Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities (original) (raw)
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DNA damage and p53-mediated cell cycle arrest: A reevaluation
Proceedings of the National Academy of Sciences, 1996
Most mammalian cells exhibit transient delays in the G 1 and G 2 phases of the cell cycle after treatment with radiation or radiomimetic compounds. p53 is required for the arrest in G 1 , which provides time for DNA repair. Recently, a role of p53 in the G 2 ͞M transition has also been suggested. However, it has been reported that the presence of functional p53 does not always correlate with the induction of these checkpoints. To precisely assess the role of p53 in activating cell cycle checkpoints and in cell survival after radiation, we studied the response of two isogenic human fibrosarcoma cell lines differing in their p53 status (wild type or mutant). We found that when irradiated cells undergo a wild-type p53-dependent G 1 arrest, they do not subsequently arrest in G 2. Moreover, wild-type p53 cells irradiated past the G 1 checkpoint arrest in G 2 but do not delay in the subsequent G 1 phase. Furthermore, in these cell lines, which do not undergo radiation-induced apoptosis, the wild-type p53 cell line exhibited a greater radioresistance in terms of clonogenic survival. These results suggest that the two checkpoints may be interrelated, perhaps through a control system that determines, depending on the extent of the damage, whether the cell needs to arrest cell cycle progression at the subsequent checkpoint for further repair. p53 could be a crucial component of this control system.
Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation
Archives of Biochemistry and Biophysics, 2003
Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.
Radiation Research, 2003
Through a detailed study of cell cycle progression, protein expression, and kinase activity in ␥-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G 2-phase and subsequent G 1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G 2 arrest is correlated with decreased cyclin B1/ CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G 2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G 2 phase, normal cells chronically arrest in the subsequent G 1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G 1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/ CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21 WAF1/CIP1) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G 1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G 1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.
Dose-dependent effects of DNA-damaging agents on p53-mediated cell cycle arrest
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1999
We examined the dose-dependent effects of DNA-damaging agents on G1 arrest in isogenic human cell lines differing in their p53 status. As expected, 5 or 20 Gy of ionizing radiation induced a p53-dependent G1 arrest. In contrast, UV light or actinomycin D induced a modest G1 arrest that was p53-dependent only at lower doses. At higher doses, cells were arrested in G1 in a p53-independent manner coinciding with inhibition of RNA synthesis and abolished cyclin E expression. Interestingly, expression of cyclin E was enhanced after exposure to moderate doses of UV light and actinomycin D, and this enhancement was suppressed by wild-type p53. We propose that agents inducing transcription-blocking DNA lesions will at higher doses inhibit the progression of cells into S phase by a p53-independent mechanism involving the attenuation of E2F-mediated transcription of genes, such as cyclin E.
Cancer research and treatment : official journal of Korean Cancer Association, 2006
Recent studies have suggested that p53 regulates the G2 checkpoint in the cell cycle and this function is required for the maintenance of genomic integrity. In this study, we addressed a role of p53 in escaping from cell cycle G2 arrest following DNA damage. Cell cycle checkpoint arrest in the human colon cancer cell line HCT116 and its derivatives carry p53 or p21 deletions, were examined by FACS analysis, immunoprecipitation, Western blot and IP-kinase assay. While the cells with functional p53 were arrested at both the G1 and G2 checkpoints, the p53-deficient cells failed to arrest at G1, but they were arrested at G2. However, the p53-deficient cells failed to sustain G2 checkpoint arrest and they entered mitosis earlier than did the p53-positive cells and so this resulted in extensive cell death. Cdc2 kinase becomes reactivated in p53-deficient cells in association with entry into mitosis, but not in the p53-positive cells. Upon DNA damage, the p21-deficient cells, like the p53-...
Oncogene, 2011
In the presence of sustained DNA damage occurring in S-phase or G2, normal cells arrest before mitosis and eventually become senescent. The checkpoint kinases Chk1/ Chk2 and the CDK inhibitor p21 are known to have important complementary roles in this process, in G2 arrest and cell cycle exit, respectively. However, additional checkpoint roles have been reported for these regulators and it is not clear to what extent their functions are redundant.
Expert Opinion on Therapeutic Targets, 2010
Importance of the field-The eukaryotic cell division cycle is a tightly regulated series of events coordinated by the periodic activation of multiple cyclin-dependent kinases (cdks). Smallmolecule cdk-inhibitory compounds have demonstrated preclinical synergism with DNAdamaging agents in solid tumor models. An improved understanding of how cdks regulate the DNA damage response now provides an opportunity for optimization of combinations of cdk inhibitors and DNA damaging chemotherapy agents that can be translated to clinical settings. Areas covered in this review-Here, we discuss novel work uncovering multiple roles for cdks in the DNA-damage-response network. First, they activate DNA damage checkpoint and repair pathways. Later their activity is turned off, resulting in cell cycle arrest, allowing time for DNA repair to occur. Recent clinical data on cdk inhibitor-DNA-damaging agent combinations are also discussed. What the reader will gain-Readers will learn about novel areas of cdk biology, the complexity of DNA damage signaling networks and clinical implications. Take home message-New data demonstrate that cdks are 'master' regulators of DNA damage checkpoint and repair pathways. Cdk inhibition may therefore provide a means of potentiating the clinical activity of DNA-damaging chemotherapeutic agents for the treatment of cancer.
Journal of Biological Chemistry, 2003
Inhibition of cyclin-dependent kinases (CDKs) by Thr 14 /Tyr 15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G 2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominantnegative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr 14 /Tyr 15 phosphorylation in normal cell cycle and during the DNA damage checkpoint. Cyclins and cyclin-dependent kinases (CDKs) 1 are key regulators of the eukaryotic cell cycle. In mammalian cells, differ