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Cell proliferation is perhaps best viewed as a combination of two distinct processes: the cell cycle, which replicates and segregates the genome, and cell growth, which doubles all the other components of the cell. The cell cycle and cell growth are intertwined in most normal and cancerous cells, but the two processes can be uncoupled, both in the laboratory and as part of normal development.
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9.2.1 The Mammalian Cell Cycle
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The cell cycle is partitioned into 4 phases: G1, S, G2, and M. This organization reflects the 2 primary goals of the cell cycle: to replicate the genome of the mother cell during DNA synthesis or S-phase, and to segregate the replicated genome into 2 daughter cells during mitosis or M-phase (Fig. 9–1). Two gap phases (G1, G2) separate these fundamental events. The combined G1, S, and G2-phases are frequently referred to as interphase. When cells cease proliferating because of insufficient nutrients, lack of growth factors, or upon differentiation, they exit the cell cycle from G1-phase and enter a quiescent state called G0. Most cells in the body are in the G0 state. If cells in G0 are instructed to start proliferating, they must transition back into G1-phase before starting another cell cycle.
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9.2.1.1 The Genome Is Duplicated During S-Phase
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Replicating the genome is the first of the cell cycle's two primary objectives. Each of the more than 6 billion base pairs in the DNA of a human cell must be replicated and replicated only once. To replicate so many base pairs, DNA synthesis is initiated at thousands of replication origins scattered throughout the genome (Fig. 9–2A,B). At each replication origin, double-stranded DNA (dsDNA) is unwound into 2 single stands and large protein complexes containing DNA polymerase load onto the single-stranded DNA (ssDNA) (Fig. 9–2C). As they start to replicate the ssDNA, these protein complexes progress away from the origin, unwinding the dsDNA in front of them and leaving 2 copies of dsDNA in their wake, thereby creating a structure called a replication fork (Fig. 9–2C). When replication forks progressing away from neighboring origins of replication collide, replication stops, the DNA replication complexes are removed, and the DNA strands are ligated together (Fig. 9–2A).
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Cells ensure that each stretch of interorigin DNA is replicated only once by allowing each DNA replication origin to initiate or "fire" only once per cell cycle. Origins can only initiate synthesis once each cell cycle because of a temporal separation between the formation of the prereplicative complex (pre-RC) on the origin and the initiation of replication at the origin (origin firing) (Arias and Walter, 2007). Pre-RCs are protein complexes containing DNA-binding proteins that assemble on origins at the end of the previous mitosis (see Fig. 9–2B). Once an origin is bound by a pre-RC, it is said to be "licensed" for replication, but these licensed origins remain inert throughout G1-phase. It is only starting in S-phase that licensed replication origins "fire," when the aforementioned replication complexes containing DNA polymerases are recruited to the pre-RC and DNA synthesis begins at the origin (see Fig. 9–2C). Pre-RC complexes cannot reassemble in S-phase, nor throughout the subsequent G2 and early M phases. This strict separation between times during which pre-RCs can assemble and hence license the origins (G1-phase) and during which the licensed origins can fire (S-phase), limits each origin to a single firing event and prevents rereplication of the DNA.
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9.2.1.2 The Duplicated Genome Is Segregated into Two Daughter Cells During Mitosis
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Segregating the replicated genomes into two daughter cells is the cell cycle's other primary objective. Chromosome segregation is powered and organized by microtubules. Microtubules and associated proteins form the mitotic spindle, a complex cellular apparatus that pulls apart the replicated chromosomes and then drags the separated sister chromatids to opposite ends of the mother cell.
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Assembly of the mitotic spindle is complicated, but is facilitated by centrosomes, organelles that sit at either spindle pole and generally act to organize microtubules. Centrosomes are small (~1 μm) and consist of an electron-dense pericentriolar material of uncertain organization centered on 2 compact (200 × 500 nm), barrel-shaped cylinders of microtubules called centrioles. Normal cells in G1-phase contain a single, centrally located centrosome with 2 separated centrioles (see Fig. 9–1). This single centrosome must replicate itself during the cell cycle. Centrosome replication begins in early S-phase when a daughter centriole grows orthogonally off the surface of each of the 2 original centrioles, leading to the formation of 2 proximal, but distinct, centrosomes, each centered on 2 orthogonally connected centrioles. Upon entering mitosis, the 2 centrosomes separate and move to opposite sides of the cell, with the spindle forming between them. Near the end of mitosis, the centrioles within each centrosome disengage, losing their orthogonal juxtaposition and drifting slightly apart to return to the G1-phase configuration.
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At the start of mitosis, the replicated chromosomes (46 in humans) are comprised of 2 sister chromatids that are glued together along their entire length by cohesive protein complexes. Although it involves multiple parallel processes that progress in a continuous fashion, mitosis continues to be described as a series of discrete phases, defined by the landmark morphological events observed under the microscope by early cell biologists (Fig. 9–3). Prophase begins with chromosome condensation in the nucleus. As long linear molecules of DNA, chromosomes must be compacted approximately 10,000-fold in order to be cleanly separated from one another and to be moved around the cell (Morgan, 2007). In the cytoplasm, the 2 centrosomes separate from one another and the microtubule spindle begins to elaborate between them, pushing the centrosomes to opposite sides of the cell. At the beginning of prometaphase, the nuclear envelope breaks down, allowing the microtubule spindle to physically interact with the chromosomes (Guttinger et al, 2009). At this point, the 2 sister chromatids that make up each chromosome have condensed into distinct rods. During prometaphase, the sister chromatids lose most, but not all, of their length-wise cohesion but remain tightly juxtaposed at their centromeres (Peters et al, 2008). The centromere is a single, long (0.2 to 7 megabases) sequence of repetitive DNA encoded in each sister chromatid which forms a platform for the kinetochore, a large protein complex that will physically link microtubules with the sister chromatid (see Fig. 9–1). During prometaphase, each kinetochore is bound by a bundle of 20 to 25 microtubules (Walczak et al, 2010). For each sister chromatid pair, the 2 bundles emanate from opposite spindle poles. The bundles apply pulling forces on each centromere toward their respective spindle pole, but the sister chromatids remain tightly cohered at their centromeres. Under the influence of these counterbalanced pulling forces, as well as other spindle forces, each of the 46 pairs of sister chromatids becomes bi-orientated in the center of the spindle, aligned in a plane called the metaphase plate (Dumont and Mitchison, 2009). The cell is now in metaphase, but progresses into anaphase when an abrupt and total loss of sister chromatid cohesion occurs, allowing the opposing microtubule bundles to pull the sister chromatids apart and drag them to opposite spindle poles. The spindle itself then elongates, driving the divided genomes to opposite ends of the mother cell. During the subsequent telophase, the events of early mitosis are reversed, as the chromosomes decondense, the nuclear envelopes assemble, and the spindle is taken apart. The process of cytokinesis, which splits the mother cell into 2 daughter cells, is initiated in late anaphase and is completed when both daughter cells are in early G1-phase.
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9.2.1.3 Gap Phases and Checkpoints Are Points of Decision Making
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The G1 and G2 gap phases are decision-making periods, during which intracellular and extracellular signals determine whether the cell is prepared to enter the subsequent S-phase and M-phase. During G2-phase, the key signal is intracellular, emanating from the newly replicated DNA. If serious DNA damage has occurred during replication, entry into mitosis is delayed. During G1-phase, many different kinds of extracellular and intracellular information are integrated into the decision to enter S-phase. Extracellularly, the appropriate signal transduction pathways need to be activated by the binding of receptor ligands. The combination of ligands required to proliferate depends on the cell type, and so we generically refer to them in this chapter as "growth factors" (see Chap. 8, Sec. 8.2). In some cell types, G1 arrest can also be imposed by excessive physical contact with neighboring cells. Together, these extracellular requirements define the special niches in which cells typically proliferate in vivo. Intracellularly, serious genomic damage will block cells in G1-phase. Cells lacking nutrients, such as essential amino acids, will also arrest in G1-phase. Supply of nutrients is unlikely to be limiting in normal animal tissues with adequate blood supply but may be limiting within solid tumors (see Chap. 12, Secs. 12.2 and 12.3). In some mammalian cell types, a minimal cell size may also be needed to enter S-phase, a requirement that would help coordinate cell growth with the cell cycle (Jorgensen and Tyers, 2004).
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When essential growth factors or nutrients are removed from cells, death by apoptosis or senescence may result (see Sec. 9.4). If the cells do not die, a transition in G1-phase called the restriction point determines the cellular response (see Fig. 9–1). If growth factors or nutrients are withdrawn before the restriction point, cells will enter the quiescent G0 state. If these factors are withdrawn after the restriction point, cells will progress through a full cell cycle before transitioning into the G0 state early in the subsequent G1-phase. The restriction point can occur early or late in G1-phase, depending upon the cell type. In most cancers, control over the restriction point appears to be loosened as the cells proliferate without the appropriate combination of extracellular signals.
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9.2.2 Molecular Mechanisms Central to Cell-Cycle Control
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The molecular biology of the cell cycle has been explored intensively with important discoveries having been made in yeasts, frog eggs, fruit flies, cultured mammalian cells, and mice. The molecular wiring of the cell cycle has largely been conserved during eukaryotic evolution and employs the full gamut of cellular regulatory mechanisms: transcription, translation, protein degradation, protein localization, protein phosphorylation, and microRNAs. But two molecular mechanisms are particularly central to cell-cycle regulation: cyclin-dependent kinases (CDKs) and E3 ubiquitin ligases.
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9.2.2.1 Cyclin-Dependent Kinase Activity Is Tightly Regulated
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CDKs are the central regulators of the cell cycle. CDK activities largely define the phases of the cell cycle, while changes in CDK activity drive transitions between these phases. CDKs exert these effects by phosphorylating hundreds of different proteins throughout the cell. But the abundance of CDKs is constant throughout the cell cycle. Multiple, overlapping, posttranslational mechanisms ensure that the catalytic activity of different CDKs is highly regulated in space and time (Fig. 9–4A).
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CDK activity is almost entirely dependent on the binding of a cyclin protein (see Fig. 9–4A). Once bound, a CYCLIN not only greatly stimulates the enzymatic activity of the CDK, it also influences substrate selection by the CDK. Based on sequence similarity, the human genome encodes at least 21 CDKs and 25 CYCLINs, but only a subset of the CYCLIN-CDK pairs have been shown to regulate the cell cycle (Malumbres et al, 2009). In normal cells, CDK1 forms complexes with A- and B-type CYCLINs, CDK2 forms complexes with E- and A-type CYCLINs, and CDK4 and CDK6 form complexes with D-type CYCLINs. In mice and humans, CYCLINs are expressed in small families of 2 to 3 proteins each, with the expression of different isoforms often being tissue-specific. For example, CYCLIN A1 is expressed solely in the male germline in mice, while CYCLIN A2 is expressed in all other cells (Kalaszczynska et al, 2009). CYCLINs D1, D2, and D3 are expressed in different tissues in mice, but with some overlap. CYCLINs also specify the intracellular location of CDK activity, as localized CYCLINs can focus CDK activity to the nucleus, the cytoplasm, or the Golgi apparatus. The location of some CYCLIN-CDK complexes changes dynamically during the cell cycle.
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Typically, A-, B-, and E-type CYCLINs oscillate in abundance during the cell cycle, defining windows of potential CDK1 and CDK2 activity. In contrast, the abundance of D-type CYCLINs does not change appreciably during the cell cycle (see Fig. 9–5). When a CYCLIN binds a CDK, a threonine in the activation loop of the CDK is phosphorylated by the CDK-activating kinase (CAK) (see Fig. 9–4A; Merrick et al, 2008). In human cells, CAK is actually another CYCLIN-CDK complex composed of CYCLIN H-CDK7, which is constitutively active throughout the cell cycle (Harper and Elledge, 1998).
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CDK activity is further shaped by repressive influences (see Fig. 9–4A). The MYT1 and WEE1 kinases inhibit CYCLIN B-CDK1 complexes by phosphorylating amino acids of CDK1. CDC25 phosphatases remove the phosphates from these amino acids. CDK activity can also be curbed by 2 families of CDK inhibitor proteins (CKIs). The INK4 family (p16INK4A, p15INK4B, p18INK4C, p19INK4D) of CKIs bind to CDK4 and CDK6 and prevent their binding to D-type CYCLINs. The CIP/KIP family (p21CIP1, p27KIP1, p57KIP2) of CKIs are more generalized inhibitors, binding and inhibiting the activity of CYCLIN E-CDK2, CYCLIN A-CDK2, and CYCLIN B-CDK1.
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9.2.2.2 Ubiquitin-Mediated Proteolysis and E3 Ligases Degrade Cell-Cycle Regulators
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Critical cell-cycle events are triggered by the destruction of regulatory proteins. Protein degradation is rapid and irreversible—these properties are advantageous when regulating a dynamic and unidirectional process such as the cell cycle. The primary cellular mechanism for targeted protein degradation is ubiquitin-mediated proteolysis in which the small protein ubiquitin is covalently attached to target proteins (see Chap. 8, Sec. 8.2.8). In this process, an E1 ubiquiting-activating enzyme forms a covalent bond with free ubiquitin and then transfers this bonded ubiquitin to an E2 ubiquitin-conjugating enzyme (see Fig. 9–4B). The ubiquitin is then transferred from the E2 enzyme to the substrate protein, a process that requires the binding of both the E2 enzyme and the substrate protein to the E3 ubiquitin-ligase. Substrate selectivity in ubiquitin-mediated proteolysis appears to be entirely conferred by the binding interaction between the substrate and the E3 ligase. The same substrate can be ubiquitinated multiple times—it is the presence of multiple ubiquitins attached to a substrate protein that result in that protein binding to and being proteolyzed by the 26S proteosome. Deubiquitinating enzymes (DUBs) can, however, hydrolyze the covalent bond between a substrate and ubiquitin, counteracting the actions of the E3 ligase. For any given ubiquitinated protein, the rate of proteolysis by the 26S proteosome is set by the relative activity of the E3 ligase and the DUB (see Fig. 9–4B; Komander et al, 2009).
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SCF, CUL4-DDB1, and the anaphase promoting complex/cyclosome (APC/C) are E3 ligases with important cell-cycle roles. These 3 protein complexes are evolutionarily related to one another and share similar modular structures, with a catalytic core binding to multiple substrate binding proteins. The catalytic core of SCF complexes binds, via an adaptor protein (SKP1), to multiple substrate-binding proteins (F-box proteins) (Willems et al, 2004). As individual F-box proteins are typically able to bind many different substrates, SCF complexes catalyze the ubiquitination of a myriad of target proteins. CUL4-DDB1 complexes are similarly organized with a catalytic core which binds, via an adaptor protein (DDB1), to multiple substrate binding proteins (DWD proteins) (Jackson and Xiong, 2009). The structure of the APC/C is more complicated: its catalytic core is bound to at least 11 other proteins, and this complex then binds to substrate-binding proteins (CDC20, CDH1) in a cell-cycle–dependent manner (Hutchins et al, 2010; Peters, 2006).
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Despite their structural similarities, SCF and APC/C differ in how they are activated to ubiquitinate their targets during the cell cycle (Peters, 2006; Willems et al, 2004). In both cases, short peptide sequences (degrons) in the targeted proteins are recognized by the E3 ligase complexes. In the case of SCF complexes, F-box proteins frequently recognize degrons only when they include a phosphorylated serine or threonine (see Fig. 9–4C). The SCF complexes are constitutively active, but they cannot target a substrate for degradation until that substrate is phosphorylated. Typically, SCF activity towards a given substrate is dependent on the activity of the kinase that phosphorylates that substrate. In sharp contrast, APC/C E3 ligases are only active during certain phases of the cell cycle. APC/CCDC20 is active during prometaphase and metaphase and APC/CCDH1 is active from anaphase to the end of G1-phase (see Fig. 9–4C). During this time, the APC/C complexes bind to degrons that are common to their substrates, most importantly destruction (D) boxes (both APC/C complexes) and KEN boxes (APC/CCDH1 only) (Pfleger and Kirschner, 2000). Because these degrons are not usually phosphorylated or otherwise modified, the degradation of these substrates doesn't rely on kinases and simply occurs during the windows of APC activity.
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9.2.3 The Molecular Underpinnings of the Human Cell Cycle
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The biochemical essence of the cell cycle is an oscillation between 2 states (Nasmyth, 1996). The first state lasts from anaphase to the end of the next G1-phase, while the second state lasts from the beginning of S-phase to metaphase (Fig. 9–5). In the first state, CDK1 and CDK2 activity is low, APC/CCDH1 activity is high, and pre-RC assembly on replication origins is permitted. In the second state, CDK2 activity is high, APC/CCDH1 activity is low, and pre-RC assembly on replication origins is not allowed. Each biochemical state is highly stable as a result of multiple positive feedback loops that continually reinforce that state. Switching between the 2 stable states requires special mechanisms that overcome the positive feedback that maintains each state. The points at which the cell switches between the 2 states, the G1/S transition and the metaphase-anaphase transition, are key points of cell-cycle control. The G2/M transition is a third key point of control at which the high CYCLIN B-CDK1 activity that controls early mitosis first appears.
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9.2.3.1 G1-Phase Is a Period of Low CDK1 and CDK2 Activity and High APC/CCDH1 Activity
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Replication origins become capable of initiating DNA replication during G1 phase when pre-RCs assemble on them (see Fig. 9–2B; Diffley, 2004). The origin DNA is bound directly by the origin recognition complex (ORC). ORC is then bound by CDC6 and CDT1, which, in turn, recruit MCM complexes to the origin (Arias and Walter, 2007). The absence of CDK1 and CDK2 kinase activity and the high activity of APC/CCDH1 during G1 phase combine to create a biochemical environment that allows pre-RC assembly (see below).
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During G1-phase, CDK1 and CDK2 are not bound to CYCLINs and so their kinase activity is very low (see Fig. 9–4A). CYCLIN A2 and CYCLIN B protein levels are very low during G1-phase because both proteins are substrates of APC/CCDH1 and because their genes are transcriptionally repressed (see Fig. 9–5). The fact that CDH1 is itself a substrate of CYCLIN A2-CDK2 and CYCLIN B-CDK1 complexes generates a self-reinforcing positive feedback loop that lasts throughout G1 phase (Fig. 9–6A, inner loop). When phosphorylated by CYCLIN A2-CDK2 or CYCLIN B-CDK1, CDH1 dissociates from the APC/C complex, abolishing APC/C activity. Therefore, the low activity of CDK1 and CDK2 in G1 phase prevents CDH1 from becoming phosphorylated and the activity of the APC/CCDH1 complex remains high, which, in turn, keeps CYCLIN A2 and CYCLIN B levels low. An additional positive feedback loop reinforces low CDK1 and CDK2 kinase activity (Fig. 9–6A, outer loop). In this loop, APC/CCDH1 targets the F-box protein SKP2 for degradation (Wei et al, 2004). As SCFSKP2 targets the CKI p27KIP1 for degradation (Malek et al, 2001; Wei et al, 2004), high APC/CCDH1 activity during G1-phase indirectly stabilizes the CKI p27KIP1. p27KIP1 can then bind to and inactivate any CYCLIN B-CDK1 or CYCLIN A2-CDK2 complexes that do manage to form.
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Quiescence or G0 is not simply an indefinitely prolonged G1 period. Like G1-phase cells, G0-phase cells have low CDK1 and CDK2 activity and high APCCDH1 activity. But in contrast to G1-phase cells, G0-phase cells have little to no CYCLIN D-CDK4/6 activity, as a result of sharply repressed CYCLIN D transcription. In G0-phase cells, pre-RCs are not assembled on replication origins, apparently because the abundance of some pre-RC components is very low (Williams et al, 1998). The entry of quiescent cells into G1-phase is a prolonged process that includes the synthesis of D-type cyclins, decreasing levels of p27KIP1, the accumulation of CDC6, CDT1, MCM, and the assembly of pre-RCs. D-type cyclins are transcriptionally induced and p27KIP1 is transcriptionally repressed downstream of growth factor signaling (Sherr and Roberts, 1999; see Chap. 8).
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9.2.3.2 The G1/S Transition Marks the Rise of CDK1/2 Activity
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At the G1/S transition, the positive feedback loops that suppress CDK2 activity and keep the APC/CCDH1 active are overturned (see Fig. 9–6B). CYCLIN D-CDK4/6 complexes are thought to have 2 key roles early in this process. First, CYCLIN D-CDK4/6 functions noncatalytically to bind p27KIP1 protein, and prevents p27KIP1 from inhibiting CYCLIN E-CDK2 and CYCLIN A-CDK2 (Sherr and Roberts, 1999). Second, CYCLIN D-CDK4/6 phosphorylates 3 related "pocket proteins," the Retinoblastoma (RB) protein, p107, and p130. Phosphorylation of the 3 pocket proteins leads to their release from the E2F family of transcription factors (Fig. 9–7). E2Fs complex with the DP protein and bind to sequences found in the promoters of a broad cohort of genes, many of which encode proteins important for S-phase entry or DNA synthesis. E2F4 and E2F5 bind primarily to p130 and p107, and these complexes act to repress target genes in G0-phase and in early G1-phase. E2F1-3 bind primarily to RB, which keeps E2F1-3 target genes inactive throughout G1-phase (Fig. 9–7). The phosphorylation of the 3 pocket proteins by CYCLIN D-CDK4/6 decreases their affinity for E2Fs, resulting in the inhibitory E2Fs (E2F4, E2F5) leaving the nucleus and the activating E2Fs (E2F1, -2, -3) inducing a broad transcriptional program in late G1-phase (Fig. 9–7).
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The E2F transcriptional program includes the genes encoding CYCLIN E, CYCLIN A2, and EMI1. Once translated, these 3 proteins collectively overturn the G1 state of inactive CDK1 and CDK2 and active APC/CCDH1. CYCLIN E-CDK2 and CYCLIN A2-CDK2 complexes phosphorylate RB and further stimulate the expression of E2F1-3 target genes in a positive feedback loop (Fig. 9–7). CYCLIN E-CDK2 and CYCLIN A2-CDK2 also phosphorylate CDH1, causing CDH1 to dissociate from the APC/C (see Fig. 9–6A, inner loop). The assault on APC/CCDH1 activity is furthered by EMI1, a binding partner and direct inhibitor of APC/CCDH1 (Di Fiore and Pines, 2008). The loss of APC/CCDH1 activity leads to the accumulation of its substrate SKP2. Rising SCFSKP2 activity in late G1 phase targets p27KIP1 for degradation, contributing to the rise in CYCLIN E-CDK2 and CYCLIN A2-CDK2 activity (see Fig. 9–6A, outer loop). These interlocking feedback loops, which are elaborated late in G1-phase and cause the G1/S transition, stabilize the new S/G2/early M-phase state of high CDK2 activity and low APC/C activity (see Fig. 9–5).
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The switch from low to high CDK2 activity and from high to low APC/CCDH1 activity is thought to be the molecular basis of the restriction point. That is, at some point in G1-phase, this switching process becomes irreversible and no longer requires growth signaling pathways. As discussed above, these signaling pathways are thought to drive the G1/S transition by (a) stimulating the synthesis and activity of CYCLIN D-CDK4/6 complexes, and (b) transcriptionally repressing and directly inactivating p27KIP1.
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9.2.3.3 The Initiation of DNA Replication and the Block to Overreplication
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The rise of CYCLIN A2-CDK2 activity at the G1/S transition triggers DNA replication. In at least some cell types, CYCLIN E-CDK2 also contributes to this process (Kalaszczynska et al, 2009). DNA replication also requires the CDC7 kinase, whose activity requires binding to the DBF4/ASK protein. Like CYCLIN A2, DBF4/ASK increases in abundance at the G1/S transition (Jiang et al, 1999). Phosphorylation by CYCLIN A2-CDK2 and CDC7-DBF4 of the pre-RC complex assembled around a particular DNA replication origin leads to the recruitment of replication proteins to that origin. The DNA double helix at the origin is subsequently unwound by the MCM helicase (Takeda and Dutta, 2005). These events attract DNA polymerase complexes, which bind to the unwound ssDNA at the origin and initiate DNA replication (see Fig. 9–2).
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The reloading of pre-RC complexes on replication origins in S-, G2-, and early M-phase is prevented primarily by eliminating CDT1 activity (Fig. 9–8A; Arias and Walter, 2007). Overlapping mechanisms are likely necessary to ensure that pre-RC complexes do not reappear on any of the tens of thousands of replication origins over the many hours it takes to complete S, G2, and early M phases. Two different E3 ligases target CDT1 for degradation. In S, G2, and early M phases, phosphorylation of CDT1 by CYCLIN A2-CDK2 leads to CDT1 being ubiquitinated by SCFSKP2. In S-phase, CDT1 also interacts with the chromatin-bound proliferating cell nuclear antigen (PCNA), which also leads to CDT1 becoming ubiquitinated. Any CDT1 that escapes ubiquitin-mediated degradation is inactivated by the Geminin protein. Geminin (GEM) binding to CDT1 prevents the recruitment of MCM complexes to DNA replication origins, thereby blocking a crucial step in pre-RC assembly. Geminin is itself an APC/C substrate that starts to accumulate at the G1/S transition when APC/CCDH1 is inactivated. Therefore, CDT1 and GEM are binding partners that have reciprocal patterns of degradation and abundance during the cell cycle (Fig. 9–8B). In addition to the mechanisms that restrict CDT1 activity in S, G2, and early M phases, CDC6 and components of the ORC complex may also be negatively regulated by CYCLIN A2-CDK2 and CYCLIN B-CDK1 phosphorylation during this same period (Arias and Walter, 2007; Diffley, 2004).
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During DNA replication, the 2 emerging sister chromatids become connected by a protein complex called Cohesin. By keeping the sister chromatids tightly connected, Cohesin is critical for the segregation of the 2 chromatids into separate daughter cells during mitosis. Cohesin is also important for the repair of dsDNA breaks (DSBs) by recombination between sister chromatids (see Chap. 5, Sec. 5.3.4; Peters et al, 2008). Cohesin may link sister chromatids by simply enclosing them within the same ring of protein (Nasmyth and Haering, 2009). How Cohesin establishes sister-chromatid cohesion during S-phase remains uncertain, but it is known to require a cohort of loading and stabilization proteins (Peters et al, 2008).
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9.2.3.4 G2 and the Entrance to Mitosis
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In late G2-phase, mammalian cells experience a surge of CYCLIN B-CDK1 activity that initiates the events of early mitosis. Throughout S- and G2-phase, the concentration of CYCLIN B rises, being induced by the FOXM1 transcription factor (see Fig. 9–5). The newly translated CYCLIN B binds to unphosphorylated CDK1. The binding of CYCLIN B allows phosphorylation of CDK1 on the activation loop, generating a momentarily active CYCLIN B-CDK1 kinase (see Fig. 9–4A; Deibler and Kirschner, 2010). This complex is quickly recognized in the cytoplasm by MYT1 and in the nucleus by WEE1, 2 protein kinases that phosphorylate other amino acids (particularly tyrosine 15) of CDK1 and thereby quell the kinase activity of the CYCLIN B-CDK1 complex (O'Farrell, 2001; see Fig. 9–4A). Throughout S- and G2-phase, these inactive, phosphorylated CYCLIN B-CDK1 complexes accumulate in the cell.
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In late G2-phase, the stockpiled complexes are suddenly activated by a rapid loss of the inhibitory phosphorylations, a process that is driven by at least 2 positive feedback loops (O'Farrell, 2001; Lindqvist et al, 2009). In the first positive feedback loop, CYCLIN B-CDK1 phosphorylates and activates the CDC25 phosphatases, which remove the inhibitory phosphate groups. In the second positive feedback loop, CYCLIN B-CDK1 inactivates WEE1 and MYT1, thereby blocking any further phosphorylations on CDK1. As inactive CYCLIN B-CDK1 complexes accumulate for many hours during S-phase and G2-phase, some trigger must be required to activate these positive feedback loops in late G2-phase. A strong candidate for the late G2-phase trigger is simply the accumulation of CYCLIN B and/or CYCLIN A2 above a threshold concentration. The time required to achieve this threshold concentration of CYCLIN B and/or CYCLIN A2 would then set the length of G2-phase.
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The elaboration of CYCLIN B-CDK1 activity in late G2-phase is blocked by severely damaged or unreplicated DNA, a checkpoint that prevents cells from attempting to segregate damaged chromosomes (see Sec. 9.3 and Chap. 5, Sec. 5.4).
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9.2.3.5 The Events of Early Mitosis Culminate in Sister Chromatid Separation
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The early events of mitosis are orchestrated by 4 mitotic kinases: CYCLIN B-CDK1, Polo-like kinase 1 (PLK1), Aurora (AUR) A, and AUR B. Correspondingly, more than 1000 proteins become phosphorylated specifically during mitosis (Dephoure et al, 2008). All 4 mitotic kinases are activated at the G2/M transition or in prophase. CYCLIN B-CDK1 activation has been discussed. Activation of PLK1, AUR A, and AUR B results from phosphorylation and from binding interactions that localize these kinases to critical locations in the mitotic cell (Fig. 9–9). At the end of mitosis, all 4 of the mitotic kinases are inactivated, in large part as a result of the resurgence of APC/C activity. CYCLIN B is targeted for degradation by APC/CCDC20 during metaphase, while the degradation of PLK1, AUR A, and AUR B by APC/CCDH1 begins in anaphase (Barr et al, 2004; Carmena et al, 2009).
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The strength and location of CYCLIN B-CDK1 activity during early mitosis helps determine the sequence of early events (see Fig. 9–9; Gavet and Pines, 2010). CYCLIN B-CDK1 activity is first apparent in the cytoplasm and on the centrosomes. In early prophase, cytoplasmic CYCLIN B-CDK1 causes adherent cells to "round up" and, in collaboration with PLK1, causes the 2 centrosomes to separate and mature (Gavet and Pines, 2010). CYCLIN B-CDK1 then moves into the nucleus and by late prophase the complex is predominantly nuclear (Takizawa and Morgan, 2000). Nuclear CYCLIN B-CDK1 triggers chromosome condensation, as well as the breakdown of the nuclear envelope, in part by phosphorylating and disassembling the meshwork of intermediate filaments called lamins that lines the inner nuclear membrane.
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Upon dissolution of the nuclear envelope, which marks the start of prometaphase, microtubules gain access to the chromatin. Microtubule interaction with the condensing chromosomes leads to the self-organization of the mitotic spindle between the 2 centrosomes (see Fig. 9–9). This process is highly complex and requires numerous proteins that move and stabilize microtubules (Gatlin and Bloom, 2010). During prometaphase, 20 to 25 microtubules bind the kinetochore of each sister chromatid (Walczak et al, 2010). By metaphase, each sister chromatid pair has formed a bipolar attachment to the mitotic spindle: one sister chromatid's kinetochore is connected by microtubules to one spindle pole, while the other sister chromatid's kinetochore is connected by microtubules to the other spindle pole (see Fig. 9–9). Each microtubule bundle exerts pulling forces on the kinetochore toward the spindle pole from which the bundle emanates. Although their kinetochores and centromeres are being pulled in opposite directions, the sister chromatids do not separate as a result of the Cohesin complexes that continue to physically connect them. The balance of these pulling and resistive forces, amongst other forces present in the mitotic spindle, cause each sister chromatid pair to align at the metaphase plate (Dumont and Mitchison, 2009). By phosphorylating multiple centrosome, spindle, and kinetochore proteins, the 4 mitotic kinases play key roles in shaping the mitotic spindle and aligning sister chromatid pairs (Carmena et al, 2009; Petronczki et al, 2008).
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The loss of cohesion between the sister chromatid pairs occurs in 2 steps. During prophase and prometaphase, most of the Cohesin along the chromosomal arms dissociates, allowing the chromosomal arms to partially separate by metaphase (Peters et al, 2008). But, Cohesin located at the centromeres does not dissociate until the onset of anaphase. The 2-step loss of cohesion explains the X-shaped structure of chromosomes in karyotypes, which are derived from cells arrested in metaphase by spindle poisons.
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The APC/C becomes active early in mitosis when CDC20 binds to the APC/C following extensive phosphorylation of the APC/C by CYCLIN B-CDK1. The bound APC/CCDC20 is active versus some substrates in prometaphase, including CYCLIN A2 (Geley et al, 2001), but remains inactive toward other critical substrates in prometaphase due to the inhibitory effects of the spindle assembly checkpoint (SAC; see Fig. 9–9). Once all of the kinetochores have attached to microtubules and each sister chromatid pair in under tension, cells are in metaphase, the SAC is inactivated, and the APC/CCDC20 becomes fully active. This fully active APC/CCDC20 then ubiquitinates 3 crucial substrates: securin and the 2 B-type Cyclins (B1 and B2). The proteolysis of these proteins is probably sufficient to explain the loss of sister chromatid cohesion that triggers anaphase (Oliveira et al, 2010). Securin is a binding partner and inhibitor of the protease Separase (Zou et al, 1999). In parallel to Securin, CYCLIN B-CDK1 phosphorylation of separase represses separase activity (Stemmann et al, 2001). Anaphase is initiated when the APC/CCDC20 simultaneously removes the 2 blocks (Securin and Cyclin Bs) to separase protease activity (Oliveira et al, 2010). Separase then cleaves the Cohesin subunit SCC1, which causes the removal of centromeric Cohesin and any Cohesin remaining on the chromosome arms. Once Cohesin is entirely removed, the sister chromatids are free to respond to the pulling forces exerted on their kinetochores and progress toward opposite spindle poles. Note that this current model of anaphase may not explain the synchrony with which the 46 pairs of sister chromatids separate.
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9.2.3.6 Late Mitosis Ends with Two Genetically Identical Daughter Cells
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The events of late anaphase and telophase are largely driven by the loss of protein phosphorylations that were applied earlier in mitosis by the CYCLIN B-CDK1, PLK1, and AUR B mitotic kinases (Sullivan and Morgan, 2007). Also key to late mitosis is the appearance of APC/CCDH1 activity (see Fig. 9–9). With the degradation of CYCLIN B during metaphase and anaphase, CYCLIN B-CDK1 kinase activity is much lower by telophase, allowing CDH1 to become dephosphorylated and bind to the APC/C (see Fig. 9–9). APC/CCDH1 has a much broader substrate range than APC/CCDC20. As 1 of the substrates of APC/CCDH1 is CDC20, APC/CCDC20 activity is lost in late mitosis (see Fig. 9–5).
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Many of the molecular details of late anaphase and telophase remain mysterious. The loss of protein phosphates applied earlier in mitosis by CYCLIN B-CDK1—most of which remain unidentified—is required to disassemble the mitotic spindle, decondense the separated chromosomes, and form nuclear envelopes around the 2 daughter genomes. One relatively well-understood event of telophase is the assembly of pre-RCs on DNA replication origins (Arias and Walter, 2007). Here the APC/C has important roles. The ubiquitin-dependent proteolysis of CYCLIN A2 in prometaphase and CYCLIN B1 and B2 in metaphase by APC/CCDC20 reestablishes the low-CDK1/2 activity state required for pre-RC formation (see above). In late anaphase and telophase, APC/CCDH1 maintains the degradation of these CYCLINs. APC/CCDH1 also initiates the destruction of GEM (the binding inhibitor of the pre-RC component CDT1) and SKP2 (which targets CDT1 for degradation via the SCFSKP2 complex) (Wei et al, 2004; Arias and Walter, 2007). As a result of APC/CCDH1 activity, CDT1 can accumulate in telophase, bind to ORC at DNA replication origins, and then recruit the remaining components of the pre-RC. The DNA replication origins are now prepared to initiate DNA synthesis in the next S-phase.
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Separation of the daughter cells (cytokinesis) is caused by the gradual constriction of a contractile ring attached to the inner face of the plasma membrane. The contractile force appears to be provided by filaments of Myosin II motor protein interacting with filaments of Actin, although exactly how these filaments are organized to constrict the ring remains uncertain. During anaphase, the contractile ring forms on the inner plasma membrane around the center of the spindle, equidistant from each mass of chromosomes. Typically, the spindle is located in the center of the cell and so the cell divides in half, but asymmetric spindle positioning leading to asymmetric cell cleavage is common during development and in stem cell divisions. During late anaphase and telophase, the spindle's morphology changes, passing through an intermediate stage called the midzone before being compacted into a bundle of microtubules called the midbody. Ingression of the contractile ring begins in late anaphase and continues throughout telophase, with membrane vesicles fusing with the cleavage furrow to provide the increased plasma membrane required by 2 smaller cells. The midbody spans the 1 to 1.5μm diameter cytoplasmic bridge connecting the 2 future daughter cells after maximal ingression of the contractile ring (Eggert et al, 2006). Membrane vesicles are then directed by the midbody to fill the remaining hole, a process which completes the physical separation of the 2 daughter cells.
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9.2.3.7 Caveats to the Current Model of the Mammalian Cell Cycle
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Studies of mice carrying null alleles of core cell-cycle regulators have raised important caveats about the current model of the human cell cycle described herein. For example, mice lacking both Cdk4 and Cdk6 die late in development as a result of poor proliferation of hematopoietic precursor cells, but most other cell types in the embryo proliferate normally (Malumbres et al, 2009). Second, genes encoding regulatory proteins that are central to this consensus model are not always essential. SKP2, CDH1, p27KIP1, CDK2, CDK4, CDK6, CYCLIN Ds, CYCLIN Es, and CYCLIN As are not required for the proliferation of many cell types in mice (eg, Geng et al, 2003; Kalaszczynska et al, 2009). Even a triple loss of CDK2, CDK4, and CDK6 still allows most cell types in the mouse embryo to proliferate (Santamaria et al, 2007). The surprising mildness of CYCLIN and CDK knockout phenotypes may be because of unusual CYCLIN-CDK complexes (eg, CYCLIN E-CDK1) that form when the usual binding partners are missing (Santamaria et al, 2007). But these mouse knockout studies also hint that current models may be far from complete.
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Although the term growth is often used loosely to refer to proliferation, the term cell growth specifically refers to increases in cell size. Fundamentally, cell growth is a net increase in biomass, primarily proteins, RNA, and membrane lipids. For cells to proliferate and maintain their size, cell growth must, on average, double the size of the mother cell by the time of mitosis. So, in addition to doubling the amount of DNA and the number of centosomes with each cell cycle, a cell must grow sufficiently to double all its other constituent parts—ribosomes, mitochondria, lysosomes, etc.
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It remains unclear how intertwined cell growth and the cell cycle are in human cells. Growth of some cells can become uncoupled from the cell cycle, arguing that they are distinct processes. For example, during the formation of the oocytes, which are giant single cells, cell growth occurs in the absence of any cell division. The uncoupling of cell growth and the cell cycle can also occur in somatic cells when the cell cycle is arrested by a DNA damage checkpoint (see Sec. 9.3). Such cell-cycle arrest usually allows cell growth to continue, resulting in oversized cells. In general, cell growth is more limiting for cell proliferation than is the cell cycle, because in animal cells the events of the cell cycle can be accomplished in far less time than it takes to double the mass of the cell. Because different amounts of time are necessary to double cell mass and to complete the cell cycle, coordination between cell growth and the cell cycle must exist at some level or else cell size would fluctuate wildly, which is not observed (Jorgensen and Tyers, 2004).
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For a given human cell, cell growth, like the cell cycle, requires the appropriate combination of growth factors to bind to the cell (see Chap. 8, Sec. 8.2). Some of the molecular regulators of cell growth that lie downstream of growth factor signaling are known. c-MYC is a transcription factor encoded by the oncogene most frequently amplified in cancers. c-MYC binds to gene promoters with its binding partner MAX (see Chap. 7, Sec. 7.5.2). The widespread transcriptional program activated by c-MYC overexpression can stimulate cell growth, in part by inducing genes involved in ribosome synthesis, a process that is intrinsically related to cell growth (Eilers and Eisenman, 2008). c-MYC/MAX can also stimulate the cell cycle by activating the transcription of cyclin D1, cyclin D2 and cdk4, while a c-MYC/MAX/MIZ1 complex represses the genes encoding the p21CIP1 and p15INK4B proteins.
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The mammalian target of rapamycin (mTOR) and phophatidylinositol-3 kinase (PI3K) signaling network (see Chap. 7, Sec. 7.5.4 and Chap. 8, Sec. 8.2.5) is a central regulator of animal cell growth and proliferation. This network integrates large amounts of extracellular and intracellular information into the decision to activate the AKT kinase and the mTORC1 kinase complex (Guertin and Sabatini, 2007). These 2 kinases then activate multiple downstream processes that drive cell growth, the cell cycle, and block cell death (Guertin and Sabatini, 2007). Genetic alterations that increase AKT and mTORC1 activity can be oncogenic. Mutations in the gene encoding PTEN (see Chap. 7, Sec. 7.6.2), a phosphatase that counteracts PI3K, can constitutively activate AKT and are common in many cancers (eg, ~40% of endometrial cancers, 30% to 40% of glioblastoma multiforme) (Liu et al, 2009; Sansal and Sellers, 2004). Amplification of the genes encoding growth factor receptors upstream of AKT and mTORC1 (eg, ERBB2 see Chap. 7, Sec. 7.5.3) are also common in cancer (Liu et al, 2009). Two rapamycin analogs (temsirolimus and everolimus, small molecular inhibitor of mTORC1) have been shown to be efficacious in the treatment of metastatic renal cell carcinoma and are in clinical trials for other types of cancer.
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To grow, cells need to either import or synthesize large amounts of amino acids, nucleotides and fatty acids. In addition, large amounts of adenosine triphosphate (ATP) are required to polymerize these building blocks into proteins, RNA, and membrane lipids. Consequently, growing cells have special metabolic properties. The anabolic state is programmed by growth factor signaling pathways and transcription factors, particularly the PI3K/mTOR network and c-MYC discussed above (Jones and Thompson, 2009). The anabolic state is characterized by a number of features, particularly rapid uptake and metabolism of glucose. The rapid uptake of glucose by some cancer cells reflects their higher rate of glycolysis. Indeed, some cancer cells produce more pyruvate—the end product of glycolysis—than can be oxidized by the tricarboxylic acid (TCA) cycle in the mitochondria. The excess pyruvate is converted to lactic acid and secreted, a phenomenon commonly known as the Warburg effect (see Chap. 12, Sec. 12.3.1). Although usually thought of as an energy generating pathway, glycolysis–via the interlinked pentose phosphate pathway–also has important roles in supplying cellular building blocks, including the reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH) which is used in many anabolic reactions, the nucleotide precursor ribose-5-phosphate, the phospholipid precursor glycerol-3-phosphate, and the precursors for 4 amino acids (Jones and Thompson, 2009). The need to produce building blocks like NADPH to allow for rapid cell growth may be the molecular explanation for why some cancer cells engage in such rapid glycolysis that lactic acid must be secreted from the cell.
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9.2.5 Modifications to Control of Cell Proliferation in Cancer
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The first tumor suppressorgene discovered in humans was Rb1, whose loss was found to be the cause of familial retinoblastoma (see Chap. 7, Sec. 7.6.4). Soon after, cyclin D1 was isolated as an oncogene in a subset of parathyroid adenomas. The cdkn2a locus, which contains the genes encoding the p15INK4B protein, the p16INK4A protein, and the p53 activator ARF, was then identified as a tumor suppressor in familial melanomas. These early findings anticipated a general truth about human cancers, which is that the vast majority contain genetic or epigenetic alterations that loosen control over the G1/S transition. Typical alterations include the amplification of genes that encode proteins that drive the G1/S transition (eg, cyclin D1), as well as genomic deletions, point mutations, and promoter methylations that remove G1/S inhibitors like RB and p16INK4A. In a genome-wide survey of 26 human cancer types, the tightly opposed loci encoding p15INK4B and p16INK4A/ARF were found to be the most frequently deleted genomic region, while the genes encoding CYCLIN D1 and CDK4 were among the 4 most frequently amplified regions (Beroukhim et al, 2010). When genetic alterations to the G1/S machinery are considered collectively, the majority of cancers contain at least 1 such change. For example, more than 90% of liver, ovarian, testicular, and lung cancers bear alterations in the genes encoding the currently known G1/S regulatory apparatus (Malumbres and Barbacid, 2001). Alterations to the genes encoding the G1/S regulatory apparatus are thought to allow cancer cells to enter the cell cycle precociously, defying the extracellular signals that limit the proliferation of their normal neighbors.
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A detailed understanding of the cell cycle could be important for developing better anticancer drugs. There is active preclinical development and clinical evaluation of agents designed to inhibit cell-cycle regulators. For example, clinical trials are evaluating specific inhibitors of EG5, PLK1, and the Aurora kinases as such drugs may kill mitotic cells without affecting microtubules in quiescent cells. Similarly, substantial effort has been dedicated to generating CDK inhibitors (Malumbres et al, 2008). Future generations of anticancer drugs could exploit the common alterations to the cell-cycle machinery shared by many cancers, such as defective control over the G1/S transition, loss of the p53 response, aneuploidy, or the presence of extra centrosomes. In particular, cancers may acquire a dependence on nonessential cell-cycle components like D- and E-type CYCLIN-CDK complexes, which could be exploited by targeted therapies. Supporting such an idea, mice lacking cyclin D1 or cdk4 can be highly resistant to tumor formation.