The process of cell reproduction is known as the cell cycle.1–3 Usually the cell cycle produces two progeny, or daughter cells, that closely resemble their parent and who are themselves capable of repeating the process. For this to occur, three things are necessary: replication of the genome; a doubling of cell mass (where cell mass refers generally to all cellular components other than chromosomes); and a precise segregation of chromosomes plus a more or less equal distribution of other cell components to the daughter cells. The execution of these events divides the cell cycle into four phases: chromosomes are replicated during S (synthetic) phase; cell constituents are segregated to daughter cells during M (mitotic) phase; and two G (gap) phases intervene between S and M. G1 precedes S phase, and G2 precedes mitosis (Fig. 23-1). Thus, chromosome replication and segregation are confined to discrete intervals of the cell cycle, whereas the third essential component of cell reproduction—growth—occurs continuously in G1, S, G2, and M. It is during G1 and G2 that cells typically respond to the proliferative and antiproliferative signals that determine whether the cell cycle ought to proceed (signals such as growth factors and cytokines). In this way, the cell cycle has the option of stopping within G1 and G2 without interrupting the critical and precarious events of chromosome replication and chromosome segregation.
The four phases of the cell cycle. Interphase is composed of S (synthesis) phase, during which time DNA replication occurs, and two G (gap) phases, during which cells respond to various proliferative and antiproliferative stimuli and cell growth occurs. Chromosomes and cellular contents are than distributed to two daughter cells during M (mitosis) phase, and the resulting progeny re-enter the cell cycle in G1.
Faithful reproduction of the cell requires that these events be coordinated with one another. Thus, mitosis ordinarily waits until all chromosomes have been replicated and the cell has doubled in size. However, there are specialized cell cycles where these processes are uncoupled from one another (Fig. 23-2). Repeated S phases with no intervening M phases, known as endocycles, result in the increased chromosome ploidy that is seen in megakaryocytes. Conversely, the basic cell-cycle logic of meiosis is the execution of two sequential M phases without an S phase. A third important variation is seen in the cleavage cycles that occur after fertilization of amphibian eggs. Amphibian eggs are huge cells, which, after fertilization, undergo extremely rapid cell cycles consisting of alternating S and M phases with no cell growth. After approximately 12 cleavage cycles, the embryo consists of 4000 cells, each containing a full complement of genetic material, and each now reduced to the size of a typical somatic cell.4,5
Specialized cell cycles. A, A normal cell cycle is depicted in which a cell gives rise to two identical daughter cells. B, During megakaryopoiesis, promegakaryocytes undergo repeated rounds of DNA replication in the absence of mitosis (endoreduplication), resulting in polyploid megakaryocytes with a DNA content greater than their progenitors. C, In meiosis, two successive cell divisions after DNA replication result in four haploid daughter cells. D, Amphibian eggs undergo 12 rapid cell cycles consisting of alternating S and M phases. No cell growth occurs during these cycles, and the large egg cell is subdivided into approximately 4000 cells, each containing a normal complement of chromosomes.
These simple examples show that each of the component processes of the cell cycle—growth, chromosome replication, and mitosis—can occur independently of the others. Because cell reproduction could not occur if these processes were executed in random order, there need to be mechanisms for establishing and enforcing the normal sequence of events. This chapter describes the molecules that control progression through the cell cycle, and illustrates how their activities are linked together to orchestrate the orderly process of cell reproduction. Based on these ideas, we suggest that cancer may be a disease of the cell cycle, a hypothesis that is elaborated on in subsequent chapters.
Origins of Modern Cell Cycle Biology
The current revolution in our understanding of cell-cycle control owes its origins to yeast genetics and amphibian reproductive cell physiology. It was through these seemingly independent lines of investigation that we came to grasp the fundamental logic of the program that controls cell reproduction, identified the genes and molecules responsible for this program, and learned how these molecules are integrated into pathways that have been evolutionarily conserved from yeast to humans.
The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been favorites of cell-cycle research since the pioneering studies of Lee Hartwell and Paul Nurse.6,7 The power of yeast as a model experimental system is in its facile genetics. It is possible to readily isolate mutations that impair the execution of specific biological processes, such as the events of the cell cycle. Identification of the mutated gene provides information about the proteins required for the execution of that biologic pathway. Of special utility are conditional mutations, because they effect the activity of the encoded protein product only under specific restrictive conditions, elevated temperatures for example. Cells harboring conditional mutations can be first collected and propagated by growth under permissive conditions, and the consequences of the mutation then determined by shifting growth to restrictive conditions.
S. cerevisiae is known as budding yeast, because it reproduces by forming a bud that grows to become the daughter cell. The regular pattern of bud development provides convenient morphologic landmarks that can be used to assemble a temporal map of the cell cycle.8 Thus, an unbudded cell is in G1, a small bud first emerges coincident with initiation of S phase, and a cell with a large bud is in G2/M. Hartwell isolated a large group of genes required for progression through the cell cycle by identifying conditional mutations that caused a cell population to arrest with a uniform morphology (e.g., all unbudded cells). Because the morphology of a yeast cell defines its position within the cell cycle, each mutant presumably had a position-specific defect in the operation of a cell cycle event. He called these cdc mutations (for “cell division cycle”).9 Thus, some cdc mutants were defective in G1-specific events (and arrested as unbudded cells), others in S phase (small-budded cells), and yet others in events that take place in G2/M (large-budded cells). Further analysis of nuclear morphology and the state of the mitotic spindle gave additional information about the specific processes affected by each mutation. In this way, Hartwell successfully identified the vast majority of genes responsible for regulating the eukaryotic cell cycle.10
The characterization of cdc mutations revealed a major principle of cell cycle regulation—that the orderly execution of cell cycle events resulted from a series of dependent relationships in which the completion of one event is required for the beginning of the next.9 For instance, a cell with a mutation in a gene required for DNA replication stops its cell cycle in S phase and does not inappropriately try to begin mitosis (even though it should be capable of doing so). Initially, these dependencies were thought to reflect underlying biochemical pathways in which the product of one event was an essential substrate for the next. Hence, only after an upstream event occurred correctly would the substrates for the next event become available. This is one way to insure that cell cycle events were not executed in random order. The idea that the cell cycle is organized by dependent relationships is still considered one of the most important in cell-cycle biology, but the explanation for dependencies has changed and is discussed shortly.
A particularly important set of dependent pathways is initiated at the transition from G1 into S phase; this is called cell-cycle START.11 Uniquely at START, the yeast cell senses the external and internal signals that control its proliferation, including mating pheromone, nutrients, and cell size (Fig. 23-3).12–14 The yeast cell responds by initiating (or failing to initiate) the three parallel pathways required for reproduction of the cell—bud emergence, DNA replication, and spindle pole body duplication (the spindle pole body being the yeast equivalent of the centrosome of the mitotic spindle). The coordinate regulation of these three parallel-reproductive pathways indicates that completion of START represents the commitment of the cell to complete the entire program of events required for cell reproduction. Thus, genes required for START must play pivotal roles in the control of cell proliferation, and among the handful of START-specific genes, the CDC28 gene has gained particular prominence.6,15,16 Analogous to START, the R POINT (restriction point) in mammalian cells defines a transition within G1 after which completion of the remainder of the cell cycle becomes independent of extracellular mitogens.
START and the R (restriction) POINT define G1 transitions after which cell cycle progression becomes mitogen independent. At START, budding yeast initiate three independent processes required for cell duplication: bud emergence, DNA synthesis, and spindle-body duplication. A variety of mitogenic and antimitogenic signals determine if cells traverse START, after which cell cycle progression no longer depends upon these stimuli. At the analogous R POINT in mammalian cells, S-phase entry is no longer mitogen dependent. The biochemical bases of START and the R-POINT are discussed in the text.
In contrast to the budding yeast cell cycle, during which cell growth and cell division are linked in G1 at START, in S. pombe (known also as fission yeast), these processes are usually coordinated at the transition between G2 and mitosis.17 Paul Nurse identified cdc mutants in S. pombe that were unable to undergo the G2/M transition and one mutant, called cdc2, received special attention.18–20 First, CDC2 is required twice during the fission yeast cell cycle, once at the G2/M transition and again at the G1/S transition (where a back-up size control exists). Second, certain dominantly acting mutations of cdc2 caused cells to shorten the length of G2, enter mitosis too quickly, and, consequently, become smaller than normal (known as a “wee” phenotype). This suggests that CDC2 activity is rate limiting for the onset of mitosis. The budding yeast CDC28 gene and the S. pombe CDC2 gene are homologs of one another.17,19 Although CDC28 was first identified through its role at G1/S and cdc2 by its role at G2/M, it is now known that these proteins are required at the G1/S and at G2/M transitions in both types of yeast.17,21,22 Because of these experiments, CDC2/CDC28 emerged as a key regulator of the cell cycle.
Complementing these genetic analyses of the yeast cell cycle were studies on the meiotic maturation of amphibian eggs. These physiological studies led to the discovery of a natural regulator of cell-cycle progression, an activity named MPF (maturation promoting factor).23–25 Amphibians produce mature eggs in response to the hormone progesterone, which induces immature oocytes to emerge from a prolonged arrest in G2 and resume the meiotic cell cycle. The progesterone-stimulated oocyte completes the reductive meiotic divisions and eventually pauses again in metaphase of meiosis II, but now as a mature egg awaiting fertilization. In other words, oocyte maturation requires cell-cycle progression from G2 of meiosis I to metaphase of meiosis II, and is under the control of MPF. Indeed, injection of MPF isolated from a mature egg into the cytoplasm of an immature oocyte is sufficient to initiate meiotic maturation independently of any hormonal trigger (Fig. 23-4). Furthermore, MPF activity is not restricted to meiosis. MPF activity oscillates during each mitotic cycle, being high in M phase and low in interphase.24,26,27 This indicates that MPF is a fundamental component of cell-cycle regulation in all cell cycles, mitotic as well as meiotic.
MPF stimulates mitosis in G2-oocytes. As described in the text, injection of cytoplasm extract from an M-phase amphibian egg is sufficient to induce maturation of the recipient egg in the absence of progesterone or protein synthesis.
Remarkably, MPF activity will continue to oscillate even in enucleated cells.28 Because MPF activity can oscillate independently of nuclear events, it has been proposed that the MPF cycle might be an autonomous mitotic clock, and that state of the clock (the “time of day”) determines which cell cycle event occurs.26,28,29 Thus, high MPF activity would permit entry into mitosis, and low MPF activity would permit cells to enter S phase. This model supplanted the earlier idea that the obligate order of cell cycle events might simply be established by substrate-product-type relationships. However, the notion of a mitotic clock would then require the existence of additional feedback controls to keep the clock entrained to actual progress through the cell cycle; the clock must stop if essential cell cycle events do not occur. These feedback controls do exist, and are known as checkpoints.30
Perhaps the most far-reaching advance in the cell-cycle field in the last 15 years has been the demonstration that cell-cycle controls are evolutionarily conserved.31,32 This first became evident when it was discovered that the CDC2 and CDC28 genes in fission and budding yeast encode homologous proteins, 17 now known as the CDC2 protein kinase. Furthermore, gene transfer experiments show that the human CDC2 gene can complement mutations in the yeast CDC2 gene.33 This was reinforced 6 years later when MPF was purified (initially from Xenopus, and later from other vertebrates), and its catalytic subunit was shown to be a homolog of CDC2.34–38 These observations set the stage for our current paradigm of eukaryotic cell cycle control, which in its simplest form depicts the cell cycle as a CDC2 cycle (Fig. 23-5).39 Thus, in organisms ranging from yeast to humans, the catalytic activity of CDC2 and related kinases is required for each of the major transitions within the cell cycle—from G1 into S, and from G2 into M.40–46
The cdc2 cycle. The enzymatic activity of cdc2 and related kinases (cdks) drives each of the key cell cycle transitions.
In budding and fission yeast, the highly regulated action of a single kinase subunit (cdc28 or cdc2, respectively) drives the cell cycle forward.47 In higher eukaryotes, cell-cycle control is more complex, and several proteins homologous to cdc2 (termed the cyclin-dependent kinases or cdks) have been identified.48 Cyclin-dependent kinases are protein kinases that vary in size between 30 and 40 kb and share greater than 40 percent sequence identity. In addition to amino acid homology, cdks share many functional and regulatory features with yeast cdc2/28.49,50 Almost all cdks require association with protein subunits called cyclins to become active kinases. Cdks also contain conserved amino acid residues that modulate kinase activity when phosphorylated or dephosphorylated. Additionally, specific regulatory molecules that bind and inhibit cdk subunits inhibit cdk activity. Each of these regulatory mechanisms is discussed in detail below. Remarkably, while differences among organisms do exist, this multi-tiered regulatory system has been conserved from yeast to humans.
Each phase of the cell cycle is characterized by a unique pattern of cdk activity (Fig. 23-6).51–53 In mammalian cells, eight cdks have been identified, and most are active (and required) only in specific phases of the cell cycle. Progression through G1 phase depends upon the activities of cdk2, cdk3, cdk4, and cdk6. The recently described cdk8 protein also may function primarily in G1, and may be involved in transcriptional regulation. Cdk2 and cdc2 are active in S-phase, and cdc2 kinase activity also governs mitotic entry and exit. In distinction to its kindred, cdk5 does not appear to be intimately involved in cell-cycle progression, but may, instead, play a role in the developing nervous system, where it associates with the non-cyclin activator p35.54,55
Patterns of cyclin-cdk activity during the mammalian cell cycle. The expression patterns of the key mammalian cyclins is superimposed upon the cell cycle, along with their respective cdk partners. The approximate position of the R POINT is shown (adapted from Sherr51).
Cyclins: Activating Subunits of CDK Enzymes
Monomeric cdk subunits are essentially devoid of enzymatic activity, and kinase activation requires the association of cdks with cyclins.49 An active cdk is thus a heterodimeric enzyme consisting of regulatory (cyclin) and enzymatic (cdk) subunits. The cyclins are a group of related proteins that contain a conserved region of homology (the cyclin box) and are usually expressed in a cell cycle specific fashion. Cyclin expression is rate-limiting for cdk activation, and control of cyclin expression is a fundamental mechanism underlying cdk periodicity. In general, cyclin levels are determined by both transcriptional control and regulated proteolysis by the ubiquitin-proteosome system.
The recently solved crystal structure of cyclin A bound to cdk2 reveals that cyclins activate cdks in at least two ways. Cyclin binding induces conformational changes in the cdk that first reorients the configuration of the ATP phosphate groups to facilitate phosphotransfer to protein substrates, and second moves the T-loop of the cdk out of a position that would otherwise block entry of protein substrates into the active site (Fig. 23-7).56,57
Crystal structure of cdk2, cyclin A-cdk2, and cyclin A-cdk2-p27 complexes. A, The structure of monomeric cdk2. The T-loop is indicated in yellow, and the PSTAIRE motif in red. An ATP molecule in indicated within the active site. B, The structure of cdk2 bound to an amino-terminal truncated version of cyclin A. Cyclin binding re-orients the PSTAIRE helix and moves the T-loop, resulting in the re-positioning of ATP-phosphate groups within the complex and allowing substrate accessibility to the active site. C, The structure of cdk-activating kinase (CAK)-phosphorylated cyclin A bound to cdk2. The yellow ball indicates the position of thr160 within the T-loop. D, The structure of a ternary complex of the amino terminus of p27 bound to cyclin A -cdk2. Separate domains of P27 interact with both cyclin A and cdk2. The structure of this complex reveals that p27 inhibits cdk activity by distorting the structure of the active site, and by binding within the catalytic cleft and preventing ATP binding.
The specificity of cdk action at different times in the cell cycle is in large part determined by its particular cyclin subunit. This functional diversity of cyclins was first established in the yeast S. cerevisiae where specific cyclins have been identified that are required for G1, S phase, and mitosis. Budding yeast express three functionally redundant G1 cyclins (cln1, cln2, and cln3), which are required for passage through START.58–62 While differences between the cln genes have been described, mutant yeast cells with mutated cln alleles can still enter S phase as long as one of these three genes remains functional. Transcription of cln1 and cln2 is controlled by the Swi4/Swi6 transcription factor, 63–65 and cln activity positively reinforces further cln expression.66–68 Thus, cln1 and cln2 mRNAs rise during G1, reaching peak levels around START.
Once START has been traversed, cln activity is no longer required for subsequent cell-cycle progression. Instead, other cyclins associate with and activate cdc28 during other phases of the cell cycle. Complexes containing cdc28 and the cyclins clb5 and clb6 are required for S phase, 69,70 and the four B-type cyclins, clb1 to clb4 are required for mitosis.71
START marks a point of transition in the yeast cell cycle where G1 cyclin expression ends and mitotic cyclin expression begins. This transition comes about because these two classes of cyclin modulate each other's expression. Cln/cdc28 kinase activity directly increases the expression of the clb genes, and conversely clb-cdc28 kinase activity represses cln expression.72 Not only do G1 cyclins promote the expression of the genes for mitotic cyclins but, as described below, they also increase the stability and functional activity of clb proteins. Furthermore, the cln proteins themselves are rapidly degraded after START. This is discussed below in the section on cell-cycle regulated proteolysis. Together, these controls insure ordered progression through the cell cycle by establishing alternating periods of the cell cycle where either G1 or mitotic cyclins are expressed and functionally active.
Mammalian cyclins C, D1, and E were first identified in a screen for mammalian genes that could complement yeast cyclin mutations.73–75 At the same time Cyclin D1 was identified by two other approaches—as a mitogen-responsive gene in a macrophage cell line76 and as a gene located at a chromosome inversion breakpoint in a parathyroid tumor (and in this guise was originally named PRAD1).77 A dozen mammalian cyclin genes have been identified that are both structurally and functionally homologous to yeast cyclins.51,52 Like the yeast cyclins, many of these molecules exhibit cell-cycle-dependent periodicity in their expression and activity (Fig. 23-6).
The primary mammalian G1 cyclins are the D-type cyclins and cyclin E. These cyclins associate with the cdk-4/6, and cdk2 subunits, respectively. There are three D-type cyclins (D1, D2, and D3), which are expressed in a cell-type specific fashion.51,52,78 The G1 role of the D-type cyclins is revealed by their pattern of expression and by their functional properties. Cyclin D expression begins in early G1 when quiescent cells are stimulated to reenter the cell cycle, and cyclin D expression remains at high levels as long as mitogens are present. In other words, the expression of these labile proteins (t½<20 min) is not intrinsically periodic, but instead depends upon the presence of cell-type specific mitogens. Inhibition of cyclin D1 function blocks the cell cycle in G1, demonstrating the necessity of cyclin D for the cell cycle.79,80 Also, enforced overexpression of cyclin D1 shortens the G1 phase of the cell cycle, and partially diminishes the mitogen requirement for cell proliferation, demonstrating that cyclin D1 levels are limiting for G1 progression.80,81
Cyclin E activity is also required in G1, although probably somewhat after cyclin D activity.81–83 Cyclin E protein expression peaks at the G1-S boundary, and then decays as S-phase progresses.82,84,85 Determinants of cyclin E periodicity include both transcriptional control by E2F and regulated proteolysis (see below). Overexpression of cyclin E results in G1 contraction and decreased mitogen requirements, 81,86 and cyclin E kinase activity is required for S-phase entry.82,83 Activation of cyclin D and cyclin E-associated kinases may biochemically constitute the restriction point, the mammalian equivalent of START control in yeast.
Later cell-cycle transitions are governed by the cyclin A and cyclin B proteins. Cyclin A associates with both the cdk2 and cdc2 subunits, and cyclin A kinase activity is required at the start of S phase and at the G2-M transition.87–89 Cyclin B associates with cdc2, and, like the yeast clb proteins, cyclin B-cdc2 kinase activity regulates both mitotic entry and exit.90
The cyclin H protein associates with cdk7, and this heterodimer constitutes the cdk-activating kinase (CAK).91 CAK is also a component of the human transcription factor TFIIH, and is capable of phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin C is classed as a G1 cyclin, although its role is not yet defined.73 Cyclin C has recently been shown to associate with cdk8, and cyclin C-cdk8 complexes also have RNA polymerase II CTD kinase activity, although they do not co-purify with TFIIH.92 Phosphorylation of the CTD by cyclin-cdk complexes may couple cell-cycle events to the cellular transcriptional machinery.
Comparatively little is known about the remaining cyclins that have been identified to date, including cyclins F, G and I. Cyclin F is the largest cyclin, with a molecular weight of 87, and is most closely related to cyclins A and B. Cyclin F mRNA peaks in G2 and cyclin F protein accumulates in interphase and is destroyed during mitosis.93 Cyclin G mRNA does not fluctuate in a cell cycle-dependent fashion, but is induced by both the p53 protein and growth stimulation of quiescent cells.94 The cyclin I protein is expressed most highly in post-mitotic tissues, including muscle and neurons, and may have a unique regulatory role.95
CDK Regulation by Phosphorylation and Dephosphorylation
In addition to cyclin binding, phosphorylation and dephosphorylation of conserved cdk residues provides another important level of control over kinase activity.49 Cdks can be either activated or inactivated by phosphorylation (Fig. 23-8). The site of activating phosphorylation is a conserved threonine residue in the so-called “T-loop” (e.g., threonine 161 in cdc2, threonine 160 in cdk2).56 The binding of cyclin to the cdk, and phosphorylation of this residues together move the T-loop away from the catalytic cleft of the enzyme, thereby providing access to protein substrates.96 Thr160 is phosphorylated by CAK (cyclin H-cdk7), and this phosphorylation is required for cdk activation.97–100 CAK activity, however, is neither cell-cycle regulated nor limiting, and the major determinant of Thr160 phosphorylation is probably cyclin binding.91
CDK regulation by cyclin binding and cdk phosphorylation. As described in the text, activation of cdks requires cyclin binding and cdk phosphorylation at thr160 by the cdk-activating kinase (CAK). Subsequent phosphorylation of Tyr15 by the wee1 and mik1 kinases and dephosphorylation by cdc25 phosphatases further regulates kinase activity.
Cdks can also be phosphorylated on a specific amino-terminal tyrosine residue (e.g., tyrosine 15 in cdc2 and cdk2). Tyrosine phosphorylated cdk2 is catalytically inactive, even if it is phosphorylated on the activating threonine within the T-loop.98,100–103 The kinases that phosphorylate tyr15 are evolutionarily conserved and are known as the wee1 and mik1 kinases.104–107 Conversely, dephosphorylation of tyrosine by the cdc25 phosphatase activates the cdk.108–112 Regulation of wee1 and cdc25 is complex, 113,114 but the bottom-line is that the relative activities of these enzymes set a threshold for cdk activation and determine mitotic entry. Three mammalian cdc25 homologues have been identified (cdc25a, cdc25b, and cdc25c), and each may have a unique cell-cycle role.115,116 Cdc25a is active in G1 and may be induced by raf-dependent pathways.117
CKIs: Inhibitory Subunits of CDK Enzymes
All organisms express proteins that directly bind to and inhibit cdk activity.118,119 These cdk-inhibitors (CKIs) provide another important strategy by which cdk activity is regulated in response to diverse stimuli. In budding yeast, two kinds of inhibitors have been described. One type is inducible and links the cell cycle to extracellular signals; the other type is an intrinsic component of the mitotic cycle. The best example of the first type of CKI is the FAR1 protein. Mating pheromones induce FAR1, a protein that binds to and inhibits the cln-cdc28 kinase and thereby causes the yeast cell cycle to arrest at START. Another important CDK inhibitor in budding yeast is Sic1, but it is a constitutive element in the mitotic clock and is not known to be induced by extrinsic proliferative signals.120,121 At the conclusion of each mitosis, Sic1 protein levels rise, inhibiting the clb-cdc28 kinases and facilitating the transition from anaphase to the next G1. Sic1 protein remains at high levels during G1 until activation of the cln-cdc28 kinases at START induce its degradation. This is one mechanism that links activation of the S-phase clb-cdc28 kinases to passage through START.
Mammalian cells express two classes of CKIs that are distinguished by their cdk targets: the Cip/Kip family of CKI's are universal cdk inhibitors, whereas the INK4 proteins are specific cdk4/6 inhibitors (Fig. 23-9).118 The Cip/Kip family consists of three members: p21, p27, and p57. Overexpression of these molecules causes a G1 arrest in cultured cells, and they are able to inhibit most cyclin-cdk complexes in vitro. These molecules bind to assembled cyclin-cdk complexes much more avidly than to monomeric cdk or cyclin subunits. p21 was first identified as a component of cyclin-CDK complexes in proliferating cells123 and as a protein induced as cells in vitro became senescent.124 Shortly thereafter, it was cloned by a number of independent approaches.125–128 The p21 protein contains two functional domains, an amino terminal cdk interaction region that is sufficient for cdk inhibition, and a carboxy-terminal region that binds PCNA, a processivity factor associated with DNA polymerase delta.129–131
Mammalian CDK-inhibitors are classed by their cyclin-cdk targets. The Cip/Kip proteins (p21, p27, p57) are universal cdk inhibitors that inactivate all cyclin-cdk complexes (with the possible exception of cyclin B-cdc2). In contrast, the INK4 proteins (p15, p16, p18, p19) specifically bind and inhibit only cdk4/6, and cyclin D-cdk4/6 complexes.
Two biological roles have been suggested for p21.118,122 The first is in contributing to the cell-cycle arrest that occurs in cells with damaged DNA.127 This is discussed in greater detail below. Additionally, p21 has been suggested as a facilitator of withdrawal from the cell cycle in cells undergoing terminal differentiation.132,133
The CKI p27Kip1 is structurally related to p21Cip1.134,135 p21 and p27 share significant amino terminal homology within the cdk inhibitory domain, but p27 does not contain the PCNA interaction region. p27 is not a p53 response gene, but p27 levels respond instead to a variety of extracellular mitogenic and antimitogenic signals.118 In general, p27 levels are high in nondividing cells and low in proliferating cells. The regulation of p27 is complex, with transcriptional, translational and post-translational mechanisms all implicated in different biological contexts.136
The mechanism of cdk inhibition by p27 has been clarified by the crystal structure of p27 bound to cyclin A-cdk2 (Fig. 23-7).96 In the ternary p27-cyclin A-cdk2 complex, separate domains in p27 interacts with the cyclin and the cdk. Although p27 does not significantly alter the structure of the cyclin, it may bind to a site on the cyclin that the cyclin would ordinarily use for interactions with protein substrates. In this way, p27 might inhibit phosphorylation of physiologically important substrates without inhibiting the catalytic activity of the cdk enzyme. In addition, p27 does have dramatic effects on cdk structure. p27 disrupts the structure of the N-terminal lobe of the cdk, widening and distorting the ATP-binding site. In fact, p27 itself inserts into the catalytic cleft and directly interacts with the amino acids that would bind ATP. This would completely prevent cdk-binding of ATP and completely inhibit catalytic activity.
Less is known about p57, the most recently isolated family member that was cloned by virtue of its homology with p27.137,138 Both the amino and carboxy-terminal domains of p57 are related to p27. Compared with p27, however, p57 expression is relatively restricted to terminally differentiated tissues.
The INK4 family of CKIs includes four structurally related proteins (p15, p16, p18, p19), each of which contains four ankyrin repeats.118 The first member of this family to be identified, p16, was found to be associated with cdk4 in transformed cells, 138 and subsequently fingered as a candidate tumor suppressor in familial melanoma.139,140 INK4 proteins bind to monomeric cdk4/6 subunits, preventing their association with D-type cyclins, and INK4 proteins can also inhibit the activity of cyclin D-cdk4/6 complexes. The other INK4 proteins are expressed ubiquitously in mouse tissues and cultured cells, and the expression of p19 does oscillate with the cell cycle.141,142 While p15 is involved in the anti-proliferative response to TGF-B, the physiological roles of the INK4 protein remain unknown. The frequent deletions of p15 and p16 in primary tumors and the high spontaneous tumor rate in p16-deficient mice indicate that these proteins play a critical role in maintaining normal growth control.143
Cdks promote progression through the cell cycle by phosphorylating a group of protein substrates.53 However, compared with the enormous amount of data concerning cdk regulation, relatively little is known about cdk substrates. The most thoroughly characterized cdk substrates are cell-cycle regulatory proteins themselves. For example, in budding yeast, phosphorylation of p40sic1 by the cln-cdc28 kinase leads to its ubiquitin-mediated proteolysis and progression from G1 to S phase.120,121 In fact, a yeast strain lacking all cln genes is viable if p40sic1 is also mutated, which suggests that phosphorylation of this CKI is a key function of the cln proteins in promoting cell cycle progression.144
A critical regulator of cell-cycle progression in higher eukaryotes, including humans, is the Rb protein. The importance of Rb in cell-cycle control became evident as a result of three separate seminal observations.145 First, the oncogenic proteins encoded by a variety of DNA tumor viruses (e.g., SV40, adenovirus, and papillomavirus) all bind to the Rb protein.146–148 Second, the Rb gene is mutated in the germ line in patients suffering hereditary retinoblastoma, and is frequently mutated in tumor cells in patients who develop spontaneous tumors.149–151 Third, the Rb protein undergoes cell-cycle dependent phosphorylation during G1, and this modifies its interaction with an essential transcription factor known as E2F.152–156 E2F transcription factors are heterodimeric proteins composed of one E2F subunit and one DP subunit that regulate the transcription of many genes required in S-phase.157–159 There are five known E2F subunits, which are designated E2F1 to E2F5, and three known DP subunits, which are designated DP1 to DP3. When complexed with Rb, E2F is inactive or it may be a transcriptional repressor, and the cell cycle arrests in the absence of these needed gene products. E2F sequestration is regulated by the phosphorylation state of Rb; unphosphorylated Rb avidly binds E2F while hyperphosphorylated does not. As cells progress through G1, Rb is progressively phosphorylated at multiple sites, ultimately releasing E2F. Viral oncoproteins specifically bind to the unphosphorylated form of Rb, and in doing so automatically release E2F from its Rb-bound inactive state.
The kinases that phosphorylate Rb are the cdks. The pRb protein contains eight consensus cdk-phosphorylation sites, and cyclin D-, E-, and A-cdk complexes have Rb kinase activity in vitro and in vivo. 160–163 Cyclin D-cdk4 complexes bind stably to Rb, and once Rb is phosphorylated, this complex disassembles.164 Cyclin D-cdk4/6 and cyclin E-cdk2 complexes probably cooperate to phosphorylate and inactivate Rb during G1. The observation that cyclin D function is dispensable in cells with mutant Rb alleles suggests that pRb phosphorylation is the critical means by which D-type cyclins promote G1 progression.165–167 Phosphorylation of Rb by cyclin E-cdk2 may also be required prior to S-phase entry, but cyclin E is essential for cell-cycle progression in Rb-mutant cells, demonstrating that other substrates of cyclin E-cdk2 must also exist.82
Rb is one member of a family of structurally related “pocket” proteins, which includes p107, p130, and Rb itself.167 All the pocket proteins bind to members of the E2F family of transcription factors and phosphorylation of these proteins by cyclin-cdk complexes liberates E2F, removing some constraints on cell proliferation. However, neither p107 nor p130 are tumor suppressors, and their function during the cell cycle is poorly understood.
E2F activity is essential for the G1-S transition, but it must also be inactivated for the ensuing S-phase to progress normally.170,171 E2F/DP heterodimers form stable complexes with cyclin A-cdk2, and phosphorylation of a specific DP residue by cyclin A-cdk2 suppresses E2F DNA-binding. Thus, G1 cyclins activate E2F via Rb phosphorylation, and cyclin A-cdk2 directly inactivates E2F through DP phosphorylation. In this way, sequentially acting cyclin-cdk complexes first initiate and then extinguish E2F activity, causing a pulse of E2F-dependent gene transcription at the G1 to S phase transition.
The elucidation of cdk substrates clearly remains incomplete.172 For instance, it is thought that proteins directly involved in the initiation of DNA replication will be phosphorylated and activated by cdks at the start of S phase, 173 although not a single such protein has been identified. Almost as short is the list of cdk substrates during mitosis. The first identified mitotic cdk substrates were the nuclear lamins.174,175 The nuclear lamina is a structure composed of intermediate filament proteins that depolymerizes at the onset of mitosis. The polymerization of lamins is regulated by phosphorylation, and lamins have been shown to be substrates for cyclin B-cdc2. Phosphorylation of lamins promotes their disassembly, and it has been proposed cyclin B-cdc2 lamin kinase activity is responsible for the breakdown of the nuclear lamina at mitosis. Cdc2 kinase activity is also required for assembly of the mitotic spindle.176 The human Eg5 protein is a kinesin-related motor protein that is needed to build a bipolar spindle. The localization of Eg5 to the spindle apparatus is dependent on phosphorylation at thr927, and this residue is phosphorylated by cyclin B-cdc2. Inhibition of Eg5 function results in a mitotic block, and one mechanism by which cyclin B-cdc2 promotes mitosis is likely to involve Eg5 phosphorylation.
Proteolysis in Cell-Cycle Regulation
A basic feature of the cell cycle is that its transitions are irreversible. Anaphase cells, for instance, cannot regress to metaphase, nor can S phase cells reverse course and go back to G1. This is accomplished by a cycle of protein destruction that complements the periodic activation of cyclin/cdk complexes. In general, protein destruction eliminates both proteins that have been used in the preceding phase of the cell cycle and proteins that would inhibit progression into the next cell cycle phase.177 The net effect is to cause the cell cycle to move irreversibly forward.
The paradigm for periodic protein degradation during the cell cycle is the destruction of cyclin B during mitosis (Fig. 23-10).35,178 The abundance of cyclin B protein oscillates during each cell division cycle, being highest as cells enter mitosis and disappearing after chromosome disjunction has occurred at the metaphase to anaphase transition.179 This is caused by changes in the rate of its degradation. Many short-lived proteins, including cyclin B, are degraded in the proteosome, a 26S complex that contains multiple proteolytic enzymes and that specifically recognizes and degrades ubiquitinated proteins.180 Conjugation of a protein to ubiquitin is the signal for its delivery to the proteosome, and this is accomplished in a multistep reaction in which ubiquitin is ultimately transferred through a thiol-ester linkage to lysine side-chains of the target protein.181,182 Ubiquitin, a 76-amino acid protein, is first attached through its carboxy terminus to an ubiquitin-activating enzyme called E1, in an ATP-dependent reaction. The E1-bound ubiquitin is then transferred to one of a family of carrier proteins called E2, or ubiquitin-conjugating enzymes, which can then transfer ubiquitin to target proteins. Most eukaryotes are thought to have a single E1 gene, but multiple E2 genes (at least 12 in budding yeast). Each of the E2 enzymes recognizes and transfers ubiquitin to only particular proteins, thereby imposing some degree of selectivity on the process of ubiquitin-dependent proteolysis. Further selectivity arises because conjugation of some proteins to ubiquitin requires collaboration between an E2 and an E3 enzyme, which is called ubiquitin ligases.
The biochemistry of cyclin destruction. See text for details (reprinted from Murray 1995190 with permission).
The E2 and E3 enzymes choose proteins for ubiquitination by recognizing specific amino acids motifs.183 A few types of motifs have been identified, each presumably recognized by certain E2 or E2/E3 combinations. The particular ubiquitination motif within cyclin B is called the cyclin destruction box.184 A mutant version of cyclin B lacking the destruction box (called cyclin BΔdb) is not conjugated to ubiquitin during mitosis and is not degraded.185 Consequently, cyclin BΔdb/cdc2 (and its MPF activity) remains active, and the cell cycle becomes blocked in mitosis.186–188 This important experiment demonstrates that destruction of cyclin B is required for inactivation of cdc2 and MPF activity in anaphase cells, and that inactivation of cdc2 is required for mitosis to be completed. Therefore, just as synthesis of cyclin B is required for a cell to enter mitosis, the destruction of cyclin B is required for a cell to exit mitosis and begin a new cell cycle.189 Cyclin A also has a destruction box and is degraded in mitosis at about the same time as cyclin B.190
The ubiquitination of cyclin B is controlled by an E3-like activity called the anaphase-promoting complex, or APC.191 As its name implies, the APC is required not only in anaphase for the destruction of cyclin B, but also earlier in mitosis at metaphase to promote the transition into anaphase.192 It is thought that at the metaphase to anaphase transition the APC is required for the targeted ubiqutination and proteolysis of a protein necessary for cohesion between sister chromatids on the metaphase plate. The APC has been characterized both genetically and biochemically, and shown to be a 20S particle comprising at least three proteins: cdc16, cdc23, and cdc27 (which were originally discovered by Hartwell in the cdc screen described above).191,193–195
The activity of the APC is regulated during the cell cycle.177 The ability of the APC to ubiquitinate B-type cyclins is turned on during mitosis and turned off in G1, leading directly to the periodic accumulation and destruction of these cyclins.195–197 In this way, the duration of APC activity defines the interval in the cell cycle where the levels of B-type cyclins are kept low; this is a key element in establishing the G1 phase of the cell cycle. Conversely, inactivation of the APC is required for accumulation of S-phase cyclins, like clb5 and clb6 in yeast, and probably cyclin A in mammalian cells, and is, therefore, a prerequisite for entry into S phase. Thus, cyclin/cdks and the APC are complementary activities that work in parallel to control major transitions within the cell cycle. The mechanisms regulating the APC are incompletely understood, but it seems as though APC activity is directly coupled to cyclin/cdk activity. First, during mitosis it is thought that cyclin B/cdc2 initiates the pathway leading to its own destruction by phosphorylating and activating the APC.177,196 Second, once activated in mitosis the APC remains active until late in G1, when it is inactivated by G1 cyclin/cdk activity.196 In yeast, this occurs coincidentally with START, and in mammalian cells, it may be one of the events that leads to cell-cycle commitment at the restriction point.
Protein destruction also controls the abundance of the cyclins and CKIs that regulate entry into S phase. Cyclin E in mammalian cells, 198,199 and the G1 cyclins in yeast (the cln proteins), 200–202 are both degraded by ubiquitin-dependent proteolysis. In both cases, phosphorylation of the cyclin triggers its ubiquitination, which is a common theme in regulated protein turnover.203–205 Often, phosphorylation of a protein is the end result of a signal transduction pathway, and can be used to allow recognition of proteins by E2 and E3 ubiquitin-conjugating enzymes. In the case of cyclin E and the cln proteins, the cyclins are directly phosphorylated by their associated cdks. Thus, cyclin E/cdk2 activity is inherently self-limited, because cdk2 activity initiates the pathway leading to cyclin E destruction. In essence, this is similar to the control of A and B-type cyclin turnover by the APC, because in each instance cyclin degradation is initiated by cdk activity.
Cdk inhibitor levels are also regulated during G1 by proteolysis. In mammalian cells, the cdk inhibitor p27Kip1 is eliminated from cells after mitogenic stimulation by the ubiquitin-proteosome pathway;206 in budding yeast, the cdk inhibitor p40Sic1 is regulated in a similar manner.121,144 In fact, biochemical reconstitution of sic1 turnover in vitro has led to the identification of an E3 complex that may rival the importance of the APC in cell-cycle control. The sic1 E3 complex is composed of three proteins, skp1, cdc53, and cdc4.206a, d–f The skp1-cdc53-cdc4 complex specifically recognizes and promotes the ubiquitination of phosphorylated sic1 in the presence of cdc34 (an E2) and E1. In addition to sic1, the skp1-cdc53-cdc4 complex also functions as an E3 for the S. cervesiae cdk inhibitor far1.206b
The components of this E3 complex are members of protein families: cdc4 belongs to a large group of proteins defined by a region of homology called an F-box, 206e, f and cdc53 belongs to the family of proteins termed cullins.208 Substitution of different family members within the E3 complex can have dramatic affects on its activity. When other F-box proteins replace cdc4, the substrate specificity of the E3 complex can change. For example, substituting Grr1 for cdc4 confers binding to phosphorylated cln1 and cln2, rather than to sic1, while the met30 F-box protein is involved in repression of genes that regulate methionine synthesis.206d, e In recognition of its combinatorial nature, this E3 has been named the SCF complex (Skp-Cullin-F-box). The vast number of SCF complexes that may form through interactions between these protein families may regulate the ubiquitination of a large number of cellular proteins. Numerous human homologs of SCF proteins have been identified, and complexes constraining cyclin A, cul1, and p45skp2 have been observed, which suggests that SCF-like complexes can form.206c Thus, this pathway for cell-cycle regulated proteolysis during G1 appears to have been evolutionarily conserved, although E3-like activities of these proteins in mammalian cells has not yet been described.
Molecular Bases of Cell-Cycle Physiology
Mitogenic and antimitogenic signals control cell proliferation by starting and stopping the cell cycle, but cells respond differently to these signals at different times in the cell cycle.209 Immediately after mitosis, cells enter a portion of G1 where the continued presence of mitogenic signals (or the absence of antimitogenic signals) is required for continued progression through the remainder of G1 and into S phase. However, at a fixed point in G1, the cell cycle becomes refractory to these signals, and cell division will be completed even if mitogens are absent or antimitogens are present.210 The restriction point is defined as the end of the mitogen-responsive portion of G1 (or the moment of transition to mitogen independence), and it reflects the execution of the fundamental proliferative decision made by the cell.211,212 It has been shown that tumor cells characteristically lose restriction point control over cell-cycle progression (they become constitutively mitogen-independent), highlighting the importance of this pathway in the normal regulation of cell proliferation.213 The restriction point is physiologically analogous to START in the yeast cell cycle, and shares many of its molecular components. Thus, the requirement for cdc28 at START is paralleled by a requirement for cdks in restriction point control.214
Mitogenic and antimitogenic signals control the cell cycle in G1 because they control the activity of the cyclins and cdks that are required for progression through G1. Growth factors and cell-substratum interactions are among the best-studied mitogenic signals, and DNA damage and TGF-β are among the best-studied antimitogenic signals. All of these proliferative signals alter the activity of essential G1 cyclin-cdk complexes and bring about either continued cell-cycle progression or cell-cycle arrest.
Mitogenic growth factors have pleiotropic effects on cell cycle proteins that stimulate progression through G1 and entry into S phase.213,215,216 They increase expression of G1 cyclins, decrease expression of cdk inhibitors, and promote assembly of G1 cyclin-cdk complexes. Growth factors increase expression of all three cyclins needed for entry into S phase—cyclin D (1, 2, and 3), cyclin E and cyclin A—at least in part by increasing transcription of their respective genes. Cyclin D1 transcription has been shown to be under the control of at least two growth-factor-modulated signal transduction pathways—the c-myc and ras pathways.217–219 The biochemical pathways linking c-myc and cyclin D1 transcription are not well defined, but the effects of ras on cyclin D1 transcription appear to be mediated by the MAP kinase pathway.219 Cyclin D1 transcription begins early in G1, and is followed by an increase in cyclin E gene expression. Cyclin E is one of a large group of genes, which includes DNA polymerase alpha, thymidine kinase, PCNA, dihydrofolate reductase, and many others, that is required for cell proliferation and whose transcription is under control of E2F.152–159 The induction of cyclin E gene transcription by E2F establishes a positive feedback loop for increasing cyclin E expression (Fig. 23-11).220 In this pathway, cyclin E-cdk2 phosphorylates Rb, releasing E2F from its Rb-bound inactive state. The free E2F promotes cyclin E gene expression, resulting in increased amounts of cyclin E-cdk2 activity, increasing Rb phosphorylation, and so on. This suggests an interesting and important physiological linkage between cyclin D and cyclin E during the mitogenic response to growth factors. Cyclin D expression is directly elevated by mitogenic growth factors, and this initiates the pathway of Rb phosphorylation and E2F-dependent gene expression. Once initiated, however, Rb phosphorylation can be maintained independently of cyclin D (and, hence, independently of mitogenic growth factors) via the autonomous feedback loop linking Rb to cyclin E. Thus, growth factors are required, through cyclin D, to start the program of E2F-dependent gene expression. These growth factors become dispensable once cyclin E-cdk2 becomes activated and substitutes for cyclin D-cdk4 in phosphorylating Rb. Inherent in this scheme is a transition from a mitogen-dependent to mitogen-independent route for maintaining Rb phosphorylation, and it may be one molecular pathway underlying commitment to cell-cycle progression at the restriction point. Cyclin A transcription also increases in growth factor-stimulated cells just prior to entry into S phase, 51 but the pathways controlling cyclin A transcription are not well understood.
A positive feedback loop reinforces cyclin E transcription. As described in the text, phosphorylation of Rb by cyclin D and cyclin E-associated kinases liberates E2F, which then stimulates cyclin E transcription. Increased cyclin E-cdk2 kinase activity then results in more Rb-phosphorylation, and greater E2F activity. Establishment of this feedback loop renders Rb-phosphorylation mitogen-independent, and may be an important component of the R-point.
The cdk inhibitor p27Kip1 is another key element in the cell-cycle response to mitogenic growth factors. p27 is required for cells to stop dividing on schedule when growth factors are withdrawn.221 p27 is expressed at very low levels in proliferating cells, but its expression greatly increases in cells starved for essential mitogenic growth factors.222–224 Under these conditions, p27 binds to and inactivates cyclin-cdk complexes and causes the cell cycle to stop (Fig. 23-12). If cells do not make p27, their exit from the cell cycle is delayed, and they will continue to proliferate in the absence of growth factors.222 Indeed, mice engineered to contain a homozygous deletion of the p27 gene grow twice as fast as control mice, and have increased numbers of cell in all lineages.225 Conversely, high levels of p27 are sufficient to prevent cell proliferation; therefore, mitogenic stimulation of non-dividing cells requires the elimination of p27.232 Both control of p27 mRNA translation rate136 and control of p27 proteolysis by the ubiquitin-proteosome pathway206 have been implicated in modulating p27 protein levels in response to mitogenic growth factors.
Induction of Cip/Kip proteins by anti-proliferative stimuli imposes a G1 arrest. The p21 and p27 proteins respond to different physiological signals. In the example depicted, DNA damage results in p53 induction and increased p21 transcription, which lead to elevated levels p21 protein and a G1 arrest via cdk inhibition. In addition, p21 inhibits DNA synthesis through the processivity factor PCNA. Similarly, mitogen-deprivation induces p27 expression and cell cycle arrest via cdk inhibition.
A third mechanism by which growth factors promote cell cycle progression is through assembly of cyclin-cdk complexes. Cyclin D-cdk4 complexes cannot assemble from their individual subunits in growth factor-starved nondividing cells.226 An assembly factor is induced by growth factors, although its molecular identity is not yet established.
The proliferative response of a cell to environmental signals depends equally on its interactions with soluble extracellular growth factors and on more local interactions with neighboring cells and with the extracellular matrix. Appropriate interactions between specific cell surface receptors (most often the integrin family of proteins) and the extracellular matrix are absolutely required for cell proliferation, a phenomenon known as anchorage-dependence. In fact, loss of anchorage-dependence is the single property of transformed cells that most closely correlates with their ability to form tumors in animals. The effect of cell anchorage on cell-cycle progression, like the effect of growth factors, occurs during G1. Cell anchorage is required for transcription and translation of cyclin D1, for activation of the cyclin E-cdk2 kinase, and for transcription of cyclin A.227,228 Anchorage regulates cyclin E-cdk2 activity by controlling the levels of the p21 and p27 cdk inhibitors. Therefore, cell anchorage controls the expression and/or activity of all three cyclin/cdk complexes required for the G1/S transition. Cell anchorage and growth factors jointly regulate the cell cycle by modulating the activity of the cyclins and cdks required for G1 and entry into S phase.
The antiproliferative action of agents like TGF-β, 229–234 cyclic-AMP, 235 and DNA damage125–128,236–238 can also be understood in terms of their effects on cell-cycle proteins. The TGF-β family of cytokines regulates diverse cellular responses, including cell proliferation, cell differentiation, and cell death.239 The antimitogenic action of TGF-β is a paradigm for the inhibition of cell proliferation by extracellular agents. The active form of TGF-β is a disulfide-linked protein dimer, which, like other members of this cytokine family, signals by bridging together type I and type II receptor serine/threonine kinases on the cell surface. The signal from the heterodimeric type I and type II receptor complex is transduced to the nucleus by a member of the Smad family of nuclear phosphoproteins. The Smad proteins are thought to be transcription factors that promote expression of genes required for the biologic effects of TGF-β or related cytokines. Mutations in DPC4, a member of the Smad protein family located on chromosome 18q21, have been detected in half of all pancreatic cancers, 240 and this may reflect a role for this protein in transmitting an antimitogenic TGF-β-like signal in pancreatic cells. Ultimately, the TGF-β signal has a plethora of effects on cell-cycle proteins, which together impose a tight blockade on progression through G1. TGF-β blocks the activation of cyclin D-cdk4 complexes by inducing expression of the cdk inhibitor p15232,241 and by inhibiting translation of cdk4 mRNA.242 TGF-β also blocks activation of cyclin E-cdk2 in some cases by directly inducing p21 and p27, and in other cases by indirectly by promoting the redistribution of p27 from cyclin D-cdk4 complexes to cyclin E-cdk2.232,233 The biochemical pathways that link activation of the Smad proteins to these diverse effects on cell-cycle proteins have not been determined.
Normal cells will not replicate a damaged chromosome. Instead, cells pause in G1 to repair the DNA lesion, thereby avoiding duplication of a damaged DNA template and preventing the propagation of genetic misinformation to daughter cells. The p53 tumor-suppressor protein controls this DNA damage response.243,244 The p53 protein is stabilized in cells containing damaged chromosomal DNA; however, it is not understood how DNA damage is detected by the cell nor how this results in decreased turnover of the p53 protein.245 p53 is a transcriptional transactivator and up-regulation of p53 leads to increased expression of p53-responsive genes.246–248 Among these genes is one encoding the cdk inhibitor p21Cip1, 127 and p21 protein levels rise in cells with damaged DNA (Fig. 23-12).238 Consequently, as p53 levels rise, G1 cyclin-cdk complexes contain elevated amounts of p21 and are inactivated. Additionally, p21 binds to and inactivates the DNA polymerase cofactor PCNA, which contributes to the G1/S phase arrest.129,130 However, p21 does not inhibit the repair functions of PCNA, which allows DNA repair to continue at the same time DNA replication is blocked.249 Mice containing an engineered homozygous deletion of the p21 gene are viable, but cells taken from these mice have a defective cell-cycle response to DNA damage.236,237
Cells are usually produced only when a new one is needed.250 Normal cells are periodically recruited into (or released from) the proliferating state by extracellular signals, and this is mediated by activation (or inactivation) of cell-cycle proteins. Tumor cells, in contrast, proliferate when normal cells would not. Current data show that many, and possibly all, cancer cells contain mutations in cell-cycle regulatory proteins, perhaps partly explaining their unregulated proliferation.251–256 In particular, one or another element in the Rb regulatory pathway, including p16, cyclin D, cdk4, E2F, or Rb itself, may be mutated in almost 100 percent of human tumors (Fig. 23-13).251–261 Altered expression of other cell-cycle regulatory proteins is also commonly observed (i.e., cyclin E and p27), but often this occurs by mechanisms other than gene mutation.262–265
The cyclin D-Rb pathway is frequently mutated in human tumors. Mutations that have been detected in human cancers include both inactivating (recessive) mutations in Rb and the INK4 proteins, and activating (dominant) mutations in the cyclin genes. All result in the deregulation of this pathway.
Although mutations in cell-cycle proteins may directly stimulate proliferation, other considerations suggest that these changes are not sufficient to explain the origin of tumorigenic cells. Epidemiologic, molecular, and genetic evidence all suggest that multiple mutations are required to transform a normal cell into a tumor cell.266–268 But simple calculations show that at normal mutation rates it is very unlikely for a cell with more than two or three mutations to arise within the lifetime of a typical person. This has suggested that increased genetic mutation rates must be a prerequisite for the evolution of malignant cells, and has given rise to the idea that the defining characteristic of a tumor cell may be genetic instability.269–272 The frequent occurrence in tumor cells of aneuploidy, DNA translocations, DNA deletions, DNA amplifications, and other genetic abnormalities may be a direct manifestation of their genetic instability.
The critical question, therefore, is what controls the genetic stability of a cell? Of course, the accuracy of the enzymes that replicate and segregate chromosomes is largely responsible for the faithful propagation of genetic information. But despite their great fidelity, these enzymes have an intrinsic, spontaneous error rate. Additionally, exogenous agents, such as chemical mutagens, can further elevate the frequency of errors. Therefore, to reduce the accumulation of genetic mistakes normal cells also continually monitor the success of DNA replication and mitosis, and bring the cell cycle to a halt if these do not occur correctly. Once active proliferation is suspended, the cell shifts from duplicating genetic information to repairing it, and only resumes proliferation once the mistakes have been corrected. Checkpoints are the pathways that make progression through the cell cycle dependent on the accurate execution of specific cell-cycle events.30,269 More specifically, they are the biochemical links between the cyclin-cdk cycle and the macromolecular events of the cell division cycle, such as DNA replication and mitosis.
Checkpoints were first identified experimentally by Weinert and Hartwell in a landmark paper describing the cell-cycle response to DNA damage.273 DNA damage causes the yeast cell cycle to pause in G2, allowing time for repair enzymes to correct the lesions, thereby preventing the cell from attempting to segregate broken chromosomes during mitosis. It was shown that the RAD9 gene controls this pause in G2, but not because the RAD9 gene was directly involved in the repair process itself. Instead, it was shown that RAD9 is part of a surveillance mechanism, and that activation of the RAD9 pathway by DNA damage prevents cyclin-cdk activity from driving a cell into mitosis.
The molecular mechanism of the DNA damage checkpoint in G2 is beginning to be understood, and it is thought to involve tyrosine phosphorylation and catalytic inactivation of cdc2.274–278 In cells containing damaged DNA, cdc2 remains inactive because an inhibitory phosphate on tyrosine 15 is not removed.279–282 The enzyme that ordinarily dephosphorylates tyrosine 15 is the cdc25C phosphatase, but in cells arrested in G2 by the DNA damage checkpoint, this does not happen. The pivotal event in this checkpoint pathway appears to be phosphorylation of cdc25C on serine 216. When cdc25C is phosphorylated on serine 216, it binds to proteins in the 14-3-3 family, and becomes sequestered in a functionally inactive state. Chk1 is the protein kinase responsible for phosphorylating cdc25C on serine 216, and Chk1 is required for the DNA damage checkpoint.283–286 It is still not understood how Chk1 is activated by DNA damage, but it is possible that this involves a signal transmitted from damaged DNA to Chk1 by the ATM tumor suppressor protein.279,287
Subsequent to the discovery of RAD9 a large number of other genes were identified in yeast that together define an intricate network of pathways that make cell-cycle progression dependent on the faithful duplication of chromosomes during S phase.288,289 A different, but equally robust, set of pathways checks for the proper attachment of chromosomes to the mitotic spindle, and delays mitosis if this has not occurred correctly.290,291 This checkpoint is triggered by the absence of spindle-induced tension on kinetochores.292 Once this checkpoint is triggered, it initiates a MAP kinase-dependent pathway293 that delays the programmed destruction of mitotic cyclins and prevents the initiation of anaphase. It is likely that this pathway modulates activation of the APC, the mitotic proteolytic machinery. Mutations in these checkpoint genes (the MAD and BUB genes) elevate rates of chromosome nondisjunction. Other less well-defined checkpoint pathways are thought to monitor cell growth, cytokinesis, and centrosome duplication, and to prevent cell-cycle progression should those events be executed incorrectly.
Normal cells use cell-cycle checkpoints as fail-safe mechanisms to avoid the accumulation of genomic errors during cell division. Inactivation of checkpoint pathways is thought to underlie the genetic instability seen in tumor cells. Thus, many tumor suppressor genes might be part of checkpoint pathways, and inactivation of these genes could contribute to the clonal evolution of cancer cells by allowing the accumulation of genomic errors that would normally have resulted in either cycle arrest (and repair) or cell death. Perhaps the best example is the p53 gene, the most commonly mutated gene in human tumors.245,249,252,255,294 p53 governs the G1 checkpoint in human cells that prevents cells from entering S phase and replicating a damaged chromosome. Also, the gene mutated in the cancer-prone syndrome ataxia-telangiectasia is thought to be required for coordinating cell-cycle progression with the repair of DNA damage during S and G2.295,296 Human homologs of various yeast checkpoint genes are now being characterized, and their relevance to genetic instability in tumor cells will soon be determined.
In conclusion, the process of tumorigenesis may begin with changes in cell-cycle regulation. Mutations in cell-cycle proteins contribute to tumorigenesis in two ways. The first is to promote cell proliferation directly by allowing the cell to override or bypass controls that ordinarily restrict proliferation. The second is to cause the cell to ignore internal “alarms” that signal the presence of errors in the duplication and segregation of genetic information. This results in genetic instability and sets the stage for evolution of malignant cells. For these reasons, cancer can be thought of as a disease of the cell cycle.
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