Chapter 17:  The Cell Cycle and Programmed Cell Death

The Cell-Cycle Control System Is Similar in All Eucaryotes

Some features of the cell cycle, including the time required to complete certain events, vary greatly from one cell type to another, even in the same organism. The basic organization of the cycle and its control system, however, are essentially the same in all eucaryotic cells. The proteins of the control system first appeared over a billion years ago. Remarkably, they have been so well conserved over the course of evolution that many of them function perfectly when transferred from a human cell to a yeast cell. We can therefore study the cell cycle and its regulation in a variety of organisms and use the findings from all of them to assemble a unified picture of how eucaryotic cells divide. In the following section, we briefly review the three eucaryotic systems in which cell-cycle control is commonly studied—yeasts, frog embryos, and cultured mammalian cells.

The Cell-Cycle Control System Can Be Dissected Genetically in Yeasts

Yeasts are tiny, single-celled fungi whose mechanisms of cell-cycle control are remarkably similar to our own. Two species are generally used in studies of the cell cycle. The fission yeast Schizosaccharomyces pombe is named after the African beer it is used to produce. It is a rod-shaped cell that grows by elongation at its ends. Division occurs by the formation of a septum, or cell plate, in the center of the rod (Figure 17-4A). The budding yeast Saccharomyces cerevisiae is used by brewers, as well as by bakers. It is an oval cell that divides by forming a bud, which first appears during G₁ and grows steadily until it separates from the mother cell after mitosis (Figure 17-4B).

Despite their outward differences, the two yeast species share a number of features that are extremely useful for genetic studies. They reproduce almost as rapidly as bacteria and have a genome size less than 1% that of a mammal. They are amenable to rapid molecular genetic manipulation, whereby genes can be deleted, replaced, or altered. Most importantly, they have the unusual ability to proliferate in a haploid state, in which only a single copy of each gene is present in the cell. When cells are haploid, it is easy to isolate and study mutations that inactivate a gene, as one avoids the complication of having a second copy of the gene in the cell.

Many important discoveries about cell-cycle control have come from systematic searches for mutations in yeasts that inactivate genes encoding essential components of the cell-cycle control system. The genes affected by these mutations are known as cell-division-cycle genes, or cdc genes. Many of these mutations cause cells to arrest at a specific point in the cell cycle, suggesting that the normal gene product is required to get the cell past this point.

A mutant that cannot complete the cell cycle cannot be propagated. Thus, cdc mutants can be selected and maintained only if their phenotype is conditional—that is, if the gene product fails to function only in certain specific conditions. Most conditional cell-cycle mutations are temperature-sensitive mutations, in which the mutant protein fails to function at high temperatures but functions well enough to allow cell division at low temperatures. A temperature-sensitive cdc mutant can be propagated at a low temperature (the permissive condition) and then raised to a higher temperature (the restrictive condition) to switch off the function of the mutant gene. At the higher temperature, the cells continue through the cell cycle until they reach the point where the function of the mutant gene is required for further progress, and at this point they halt (Figure 17-5). In budding yeasts, a uniform cell-cycle arrest of this type can be detected by just looking at the cells: the presence or absence of a bud, and bud size, indicate the point in the cycle at which the mutant is arrested (Figure 17-6).

The Cell-Cycle Control System Can Be Analyzed Biochemically in Animal Embryos

While yeasts are ideal for studying the genetics of the cell cycle, the biochemistry of the cycle is most easily analyzed in the giant fertilized eggs of many animals, which carry large stockpiles of the proteins needed for cell division. The egg of the frog Xenopus, for example, is over 1 mm in diameter and carries 100,000 times more cytoplasm than an average cell in the human body (Figure 17-7). Fertilization of the Xenopus egg triggers an astonishingly rapid sequence of cell divisions, called cleavage divisions, in which the single giant cell divides, without growing, to generate an embryo containing thousands of smaller cells (Figure 17-8). In this process, almost the only macromolecules synthesized are DNA—required to produce the thousands of new nuclei—and a small amount of protein. After a first division that takes about 90 minutes, the next 11 divisions occur, more or less synchronously, at 30-minute intervals, producing about 4096 (2¹²) cells within 7 hours. Each cycle is divided into S and M phases of about 15 minutes each, without detectable G₁ or G₂ phases.

The cells in early embryos of Xenopus, as well as those of the clam Spisula and the fruit fly Drosophila, are thus capable of exceedingly rapid division in the absence of either growth or many of the control mechanisms that operate in more complex cell cycles. These early embryonic cell cycles therefore reveal the workings of the cell-cycle control system stripped down and simplified to the minimum needed to achieve the most fundamental requirements—the duplication of the genome and its segregation into two daughter cells. Another advantage of these early embryos for cell-cycle analysis is their large size. It is relatively easy to inject test substances into an egg to determine their effect on cell-cycle progression. It is also possible to prepare almost pure cytoplasm from Xenopus eggs and reconstitute many events of the cell cycle in a test tube (Figure 17-9). In such cell extracts, one can observe and manipulate cell-cycle events under highly simplified and controllable conditions.

The Cell-Cycle Control System of Mammals Can Be Studied in Culture

It is not easy to observe individual cells in an intact mammal. Most studies on mammalian cell-cycle control therefore use cells that have been isolated from normal tissues or tumors and grown in plastic culture dishes in the presence of essential nutrients and other factors (Figure 17-10). There is a complication, however. When cells from normal mammalian tissues are cultured in standard conditions, they often stop dividing after a limited number of division cycles. Human fibroblasts, for example, permanently cease dividing after 25–40 divisions, a process called replicative cell senescence, which we discuss later.

Mammalian cells occasionally undergo mutations that allow them to proliferate readily and indefinitely in culture as “immortalized” cell lines. Although they are not normal, such cell lines are used widely for cell-cycle studies—and for cell biology generally—because they provide an unlimited source of genetically homogeneous cells. In addition, these cells are sufficiently large to allow detailed cytological observations of cell-cycle events, and they are amenable to biochemical analysis of the proteins involved in cell-cycle control.

Studies of cultured mammalian cells have been especially useful for examining the molecular mechanisms governing the control of cell proliferation in multicellular organisms. Such studies are important not only for understanding the normal controls of cell numbers in tissues but also for understanding the loss of these controls in cancer (discussed in Chapter 23).

Cell-Cycle Progression Can Be Studied in Various Ways

How can one tell at what stage an animal cell is in the cell cycle? One way is to simply look at living cells with a microscope. A glance at a population of mammalian cells proliferating in culture reveals that a fraction of the cells have rounded up and are in mitosis. Others can be observed in the process of cytokinesis. The S-phase cells, however, cannot be detected by simple observation. They can be recognized, however, by supplying them with visualizable molecules that are incorporated into newly synthesized DNA, such as ³H-thymidine or the artificial thymidine analog bromo-deoxyuridine (BrdU). Cell nuclei that have incorporated ³H-thymidine are visualized by autoradiography (Figure 17-11A), whereas those that have incorporated BrdU are visualized by staining with anti-BrdU antibodies (Figure 17-11B).

Typically, in a population of cells that are all proliferating rapidly but asynchronously, about 30–40% will be in S phase at any instant and become labeled by a brief pulse of ³H-thymidine or BrdU. From the proportion of cells in such a population that are labeled (the labeling index), one can estimate the duration of S phase as a fraction of the whole cell cycle duration. Similarly, from the proportion of these cells in mitosis (the mitotic index), one can estimate the duration of M phase. In addition, by giving a pulse of ³H-thymidine or BrdU and allowing the cells to continue around the cycle for measured lengths of time, one can determine how long it takes for an S-phase cell to progress through G₂ into M phase, through M phase into G₁, and finally through G₁ back into S phase.

Another way to assess the stage that a cell has reached in the cell cycle is by measuring its DNA content, which doubles during S phase. This approach is greatly facilitated by the use of DNA-binding fluorescent dyes and a flow cytometer, which allows large numbers of cells to be analyzed rapidly and automatically (Figure 17-12). One can also use flow cytometry to determine the lengths of G₁, S, and G₂ + M phases, by following over time a population of cells that have been preselected to be in one particular phase of the cell cycle: DNA content measurements on such a synchronized population of cells reveal how the cells progress through the cycle.

Summary

Cell reproduction begins with duplication of the cell's contents, followed by distribution of those contents into two daughter cells. Chromosome duplication occurs during S phase of the cell cycle, whereas most other cell components are duplicated continuously throughout the cycle. During M phase, the replicated chromosomes are segregated into individual nuclei (mitosis), and the cell then splits in two (cytokinesis). S phase and M phase are usually separated by gap phases called G₁ and G₂, when cell-cycle progression can be regulated by various intracellular and extracellular signals. Cell-cycle organization and control have been highly conserved during evolution, and studies in a wide range of systems—including yeasts, frog embryos, and mammalian cells in culture—have led to a unified view of eucaryotic cell-cycle control.

The Cell-Cycle Control System Triggers the Major Processes of the Cell Cycle

The cell-cycle control system operates much like the control system of an automatic clothes-washing machine. The washing machine functions in a series of stages: it takes in water, mixes it with detergent, washes the clothes, rinses them, and spins them dry. These essential processes of the wash cycle are analogous to the essential processes of the cell cycle—DNA replication, mitosis, and so on. In both cases, a central controller triggers each process in a set sequence (Figure 17-13).

How might one design a control system that safely guides the cell through the events of the cell cycle (or a wash cycle, for that matter)? In principle, one can imagine that the most basic control system should possess the following features:

• A clock, or timer, that turns on each event at a specific time, thus providing a fixed amount of time for the completion of each event.

• A mechanism for initiating events in the correct order; entry into mitosis, for example, must always come after DNA replication.

• A mechanism to ensure that each event is triggered only once per cycle.

• Binary (on/off) switches that trigger events in a complete, irreversible fashion. It would clearly be disastrous, for example, if events like chromosome condensation or nuclear envelope breakdown were initiated but not completed.

• Robustness: backup mechanisms to ensure that the cycle can work properly even when parts of the system malfunction.

• Adaptability, so that the system's behavior can be modified to suit specific cell types or environmental conditions.

We shall see in this chapter that the cell-cycle control system possesses all of these features, and that we are now beginning to understand the molecular mechanisms involved.

The Control System Can Arrest the Cell Cycle at Specific Checkpoints

We can illustrate the importance of an adjustable cell-cycle control system by extending our washing machine analogy. The control system of simple embryonic cell cycles, like the controller in a simple washing machine, is based on a clock. The clock is unaffected by the events it regulates and will progress through the whole sequence of events even if one of those events has not been successfully completed. In contrast, the control system of most cell cycles (and sophisticated washing machines) is responsive to information received back from the processes it is controlling. Sensors, for example, detect the completion of DNA synthesis (or the successful filling of the washtub), and, if some malfunction prevents the successful completion of this process, signals are sent to the control system to delay progression to the next phase. These delays provide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressed prematurely to the next stage.

In most cells there are several points in the cell cycle, called checkpoints, at which the cycle can be arrested if previous events have not been completed (Figure 17-14). Entry into mitosis is prevented, for example, when DNA replication is not complete, and chromosome separation in mitosis is delayed if some chromosomes are not properly attached to the mitotic spindle.

Progression through G₁ and G₂ is delayed by braking mechanisms if the DNA in the chromosomes is damaged by radiation or chemicals. Delays at these DNA damage checkpoints provide time for the damaged DNA to be repaired, after which the cell-cycle brakes are released and progress resumes.

Checkpoints are important in another way as well. They are points in the cell cycle at which the control system can be regulated by extracellular signals from other cells. These signals—which can either promote or inhibit cell proliferation—tend to act by regulating progression through a G₁ checkpoint, using mechanisms discussed later in the chapter.

Checkpoints Generally Operate Through Negative Intracellular Signals

Checkpoint mechanisms like those just described tend to act through negative intracellular signals that arrest the cell cycle, rather than through the removal of positive signals that normally stimulate cell-cycle progression. The following argument suggests why this is so.

Consider, for example, the checkpoint that monitors the attachment of chromosomes to the mitotic spindle. If a cell proceeds into anaphase and starts to segregate its chromosomes into separate daughter cells before all chromosomes are appropriately attached, one daughter receives an incomplete chromosome set, while the other daughter receives a surplus. The cell therefore needs to be able to detect the attachment of the last unattached chromosome to the microtubules of the spindle. In a cell with many chromosomes, if each chromosome sends a positive signal to the cell-cycle control system once it is attached, the attachment of the last chromosome will be hard to detect, as it will be signaled by only a small fractional change in the total intensity of the “go” signal. On the other hand, if each unattached chromosome sends a negative signal to inhibit progress through the cell cycle, the attachment of the last chromosome will be easily detected because it will cause a change from some “stop” signal to none. A similar argument would imply that unreplicated DNA inhibits the initiation of mitosis, creating a stop signal that persists until the completion of DNA replication.

The most convincing evidence that checkpoints operate through negative signals comes from studies of cells in which a checkpoint is inactivated by either mutation or chemical treatment. In these cells, the cell cycle continues to progress even if DNA replication or spindle assembly is incomplete, indicating that checkpoints are generally not essential for cell-cycle progression. Checkpoints are best viewed as accessory braking systems that have been added to the cell-cycle control system to provide a more sophisticated form of regulation.

Although most checkpoints are not essential for normal cell-cycle progression under ideal conditions, populations of cells with checkpoint defects often accumulate mutations due to occasional malfunctions in DNA replication, DNA repair, or spindle assembly. Some of these mutations can promote the development of cancer, as we discuss later and in Chapter 23.

The Cell-Cycle Control System Is Based on Cyclically Activated Protein Kinases

At the heart of the cell-cycle control system is a family of protein kinases known as cyclin-dependent kinases (Cdks). The activity of these kinases rises and falls as the cell progresses through the cycle. The oscillations lead directly to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle—DNA replication, mitosis, and cytokinesis. An increase in Cdk activity at the beginning of mitosis, for example, leads to increased phosphorylation of proteins that control chromosome condensation, nuclear envelope breakdown, and spindle assembly.

Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins. The most important of these Cdk regulators are proteins known as cyclins. Cdks, as their name implies, are dependent on cyclins for their activity: unless they are tightly bound to a cyclin, they have no protein kinase activity (Figure 17-15). Cyclins were originally named as such because they undergo a cycle of synthesis and degradation in each cell cycle. Cdk levels, by contrast, are constant, at least in the simplest cell cycles. Cyclical changes in cyclin levels result in the cyclic assembly and activation of the cyclin-Cdk complexes; this activation in turn triggers cell-cycle events (Figure 17-16).

There are four classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and function. Three of these classes are required in all eucaryotic cells:

• 1

  G₁/S-cyclins bind Cdks at the end of G₁ and commit the cell to DNA replication.

• 2

  S-cyclins bind Cdks during S phase and are required for the initiation of DNA replication.

• 3

  M-cyclins promote the events of mitosis.

In most cells, a fourth class of cyclins, the G₁-cyclins, helps promote passage through Start or the restriction point in late G₁.

In yeast cells, a single Cdk protein binds all classes of cyclins and drives all cell-cycle events by changing cyclin partners at different stages of the cycle. In vertebrate cells, by contrast, there are four Cdks. Two interact with G₁-cyclins, one with G₁/S- and S-cyclins, and one with M-cyclins. In this chapter, we simply refer to the different cyclin-Cdk complexes as G ₁-Cdk, G₁/S-Cdk, S-Cdk, and M-Cdk. The names of the individual Cdks and cyclins are given in Table 17-1.

How do different cyclin-Cdk complexes drive different cell-cycle events? The answer, at least in part, seems to be that the cyclin protein does not simply activate its Cdk partner but also directs it to specific target proteins. As a result, each cyclin-Cdk complex phosphorylates a different set of substrate proteins. The same cyclin-Cdk complex can also induce different effects at different times in the cycle, probably because the accessibility of some Cdk substrates changes during the cell cycle. Certain proteins that function in mitosis, for example, may become available for phosphorylation only in G₂.

Studies of the three-dimensional structures of Cdk and cyclin proteins have revealed that, in the absence of cyclin, the active site in the Cdk protein is partly obscured by a slab of protein, like a stone blocking the entrance to a cave (Figure 17-17A). Cyclin binding causes the slab to move away from the active site, resulting in partial activation of the Cdk enzyme (Figure 17-17B). Full activation of the cyclin-Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site. This causes a small conformational change that further increases the activity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events (Figure 17-17C).

Cdk Activity Can Be Suppressed Both by Inhibitory Phosphorylation and by Inhibitory Proteins

The rise and fall of cyclin levels is the primary determinant of Cdk activity during the cell cycle. Several additional mechanisms, however, are important for fine-tuning Cdk activity at specific stages in the cell cycle.

The activity of a cyclin-Cdk complex can be inhibited by phosphorylation at a pair of amino acids in the roof of the active site. Phosphorylation of these sites by a protein kinase known as Wee1 inhibits Cdk activity, while dephosphorylation of these sites by a phosphatase known as Cdc25 increases Cdk activity (Figure 17-18). We see later that this regulatory mechanism is particularly important in the control of M-Cdk activity at the onset of mitosis.

Cyclin-Cdk complexes can also be regulated by the binding of Cdk inhibitor proteins (CKIs). There are a variety of CKI proteins, and they are primarily employed in the control of G₁ and S phase. The three-dimensional structure of a cyclin-Cdk-CKI complex reveals that CKI binding dramatically rearranges the structure of the Cdk active site, rendering it inactive (Figure 17-19).

The Cell-Cycle Control System Depends on Cyclical Proteolysis

Cell-cycle control depends crucially on at least two distinct enzyme complexes that act at different times in the cycle to cause the proteolysis of key proteins of the cell-cycle control system, thereby inactivating them. Most notably, cyclin-Cdk complexes are inactivated by regulated proteolysis of cyclins at certain cell-cycle stages. This cyclin destruction occurs by a ubiquitin-dependent mechanism, like that involved in the proteolysis of many other intracellular proteins (discussed in Chapter 6). An activated enzyme complex recognizes specific amino-acid sequences on the cyclin and attaches multiple copies of ubiquitin to it, marking the protein for complete destruction in proteasomes.

The rate-limiting step in cyclin destruction is the final ubiquitin-transfer reaction catalyzed by enzymes known as ubiquitin ligases (see Figure 6-87B). Two ubiquitin ligases are important in the destruction of cyclins and other cell-cycle regulators. In G₁ and S phase, an enzyme complex called SCF (after its three main protein subunits) is responsible for the ubiquitylation and destruction of G₁/S-cyclins and certain CKI proteins that control S-phase initiation. In M phase, the anaphase-promoting complex (APC) is responsible for the ubiquitylation and proteolysis of M-cyclins and other regulators of mitosis.

These two large, multisubunit complexes contain some related components, but they are regulated in different ways. SCF activity is constant during the cell cycle. Ubiquitylation by SCF is controlled by changes in the phosphorylation state of its target proteins: only specifically phosphorylated proteins are recognized, ubiquitylated, and destroyed (Figure 17-20A). APC activity, by contrast, changes at different stages of the cell cycle. APC is turned on mainly by the addition of activating subunits to the complex (Figure 17-20B). We discuss the functions of SCF and APC in more detail later.

Cell-Cycle Control Also Depends on Transcriptional Regulation

In the frog embryonic cell cycle discussed earlier, gene transcription does not occur. Cell-cycle control depends exclusively on post-transcriptional mechanisms that involve the regulation of Cdk activity by phosphorylation and the binding of regulatory proteins such as cyclins, which are themselves regulated by proteolysis. In the more complex cell cycles of most cell types, however, transcriptional control provides an added level of regulation. Cyclin levels in most cells, for example, are controlled not only by changes in cyclin degradation but also by changes in cyclin gene transcription and cyclin synthesis.

In certain organisms, such as budding yeasts, one can use DNA arrays (discussed in Chapter 8) to analyze changes in the expression of all of the genes in the genome as the cell progresses through the cell cycle. The results of these studies are surprising. About 10% of the yeast genes encode mRNAs whose levels oscillate during the cell cycle. Some of these genes encode proteins with known cell-cycle functions, but the functions of many others are unknown. It seems likely that these oscillations in gene expression are controlled by the cyclin-Cdk-dependent phosphorylation of gene regulatory proteins, but the details of this regulation remain unknown.

Summary

Events of the cell cycle are triggered by an independent cell-cycle control system, which ensures that the events are properly timed, occur in the correct order, and occur only once per cell cycle. The control system is responsive to various intracellular and extracellular signals, so that cell-cycle progression can be arrested when the cell either fails to complete an essential cell-cycle process or encounters unfavorable environmental conditions.

The central components of the cell-cycle control system are cyclin-dependent protein kinases (Cdks), whose activity depends on association with regulatory subunits called cyclins. Oscillations in the activities of various cyclin-Cdk complexes leads to the initiation of various cell-cycle events. Thus, activation of S-phase cyclin-Cdk complexes initiates S phase, while activation of M-phase cyclin-Cdk complexes triggers mitosis. The activities of cyclin-Cdk complexes are influenced by several mechanisms, including phosphorylation of the Cdk subunit, the binding of special inhibitory proteins (CKIs), proteolysis of cyclins, and changes in the transcription of genes encoding Cdk regulators. Two enzyme complexes, SCF and APC, are also crucial components of the cell-cycle control system; they induce the proteolysis of specific cell-cycle regulators by ubiquitylating them and thereby trigger several critical events in the cycle.

S-Phase Cyclin-Cdk Complexes (S-Cdks) Initiate DNA Replication Once Per Cycle

A cell must solve several problems in controlling the initiation and completion of DNA replication. Not only must replication occur with extreme accuracy to minimize the risk of mutations in the next cell generation, but every nucleotide in the genome must be copied once, and only once, to prevent the damaging effects of gene amplification. In Chapter 5, we discuss the sophisticated protein machinery that performs DNA replication with astonishing speed and accuracy. In this chapter, we consider the elegant mechanisms by which the cell-cycle control system initiates the replication process and, at the same time, prevents it from happening more than once per cycle.

Early clues about the regulation of S phase came from studies in which human cells at various cell-cycle stages were fused to form single cells with two nuclei. These experiments revealed that when a G₁ cell is fused with an S-phase cell, DNA replication occurs in the G₁ nucleus (presumably triggered by S-Cdk activity in the S-phase cell). Fusion of a G₂ cell with an S-phase cell, however, does not cause DNA synthesis in the G₂ nucleus (Figure 17-21). These studies provided a clear hint that only G₁ cells are competent to initiate DNA replication and that cells that have completed S phase (i.e. G₂ cells) are not able to rereplicate their DNA, even when provided with S-Cdk activity. Apparently, passage through mitosis is required for the cell to regain the ability to undergo S phase.

We have begun to decipher the molecular basis of these cell fusion experiments only recently. DNA replication begins at origins of replication, which are scattered at various locations in the chromosome. Replication origins are simple and well defined in the budding yeast S. cerevisiae, and most of our understanding of the initiation machinery comes from studies of this organism. Analyses of proteins that bind to the yeast replication origin have identified a large, multiprotein complex known as the origin recognition complex (ORC). These complexes bind to replication origins throughout the cell cycle and serve as landing pads for several additional regulatory proteins.

One of these regulatory proteins is Cdc6. It is present at low levels during most of the cell cycle but increases transiently in early G₁. It binds to ORC at replication origins in early G₁, where it is required for the binding of a complex composed of a group of closely related proteins, the Mcm proteins. The resulting large protein complex formed at an origin is known as the pre-replicative complex, or pre-RC (Figure 17-22).

Once the pre-RC has been assembled in G₁, the replication origin is ready to fire. The activation of S-Cdk in late G₁ pulls the trigger and initiates DNA replication. The initiation of replication also requires the activity of a second protein kinase, which collaborates with S-Cdk to cause the phosphorylation of ORC.

The S-Cdk not only initiates origin firing, but also helps to prevent rereplication in several ways. First, it causes the Cdc6 protein to dissociate from ORC after an origin has fired. This results in the disassembly of the pre-RC, which prevents replication from occurring again at the same origin. Second, it prevents the Cdc6 and Mcm proteins from reassembling at any origin. By phosphorylating Cdc6, it triggers Cdc6 ubiquitylation by the SCF enzyme complex discussed earlier. As a result, any Cdc6 protein that is not bound to an origin is rapidly degraded in proteasomes. S-Cdk also phosphorylates certain Mcm proteins, which triggers their export from the nucleus, further ensuring that the Mcm protein complex cannot bind to a replication origin (see Figure 17-22).

S-Cdk activity remains high during G₂ and early mitosis, preventing rereplication from occurring after the completion of S phase. M-Cdk also helps ensure that rereplication does not occur during mitosis by phosphorylating the Cdc6 and Mcm proteins. The G₁/S-Cdks help as well, by inducing Mcm export from the nucleus, ensuring that excess Mcm proteins that have not bound to origins in late G₁ are taken out of action before replication begins.

Thus, several cyclin-Cdk complexes cooperate to restrain pre-RC assembly and prevent DNA rereplication after S phase. How, then, is the cell-cycle control system reset to allow replication to occur in the next cell cycle? The answer is simple. At the end of mitosis, all Cdk activity in the cell is reduced to zero. The resulting dephosphorylation of the Cdc6 and Mcm proteins allows pre-RC assembly to occur once again, readying the chromosomes for a new round of replication.

The Activation of M-Phase Cyclin-Cdk Complexes (M-Cdks) Triggers Entry into Mitosis

The completion of DNA replication leaves the G₂ cell with two accurate copies of the entire genome, with each replicated chromosome consisting of two identical sister chromatids glued together along their length. The cell then undergoes the dramatic upheaval of M phase, in which the duplicated chromosomes and other cell contents are distributed equally to the two daughter cells. The events of mitosis are triggered by M-Cdk, which is activated after S phase is complete.

The activation of M-Cdk begins with the accumulation of M-cyclin (cyclin B in vertebrate cells, see Table 17-1). In embryonic cell cycles, the synthesis of M-cyclin is constant throughout the cell cycle, and M-cyclin accumulation results from a decrease in its degradation. In most cell types, however, M-cyclin synthesis increases during G₂ and M, owing primarily to an increase in M-cyclin gene transcription. This increase in M-cyclin protein leads to a gradual accumulation of M-Cdk (the complex of Cdk1 and M-cyclin) as the cell approaches mitosis. Although the Cdk in these complexes is phosphorylated at an activating site by the enzyme CAK discussed earlier, it is held in an inactive state by inhibitory phosphorylation at two neighboring sites by the protein kinase Wee1 (see Figure 17-18). Thus, by the time the cell reaches the end of G₂, it contains an abundant stockpile of M-Cdk that is primed and ready to act, but the M-Cdk activity is repressed by the presence of two phosphate groups that block the active site of the kinase.

What, then, triggers the activation of the M-Cdk stockpile? The crucial event is the activation in late G₂ of the protein phosphatase Cdc25, which removes the inhibitory phosphates that restrain M-Cdk (Figure 17-23). At the same time, the activity of the inhibitory kinase Wee1 is also suppressed, further ensuring that M-Cdk activity increases abruptly. Two protein kinases activate Cdc25. One, known as Polo kinase, phosphorylates Cdc25 at one set of sites. The other activating kinase is M-Cdk itself, which phosphorylates a different set of sites on Cdc25. M-Cdk also phosphorylates and inhibits Wee1.

The ability of M-Cdk to activate its own activator (Cdc25) and inhibit its own inhibitor (Wee1) suggests that M-Cdk activation in mitosis involves a positive feedback loop (see Figure 17-23). According to this attractive model, the partial activation of Cdc25, perhaps by Polo kinase, leads to the partial activation of a subpopulation of M-Cdk complexes, which then phosphorylate more Cdc25 and Wee1 molecules. This leads to more M-Cdk dephosphorylation and activation, and so on. Such a mechanism would quickly promote the complete activation of all the M-Cdk complexes in the cell, converting a gradual increase in M-cyclin levels into a switchlike, abrupt rise in M-Cdk activity. As mentioned earlier, similar molecular switches operate at various points in the cell cycle to ensure that events such as entry into mitosis occur in an all-or-none fashion.

Entry into Mitosis Is Blocked by Incomplete DNA Replication: The DNA Replication Checkpoint

If a cell is driven into mitosis before it has finished replicating its DNA, it will pass on broken or incomplete sets of chromosomes to its daughter cells. This disaster is avoided in most cells by a DNA replication checkpoint mechanism, which ensures that the initiation of mitosis cannot occur until the last nucleotide in the genome has been copied. Sensor mechanisms, of unknown molecular nature, detect either the unreplicated DNA or the corresponding unfinished replication forks and send a negative signal to the cell-cycle control system, blocking the activation of M-Cdk. Thus, normal cells treated with chemical inhibitors of DNA synthesis, such as hydroxyurea, do not progress into mitosis. If the checkpoint mechanism is defective, however, as in yeast cells with certain mutations or in mammalian cells treated with high doses of caffeine, the cells plunge into a suicidal mitosis despite the failure to complete DNA replication (Figure 17-24).

The final targets of the negative checkpoint signal are the enzymes that control M-Cdk activation. The negative signal activates a protein kinase that inhibits the Cdc25 protein phosphatase (see Figures 17-18 and 17-23). As a result, M-Cdk remains phosphorylated and inactive until DNA replication is complete.

M-Cdk Prepares the Duplicated Chromosomes for Separation

One of the most remarkable features of cell-cycle control is that a single protein kinase, M-Cdk, is able to bring about all of the diverse and complex rearrangements that occur in the early stages of mitosis (discussed in Chapter 18). At a minimum, M-Cdk must induce the assembly of the mitotic spindle and ensure that replicated chromosomes attach to the spindle. In many organisms, M-Cdk also triggers chromosome condensation, nuclear envelope breakdown, actin cytoskeleton rearrangement, and the reorganization of the Golgi apparatus and endoplasmic reticulum. Each of these events is thought to be triggered when M-Cdk phosphorylates specific structural or regulatory proteins involved in the event, although most of these proteins have not yet been identified.

The breakdown of the nuclear envelope, for example, requires the disassembly of the nuclear lamina—the underlying shell of polymerized lamin filaments that gives the nuclear envelope its structural rigidity. Direct phosphorylation of lamin proteins by M-Cdk results in their depolymerization, which is an essential first step in the dismantling of the envelope (see Figure 12-21).

Chromosome condensation also seems to be a direct consequence of phosphorylation by M-Cdk. A complex of five proteins, known as the condensin complex, is required for chromosome condensation in Xenopus embryos. After M-Cdk has phosphorylated several subunits in the complex, two of the subunits are able to change the coiling of DNA molecules in a test tube. It is thought that this coiling activity is important for chromosome condensation during mitosis (see Figure 4-56).

Phosphorylation by M-Cdk also triggers the complex microtubule rearrangements and other events that lead to the assembly of the mitotic spindle. As discussed in Chapter 18, M-Cdk is known to phosphorylate a number of proteins that regulate microtubule behavior, causing the increase in microtubule instability that is required for spindle assembly.

Sister Chromatid Separation Is Triggered by Proteolysis

After M-Cdk has triggered the complex rearrangements that occur in early mitosis, the cell cycle reaches its culmination with the separation of the sister chromatids at the metaphase-to-anaphase transition. Although M-Cdk activity sets the stage for this event, an entirely different enzyme complex—the anaphase-promoting complex (APC) introduced earlier—throws the switch that initiates sister-chromatid separation. The APC is a highly regulated ubiquitin ligase that promotes the destruction of several mitotic regulatory proteins (see Figure 17-20B).

The attachment of the two sister chromatids to opposite poles of the mitotic spindle early in mitosis results in forces tending to pull the two chromatids apart. These pulling forces are initially resisted because the sister chromatids are bound tightly together, both at their centromeres and all along their arms. This sister-chromatid cohesion depends on a complex of proteins, the cohesin complex, that is deposited along the chromosomes as they are duplicated in S phase. The cohesin proteins (cohesins) are closely related to the proteins of the condensin complex involved in chromosome condensation, suggesting a common evolutionary origin for the two processes (see Figure 18-3).

Anaphase begins with a sudden disruption of the cohesion between sister chromatids, which allows them to separate and move to opposite poles of the spindle. This process is initiated by a remarkable cascade of signaling events. The sister-chromatid separation requires the activation of the APC enzyme complex, suggesting that proteolysis is central to the process (Figure 17-25). The relevant target of the APC is the protein securin. Before anaphase, securin binds to and inhibits the activity of a protease called separase. The destruction of securin at the end of metaphase releases separase, which is then free to cleave one of the subunits of the cohesin complex. In an instant, the cohesin complex falls away from the chromosomes, and the sister chromatids separate (Figure 17-26).

If the APC triggers anaphase, what triggers the APC? The answer is only partly known. APC activation requires the protein Cdc20, which binds to and activates the APC at mitosis (see Figures 17-26 and 17-20B). At least two processes regulate Cdc20 and its association with the APC. First, Cdc20 synthesis increases as the cell approaches mitosis, owing to an increase in the transcription of its gene. Second, phosphorylation of the APC helps Cdc20 bind to the APC, thereby helping to create an active complex.

It is not clear what kinases phosphorylate and activate the Cdc20-APC complex. M-Cdk activity is required for the activity of these kinases, but there is a significant delay, or lag phase, between M-Cdk activation and the activation of the Cdc20-APC complex. The molecular basis of this delay is still mysterious, but it is likely to hold the key to how anaphase is initiated at the correct time in M phase.

Unattached Chromosomes Block Sister-Chromatid Separation: The Spindle-Attachment Checkpoint

The cell does not commit itself to the momentous events of anaphase before it is fully prepared. In most cell types, a spindle-attachment checkpoint mechanism operates to ensure that all chromosomes are properly attached to the spindle before sister-chromatid separation occurs. The checkpoint depends on a sensor mechanism that monitors the state of the kinetochore, the specialized region of the chromosome that attaches to microtubules of the spindle. Any kinetochore that is not properly attached to the spindle sends out a negative signal to the cell-cycle control system, blocking Cdc20-APC activation and sister-chromatid separation.

The nature of the signal generated by an unattached kinetochore is not clear, although several proteins, including Mad2, are recruited to unattached kinetochores and are required for the spindle-attachment checkpoint to function. Even a single unattached kinetochore in the cell results in Mad2 binding and the inhibition of Cdc20-APC activity and Securin destruction (Figure 17-27). Thus, sister-chromatid separation cannot occur until the last kinetochore is attached.

Surprisingly, the normal timing of anaphase does not require a functional spindle-attachment checkpoint, at least in frog embryos and yeasts. Mutant yeast cells with a defective checkpoint undergo anaphase with normal timing, indicating that some other mechanism normally determines the timing of anaphase in these cells. In mammalian cells, however, a defect in the spindle-attachment checkpoint causes anaphase to occur slightly earlier than normal. This finding suggests that, in our cells, the checkpoint has evolved from a useful accessory to an essential component of the cell-cycle control system.

Exit from Mitosis Requires the Inactivation of M-Cdk

After the chromosomes have been segregated to the poles of the spindle, the cell must reverse the complex changes of early mitosis. The spindle must be disassembled, the chromosomes decondensed, and the nuclear envelope reformed. Because the phosphorylation of various proteins is responsible for getting cells into mitosis in the first place, it is not surprising that the dephosphorylation of these same proteins is required to get them out. In principle, these dephosphorylations and the exit from mitosis could be triggered by the inactivation of M-Cdk, the activation of phosphatases, or both. Evidence suggests that M-Cdk inactivation is primarily responsible.

M-Cdk inactivation occurs mainly by ubiquitin-dependent proteolysis of M-cyclins. Ubiquitylation of the cyclin is usually triggered by the same Cdc20-APC complex that promotes the destruction of Securin at the metaphase-to-anaphase transition (see Figure 17-20B). Thus, the activation of the Cdc20-APC complex leads not only to anaphase, but also to M-Cdk inactivation—which in turn leads to all of the other events that take the cell out of mitosis.

The G₁ Phase Is a State of Stable Cdk Inactivity

In early animal embryos, the inactivation of M-Cdk in late mitosis is due almost entirely to the action of Cdc20-APC. Recall, however, that M-Cdk stimulates Cdc20-APC activity (see Figure 17-26). Thus, the destruction of M-cyclin in late mitosis soon leads to the inactivation of all APC activity in an embryonic cell. This is a useful arrangement in rapid embryonic cell cycles, as APC inactivation immediately after mitosis allows the cell to quickly begin accumulating new M-cyclin for the next cycle (Figure 17-28A).

Rapid cyclin accumulation immediately after mitosis is not useful, however, in cell cycles containing a G₁ phase. In these cycles, progression into the next S phase is delayed in G₁ to allow for cell growth and for the cycle to be regulated by extracellular signals. Thus, most cells employ several mechanisms to ensure that Cdk reactivation is prevented after mitosis. One mechanism makes use of another APC-activating protein called Hct1, a close relative of Cdc20. Although both Hct1 and Cdc20 bind and activate the APC, they differ in one important respect. Whereas the Cdc20-APC complex is activated by M-Cdk, the Hct1-APC complex is inhibited by M-Cdk, which directly phosphorylates Hct1. As a result of this relationship, Hct1-APC activity increases in late mitosis after the Cdc20-APC complex has initiated the destruction of M-cyclin. M-cyclin destruction therefore continues after mitosis: although Cdc20-APC activity has declined, Hct1-APC activity is high (Figure 17-28B).

A second mechanism that suppresses Cdk activity in G₁ depends on the increased production of CKIs, the Cdk inhibitory proteins discussed earlier. Budding yeast cells, in which this mechanism is best understood, contain a CKI protein called Sic1, which binds to and inactivates M-Cdk in late mitosis and G₁. Like Hct1, Sic1 is inhibited by M-Cdk, which phosphorylates Sic1 during mitosis. M-Cdk also phosphorylates and inhibits a gene regulatory protein required for Sic1 synthesis, resulting in decreased Sic1 production. Thus, Sic1 and M-Cdk, like Hct1 and M-Cdk, mutually inhibit each other. As a result, the decline in M-Cdk activity that occurs in late mitosis triggers the rapid accumulation of Sic1 protein, and this CKI helps ensure that M-Cdk activity is stably inhibited after mitosis.

In most cells, M-Cdk inactivation in late mitosis also results from decreased transcription of M-cyclin genes. In budding yeast, for example, M-Cdk promotes the expression of these genes, resulting in a positive feedback loop. This loop is turned off as cells exit from mitosis: the inactivation of M-Cdk by Hct1 and Sic1 leads to decreased M-cyclin gene transcription and thus decreased M-cyclin synthesis.

In summary Hct1-APC activation, CKI accumulation, and decreased cyclin production act together to ensure that the early G₁ phase is a time when essentially all Cdk activity is suppressed. As in many other aspects of cell-cycle control, the use of multiple regulatory mechanisms makes the suppression system robust, so that it still operates with reasonable efficiency even if one mechanism fails.

How does the cell escape from this stable G₁ state to initiate S phase? As we describe later, escape usually occurs through the accumulation of G₁-cyclins. In budding yeast, for example, these cyclins are not targeted for destruction by Hct1-APC and are not inhibited by Sic1. As a result, the accumulation of G₁ cyclins leads to an unopposed increase in G₁-Cdk activity (Figure 17-29). In animal cells, the accumulation of G₁-cyclins is stimulated by the extracellular signals that promote cell proliferation, as we discuss later.

In budding yeast, G₁-Cdk activity triggers the transcription of G₁/S-cyclin genes, leading to increased synthesis of G₁/S-cyclins and the formation of G₁/S-Cdk complexes, which are also resistant to Hct1-APC and Sic1. The increased G₁/S-Cdk activity initiates the events that commit the cell to enter S phase. It stimulates the transcription of S-cyclin genes, leading to the synthesis of S-cyclins and the formation of S-Cdk complexes. These complexes are inhibited by Sic1, but G₁/S-Cdk phosphorylates and inactivates Sic1, thereby causing S-Cdk activation. G₁/S-Cdk and S-Cdk also phosphorylate and inactivate Hct1-APC. Thus, the same feedback loops that trigger rapid M-Cdk inactivation in late mitosis now work in reverse at the end of G₁ to ensure the rapid and complete activation of S-Cdk activity.

The Rb Protein Acts as a Brake in Mammalian G₁ Cells

The control of G₁ progression and S-phase initiation is often disrupted in cancer cells, leading to unrestrained cell-cycle entry and cell proliferation (discussed in Chapter 23). To develop improved methods for controlling cancer growth, we need a better understanding of the proteins that control G₁ progression in mammalian cells.

Animal cells suppress Cdk activity in G₁ by the same three mechanisms mentioned earlier for budding yeast: Hct1 activation, the accumulation of a CKI protein (p27 in mammalian cells), and the inhibition of cyclin gene transcription. As in yeasts, the activation of G₁-Cdk complexes reverses all three inhibitory mechanisms in late G₁.

The best understood effects of G₁-Cdk activity in animal cells are mediated by a gene regulatory protein called E2F. It binds to specific DNA sequences in the promoters of many genes that encode proteins required for S-phase entry, including G₁/S-cyclins and S-cyclins. E2F function is controlled primarily by an interaction with the retinoblastoma protein (Rb), an inhibitor of cell-cycle progression. During G₁, Rb binds to E2F and blocks the transcription of S-phase genes. When cells are stimulated to divide by extracellular signals, active G₁-Cdk accumulates and phosphorylates Rb, reducing its affinity for E2F. The Rb then dissociates, allowing E2F to activate S-phase gene expression (Figure 17-30).

This transcriptional control system, like so many other control systems that regulate the cell cycle, includes feedback loops that sharpen the G₁/S transition (see Figure 17-30):

• The liberated E2F increases the transcription of its own gene.

• E2F-dependent transcription of G₁/S-cyclin and S-cyclin genes leads to increased G₁/S-Cdk and S-Cdk activities, which in turn increase Rb phosphorylation and promote further E2F release.

• The increase in G₁/S-Cdk and S-Cdk activities enhances the phosphorylation of Hct1 and p27, leading to their inactivation or destruction.

As in yeast cells, the result of all these interactions is the rapid and complete activation of the S-Cdk complexes required for S-phase initiation.

The Rb protein was identified originally through studies of an inherited form of eye cancer in children, known as retinoblastoma (discussed in Chapter 23). The loss of both copies of the Rb gene leads to excessive cell proliferation in the immature retina, suggesting that the Rb protein is particularly important for restraining the rate of cell division in the developing retina. The complete loss of Rb does not immediately cause increased proliferation of other cell types, in part because Hct1 and p27 provide assistance in G₁ control, and in part because other cell types contain Rb-related proteins that provide backup support in the absence of Rb. It is also likely that other proteins, unrelated to Rb, help to regulate the activity of E2F.

Cell-Cycle Progression Is Somehow Coordinated With Cell Growth

For proliferating cells to maintain a relatively constant size, the length of the cell cycle must match the time it takes the cell to double in size. If the cycle time is shorter than this, the cells will get smaller with each division; if it is longer, the cells will get bigger with each division. Because cell growth depends on nutrients and growth signals in the environment, the length of the cell cycle has to be able to adjust to varying environmental conditions (Figure 17-31). It is not clear how proliferating cells coordinate their growth with the rate of cell-cycle progression to maintain their size.

There is evidence that budding yeasts coordinate their growth and cell-cycle progression by monitoring the total amount of a G₁ cyclin called Cln3 (see Table 17-1, p. 994). Because Cln3 is synthesized in parallel with cell growth, its concentration remains constant while its total amount increases as the cell grows. If the amount of Cln3 is artificially increased, the cells divide at a smaller size than normal, whereas if it is artificially decreased, the cells divide at a larger size than normal. These experiments are consistent with the idea that the cells commit themselves to division when the total amount of Cln3 reaches some threshold value. How, then, can the cell monitor the total amount of Cln3, rather than its concentration? One possibility is that cells inherit a fixed amount of an inhibitor that can bind to Cln3 and block its activity. When the amount of Cln3 exceeds the amount of this inhibitor, the extra Cln3 triggers G₁-Cdk activation and a new cell cycle. Since all cells receive a fixed and equal quantity of DNA, it has been speculated that the Cln3 inhibitor could be DNA itself, or some protein bound to DNA (Figure 17-32). Such a mechanism would also explain why cell size in all organisms is proportional to ploidy (the number of copies of the nuclear genome per cell).

Whereas yeast cells grow and proliferate constitutively if nutrients are plentiful, animal cells generally grow and proliferate only when they are stimulated to do so by signals from other cells. The size at which an animal cell divides depends, at least in part, on these extracellular signals, which can regulate cell growth and proliferation independently. Animal cells can also completely uncouple cell growth and division so as to grow without dividing or to divide without growing. The eggs of many animals, for example, grow to an extremely large size without dividing. After fertilization, this relationship is reversed, and many rounds of division occur without growth (see Figure 17-8). Thus, although cell growth and cell division are usually coordinated, they can be regulated independently. Cell growth does not depend on cell-cycle progression. Yeast cells continue to grow when cell-cycle progression is blocked by a mutation; and many animal cells, including neurons and muscle cells, grow large after they have withdrawn permanently from the cell cycle.

Cell-Cycle Progression is Blocked by DNA Damage and p53: DNA Damage Checkpoints

When chromosomes are damaged, as can occur after exposure to radiation or certain chemicals, it is essential that they be repaired before the cell attempts to duplicate or segregate them. The cell-cycle control system can readily detect DNA damage and arrest the cycle at DNA damage checkpoints. Most cells have at least two such checkpoints—one in late G₁, which prevents entry into S phase, and one in late G₂, which prevents entry into mitosis.

The G₂ checkpoint depends on a mechanism similar to the one discussed earlier that delays entry into mitosis in response to incomplete DNA replication. When cells in G₂ are exposed to damaging radiation, for example, the damaged DNA sends a signal to a series of protein kinases that phosphorylate and inactivate the phosphatase Cdc25. This blocks the dephosphorylation and activation of M-Cdk, thereby blocking entry into mitosis. When the DNA damage is repaired, the inhibitory signal is turned off, and cell-cycle progression resumes.

The G₁ checkpoint blocks progression into S phase by inhibiting the activation of G₁/S-Cdk and S-Cdk complexes. In mammalian cells, for example, DNA damage leads to the activation of the gene regulatory protein p53, which stimulates the transcription of several genes. One of these genes encodes a CKI protein called p21, which binds to G₁/S-Cdk and S-Cdk and inhibits their activities, thereby helping to block entry into S phase.

DNA damage activates p53 by an indirect mechanism. In undamaged cells, p53 is highly unstable and is present at very low concentrations. This is because it interacts with another protein, Mdm2, that acts as a ubiquitin ligase that targets p53 for destruction by proteasomes. DNA damage activates protein kinases that phosphorylate p53 and thereby reduce its binding to Mdm2. This decreases p53 degradation, which results in a marked increase in p53 concentration in the cell. In addition, the decreased binding to Mdm2 enhances the ability of p53 to stimulate gene transcription (Figure 17-33).

Like many other checkpoints, DNA damage checkpoints are not essential for normal cell division if environmental conditions are ideal. Conditions are rarely ideal, however: a low level of DNA damage occurs in the normal life of any cell, and this damage accumulates in the cell's progeny if the damage checkpoints are not functioning. Over the long term, the accumulation of genetic damage in cells lacking checkpoints leads to an increased frequency of cancer-promoting mutations. Indeed, mutations in the p53 gene occur in at least half of all human cancers (discussed in Chapter 23). This loss of p53 function allows the cancer cell to accumulate mutations more readily. Similarly, a rare genetic disease known as ataxia telangiectasia is caused by a defect in one of the protein kinases that phosphorylates and activates p53 in response to x-ray-induced DNA damage; patients with this disease are very sensitive to x-rays due to the loss of the DNA damage checkpoints, and they consequently suffer from increased rates of cancer.

What if DNA damage is so severe that repair is not possible? In this case, the response is different in different organisms. Unicellular organisms such as budding yeast transiently arrest their cell cycle to repair the damage. If repair cannot be completed, the cycle resumes despite any damage. For a single-celled organism, life with mutations is apparently better than no life at all. In multicellular organisms, however, the health of the organism takes precedence over the life of an individual cell. Cells that divide with severe DNA damage threaten the life of the organism, since genetic damage can often lead to cancer and other lethal defects. Thus, animal cells with severe DNA damage do not attempt to continue division, but instead commit suicide by undergoing programmed cell death, or apoptosis, as we discuss in the next section. The decision to die in this way also depends on the activation of p53, and it is this function of p53 that is apparently most important in protecting us against cancer.

As a review, the major cell-cycle regulatory proteins are summarized in Table 17-2, with the general structure of the cell-cycle control system shown in Figure 17-34.

Summary

An ordered sequence of cyclin-Cdk activities triggers most of the events of the cell cycle. During G₁ phase, Cdk activity is reduced to a minimum by Cdk inhibitors (CKIs), cyclin proteolysis, and decreased cyclin gene transcription. When environmental conditions are favorable, G₁- and G₁/S-Cdks increase in concentration, overcoming these inhibitory barriers in late G₁ and triggering the activation of S-Cdk. The S-Cdk phosphorylates proteins at DNA replication origins, initiating DNA synthesis through a mechanism that ensures that the DNA is duplicated only once per cell cycle.

Once S phase is completed, the activation of M-Cdk leads to the events of early mitosis, whereby the cell assembles a mitotic spindle and prepares for segregation of the duplicated chromosomes—which consist of sister chromatids glued together. Anaphase is triggered by the destruction of the proteins that hold the sisters together. The M-Cdk is then inactivated by cyclin proteolysis, which leads to cytokinesis and the end of M phase. Progression through the cell cycle is regulated precisely by various inhibitory mechanisms that arrest the cell cycle at specific checkpoints when events are not completed successfully, when DNA damage occurs, or when extracellular conditions are unfavorable.

Apoptosis Is Mediated by an Intracellular Proteolytic Cascade

Cells that die as a result of acute injury typically swell and burst. They spill their contents all over their neighbors—a process called cell necrosis—causing a potentially damaging inflammatory response. By contrast, a cell that undergoes apoptosis dies neatly, without damaging its neighbors. The cell shrinks and condenses. The cytoskeleton collapses, the nuclear envelope disassembles, and 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 (a specialized phagocytic cell, discussed in Chapter 24), before any leakage of its contents occurs (Figure 17-37). 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.

The intracellular machinery responsible for apoptosis seems to be similar in all animal cells. This machinery depends on a family of proteases that have a cysteine at their active site and cleave their target proteins at specific aspartic acids. They are therefore called caspases. Caspases are synthesized in the cell as inactive precursors, or procaspases, which are usually activated by cleavage at aspartic acids by other caspases (Figure 17-38A). Once activated, caspases cleave, and thereby activate, other procaspases, resulting in an amplifying proteolytic cascade (Figure 17-38B). Some of the activated caspases then cleave other key proteins in the cell. Some cleave the nuclear lamins, for example, causing the irreversible breakdown of the nuclear lamina; another cleaves a protein that normally holds a DNA-degrading enzyme (a DNAse) in an inactive form, freeing the DNAse to cut up the DNA in the cell nucleus. In this way, the cell dismantles itself quickly and neatly, and its corpse is rapidly taken up and digested by another cell.

Activation of the intracellular cell death pathway, like entry into a new stage of the cell cycle, is usually triggered in a complete, all-or-none fashion. The protease cascade is not only destructive and self-amplifying but also irreversible, so that once a cell reaches a critical point along the path to destruction, it cannot turn back.

Procaspases Are Activated by Binding to Adaptor Proteins

All nucleated animal cells contain the seeds of their own destruction, in the form of various inactive procaspases that lie waiting for a signal to destroy the cell. It is therefore not surprising that caspase activity is tightly regulated inside the cell to ensure that the death program is held in check until needed.

How are procaspases activated to initiate the caspase cascade? A general principle is that the activation is triggered by adaptor proteins that bring multiple copies of specific procaspases, known as initiator procaspases, close together in a complex or aggregate. In some cases, the initiator procaspases have a small amount of protease activity, and forcing them together into a complex causes them to cleave each other, triggering their mutual activation. In other cases, the aggregation is thought to cause a conformational change that activates the procaspase. Within moments, the activated caspase at the top of the cascade cleaves downstream procaspases to amplify the death signal and spread it throughout the cell (see Figure 17-38B).

Procaspase activation can be triggered from outside the cell by the activation of death receptors on the cell surface. Killer lymphocytes (discussed in Chapter 24), for example, can induce apoptosis by producing a protein called Fas ligand, which binds to the death receptor protein Fas on the surface of the target cell. The clustered Fas proteins then recruit intracellular adaptor proteins that bind and aggregate procaspase-8 molecules, which cleave and activate one another. The activated caspase-8 molecules then activate downstream procaspases to induce apoptosis (Figure 17-39A). Some stressed or damaged cells kill themselves by producing both the Fas ligand and the Fas protein, thereby triggering an intracellular caspase cascade.

When cells are damaged or stressed, they can also kill themselves by triggering procaspase aggregation and activation from within the cell. In the best understood pathway, mitochondria are induced to release the electron carrier protein cytochrome c (see Figure 14-26) into the cytosol, where it binds and activates an adaptor protein called Apaf-1 (Figure 17-39B). This mitochondrial pathway of procaspase activation is recruited in most forms of apoptosis to initiate or to accelerate and amplify the caspase cascade. DNA damage, for example, as discussed earlier, can trigger apoptosis. This response usually requires p53, which can activate the transcription of genes that encode proteins that promote the release of cytochrome c from mitochondria. These proteins belong to the Bcl-2 family.

Bcl-2 Family Proteins and IAP Proteins Are the Main Intracellular Regulators of the Cell Death Program

The Bcl-2 family of intracellular proteins helps regulate the activation of procaspases. 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.

Another important family of intracellular apoptosis regulators is the IAP (inhibitor of apoptosis) family. These proteins are thought to inhibit apoptosis in two ways: they bind to some procaspases to prevent their activation, and they bind to caspases to inhibit their activity. IAP proteins were originally discovered as proteins produced by certain insect viruses, which use them to prevent the infected cell from killing itself before the virus has had time to replicate. When mitochondria release cytochrome c to activate Apaf-1, they also release a protein that blocks IAPs, thereby greatly increasing the efficiency of the death activation process.

The intracellular cell death program is also regulated by extracellular signals, which can either activate apoptosis or inhibit it. These signal molecules mainly act by regulating the levels or activity of members of the Bcl-2 and IAP families. We see in the next section how these signal molecules help multicellular organisms regulate their cell numbers.

Summary

In multicellular organisms, cells that are no longer needed or are a threat to the organism are destroyed by a tightly regulated cell suicide process known as programmed cell death, or apoptosis. Apoptosis is mediated by proteolytic enzymes called caspases, which trigger cell death by cleaving specific proteins in the cytoplasm and nucleus. Caspases exist in all cells as inactive precursors, or procaspases, which are usually activated by cleavage by other caspases, producing a proteolytic caspase cascade. The activation process is initiated by either extracellular or intracellular death signals, which cause intracellular adaptor molecules to aggregate and activate procaspases. Caspase activation is regulated by members of the Bcl-2 and IAP protein families.

Mitogens Stimulate Cell Division

Unicellular organisms tend to grow and divide as fast as they can, and their rate of proliferation depends largely on the availability of nutrients in the environment. The cells of a multicellular organism, however, divide only when more cells are needed by the organism. Thus, for an animal cell to proliferate, nutrients are not enough. It must also receive stimulatory extracellular signals, in the form of mitogens, from other cells, usually its neighbors. Mitogens act to overcome intracellular braking mechanisms that block progress through the cell cycle.

One of the first mitogens to be identified was platelet-derived growth factor (PDGF), and it is typical of many others discovered since. The path to its isolation began with the observation that fibroblasts in a culture dish proliferate when provided with serum but not when provided with plasma. Plasma is prepared by removing the cells from blood without allowing clotting to occur; serum is prepared by allowing blood to clot and taking the cell-free liquid that remains. When blood clots, platelets incorporated in the clot are triggered to release the contents of their secretory vesicles (Figure 17-40). The superior ability of serum to support cell proliferation suggested that platelets contain one or more mitogens. This hypothesis was confirmed by showing that extracts of platelets could serve instead of serum to stimulate fibroblast proliferation. The crucial factor in the extracts was shown to be a protein, which was subsequently purified and named PDGF. In the body, PDGF liberated from blood clots probably has a major role in stimulating cell division during wound healing.

PDGF is only one of over 50 proteins that are known to act as mitogens. Most of these proteins are broad-specificity factors, like PDGF and epidermal growth factor (EGF), that can stimulate many types of cells to divide. Thus, PDGF acts on a range of cell types, including fibroblasts, smooth muscle cells, and neuroglial cells. Similarly, EGF acts not only on epidermal cells but also on many other cell types, including both epithelial and nonepithelial cells. At the opposite extreme lie narrow-specificity factors such as erythropoietin, which induces the proliferation of red blood cell precursors only.

In addition to mitogens that stimulate cell division, there are factors, such as some members of the transforming growth factor-β (TGF-β) family, that act on some cells to stimulate cell proliferation and others to inhibit it, or that stimulate at one concentration and inhibit at another. Indeed, like PDGF, many mitogens have other actions beside the stimulation of cell division: they can stimulate cell growth, survival, differentiation, or migration, depending on the circumstances and the cell type.

Cells Can Delay Division by Entering a Specialized Nondividing State

In the absence of a mitogenic signal to proliferate, Cdk inhibition in G₁ is maintained, and the cell cycle arrests. In some cases, cells partly disassemble their cell-cycle control system and exit from the cycle to a specialized, nondividing state called G₀.

Most cells in our body are in G₀, but the molecular basis and reversibility of this state vary in different cell types. Neurons and skeletal muscle cells, for example, are in a terminally differentiated G₀ state, in which their cell-cycle control system is completely dismantled: the expression of the genes encoding various Cdks and cyclins are permanently turned off, and cell division never occurs. Other cell types withdraw from the cell cycle only transiently and retain the ability to reassemble the cell-cycle control system quickly and reenter the cycle. Most liver cells, for example, are in G₀, but they can be stimulated to divide if the liver is damaged. Still other types of cells, including some lymphocytes, withdraw from and re-enter the cell cycle repeatedly throughout their lifetime.

Almost all the variation in cell-cycle length in the adult body occurs during the time the cell spends in G₁ or G₀. By contrast, the time taken for a cell to progress from the beginning of S phase through mitosis is usually brief (typically 12–24 hours in mammals) and relatively constant, regardless of the interval from one division to the next.

Mitogens Stimulate G₁-Cdk and G₁/S-Cdk Activities

For the vast majority of animal cells, mitogens control the rate of cell division by acting in the G₁ phase of the cell cycle. As discussed earlier, multiple mechanisms act during G₁ to suppress Cdk activity and thereby hinder entry into S phase. Mitogens act to release the brakes on Cdk activity, thereby allowing S phase to begin. They do so by binding to cell-surface receptors to initiate a complex array of intracellular signals that penetrate deep into the cytoplasm and nucleus (discussed in Chapter 15). The ultimate result is the activation of G₁-Cdk and G₁/S-Cdk complexes, which overcome the inhibitory barriers that normally block progression into S phase.

As we discuss in Chapter 15, an early step in mitogen signaling is often the activation of the small GTPase Ras, which leads to the activation of a MAP kinase cascade. By uncertain mechanisms, this leads to increased levels of the gene regulatory protein Myc. Myc promotes cell-cycle entry by several overlapping mechanisms (Figure 17-41). It increases the transcription of genes that encode G₁ cyclins (D cyclins), thereby increasing G₁-Cdk (cyclin D-Cdk4) activity. In addition, Myc increases the transcription of a gene that encodes a component of the SCF ubiquitin ligase. This mechanism promotes the degradation of the CKI protein p27, leading to increased G₁/S-Cdk (cyclin E-Cdk2) activity. As discussed earlier, increased G₁-Cdk and G₁/S-Cdk activities stimulate phosphorylation of the inhibitory protein Rb, which then leads to activation of the gene regulatory protein E2F. Myc may also stimulate the transcription of the gene encoding E2F, further promoting E2F activity in the cell. The end result is the increased transcription of genes required for entry into S phase (see Figure 17-30). As we discuss later, Myc also has a major role in stimulating the transcription of genes that increase cell growth.

Abnormal Proliferation Signals Cause Cell-Cycle Arrest or Cell Death

As we discuss in Chapter 23, many of the components of intracellular signaling pathways are encoded by genes that were originally identified as cancer-promoting genes, or oncogenes, because mutations in them contribute to the development of cancer. The mutation of a single amino acid in Ras, for example, causes the protein to become permanently overactive, leading to constant stimulation of Ras-dependent signaling pathways, even in the absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc promote excessive cell growth and proliferation and thereby promote the development of cancer.

Surprisingly, however, when Ras or Myc is experimentally hyperactivated in most normal cells, the result is not excessive proliferation but the opposite: the activation of checkpoint mechanisms causes the cells to undergo either cell-cycle arrest or apoptosis. The normal cell seems able to detect abnormal mitogenic stimulation, and it responds by preventing further division. Such checkpoint responses help prevent the survival and proliferation of cells with various cancer-promoting mutations.

Although it is not known how a cell detects excessive mitogenic stimulation, such stimulation often leads to the production of a cell-cycle inhibitor protein called p19 ^ARF, which binds and inhibits Mdm2. As discussed earlier, Mdm2 normally promotes p53 degradation. Activation of p19^ARF therefore causes p53 levels to increase, thereby inducing either cell-cycle arrest or apoptosis (Figure 17-42).

How do cancer cells ever arise if these mechanisms block the division or survival of mutant cells with overactive proliferation signals? The answer is that the protective system is often inactivated in cancer cells by mutations in the genes that encode essential components of the checkpoint responses, such as p19^ARF or p53.

Human Cells Have a Built-in Limitation on the Number of Times They Can Divide

Cell division is controlled not only by extracellular mitogens but also by intracellular mechanisms that can limit cell proliferation. Many animal precursor cells, for example, divide a limited number of times before they stop and terminally differentiate into permanently arrested, specialized cells. Although the stopping mechanisms are poorly understood, a progressive increase in CKI proteins probably contributes in some cases. Mice that are deficient in the CKI p27, for example, have more cells than normal in all of their organs because the stopping mechanisms are apparently defective.

The best-understood intracellular mechanism that limits cell proliferation occurs in human fibroblasts. Fibroblasts taken from a normal human tissue go through only about 25–50 population doublings when cultured in a standard mitogenic medium. Toward the end of this time, proliferation slows down and finally halts, and the cells enter a nondividing state from which they never recover. This phenomenon is called replicative cell senescence, although it is unlikely to be responsible for the senescence (aging) of the organism. Organism senescence is thought to depend, in part at least, on progressive oxidative damage to macromolecules, in as much as strategies that reduce metabolism (such as reduced food intake), and thereby reduce the production of reactive oxygen species, can extend the lifespan of experimental animals.

Replicative cell senescence in human fibroblasts seems to be caused by changes in the structure of the telomeres, the repetitive DNA sequences and associated proteins at the ends of chromosomes. As discussed in Chapter 5, when a cell divides, telomeric DNA sequences are not replicated in the same manner as the rest of the genome but instead are synthesized by the enzyme telomerase. By mechanisms that remain unclear, telomerase also promotes the formation of protein cap structures that protect the chromosome ends. Because human fibroblasts, and many other human somatic cells, are deficient in telomerase, their telomeres become shorter with every cell division, and their protective protein caps progressively deteriorate. Eventually, DNA damage occurs at chromosome ends. The damage activates a p53-dependent cell-cycle arrest that resembles the arrest caused by other types of DNA damage (see Figure 17-33).

The lack of telomerase in most somatic cells has been proposed to help protect humans from the potentially damaging effects of runaway cell proliferation, as occurs in cancer. Unfortunately, most cancer cells have regained the ability to produce telomerase and therefore maintain telomere function as they proliferate; as a result, they do not undergo replicative cell senescence (discussed in Chapter 23). The forced expression of telomerase in normal human fibroblasts, using genetic engineering techniques, has the same effect (Figure 17-43).

Normal rodent cells, by contrast, usually maintain telomerase activity and telomere function as they proliferate and therefore do not undergo this type of replicative senescence. When overstimulated to proliferate in culture, however, they frequently activate the p19^ARF-dependent checkpoint mechanism described earlier and eventually stop dividing. Mutations that inactivate these checkpoints make it easier for rodent cells to proliferate indefinitely in culture. Such mutant cells are often described as “immortalized”. If cultured in optimal conditions that avoid the activation of checkpoint responses, however, at least some normal rodent cells also seem able to proliferate indefinitely. Nevertheless, rodents age much more rapidly than humans.

Extracellular Growth Factors Stimulate Cell Growth

The growth of an organism or organ depends on cell growth: cell division alone cannot increase total cell mass without cell growth. In single-celled organisms such as yeasts, cell growth (like cell division) requires only nutrients. In animals, by contrast, cell growth and cell division both depend on signals from other cells.

The extracellular growth factors that stimulate cell growth bind to receptors on the cell surface and activate intracellular signaling pathways. These pathways stimulate the accumulation of proteins and other macromolecules, and they do so by both increasing their rate of synthesis and decreasing their rate of degradation.

One of the most important intracellular signaling pathways activated by growth factor receptors involves the enzyme PI 3-kinase, which adds a phosphate from ATP to the 3 position of inositol phospholipids in the plasma membrane. As discussed in Chapter 15, the activation of PI 3-kinase leads to the activation of several protein kinases, including S6 kinase. The S6 kinase phosphorylates ribosomal protein S6, increasing the ability of ribosomes to translate a subset of mRNAs, most of which encode ribosomal components. Protein synthesis therefore increases. When the gene encoding S6 kinase is inactivated in Drosophila, the mutant flies are small; whereas cell numbers are normal, cell size is abnormally small. Growth factors also activate a translation initiation factor called eIF4E, further increasing protein synthesis and cell growth (Figure 17-44).

Growth factor stimulation also leads to increased production of the gene regulatory protein Myc, which also plays an important part in signaling by mitogens (see Figure 17-41). Myc increases the transcription of a number of genes that encode proteins involved in cell metabolism and macromolecular synthesis. In this way, it stimulates both cell metabolism and cell growth.

Some extracellular signal proteins, including PDGF, can act as both growth factors and mitogens, stimulating both cell growth and cell-cycle progression. This functional overlap is achieved in part by overlaps in the intracellular signaling pathways that control these two processes. The signaling protein Ras, for example, is activated by both growth factors and mitogens. It can stimulate the PI3-kinase pathway to promote cell growth and the MAP-kinase pathway to trigger cell-cycle progression. Similarly, as described above, Myc stimulates both cell growth and cell-cycle progression. Extracellular factors that act as both growth factors and mitogens help ensure that cells maintain their appropriate size as they proliferate.

Cell growth and division, however, can be controlled by separate extracellular signal proteins in some cell types. Such independent control may be particularly important during embryonic development, when dramatic changes in the size of certain cell types can occur. Even in adult animals, however, growth factors can stimulate cell growth without affecting cell division. The size of a sympathetic neuron, for example, which has permanently withdrawn from the cell cycle, depends on the amount of nerve growth factor (NGF) secreted by the target cells it innervates. The greater the amount of NGF the neuron has access to, the larger it becomes. It remains a mystery, however, how different cell types in the same animal grow to be so different in size (Figure 17-45).

Extracellular Survival Factors Suppress Apoptosis

Animal cells need signals from other cells—not only to grow and proliferate, but also to survive. If deprived of such survival factors, cells activate their intracellular death program and die by apoptosis. This arrangement ensures that cells survive only when and where they are needed. Nerve cells, for example, are produced in excess in the developing nervous system and then compete for limited amounts of survival factors that are secreted by the target cells they contact. Nerve cells that receive enough survival factor live, while the others die by apoptosis (Figure 17-46). A similar dependence on survival signals from neighboring cells is thought to control cell numbers in other tissues, both during development and in adulthood.

Survival factors, just like mitogens and growth factors, usually bind to cell-surface receptors. Binding activates signaling pathways that keep the death program suppressed, often by regulating members of the Bcl-2 family of proteins. Some factors, for example, stimulate the increased production of apoptosis-suppressing members of this family. Others act by inhibiting the function of apoptosis-promoting members of the family (Figure 17-47A). In Drosophila, and probably in vertebrates as well, some survival factors also act by stimulating the activity of IAPs, which suppress apoptosis (Figure 17-47B).

Neighboring Cells Compete for Extracellular Signal Proteins

When most types of mammalian cells are cultured in a dish in the presence of serum, they adhere to the bottom of the dish, spread out, and divide until a confluent monolayer is formed. Each cell is attached to the dish and contacts its neighbors on all sides. At this point, normal cells, unlike cancer cells, stop proliferating—a phenomenon known as density-dependent inhibition of cell division. This phenomenon was originally described in terms of “contact inhibition” of cell division, but it is unlikely that cell-cell contact interactions are solely responsible. The cell population density at which cell proliferation ceases in the confluent monolayer increases with increasing concentration of serum in the medium. Moreover, passing a stream of fresh culture medium over a confluent layer of fibroblasts reduces the diffusional limitation to the supply of mitogens, and it induces the cells under the stream of medium to divide at densities at which they would normally be inhibited from doing so (Figure 17-48). Thus, density-dependent inhibition of cell proliferation seems to reflect, in part at least, the ability of a cell to deplete the medium locally of extracellular mitogens, thereby depriving its neighbors.

This type of competition could be important for cells in tissues as well as in culture, because it prevents them from proliferating beyond a certain population density, determined by the available amounts of mitogens, growth factors, and survival factors. The amounts of these factors in tissues is usually limited, and increasing their amounts results in an increase in cell number, cell size, or both. Thus, the concentrations of these factors in tissues have important roles in determining cell size and number.

Many Types of Normal Animal Cells Need Anchorage to Grow and Proliferate

The shape of a cell changes as it spreads and crawls out over a substratum to occupy vacant space, and this can have a major impact on cell growth, cell division, and cell survival. When normal fibroblasts or epithelial cells, for example, are cultured in suspension, unattached to any solid surface and therefore rounded up, they almost never divide—a phenomenon known as anchorage dependence of cell division (Figure 17-49). But when these cells are allowed to settle and adhere to a sticky substrate, they rapidly form focal adhesions at sites of attachment, and then begin to grow and proliferate.

How are the growth and proliferation signals generated by cell attachments? Focal adhesions are places where extracellular matrix molecules, such as laminin or fibronectin, interact with cell-surface matrix receptors called integrins, which are linked to the actin cytoskeleton (discussed in Chapter 19). The binding of extracellular matrix molecules to integrins leads to the local activation of protein kinases, including focal adhesion kinase (FAK), which in turn leads to the activation of intracellular signaling pathways that can promote the survival, growth, and division of cells (Figure 17-50).

Like other controls on cell division, anchorage control operates in G₁. Cells require anchorage to progress through G₁ into S phase, but anchorage is not required for completing the cycle. In fact, cells commonly loosen their attachments and round up as they pass through M phase. This cycle of attachment and detachment presumably allows cells in tissues to rearrange their contacts with other cells and with the extracellular matrix. In this way, tissues can accommodate the daughter cells produced by cell division and then bind them securely into the tissue before they are allowed to begin the next division cycle.

Some Extracellular Signal Proteins Inhibit Cell Growth, Cell Division, and Survival

The extracellular signal proteins discussed in this chapter—mitogens, growth factors and survival factors—are positive regulators of cell-cycle progression, cell growth, and cell survival, respectively. They therefore tend to increase the size of organs and organisms. In some tissues, however, cell and tissue size also is influenced by inhibitory extracellular signal proteins that oppose the positive regulators and thereby inhibit organ growth.

The best-understood inhibitory signal proteins are TGF-β and its relatives. TGF-β inhibits the proliferation of several cell types, either by blocking cell-cycle progression in G₁ or by stimulating apoptosis. As discussed in Chapter 15, TGF-β binds to cell-surface receptors and initiates an intracellular signaling pathway that leads to changes in the activities of gene regulatory proteins called Smads. This results in complex and poorly understood changes in the transcription of genes encoding regulators of cell division and cell death.

One example of an apoptosis-inducing extracellular signal is bone morphogenetic protein (BMP), a TGF-β family member. BMP helps trigger the apoptosis that removes the tissue between the developing digits in the mouse paw (see Figure 17-35). Like TGF-β, BMP stimulates changes in the transcription of genes that regulate cell death, although the nature of these genes remains unclear.

The overall size of an organ may be limited in some cases by inhibitory signaling proteins. Myostatin, for example, is a TGF-β family member that normally inhibits the proliferation of myoblasts that fuse to form skeletal muscle cells. When the gene that encodes myostatin is deleted in mice, muscles grow to be several times larger than normal (see Figure 22-43). Both the number and the size of muscle cells increase. Remarkably, two breeds of cattle that were bred for large muscles have both turned out to have mutations in the gene encoding myostatin (Figure 17-51).

Intricately Regulated Patterns of Cell Division Generate and Maintain Body Form

The life of multicellular organisms begins with a series of division cycles that are controlled according to intricate rules. This is strikingly illustrated by the nematode Caenorhabditis elegans. The fertilized egg of C. elegans divides to produce an adult worm with precisely 959 somatic cell nuclei (in the male), each of which is generated by its own characteristic and absolutely predictable sequence of cell divisions. (The initial cell number is greater than this, but more than 100 cells die by apoptosis during development.) In general, the controls that generate such precise cell numbers do not operate by merely counting cell divisions according to a clocklike schedule. Instead, the organism seems mainly to control total cell mass, which depends not only on cell numbers but also on cell size. Salamanders of different ploidies, for example, are the same size but have different numbers of cells. Individual cells in a pentaploid salamander are about five times the volume of those in a haploid salamander, and in each organ the pentaploids have generated only one-fifth as many cells as their haploid cousins, so that the organs are about the same size in the two animals (Figures 17-52 and 17-53). Evidently, in this case (and in many others) the size of organs and organisms depends on mechanisms that can somehow measure total cell mass.

The development of limbs and organs of specific size and shape depends on complex positional controls, as well as on local concentrations of extracellular signal proteins that stimulate or inhibit cell growth, division, and survival. As we discuss in Chapter 21, some of the genes that help pattern these processes in the embryo are now known. A great deal remains to be learned, however, about how these genes regulate cell growth, division, survival, and differentiation to generate a complex organism (discussed in Chapter 21).

The controls that govern these processes in an adult body are also poorly understood. When a skin wound heals in a vertebrate, for example, about a dozen cell types, ranging from fibroblasts to Schwann cells, must be regenerated in appropriate numbers and in appropriate positions to reconstruct the lost tissue. The mechanisms that control cell proliferation in tissues are likewise central to the understanding of cancer, a disease in which the controls go wrong, as discussed in Chapter 23.

Summary

In multicellular animals, cell size, cell division, and cell death are carefully controlled to ensure that the organism and its organs achieve and maintain an appropriate size. Three classes of extracellular signal proteins contribute to this control, although many of them affect two or more of these processes. Mitogens stimulate the rate of cell division by removing intracellular molecular brakes that restrain cell-cycle progression in G₁. Growth factors promote an increase in cell mass by stimulating the synthesis and inhibiting the degradation of macromolecules. Survival factors increase cell numbers by inhibiting apoptosis. Extracellular signals that inhibit cell division or cell growth, or induce cells to undergo apoptosis, also contribute to size control.