Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

Requirement of Cyclin E-Cdk2 Inhibition in p16INK4a-Mediated Growth Suppression

Loss-of-function mutations of p16^INK4a have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G₁ cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4^N158, designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050–2054, 1993). In this study, we determined functional differences between p16 and Cdk4^N158. We show that p16 and Cdk4^N158 inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27^KIP1, while Cdk4^N158 formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G₁ arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.     

Cyclin D1 Promotes Cell Cycle Progression through Enhancing NDR1/2 Kinase Activity Independent of Cyclin-dependent Kinase 4

Cyclin/cyclin-dependent kinases (Cdks) are critical protein kinases in regulating cell cycle progression. Among them, cyclin D1/Cdk4 exerts its function mainly in the G₁ phase. By using the tandem affinity purification tag approach, we identified a set of proteins interacting with Cdk4, including NDR1/2. Interestingly, confirming the interactions between NDR1/2 and cyclin D1/Cdk4, we observed that NDR1/2 interacted with cyclin D1 independent of Cdk4, but NDR1/2 and cyclin D1/Cdk4 did not phosphorylate each other. In addition, we found that NDR1/2 did not affect the kinase activity of cyclin D1/Cdk4 upon phosphorylation of GST-Rb. However, cyclin D1 but not Cdk4 promoted the kinase activity of NDR1/2. We also demonstrated that cyclin D1 K112E, which could not bind Cdk4, enhanced the kinase activity of NDR1/2. To test whether cyclin D1 promotes G₁/S transition though enhancing NDR1/2 kinase activity, we performed flow cytometry analysis using cyclin D1 and cyclin D1 K112E Tet-On inducible cell lines. The data show that both cyclin D1 and cyclin D1 K112E promoted G₁/S transition. Importantly, knockdown of NDR1/2 almost completely abolished the function of cyclin D1 K112E in promoting G₁/S transition. Consistently, we found that the protein level of p21 was reduced in cells overexpressing cyclin D1 K112E but not when NDR1/2 was knocked down. Taken together, these results reveal a novel function of cyclin D1 in promoting cell cycle progression by enhancing NDR kinase activity independent of Cdk4.     

Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr¹⁶¹ by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr¹⁴ and Tyr¹⁵ phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G₂/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr¹⁵. In addition, Tyr¹⁵-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G₂ and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr¹⁶¹ phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G₂/M is tightly coupled and regulated by Cdc25 phosphatases.     

Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G₁ for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G₁ cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G₁ Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G₁.     

The Auto-ubiquitylation of E3 Ubiquitin-protein Ligase Chfr at G2 Phase Is Required for Accumulation of Polo-like Kinase 1 and Mitotic Entry in Mammalian Cells

The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G₂/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G₂/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G₂ phase, but not at S phase, were ∼2–3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G₂ phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G₂/M transition.     

p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G₁. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21^Waf1/Cip1 enter S phase with a ≥4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G₁/S and G₂/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21^+/+ and p21^−/− cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21^−/− cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G₁-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21^+/+ cells paralleled the onset of endoreduplication in HCT116 p21^−/− cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts

The irreversible G1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G1 cyclin-cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21(Sdi1,Cip1,Waf1), which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16(Ink4a), suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by 相似文献   

Induction of a G2-Phase Arrest in Xenopus Egg Extracts by Activation of p42 Mitogen-activated Protein Kinase

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G₂-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G₂-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G₂ arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G₂-phase during the first mitotic cell cycle.     

Regulation of glioblastoma progression by cord blood stem cells is mediated by downregulation of cyclin D1

Background

The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G₁ phase.

Methodology/Principal Findings

In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G₀-G₁ phase arrest, but also reduced the number of cells at S and G₂-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G₀-G₁ phase despite the reduction of G₀-G₁ regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G₀-G₁ regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC.

Conclusions/Significance

This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G₀-G₁ phase.     

Phospho-Bcl-xL(Ser62) plays a key role at DNA damage-induced G2 checkpoint

Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G₂ checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G₂ arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G₂ arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G₂ checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G₂ checkpoint, phospho-Bcl-xL(Ser62) stabilizes G₂ arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.     

Phospho-Bcl-xL(Ser62) plays a key role at DNA damage-induced G2 checkpoint

Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G₂ checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G₂ arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G₂ arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G₂ checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G₂ checkpoint, phospho-Bcl-xL(Ser62) stabilizes G₂ arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.     

Cyclin E2, a Novel G1 Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27^Kip1 and p21^Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G₁/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G₁, demonstrating that cyclin E2 may be rate limiting for G₁ progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.     

Complex mechanisms underlying impaired activation of Cdk4 and Cdk2 in replicative senescence: roles of p16, p21, and cyclin D1

Numerous changes in gene expression are known to occur during replicative senescence, including changes in genes involved in the cell cycle control. In the present study, we have found a severe impairment in the activation of Cdk2 and Cdk4 in response to mitogens in senescent human fibroblasts and determined the molecular basis for this. Although Cdk4 protein was constitutively expressed in senescent cells at the same level as in early-passage young cells, it was found to be complexed with a distinct set of Cdk inhibitors. Cdk4 derived from early passage quiescent cells was effectively activated by incubation with cyclin D1 and Cdk-activating kinase (CAK) in vitro, whereas Cdk4 from senescent cells was not. Cdk2 protein was dramatically decreased in senescent cells and complexed primarily with cyclin D1 and p21. This cyclin D1-bound Cdk2 was not activated by CAK either in vivo or in vitro, implicating cyclin D1 as an inhibitor of Cdk2 activation. Thus, one of the underlying molecular events involved in replicative senescence is the impaired activation of Cdk4 and Cdk2 due to increased binding of p16 to Cdk4 and increased association of Cdk2 with cyclin D1 and p21.     

Phosphorylation of cyclin D1 regulated by ATM or ATR controls cell cycle progression

Cyclin D1 is required at high levels for passage through G₁ phase but must be reduced to low levels during S phase to avoid the inhibition of DNA synthesis. This suppression requires the phosphorylation of Thr286, which is induced directly by DNA synthesis. Because the checkpoint kinase ATR is activated by normal replication as well as by DNA damage, its potential role in regulating cyclin D1 phosphorylation was tested. We found that ATR, activated by either UV irradiation or the topoisomerase IIβ binding protein 1 activator, promoted cyclin D1 phosphorylation. Small interfering RNA against ATR inhibited UV-induced Thr286 phosphorylation, together with that seen in normally cycling cells, indicating that ATR regulates cyclin D1 phosphorylation in normal as well as stressed cells. Following double-stranded DNA (dsDNA) breakage, the related checkpoint kinase ATM was also able to promote the phosphorylation of cyclin D1 Thr286. The relationship between these checkpoint kinases and cyclin D1 was extended when we found that normal cell cycle blockage in G₁ phase observed following dsDNA damage was efficiently overcome when exogenous cyclin D1 was expressed within the cells. These results indicate that checkpoint kinases play a critical role in regulating cell cycle progression in normal and stressed cells by directing the phosphorylation of cyclin D1.     

Regulation of Cyclin A-Cdk2 by SCF Component Skp1 and F-Box Protein Skp2

Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21^Cip1/WAF1 bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G₁/S cell cycle arrest.     

Nuclear Accumulation of p21Cip1 at the Onset of Mitosis: a Role at the G2/M-Phase Transition

Cell cycle arrest in G₁ in response to ionizing radiation or senescence is believed to be provoked by inactivation of G₁ cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21^{Cip1/Waf1/Sdi1}. We provide evidence that in addition to exerting negative control of the G₁/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G₂/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21^−/− mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G₂ that may contribute to the implementation of late cell cycle checkpoint controls.     

The Protein Kinase C�� Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint

Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G₂/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G₂/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr¹⁵), a critical event in the G₂/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G₂/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G₂/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G₂/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G₂/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G₂/M cell cycle checkpoint may be a critical component of its tumor suppressor function.