Sustained activation of the JNK cascade and rapamycin-induced apoptosis are suppressed by p53/p21(Cip1)

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21(Cip1)arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21(Cip1) independent of cell cycle arrest. Rapamycin induces p21(Cip1) binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21(Cip1).     

p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia

X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.     

Different cell cycle mechanisms between UV-induced and X-ray-induced apoptosis in WiDr colorectal carcinoma cells

Although DNA-damaging agents such as ultraviolet (UV) and X-ray can induce apoptosis, the difference in the apoptotic mechanism is not clearly understood. In the present study, we investigated the effects of these two genotoxic agents on the induction of DNA damage and subsequent apoptotic cell death from the viewpoint of cell cycle regulation by using WiDr cells. Transient G1 arrest was observed after UV exposure, whereas G2 but not G1 arrest was induced after X-ray irradiation. UV-exposure could induce G1 arrest in both mutant-type (mt-p53) and wild-type p53 (wt-p53) cells, but obvious G1 arrest was not observed in the cells lacking in p53 expression. An increase in the DNA fragmentation was observed at S phase in UV-irradiated cells and at G2 phase in X-irradiated cells, respectively. UV-irradiated cells showed an increase production of p53 protein and accumulation of p21 protein. On the contrary, both p53 and p21 proteins remained at a low level in X-irradiated cells. Treatment with aphidicolin, an S phase blocking agent, prolonged cell cycle arrest and reduced the rate of apoptotic cell death in both UV-irradiated and X-irradiated cells. From these results, it is suggested that UV-induced apoptosis occurs mainly at S phase and is regulated by increased production of p53 and p21 proteins, while X-ray-induced apoptosis occurs after G2 blockade and may be independent of p53.     

Cdc6 determines utilization of p21(WAF1/CIP1)-dependent damage checkpoint in S phase cells

When cells traversing G(1) are irradiated with UV light, two parallel damage checkpoint pathways are activated: Chk1-Cdc25A and p53-p21(WAF1/CIP1), both targeting Cdk2, but the latter inducing a long lasting arrest. In similarly treated S phase-progressing cells, however, only the Cdc25A-dependent checkpoint is active. We have recently found that the p21-dependent checkpoint can be activated and induce a prolonged arrest if S phase cells are damaged with a base-modifying agent, such as methyl methanesulfonate (MMS) and cisplatin. But the mechanistic basis for the differential activation of the p21-dependent checkpoint by different DNA damaging agents is not understood. Here we report that treatment of S phase cells with MMS but not a comparable dose of UV light elicits proteasome-mediated degradation of Cdc6, the assembler of pre-replicative complexes, which allows induced p21 to bind Cdk2, thereby extending inactivation of Cdk2 and S phase arrest. Consistently, enforced expression of Cdc6 largely eliminates the prolonged S phase arrest and Cdk2 inactivation induced with MMS, whereas RNA interference-mediated Cdc6 knockdown not only prolongs such arrest and inactivation but also effectively activates the p21-dependent checkpoint in the UV-irradiated S phase cells.     

Cell-cycle arrest in TrkA-expressing NIH3T3 cells involves nitric oxide synthase

We have examined nerve growth factor (NGF)-triggered signaling in two NIH3T3 cell lines exogenously expressing the NGF receptor, TrkA. TRK1 cells cease to proliferate and extend long processes in response to NGF, while E25 cells continue to proliferate in the presence of NGF. These two cell lines express similar levels of TrkA and respond to NGF with rapid elevation of mitogen-activated protein kinase (MAPK) activity. MAPK activation is slightly more sustained for E25 cells than for TRK1 cells, although sustained activation of MAPK has been suggested to cause cell-cycle arrest. As judged by NADPH-diaphorase staining, nitric oxide synthase (NOS) activity is increased in TRK1 cells upon exposure to NGF. In contrast, diaphorase staining in E25 cells is unaffected by NGF treatment. Immunocytochemistry shows that levels of the brain NOS (bNOS) isoform are increased in TRK1, but not E25, cells exposed to NGF. Furthermore, Western blots show that NGF elevated cyclin-dependent kinase inhibitor, p21(WAF1), in TRK1 cells only. NGF-induced p21(WAF1) expression, cell-cycle arrest and process extension are abolished by N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS. The inactive enantiomer, D-NAME, did not inhibit these responses. Furthermore, even though E25 cells do not respond to NGF or nitric oxide donors, they do undergo a morphological change in response to NGF plus a nitric oxide donor. Therefore, NOS and p21(WAF1) are induced only in the TrkA-expressing NIH3T3 cell line that undergoes cell-cycle arrest and morphological changes in response to NGF. These results demonstrate that sustained activation of MAPK is not the sole determining factor for NGF-induced cell-cycle arrest and implicate NO in the cascade of events leading to NGF-induced morphological changes and cell-cycle arrest.     

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.     

p21 functions in a post-mitotic block checkpoint in the apoptotic response to vinblastine

We have shown previously that in KB-3 (HeLa) cells vinblastine causes downregulation of the CDK inhibitor p21 through a c-Jun regulated pathway. To test the hypothesis that p21 downregulation is necessary to alleviate a protective function, we transfected p21 in KB-3 cells and examined the apoptotic response to vinblastine. The results showed that cells overexpressing p21 were apoptosis-resistant, not through an ability of p21 to cause cell cycle arrest prior to mitotic arrest, but through altering the fate of mitotically arrested cells after drug treatment. Moreover, p21 null HCT116 cells were more prone to vinblastine-induced apoptosis relative to wild-type cells. The results provide support for a model whereby p21 downregulation promotes vinblastine-induced apoptosis by alleviating its protective function following mitotic arrest.     

Regulation of DNA repair throughout the cell cycle

The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.     

A role for mitochondria as potential regulators of cellular life span

We demonstrate that by simply raising extracellular pyruvate levels, and hence increasing metabolic supply, human diploid fibroblasts undergo a concentration-dependent induction of cellular senescence. Fibroblasts treated with pyruvate undergo a rapid growth arrest accompanied by elevated levels of the cell-cycle regulatory molecules p53, p21, and p16. These cells also exhibit a rise in mitochondrial oxidant production and a fall in intracellular glutathione levels. Exposure of pyruvate treated cells to the antioxidant and glutathione precursor N-acetylcysteine restores cell growth and reverses the increase in senescence-associated beta-galactosidase activity. Similarly, we demonstrate that by increasing mitochondrial number via retroviral-mediated expression of the mitochondrial biogenesis regulator PGC-1 there is also a reduction in cell growth and the more rapid induction of senescence. These results suggest that mitochondria appear to play a central role in regulating cellular life span.     

Role of p21WAF1 in the Cellular Response to UV

UV or gamma irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin-dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21 function.     

Role of p21WAF1 in the cellular response to UV

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.     

DNA methyltransferase inhibition in normal human fibroblasts induces a p21-dependent cell cycle withdrawal

Maintenance of methylation patterns in the mammalian genome by DNA (cytosine-5) methyltransferases (DNAMeTase) is required for normal cell and tissue function. Inhibition of DNAMeTase in cultured cells induces the expression of p21, a cyclin-dependent kinase (Cdk) inhibitor critical for cells to enter replicative senescence. We investigated the effects of DNAMeTase inhibition in normal human fibroblasts and found that it induces an irreversible growth arrest. Cells arrested by DNAMeTase inhibition became enlarged and had a flat morphology, exhibited an increased expression of collagenase and p21, and the DNA synthesis block could be overcome by the introduction of the SV40 large T antigen, all characteristics of senescent cells. In contrast, normal human fibroblasts lacking a functional p21 gene fail to undergo cell cycle arrest following DNAMeTase inhibition, indicating that p21 is an essential component of this arrest. Furthermore, DNAMeTase activity was reduced as cells approached the end of their proliferative potential. These data suggest that DNAMeTase could be an integral part of the mechanisms by which cells count the number of cell divisions completed and initiate a signaling cascade that ultimately results in the senescent phenotype.