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Cell Cycle
Cell Cycle
1
Cell Cycle Alice M Sheridan, Vishal S Vaidya, and Harihara M Mehendale & 2005 Elsevier Inc. All rights reserved.
The cell cycle is the orderly progression of cells through specific stages during which DNA is replicated and distributed to two daughter cells resulting in cell proliferation. Precise regulation of the passage of cells through this cycle is necessary to assure the maintenance of DNA integrity through multiple generations. Cell cycle regulation also ensures that cell proliferation occurs only under defined conditions in response to growth factors and in the presence of a suitable environment. Loss of cell cycle regulation is a characteristic of cancer. The cell cycle comprises four stages, which are called G1, S, G2, and M phases (Figure 1). S (for DNA synthesis) is the stage in which DNA is duplicated. G1 is the stage immediately prior to S during which the cell prepares for DNA synthesis. M (for mitosis) is the stage in which the cell divides and G2 is the stage preceding M during which the cell prepares for cell division. Two major points of regulation are at the transitions between G1 and S and between G2 and M phases. The progression of cells through late G1/S requires the presence of growth factors. A restriction point in late G1 marks the point Cyclin B-cdk1
G0 M Rb-E2F P E2F E2F-DP S
G1 pRb
p18INK4c p19INK4d
Re
G2
p16INK4a p15INK4b
str po ictio int n
Cyclin Dcdk4/6
Cyclin E-cdk2
Cyclin A-cdk2
p21Kip1 p27Kip1 p57Kip2
p53
Figure 1 Overview of the different phases of the cell cycle. Quiescent cells are in G0 phase and reenter the cell cycle at G1 during which cells prepare for DNA synthesis. After passing the restriction point in late G1 cells are committed to enter S phase, during which DNA replication occurs. Cells in G2 phase prepare for mitosis (M phase). Cell cycle progression is controlled by various positive and negative cell cycle regulatory proteins including cyclins (A, B, D, E); cyclin dependent kinases (cdk 1, 2, 4, 6); cdk inhibitors (p15, p16, p18, p19, p21, p27, p57), retinoblastoma (Rb) and p53.
at which cycle progression becomes growth factor independent. Cells that are actively proliferating progress from M phase back to G1 where preparations for DNA synthesis immediately start anew. Cells that are not actively proliferating are said to be quiescent and are in G0 phase. The entry of cells from G0 into the cell cycle is also a closely regulated step and requires an extracellular stimulus or growth factor. We describe below the critical proteins that have been identified to date that regulate G1/S and G2/M transitions. We emphasize that the cell cycle paradigm is rapidly evolving and expanding and that this description is likely incomplete.
Cyclins and Cyclin-Dependent Kinases Numerous proteins have been identified that stringently regulate the passage of cells at G1/S and G2/M phase transitions. Conserved serine/threonine kinases, called cyclin-dependent kinases (cdks), phosphorylate and activate specific regulatory proteins that drive cell cycle progression. The activity of cdk is controlled at three levels. First, cdks are activated by their interaction with proteins, called cyclins. Cyclins are proteins with very short half-lives of less than 30–60 min. Whereas the cdks are constitutively expressed throughout the cell cycle, the level of the cyclins varies throughout. Cyclin levels are controlled by both regulated synthesis and ubiquitin-mediated proteolysis. Specific cyclin–cdk complexes function at different cell cycle phases. Formation of the heterodimers cyclin D/cdk4, cyclin D/cdk6, and cyclin E/cdk2 are necessary for entry into and progression through G1. The induction of cyclin D family members is provoked by an extracellular signal or growth factor and initiates the entry of quiescent cells from G0 into G1. Cyclin D/cdk heterodimers phosphorylate and inactivate retinoblastoma protein (pRb) causing the release and activation of the E2F family of transcription factors. This family of transcription factors drives transcription of genes necessary for the G1/S transition, including cyclin E. Cyclin E/cdk2 also phosphorylates pRb but unlike cyclin D heterodimers, its activity is mitogen-independent. Both cyclin E/cdk2 and cyclin A/cdk2 drive entry and progression through S phase via the phosphorylation of non-Rb proteins that initiate DNA synthesis. Cyclins A and B form complexes with cdk1 (also called cdc2) and are called the mitotic cylins since these complexes regulate mitosis. Cyclin B/cdk1 controls the G2/M transition. Cyclin B is synthesized as the cell progresses through G2. Upon binding of
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Cell Cycle
cyclin B to cdk1, the activated heterodimer phosphorylates proteins that are involved in mitosis. Activity of the cyclin/cdk complexes is also regulated by phosphorylation/dephosphorylation by cdk activating kinases (CAKs) and phosphatases. A third level of regulation is achieved by control of protein levels of cdk inhibitors. Cdk inhibitors are proteins that accumulate in response to multiple environmental stimuli including DNA damage, hypoxia, cell–cell contact and cytokines, and inhibit the activity of cyclin/cdk heterodimers. The cdk inhibitors include two classes of proteins. The INK 4 proteins, which include p16INK4a, p15INK4b, p18INK4c, and p19INK4d, specifically inhibit the activity of cdk4 and cdk6 by competitive inhibition of cyclin D binding to the monomeric kinases. Mutations and deletions of the p16INK4a gene and inactivation by hypermethylation, have been shown to play a role in tumorigenesis in many different types of tumors. The Kip/Cip proteins include three structurally related proteins, p21, p27, and p57. In contrast to the INK4 proteins, the Kip/Cip proteins inhibit most cyclin/cdk heterodimers. Specific Kip/Cip proteins are induced by upstream events. p21 is induced in response to DNA damage and specifically inhibits cyclin E/cdk2. Protein levels of p27 are highest in quiescent cells and induce G1 arrest in response to conditions that typically result in cell quiescence such as growth factor deprivation or contact inhibition. Both the INK4 and the Kip/Cip proteins inhibit the phosphorylation and inactivation of pRb.
Retinoblastoma The retinoblastoma gene (Rb) was the first tumor suppressor to be identified. Rb mutations were first shown to be causal in familial and sporadic retinoblastoma, a rare tumor of the eye, but have since been associated with many other tumors including osteosarcoma, small cell lung cancer, and prostate and breast cancer. In addition, mutations in the upstream Rb signaling pathway that result in the functional inactivation of the Rb gene product, pRb, are found in virtually all malignancies. Three Rb homologs have been described, including Rb, Rb 107, and Rb 130. All Rb homologs are characterized by a ‘pocket’ domain, which is highly conserved and necessary for pRb’s tumor suppressor function. All the Rb homologs bind viral oncoproteins as well as E2F family members. Binding of viral oncoproteins disrupts the pocket domain of pRb and impairs pRb’s tumor suppressor function. All pRb homologs cause G1 arrest.
The primary role of pRb is the inhibition of transcription of genes that mediate passage across the G1/S transition. There are two mechanisms by which pRb inhibits transcription. First, pRb binds to and inhibits the E2F family of transcription factors. The binding characteristics of the homologs vary slightly as, whereas pRb binds preferentially to E2F1-4, p107 and p130 bind preferentially to E2F4 and E2F5. Phosphorylation of pRb regulates its interaction with E2F. The phosphorylation status of pRb fluctuates throughout the cell cycle. Hypophosphorylated pRb is active and binds to E2F family members thus sequestering E2F and inhibiting its transcriptional activity. Hyperphosphorylated pRb is inactive and releases E2F, which results in the transcription of genes that allow the cell to progress to S phase. Upon release from pRb, E2F binds to DP-1 or DP-2 and the resulting heterodimer activates genes necessary for DNA replication. The mechanism by which pRb inhibits E2F transcriptional activity is still debated but it may be via the recruitment of chromatin remodeling enzymes such as histone deacetylases (HDACs) which directly repress transcription by removing acetyl groups from chromatin which causes the chromatin to be less accessible to transcription factors. The role of the pRb-bound HDAC may be to counteract the activity of the E2F-bound acetyltransferase protein, p300/CBP, which transfers acetyl groups to chromatin and enhances transcriptional activity. In addition to its inactivation of E2F resulting in a decrease in transcription of E2F-responsive genes, the complex of pRb and E2F actively represses transcription, which may also be via the recruitment of HDACs to the promoter regions. The regulation of pRb activity is complex. There are 16 possible sites for cdk-mediated phosphorylation and data suggest that phosphorylation at each different site regulates a distinct pRb function. pRb is phosphorylated by multiple cyclin/cdk complexes. Cyclin D/cdk4/6 initiates phosphorylation in early G1 and cyclin E/cdk2 hyperphosphorylates pRb in late G1. Cyclin A/cdk2 maintains phosphorylation of pRb thoughout S phase. pRb may perform other roles in addition to regulation of G1/S including the regulation of apoptosis. A decrease in functional pRb results in the activation of p53-induced apoptosis, which appears to be mediated via the release of E2F1. Free E2F1 activates transcription of ARF (alternate reading frame of the p16INK4a locus), which inhibits a protein called mdm-2 ubiquitin ligase (mdm-2). mdm-2 targets proteins for ubituitin-mediated proteolysis. Since mdm-2 initiates the degradation of p53, its inhibition results in an increase in p53 and a corresponding increase in apoptosis. Thus, a decrease
Cell Cycle
in functional pRb, which could otherwise result in unchecked cell proliferation, triggers an apoptotic response. A decrease in functional pRb also creates a selection pressure for p53 mutations, since only cells that have mutated dysfunctional p53 survive. Not surprisingly, p53 mutations are often found to coexist with Rb mutations in malignant tumors.
Checkpoints Checkpoints are surveillance mechanisms comprising numerous genes that detect DNA damage and induce either cell cycle arrest and DNA repair mechanisms, or, in the presence of extensive DNA damage, apoptosis. The data elucidating this surveillance network are very incomplete but have been advanced significantly since the isolation of the mutation that is associated with ataxia telangiectasia (AT). AT is a rare pediatric disease that is associated with immune deficiency and an increased susceptibility to cancer. Prior to the isolation of the AT mutation, it had long been observed that stimuli that induce DNA damage delay progression through the cell cycle. For years this phenomenon was assumed to be the passive response of the cell as a direct result of the DNA damage itself. By contrast, cells that harbor the AT mutation demonstrate a marked decrease in cell cycle arrest after DNA damaging radiation. These data suggested that an active system exists in normal cells that retards cycle progression in the presence of DNA damage. The checkpoint surveillance system comprises sensor proteins (proteins that detect DNA damage and initiate a signaling cascade); transducers (modifying enzymes such as kinases that relay the signal to effector proteins); and effectors (downstream target proteins that, upon activation by modifying enzymes, cause cycle arrest). Of these proteins, the least is known about sensor proteins, although several candidate genes have been suggested. The effector proteins include kinase inhibitors such as p21, or cyclin/cdk heterodimers that are either activated or inhibited to cause cycle arrest. Major transducer proteins include p53, ATM (AT mutated) and ATR (ATM and RAD-3 related). p53 is a transcription factor that activates the transcription of genes that cause cell cycle arrest at either G1/S or G2. In addition, p53 activates genes that initiate DNA repair and cause apoptosis. Mutations of p53 are commonly described in association with human tumors. The result of p53 activation is cell type-specific and depends on the type and severity of injury. p53-induced G1 cell cycle arrest is mediated via the induction of p21 and p16. p53 has a very short half-life and is generally undetectable in healthy cells. In the presence of DNA damage
3
induced by either ultraviolet or g-irradiation, p53 is activated by posttranscriptional modifications including phosphorylation and acetylation, that either enhance its stability or alter its affinity for binding proteins. ATM and its related protein, ATR, phosphorylate p53 which decreases its binding to mdm-2. A decrease in the interaction between p53 and mdm2 causes a decrease in ubiquitin-mediated proteolysis of p53 and a resulting increase in p53 protein levels. As described previously, ARF also activates p53 via the inactivation of mdm-2. In addition to phosphorylation, the acetylation status of p53 also determines its stability. p53 is acetylated and stabilized by p300/ CBP which increases apoptosis. The recently described NAD-dependent deacetylase protein, SIRT1, removes acetyl groups from p53 and decreases apoptosis. ATM and ATR are closely related phosphoinositide 3-kinases that are activated by DNA damage. Upon activation, ATM phosphorylates and activates multiple proteins in addition to p53, including mdm2 and a serine/threonine kinase called Chk-2. Activated Chk-2 phosphorylates p53. ATR phosphorylates many but not all of the same substrates as ATM. ATR phosphorylates and activates Chk-1, which also phosphorylates p53. p53, ATM, and ATR also contribute to G2 arrest. Upon activation, cdk1 initiates mitosis. cdk1 is activated via its interaction with cyclin B and via dephosphorylation by cdc25C phosphatase. Upon phosphorylation by ATM and ATR, Chk-2 and Chk-1 phosphorylate and inhibit cdc25C, which prevents the activation of cdk1. p53 activates transcription of two genes that inhibit cdk1 activity including GADD45 and 14-3-3 s. GADD45 disrupts the cyclin B/cdk1 heterodimer. The protein product of 14-3-3 s sequesters cdc25C, which prevents the dephosphorylation of cdk1. In the face of overwhelming DNA damage, checkpoints, in particular p53, induce cell death by apoptosis rather than cell cycle arrest. Apoptosis, or programmed cell death is an evolutionarily conserved, energy-requiring mechanism by which unwanted or irreparably damaged cells are removed from the organism. Apoptosis is a fundamental component of both normal embryogenesis and adult homeostasis. Apoptosis is also a physiologic response to diverse toxic stimuli including viral infection, DNA damage induced by irradiation or reactive oxygen species, hypoxia, growth factor deficiency or genetic aberration. Apoptosis is carried out by caspases, which are proteases that contain a cysteine nucleophile and cleave proteins whose sequence contains specific motifs that include an aspartic acid residue. Upstream or initiator caspases are activated
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Cell Cycle
by the binding of an extracellular ligand to a death receptor. Death receptors are members of the tumor necrosis superfamily and are characterized by an intracellular death domain. An important example of a death receptor is CD95, or fas, which binds fas ligand. Upon binding of a ligand, the death receptor binds to intracellular adaptor proteins. Adapter proteins bind to initiator caspases 2, 8, 9, or 10, which provokes their autocleavage and activation. Initiator caspases activate downstream effector or executioner caspases, such as caspase 3 or 7, or proapoptotic BCL-2 proteins. The BCL-2 family includes proteins that contain BCL-2 homology domains. These domains allow for heterdimerization by which BCL-2 proteins activate other family members. BCL-2 proteins modulate the intrinsic apoptotic pathway and may have either proor anti-apoptotic effects. Proapoptotic BCL-2 proteins increase mitochondrial membrane permeability which allows for the release of cytochrome c. Cytochrome c release from mitochondria results in dimerization of an adaptor protein called Apaf-1 (apoptotic protease activating factor) which binds procaspase 9 resulting in its cleavage and activation. Caspase 9 activates the downstream effector, caspase 3. Antiapoptotic proteins in the BCL-2 family inhibit the proapoptotic members and prevent the increase in mitochondrial membrane permeability. The downstream effector caspases target multiple proteins for degradation including enzymes, nuclear structural proteins such as lamins, cytoskeletal proteins such as actin, proteins critical for cell–cell interaction such as b-catenin, and DNA repair enzymes. p53 activates multiple genes that are involved in apoptosis, including genes that encode proteins that function via receptor-mediated signaling and those which encode proteins that modulate downstream effectors. p53-activated IGF-BP3 inhibits binding of IGF-1 to the IGF-1 receptor, which can induce apoptosis. p53 activates transcription of the death receptor ligands fas/Apo1/CD95 and the death receptor KILLER/DR5. p53 also induces the proapoptotic BCL-2 protein bax, as well as other proteins that enhance cytochrome c release from mitochondria, including p53, AIP1, PUMA, and Noxa. Apoptosis may also be induced via an increase in oxidative stress generated by multiple p53-induced genes that are homologous to NADPH-quinone oxidoreductase. Importantly, no singular p53-activated gene product has been conclusively shown to initiate apoptosis. It appears that many p53-induced proapoptotic genes need to be activated concurrently in order for apoptosis to occur. It is not clear which variables determine whether p53 induces cell cycle arrest or apoptosis. Certain cell
types, such as T lymphocytes, are especially sensitive to apoptosis whereas fibroblasts are more likely to undergo cell cycle arrest. Whereas p53 induces arrest or senescence in normal cells, p53 activation usually causes apoptosis in transformed cells. The reason for the enhanced sensitivity to p53-induced apoptosis in transformed cells may be related to the deregulation of E2F due to the inactivation of pRb. Cycle arrest induced by p21 may protect the cell from apoptosis. Other factors that may predispose the cell toward p53-induced apoptosis include alterations in the bax/ bcl-2 ratio, concurrent absence of growth factors, a greater intensity of stress and higher protein levels of p53. Posttranslational modifications may also determine p53 promoter specificity, which may play a major role in determining whether p53 expression results in cell cycle arrest or apoptosis.
Clinical Application The normal regulation of the cell cycle plays an important role in tissue repair and inflammation. All tissues may be stratified by proliferative capability into three categories including labile, quiescent or permanently nondividing cells. Labile cells are continuously dividing and include surface epithelial cells such as stratified squamous epithelial cells of the skin and columnar epithelial cells of the gastrointestinal tract. Quiescent cells are nondividing under normal circumstances but can be induced to reenter the cell cycle by exposure to growth factors. Quiescent cells include parenchymal cells of the liver, kidney, and pancreas and mesenchymal cells such as fibroblasts. The cytokine-induced reentry of quiescent cells into G1 phase is an important component of the inflammatory response, which has been well characterized in the kidney. Glomerular mesangial cells proliferate in many models of glomerular disease, including lupus nephritis and diabetes. The proliferation of mesangial cells occurs in response to cytokines such as platelet-derived growth factor and basic fibroblast growth factor. Inhibition of mesangial cell proliferation may abrogate the glomerulosclerosis or the glomerular scarring that occurs as a result of inflammation. Permanently nondividing cells have lost all capacity for proliferation and include nerve cells and cardiac muscle cells. The deregulation of the cell cycle resulting in unchecked cell proliferation is a hallmark of cancer. All human cancers are characterized by defects of restriction point control, checkpoints, DNA repair, or apoptosis. Defects of restriction point control allow for uncontrolled proliferation and result in loss of
Cell Cycle
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terminal differentiation. While some cancers are characterized by loss of function mutations of Rb or by disruption of the Rb pocket domain by viral oncoproteins, many more are caused by functional inactivation of pRb through cyclin D overexpression or INK4a mutations. Mutations of p53 are the most common mutations associated with cancer and occur in almost 50% of all human cancers.
understanding of cell cycle regulation remains incomplete. Further studies may allow better understanding of diseases that result from deregulation of these pathways.
Conclusion
Further Reading
The regulation of the cell cycle plays an important role in normal tissue repair and regeneration. Loss of cell cycle regulation is a chief characteristic of cancer. Cell cycle regulation involves numerous signaling pathways that determine whether cells will proliferate, remain quiescent, arrest or undergo apoptosis. While enormous progress has been made in the elucidation of these signaling pathways, our
Griffin SV, Pichler R, Wada T, et al. (2003) The role of cell cycle proteins in glomerular disease. Seminars in Nephrology 23: 569–582. Iliakis G, Wang Y, Guan J, and Wang H (2003) DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene 22: 5834–5847. Sionov RV and Haupt Y (1999) The cellular response to p53: The decision between life and death. Oncogene 18: 6145–6157.
See also: Tissue Repair.
1
Cell Cycle Alice M Sheridan, Vishal S Vaidya, and Harihara M Mehendale & 2005 Elsevier Inc. All rights reserved.
The cell cycle is the orderly progression of cells through specific stages during which DNA is replicated and distributed to two daughter cells resulting in cell proliferation. Precise regulation of the passage of cells through this cycle is necessary to assure the maintenance of DNA integrity through multiple generations. Cell cycle regulation also ensures that cell proliferation occurs only under defined conditions in response to growth factors and in the presence of a suitable environment. Loss of cell cycle regulation is a characteristic of cancer. The cell cycle comprises four stages, which are called G1, S, G2, and M phases (Figure 1). S (for DNA synthesis) is the stage in which DNA is duplicated. G1 is the stage immediately prior to S during which the cell prepares for DNA synthesis. M (for mitosis) is the stage in which the cell divides and G2 is the stage preceding M during which the cell prepares for cell division. Two major points of regulation are at the transitions between G1 and S and between G2 and M phases. The progression of cells through late G1/S requires the presence of growth factors. A restriction point in late G1 marks the point Cyclin B-cdk1
G0 M Rb-E2F P E2F E2F-DP S
G1 pRb
p18INK4c p19INK4d
Re
G2
p16INK4a p15INK4b
str po ictio int n
Cyclin Dcdk4/6
Cyclin E-cdk2
Cyclin A-cdk2
p21Kip1 p27Kip1 p57Kip2
p53
Figure 1 Overview of the different phases of the cell cycle. Quiescent cells are in G0 phase and reenter the cell cycle at G1 during which cells prepare for DNA synthesis. After passing the restriction point in late G1 cells are committed to enter S phase, during which DNA replication occurs. Cells in G2 phase prepare for mitosis (M phase). Cell cycle progression is controlled by various positive and negative cell cycle regulatory proteins including cyclins (A, B, D, E); cyclin dependent kinases (cdk 1, 2, 4, 6); cdk inhibitors (p15, p16, p18, p19, p21, p27, p57), retinoblastoma (Rb) and p53.
at which cycle progression becomes growth factor independent. Cells that are actively proliferating progress from M phase back to G1 where preparations for DNA synthesis immediately start anew. Cells that are not actively proliferating are said to be quiescent and are in G0 phase. The entry of cells from G0 into the cell cycle is also a closely regulated step and requires an extracellular stimulus or growth factor. We describe below the critical proteins that have been identified to date that regulate G1/S and G2/M transitions. We emphasize that the cell cycle paradigm is rapidly evolving and expanding and that this description is likely incomplete.
Cyclins and Cyclin-Dependent Kinases Numerous proteins have been identified that stringently regulate the passage of cells at G1/S and G2/M phase transitions. Conserved serine/threonine kinases, called cyclin-dependent kinases (cdks), phosphorylate and activate specific regulatory proteins that drive cell cycle progression. The activity of cdk is controlled at three levels. First, cdks are activated by their interaction with proteins, called cyclins. Cyclins are proteins with very short half-lives of less than 30–60 min. Whereas the cdks are constitutively expressed throughout the cell cycle, the level of the cyclins varies throughout. Cyclin levels are controlled by both regulated synthesis and ubiquitin-mediated proteolysis. Specific cyclin–cdk complexes function at different cell cycle phases. Formation of the heterodimers cyclin D/cdk4, cyclin D/cdk6, and cyclin E/cdk2 are necessary for entry into and progression through G1. The induction of cyclin D family members is provoked by an extracellular signal or growth factor and initiates the entry of quiescent cells from G0 into G1. Cyclin D/cdk heterodimers phosphorylate and inactivate retinoblastoma protein (pRb) causing the release and activation of the E2F family of transcription factors. This family of transcription factors drives transcription of genes necessary for the G1/S transition, including cyclin E. Cyclin E/cdk2 also phosphorylates pRb but unlike cyclin D heterodimers, its activity is mitogen-independent. Both cyclin E/cdk2 and cyclin A/cdk2 drive entry and progression through S phase via the phosphorylation of non-Rb proteins that initiate DNA synthesis. Cyclins A and B form complexes with cdk1 (also called cdc2) and are called the mitotic cylins since these complexes regulate mitosis. Cyclin B/cdk1 controls the G2/M transition. Cyclin B is synthesized as the cell progresses through G2. Upon binding of
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Cell Cycle
cyclin B to cdk1, the activated heterodimer phosphorylates proteins that are involved in mitosis. Activity of the cyclin/cdk complexes is also regulated by phosphorylation/dephosphorylation by cdk activating kinases (CAKs) and phosphatases. A third level of regulation is achieved by control of protein levels of cdk inhibitors. Cdk inhibitors are proteins that accumulate in response to multiple environmental stimuli including DNA damage, hypoxia, cell–cell contact and cytokines, and inhibit the activity of cyclin/cdk heterodimers. The cdk inhibitors include two classes of proteins. The INK 4 proteins, which include p16INK4a, p15INK4b, p18INK4c, and p19INK4d, specifically inhibit the activity of cdk4 and cdk6 by competitive inhibition of cyclin D binding to the monomeric kinases. Mutations and deletions of the p16INK4a gene and inactivation by hypermethylation, have been shown to play a role in tumorigenesis in many different types of tumors. The Kip/Cip proteins include three structurally related proteins, p21, p27, and p57. In contrast to the INK4 proteins, the Kip/Cip proteins inhibit most cyclin/cdk heterodimers. Specific Kip/Cip proteins are induced by upstream events. p21 is induced in response to DNA damage and specifically inhibits cyclin E/cdk2. Protein levels of p27 are highest in quiescent cells and induce G1 arrest in response to conditions that typically result in cell quiescence such as growth factor deprivation or contact inhibition. Both the INK4 and the Kip/Cip proteins inhibit the phosphorylation and inactivation of pRb.
Retinoblastoma The retinoblastoma gene (Rb) was the first tumor suppressor to be identified. Rb mutations were first shown to be causal in familial and sporadic retinoblastoma, a rare tumor of the eye, but have since been associated with many other tumors including osteosarcoma, small cell lung cancer, and prostate and breast cancer. In addition, mutations in the upstream Rb signaling pathway that result in the functional inactivation of the Rb gene product, pRb, are found in virtually all malignancies. Three Rb homologs have been described, including Rb, Rb 107, and Rb 130. All Rb homologs are characterized by a ‘pocket’ domain, which is highly conserved and necessary for pRb’s tumor suppressor function. All the Rb homologs bind viral oncoproteins as well as E2F family members. Binding of viral oncoproteins disrupts the pocket domain of pRb and impairs pRb’s tumor suppressor function. All pRb homologs cause G1 arrest.
The primary role of pRb is the inhibition of transcription of genes that mediate passage across the G1/S transition. There are two mechanisms by which pRb inhibits transcription. First, pRb binds to and inhibits the E2F family of transcription factors. The binding characteristics of the homologs vary slightly as, whereas pRb binds preferentially to E2F1-4, p107 and p130 bind preferentially to E2F4 and E2F5. Phosphorylation of pRb regulates its interaction with E2F. The phosphorylation status of pRb fluctuates throughout the cell cycle. Hypophosphorylated pRb is active and binds to E2F family members thus sequestering E2F and inhibiting its transcriptional activity. Hyperphosphorylated pRb is inactive and releases E2F, which results in the transcription of genes that allow the cell to progress to S phase. Upon release from pRb, E2F binds to DP-1 or DP-2 and the resulting heterodimer activates genes necessary for DNA replication. The mechanism by which pRb inhibits E2F transcriptional activity is still debated but it may be via the recruitment of chromatin remodeling enzymes such as histone deacetylases (HDACs) which directly repress transcription by removing acetyl groups from chromatin which causes the chromatin to be less accessible to transcription factors. The role of the pRb-bound HDAC may be to counteract the activity of the E2F-bound acetyltransferase protein, p300/CBP, which transfers acetyl groups to chromatin and enhances transcriptional activity. In addition to its inactivation of E2F resulting in a decrease in transcription of E2F-responsive genes, the complex of pRb and E2F actively represses transcription, which may also be via the recruitment of HDACs to the promoter regions. The regulation of pRb activity is complex. There are 16 possible sites for cdk-mediated phosphorylation and data suggest that phosphorylation at each different site regulates a distinct pRb function. pRb is phosphorylated by multiple cyclin/cdk complexes. Cyclin D/cdk4/6 initiates phosphorylation in early G1 and cyclin E/cdk2 hyperphosphorylates pRb in late G1. Cyclin A/cdk2 maintains phosphorylation of pRb thoughout S phase. pRb may perform other roles in addition to regulation of G1/S including the regulation of apoptosis. A decrease in functional pRb results in the activation of p53-induced apoptosis, which appears to be mediated via the release of E2F1. Free E2F1 activates transcription of ARF (alternate reading frame of the p16INK4a locus), which inhibits a protein called mdm-2 ubiquitin ligase (mdm-2). mdm-2 targets proteins for ubituitin-mediated proteolysis. Since mdm-2 initiates the degradation of p53, its inhibition results in an increase in p53 and a corresponding increase in apoptosis. Thus, a decrease
Cell Cycle
in functional pRb, which could otherwise result in unchecked cell proliferation, triggers an apoptotic response. A decrease in functional pRb also creates a selection pressure for p53 mutations, since only cells that have mutated dysfunctional p53 survive. Not surprisingly, p53 mutations are often found to coexist with Rb mutations in malignant tumors.
Checkpoints Checkpoints are surveillance mechanisms comprising numerous genes that detect DNA damage and induce either cell cycle arrest and DNA repair mechanisms, or, in the presence of extensive DNA damage, apoptosis. The data elucidating this surveillance network are very incomplete but have been advanced significantly since the isolation of the mutation that is associated with ataxia telangiectasia (AT). AT is a rare pediatric disease that is associated with immune deficiency and an increased susceptibility to cancer. Prior to the isolation of the AT mutation, it had long been observed that stimuli that induce DNA damage delay progression through the cell cycle. For years this phenomenon was assumed to be the passive response of the cell as a direct result of the DNA damage itself. By contrast, cells that harbor the AT mutation demonstrate a marked decrease in cell cycle arrest after DNA damaging radiation. These data suggested that an active system exists in normal cells that retards cycle progression in the presence of DNA damage. The checkpoint surveillance system comprises sensor proteins (proteins that detect DNA damage and initiate a signaling cascade); transducers (modifying enzymes such as kinases that relay the signal to effector proteins); and effectors (downstream target proteins that, upon activation by modifying enzymes, cause cycle arrest). Of these proteins, the least is known about sensor proteins, although several candidate genes have been suggested. The effector proteins include kinase inhibitors such as p21, or cyclin/cdk heterodimers that are either activated or inhibited to cause cycle arrest. Major transducer proteins include p53, ATM (AT mutated) and ATR (ATM and RAD-3 related). p53 is a transcription factor that activates the transcription of genes that cause cell cycle arrest at either G1/S or G2. In addition, p53 activates genes that initiate DNA repair and cause apoptosis. Mutations of p53 are commonly described in association with human tumors. The result of p53 activation is cell type-specific and depends on the type and severity of injury. p53-induced G1 cell cycle arrest is mediated via the induction of p21 and p16. p53 has a very short half-life and is generally undetectable in healthy cells. In the presence of DNA damage
3
induced by either ultraviolet or g-irradiation, p53 is activated by posttranscriptional modifications including phosphorylation and acetylation, that either enhance its stability or alter its affinity for binding proteins. ATM and its related protein, ATR, phosphorylate p53 which decreases its binding to mdm-2. A decrease in the interaction between p53 and mdm2 causes a decrease in ubiquitin-mediated proteolysis of p53 and a resulting increase in p53 protein levels. As described previously, ARF also activates p53 via the inactivation of mdm-2. In addition to phosphorylation, the acetylation status of p53 also determines its stability. p53 is acetylated and stabilized by p300/ CBP which increases apoptosis. The recently described NAD-dependent deacetylase protein, SIRT1, removes acetyl groups from p53 and decreases apoptosis. ATM and ATR are closely related phosphoinositide 3-kinases that are activated by DNA damage. Upon activation, ATM phosphorylates and activates multiple proteins in addition to p53, including mdm2 and a serine/threonine kinase called Chk-2. Activated Chk-2 phosphorylates p53. ATR phosphorylates many but not all of the same substrates as ATM. ATR phosphorylates and activates Chk-1, which also phosphorylates p53. p53, ATM, and ATR also contribute to G2 arrest. Upon activation, cdk1 initiates mitosis. cdk1 is activated via its interaction with cyclin B and via dephosphorylation by cdc25C phosphatase. Upon phosphorylation by ATM and ATR, Chk-2 and Chk-1 phosphorylate and inhibit cdc25C, which prevents the activation of cdk1. p53 activates transcription of two genes that inhibit cdk1 activity including GADD45 and 14-3-3 s. GADD45 disrupts the cyclin B/cdk1 heterodimer. The protein product of 14-3-3 s sequesters cdc25C, which prevents the dephosphorylation of cdk1. In the face of overwhelming DNA damage, checkpoints, in particular p53, induce cell death by apoptosis rather than cell cycle arrest. Apoptosis, or programmed cell death is an evolutionarily conserved, energy-requiring mechanism by which unwanted or irreparably damaged cells are removed from the organism. Apoptosis is a fundamental component of both normal embryogenesis and adult homeostasis. Apoptosis is also a physiologic response to diverse toxic stimuli including viral infection, DNA damage induced by irradiation or reactive oxygen species, hypoxia, growth factor deficiency or genetic aberration. Apoptosis is carried out by caspases, which are proteases that contain a cysteine nucleophile and cleave proteins whose sequence contains specific motifs that include an aspartic acid residue. Upstream or initiator caspases are activated
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Cell Cycle
by the binding of an extracellular ligand to a death receptor. Death receptors are members of the tumor necrosis superfamily and are characterized by an intracellular death domain. An important example of a death receptor is CD95, or fas, which binds fas ligand. Upon binding of a ligand, the death receptor binds to intracellular adaptor proteins. Adapter proteins bind to initiator caspases 2, 8, 9, or 10, which provokes their autocleavage and activation. Initiator caspases activate downstream effector or executioner caspases, such as caspase 3 or 7, or proapoptotic BCL-2 proteins. The BCL-2 family includes proteins that contain BCL-2 homology domains. These domains allow for heterdimerization by which BCL-2 proteins activate other family members. BCL-2 proteins modulate the intrinsic apoptotic pathway and may have either proor anti-apoptotic effects. Proapoptotic BCL-2 proteins increase mitochondrial membrane permeability which allows for the release of cytochrome c. Cytochrome c release from mitochondria results in dimerization of an adaptor protein called Apaf-1 (apoptotic protease activating factor) which binds procaspase 9 resulting in its cleavage and activation. Caspase 9 activates the downstream effector, caspase 3. Antiapoptotic proteins in the BCL-2 family inhibit the proapoptotic members and prevent the increase in mitochondrial membrane permeability. The downstream effector caspases target multiple proteins for degradation including enzymes, nuclear structural proteins such as lamins, cytoskeletal proteins such as actin, proteins critical for cell–cell interaction such as b-catenin, and DNA repair enzymes. p53 activates multiple genes that are involved in apoptosis, including genes that encode proteins that function via receptor-mediated signaling and those which encode proteins that modulate downstream effectors. p53-activated IGF-BP3 inhibits binding of IGF-1 to the IGF-1 receptor, which can induce apoptosis. p53 activates transcription of the death receptor ligands fas/Apo1/CD95 and the death receptor KILLER/DR5. p53 also induces the proapoptotic BCL-2 protein bax, as well as other proteins that enhance cytochrome c release from mitochondria, including p53, AIP1, PUMA, and Noxa. Apoptosis may also be induced via an increase in oxidative stress generated by multiple p53-induced genes that are homologous to NADPH-quinone oxidoreductase. Importantly, no singular p53-activated gene product has been conclusively shown to initiate apoptosis. It appears that many p53-induced proapoptotic genes need to be activated concurrently in order for apoptosis to occur. It is not clear which variables determine whether p53 induces cell cycle arrest or apoptosis. Certain cell
types, such as T lymphocytes, are especially sensitive to apoptosis whereas fibroblasts are more likely to undergo cell cycle arrest. Whereas p53 induces arrest or senescence in normal cells, p53 activation usually causes apoptosis in transformed cells. The reason for the enhanced sensitivity to p53-induced apoptosis in transformed cells may be related to the deregulation of E2F due to the inactivation of pRb. Cycle arrest induced by p21 may protect the cell from apoptosis. Other factors that may predispose the cell toward p53-induced apoptosis include alterations in the bax/ bcl-2 ratio, concurrent absence of growth factors, a greater intensity of stress and higher protein levels of p53. Posttranslational modifications may also determine p53 promoter specificity, which may play a major role in determining whether p53 expression results in cell cycle arrest or apoptosis.
Clinical Application The normal regulation of the cell cycle plays an important role in tissue repair and inflammation. All tissues may be stratified by proliferative capability into three categories including labile, quiescent or permanently nondividing cells. Labile cells are continuously dividing and include surface epithelial cells such as stratified squamous epithelial cells of the skin and columnar epithelial cells of the gastrointestinal tract. Quiescent cells are nondividing under normal circumstances but can be induced to reenter the cell cycle by exposure to growth factors. Quiescent cells include parenchymal cells of the liver, kidney, and pancreas and mesenchymal cells such as fibroblasts. The cytokine-induced reentry of quiescent cells into G1 phase is an important component of the inflammatory response, which has been well characterized in the kidney. Glomerular mesangial cells proliferate in many models of glomerular disease, including lupus nephritis and diabetes. The proliferation of mesangial cells occurs in response to cytokines such as platelet-derived growth factor and basic fibroblast growth factor. Inhibition of mesangial cell proliferation may abrogate the glomerulosclerosis or the glomerular scarring that occurs as a result of inflammation. Permanently nondividing cells have lost all capacity for proliferation and include nerve cells and cardiac muscle cells. The deregulation of the cell cycle resulting in unchecked cell proliferation is a hallmark of cancer. All human cancers are characterized by defects of restriction point control, checkpoints, DNA repair, or apoptosis. Defects of restriction point control allow for uncontrolled proliferation and result in loss of
Cell Cycle
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terminal differentiation. While some cancers are characterized by loss of function mutations of Rb or by disruption of the Rb pocket domain by viral oncoproteins, many more are caused by functional inactivation of pRb through cyclin D overexpression or INK4a mutations. Mutations of p53 are the most common mutations associated with cancer and occur in almost 50% of all human cancers.
understanding of cell cycle regulation remains incomplete. Further studies may allow better understanding of diseases that result from deregulation of these pathways.
Conclusion
Further Reading
The regulation of the cell cycle plays an important role in normal tissue repair and regeneration. Loss of cell cycle regulation is a chief characteristic of cancer. Cell cycle regulation involves numerous signaling pathways that determine whether cells will proliferate, remain quiescent, arrest or undergo apoptosis. While enormous progress has been made in the elucidation of these signaling pathways, our
Griffin SV, Pichler R, Wada T, et al. (2003) The role of cell cycle proteins in glomerular disease. Seminars in Nephrology 23: 569–582. Iliakis G, Wang Y, Guan J, and Wang H (2003) DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene 22: 5834–5847. Sionov RV and Haupt Y (1999) The cellular response to p53: The decision between life and death. Oncogene 18: 6145–6157.
See also: Tissue Repair.