p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest

We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.     

Nerve growth factor-induced cell cycle reentry in newborn neurons is triggered by p38MAPK-dependent E2F4 phosphorylation

Cumulative evidence indicates that activation of cyclin D-dependent kinase 4/6 (cdk4/6) represents a major trigger of cell cycle reentry and apoptosis in vertebrate neurons. We show here the existence of another mechanism triggering cell cycle reentry in differentiating chick retinal neurons (DCRNs), based on phosphorylation of E2F4 by p38(MAPK). We demonstrate that the activation of p75(NTR) by nerve growth factor (NGF) induces nuclear p38(MAPK) kinase activity, which leads to Thr phosphorylation and subsequent recruitment of E2F4 to the E2F-responsive cdc2 promoter. Inhibition of p38(MAPK), but not of cdk4/6, specifically prevents NGF-dependent cell cycle reentry and apoptosis in DCRNs. Moreover, a constitutively active form of chick E2F4 (Thr261Glu/Thr263Glu) stimulates G(1)/S transition and apoptosis, even after inhibition of p38(MAPK) activity. In contrast, a dominant-negative E2F4 form (Thr261Ala/Thr263Ala) prevents NGF-induced cell cycle reactivation and cell death in DCRNs. These results indicate that NGF-induced cell cycle reentry in neurons depends on the activation of a novel, cdk4/6-independent pathway that may participate in neurodegeneration.     

Down-regulation of p21WAF1 promotes apoptosis in senescent human fibroblasts: involvement of retinoblastoma protein phosphorylation and delay of cellular aging

It has been suggested that genes which exercise checkpoint control during cell cycle traverse are equally important to the process of apoptotic cell death. In this study, we show that the key cell cycle regulatory gene p21(WAF1) is also involved in the execution of apoptosis. p21(WAF1) expression was down-regulated during NaBu-induced apoptosis of senescent normal diploid human 2BS fibroblasts. Conversely, when p21(WAF1) expression was actively suppressed in 2BS cells by a stably transfected antisense p21(WAF1) construct, apoptosis was accelerated and senescence was delayed, as shown by several markers of cell aging. Down-regulation of p21(WAF1) by antisense caused an increase in the phosphorylation and inactivation of pRb. Phosphorylation of pRb was further enhanced upon induction of apoptosis by NaBu. Our results suggest that p21(WAF1), acting through the phosphorylation of pRb, regulates whether 2BS cells cease to proliferate and become senescent but resistant to apoptosis, or whether they accelerate proliferation while becoming more susceptible to apoptotic stimuli.     

Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts

Irreversible G(1) arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21(Cip1/SDI1/WAF1) and p16(Ink4A). To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G(1) arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21(-/-) mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G(1) arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.     

Cyclin E and c-Myc promote cell proliferation in the presence of p16INK4a and of hypophosphorylated retinoblastoma family proteins.

Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16. Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation. Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However, cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with effects indistinguishable from those of cyclin E. We suggest that this effect of Myc is mediated by its action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins, rather than induction of Cdc25A. Our data imply that oncogenic activation of c-Myc, and possibly also of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis.     

Cooperation between p53 and p130(Rb2) in induction of cellular senescence

To determine pathways cooperating with p53 in cellular senescence when the retinoblastoma protein (pRb)/p16INK4a pathway is defunct, we stably transfected the p16INK4a-negative C6 rat glioma cell line with a temperature-sensitive mutant p53. Activation of p53(Val-135) induces a switch in pocket protein expression from pRb and p107 to p130(Rb2) and stalls the cells in late G1, early S-phase at high levels of cyclin E. Maintenance of the arrest depends on the functions of p130(Rb2) repressing cyclin A. Inactivation of p53 in senescent cultures restores the pocket proteins to initial levels and initiates progression into S-phase, but the cells fail to resume proliferation, likely due to DNA damage becoming apparent in the arrest and activating apoptosis subsequent to the release from p53-dependent growth suppression. The data indicate that p53 can cooperate selectively with p130(Rb2) to induce cellular senescence, a pathway that may be relevant when the pRb/p16INK4a pathway is defunct.     

Fez1/Lzts1 a new mitotic regulator implicated in cancer development

The retinoblastoma (Rb) tumor suppressor gene product, pRb, has an established role in the implementation of cellular senescence, the state of irreversible G1 cell cycle arrest provoked by diverse oncogenic stresses. In murine cells, senescence cell cycle arrest can be reversed by subsequent inactivation of pRb, indicating that pRb is required not only for the onset of cellular senescence, but also for the maintenance of senescence program in murine cells. However, in human cells, once pRb is fully activated by p16^INK4a, senescence cell cycle arrest becomes irreversible and is no longer revoked by subsequent inactivation of pRb, suggesting that p16^INK4a/Rb-pathway activates an alternative mechanism to irreversibly block the cell cycle in human senescent cells. Here, we discuss the molecular mechanism underlying the irreversibility of senescence cell cycle arrest and its potential towards tumor suppression.     

Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb

Adeno-associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G(1) and G(2), with elevated CDK inhibitor p21 and reduced cyclin E-, A- and B1-associated kinase activity. Rep78-expressing cells were also impaired in S-phase progression and accumu lated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C-terminal zinc finger domain of Rep78. Rep78-induced S-phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb(-/-) primary mouse embryonic fibroblasts displayed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb(+/+) controls. These results suggest that physiological levels of active pRb can interfere with S-phase progression. We propose that the AAV Rep78 protein arrests cells within S-phase by a novel mechanism involving the ectopic accumulation of active pRb.     

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   

Perturbation of the p53 response by human papillomavirus type 16 E7.

The p53 tumor suppressor protein can induce both cell cycle arrest and apoptosis in DNA-damaged cells. In human carcinoma cell lines expressing wild-type p53, expression of E7 allowed the continuation of full cell cycle progression following DNA damage, indicating that E7 can overcome both G1 and G2 blocks imposed by p53. E7 does not interfere with the initial steps of the p53 response, however, and E7 expressing cells showed enhanced expression of p21(waf1/cip1) and reductions in cyclin E- and A-associated kinase activities following DNA damage. One function of cyclin-dependent kinases is to phosphorylate pRB and activate E2F, thus allowing entry into DNA synthesis. Although E7 may substitute for this activity during cell division by directly targeting pRB, continued cell cycle progression in E7-expressing cells was associated with phosphorylation of pRB, suggesting that E7 permits the retention of some cyclin-dependent kinase activity. One source of this activity may be the E7-associated kinase, which was not inhibited following DNA damage. Despite allowing cell cycle progression, E7 was unable to protect cells from p53-induced apoptosis, and the elevated apoptotic response seen in these cells correlated with the reduction of cyclin A-associated kinase activity. It is possible that inefficient cyclin A-dependent inactivation of E2F at the end of DNA synthesis contributes to the enhanced apoptosis displayed by E7-expressing cells.     

Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated beta-galactosidase (SA-beta-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-beta-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest.     

DNA mismatch repair-dependent activation of c-Abl/p73alpha/GADD45alpha-mediated apoptosis

Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.     

Significance of MDM2 protein in the cell cycle

The basis of oncogenesis underlies the modification of the control of the cell cycle, which leads to disturb balance between proliferation and apoptosis. The MDM2 protein suppresses the ability of p53 to activate genes responsible for repairing or apoptosis, but also promotes p53 degradation by ubiquitination. MDM2 inhibits tumor suppressor property of pRb, by releasing E2F1, which stimulates DNA synthesis in S-phase. MDM2 influences on the neuronal and muscle differentiation. Quantity and stability of the MDM2 protein is regulated by p73, p53, TSG101, p14ARF and Ras-Raf-MEK-ERK pathway. Changes of the level of the MDM2 can disturb control of cell cycle and contribute to oncogenesis.     

Topoisomerase IIα mediates E2F-1-induced chemosensitivity and is a target for p53-mediated transcriptional repression

Mutations of the retinoblastoma tumor suppressor, pRb, or its cyclin-cyclin-dependent kinase (CDK) regulatory kinases or CDK inhibitors, allows unrestrained E2F activity, leading to unregulated cell cycle progression. However, overexpression of E2F-1 also sensitizes cells to apoptosis, suggesting that targeting this pathway may be of therapeutic benefit. Enforced expression of E2F-1 in interleukin-3-dependent myeloid cells led to preferential sensitivity to the topoisomerase II inhibitor, etoposide, which was independent of p53 accumulation. Pretreatment of the E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not cause DNA damage, protected these cells against etoposide-induced apoptosis. However, ICRF-193 cooperated with other DNA-damaging agents to induce apoptosis. Enforced expression of E2F-1 led to accumulation of p53 protein. An E2F-1 mutant that is defective in inducing cell cycle progression also induced p53, suggesting that p53 was responding directly to E2F, and not to secondary events caused by inappropriate cell cycle progression (i.e., DNA damage). Thus, topoisomerase II inhibition and DNA damage cooperate to selectively induce apoptosis in cells that have mutations in the pRb pathway.