of the current model of the cell death pathways is shown in Fir. 23-50. This is the situation when a cell requires the presence of a growth factor (or "trophic=feeding factor"). The crucial step in activation of the cell death program is activation of a protein called caspase 3 which then sets in motion a series of irreversible, ultimately fatal reactions. For a cell to survive and prosper then, caspase 3 must be inactive. It is made as an inactive precursor protein that only becomes active when it is split by another caspase, caspase 9. Thus, to keep caspase 3 from being active, caspase 9 (and a related protein called caspase 8) must be inactive. In a healthy cell, caspase 9 is essentially pinned to the surface of a mitochondrial cell by a protein complex; in an unhealthy cell the protein is released and can cleave and activate caspase 3. As Fig. 23-50 shows a variety of proteins are involved in this complex process. Two are integral proteins of the outer mitochondrial membrane called Bcl-2 and Bcl-xl (which stands for B cell lymphoma mutant #2, and B cell lymphoma mutant, extra long, respectively; they were originally identified as proteins that are mutated in certain blood cancers called lymphomas). These proteins, when activated by binding to a related protein called Bad, deactivate a protein called Apaf-1, by releasing the cytochrome C protein (which is normally involved in the electron transport pathway). Cytochrome C binds Apaf-1, Apaf-1 lets go of caspase 9, caspase 9 chops caspase 3 in half to activate it, and the cell is as good as dead. In addition the Bcl-2/Bcl-xl complex activates an ion channel in the mitochondrial outer membrane, allowing the influx of ions, followed by water, which causes the mitochondria to swell and disintegrate, depriving the cell of the ATP it requires. The activity of the Bad protein is regulated by growth or trophic factors that act through receptor tyrosine kinases, that can activate the PI-3 kinase pathway. This in turn activates the protein kinase called Protein kinase B (PKB) or Akt, and that phosphorylates Bad, which allows Bad to be sequestered away from mitochondria, thus blocking activation of caspase 9. Thus a delicate balance between apoptosis-inducing molecules and apoptosis-inhibiting molecules keeps the cell alive most of the time, but sometimes tips the scales toward death.
In other words, many cells are kept alive by a continuous supply of an essential signaling molecule (nerve growth factor is used as an example by Lodish et al., but there are many such proteins); if the cell is deprived of the factor, it essentially self-destructs.
A second way the apoptotic pathway can be activated is not by absence of a needed factor, but by the presence of an external signal that says essentially "kill yourself". An example of such a signal is called Fas ligand (FasL) because it binds to a receptor protein that was first identified as an oncogene (that is, mutations in this protein caused cancer) and named Fas. Activation of this cellular pathway seems to cause a signal cascade in the cell leading to activation of caspase 8, which can also cleave caspase 3 into an active form. In addition certain kinds of cellular stress can be so severe, that they disrupt the endoplasmic reticulum and lead to excessive release of calcium, which activates caspase 12, a protein that can also cleave, and activate, caspase 3. Thus, absence of a necessary factor, presence of a nasty factor, or stress can all activate apoptosis.
Finally, another stimulus that can cause apoptosis is p53. If a cell has active p53, which leads to inhibition of its cyclin/cdks, it is prevented from proceeding through the cell cycle until it can repair whatever problem caused stabilization of the p53. But the cell has only a limited time to do that. After a few hours, if the problem is not repaired, p53 can activate the apoptotic pathway, and cause the cell to commit suicide rather than reproduce and cause harm to the organism.
I hope you can see the overarching logic of all this. In order to survive an organism must keep all it's parts operating in a balanced way. Cells that are stressed so that they can't function, that need to be removed for proper development (such as removal of the tail of tadpole so it can metomorphose into a frog), that can't be sustained because of insufficient levels of trophic factors, or that are defective and may harm the organism if they replicate, are a hazard to this balance. Thus, there is an active apoptotic pathway to remove such cells with minimal damage to the organism, in order that the remaining cells to survive and thrive.