DNA-DAMAGE TRIGGERS A PROLONGED P53-DEPENDENT G(1) 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 G(1) 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 G(1) 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 NDE 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, G(1) induced the arrest, suggesting that the p53 checkpoint is only active in G(1) until cells commit to enter S phase at the G(1) restriction point. A log-linear plot of the fraction of irradiated G(0) 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 NDE by mediating a permanent cell cycle arrest through long-term induction of Cip1 when low amounts of unrepaired DNA damage are present in G(1) before the restriction point.