DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts.
The tumor suppressor p53 is a cell cycle checkpoint protein that contributes to the preservation of genetic stability by mediating either a G1 arrest or apoptosis in response to DNA damage. Recent reports suggest that p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase (Cdk)-inhibitor Cip1. Here, we characterize the p53-dependent G1 arrest in several normal human diploid fibroblast (NDF) strains and p53-deficient cell lines treated with 0.1-6 Gy gamma radiation. DNA damage and cell cycle progression analyses showed that NDF entered a prolonged arrest state resembling senescence, even at low doses of radiation. This contrasts with the view that p53 ensures genetic stability by inducing a transient arrest to enable repair of DNA damage, as reported for some myeloid leukemia lines. Gamma radiation administered in early to mid-, but not late, G1 induced the arrest, suggesting that the p53 checkpoint is only active in G1 until cells commit to enter S phase at the G1 restriction point. A log-linear plot of the fraction of irradiated G0 cells able to enter S phase as a function of dose is consistent with single-hit kinetics. Cytogenetic analyses combined with radiation dosage data indicate that only one or a small number of unrepaired DNA breaks may be sufficient to cause arrest. The arrest also correlated with long-term elevations of p53 protein, Cip1 mRNA, and Cip1 protein. We propose that p53 helps maintain genetic stability in NDF by mediating a permanent cell cycle arrest through long-term induction of Cip1 when low amounts of unrepaired DNA damage are present in G1 before the restriction point.
Gene Expression Lab, Salk Institute, La Jolla, California 92037.
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Di Leonardo
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Linke
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Clarkin
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Wahl
D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology* / Q000528:radiation effects; D002460:Cell Line; D002869:Chromosome Aberrations; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis*; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D005434:Flow Cytometry; D016193:G1 Phase / Q000502:physiology / Q000528:radiation effects; D005720:Gamma Rays; D006801:Humans; D007700:Kinetics; D007951:Leukemia, Myeloid; D008677:Metaphase; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D012867:Skin; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53