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 contr ibutes to the preservation of genetic stability by mediating either a G(1) arrest or apoptosis in response to DNA damage. Recent reports sug gest that p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase (Cdk)-inhibitor Cip1. Here, we characte rize the p53-dependent G(1) arrest in several normal human diploid fib roblast (NDF) strains and p53 deficient cell lines treated with 0.1-6 Gy gamma radiation. DNA damage and cell cycle progression analyses sho wed that NDE entered a prolonged arrest state resembling senescence, e ven at low doses of radiation. This contrasts with the view that p53 e nsures genetic stability by inducing a transient arrest to enable repa ir of DNA damage, as reported for some myeloid leukemia lines. Gamma r adiation administered in early to mid-, but not late, G(1) induced the arrest, suggesting that the p53 checkpoint is only active in G(1) unt il 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 p hase as a function of dose is consistent with single-hit kinetics. Cyt ogenetic analyses combined with radiation dosage data indicate that on ly 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 m aintain genetic stability in NDE by mediating a permanent cell cycle a rrest through long-term induction of Cip1 when low amounts of unrepair ed DNA damage are present in G(1) before the restriction point.