A role for p53 - p21(WAF1/CIP1) in the G2/M transition following exposure to ionizing radiation

Purpose: The tumor suppressor gene p53 is known to mediate cell cycle arrest at the G1/S checkpoint in response to DNA damage. We have previously shown that p53(-/-) cells are more sensitive to the effects of caffeine, and suggested that the G2/M arrest in response to DNA damage was more easily overridden by caffeine in p53(-/-) cells. This study was designed to investigate the mechanism of differential sensitivity in p53(-/-) cells. Materials and Methods: Clonogenic survival assays of p21(-/-) cells compared with p21(+/+) cells exposed to ionizing radiation (IR) were done with and without caffeine as previously for p53(-/-) cells. Caffeine treatment was started at the time of irradiation and maintained for 24 hours. For cell-cycle analysis, cells were initially evaluated in exponential asynchronous growth at the time of irradiation. The proportion of cells in G1, S and G2/M phases of the cell cycle were recorded immediately before and following irradiation and subsequently at 3,6,9,12,24 and 48 hours following irradiation. Subsequent studies used aphidicolin arrest and irradiation 3-4 hours after release from aphidicolin to evaluate a G2-enriched population. H1 kinase assays are in progress to evaluate the effect of p21 genotype and p21 induction upon H1 kinase inhibition. A primary rodent embryo fibroblast (REF) culture with wild-type p53 and the same REF transfected with a dominant mutant p53 (alanine-143) were also transfected with a plasmid containing wild type p21 under the control of a tetracycline inducible promoter, to investigate the effects of p21 upon G2 arrest. Results: Caffeine sensitization of ionizing radiation cell survival was seen only in the p21(-/-) cells, as was found for p53(-/-) cells, implying that the p53 - p21 pathway was responsible for the differential sensitivity. The sensitization enhancement ratio (SER) was 1.65 which was similar to the SER seen in p53(-/-) cells. Furthermore, in asynchronous cell culture, override of G2 block was seen for p21(-/-) cells, but not for p21(+/+) cells, using 2mM caffeine. These data suggested that the p53-p21 transactivation pathway was responsible for the differential sensitivity because of the effect on the G2 arrest in response to DNA damage. To confirm these observations, synchronized cells showed a clear difference in the duration of G2 arrest following caffeine treatment: p21(+/+) cells showed exit from G2/M by flow cytometry at 16-18 hours following release from aphidicolin, whereas p21(-/-) cells entered G1 at 10-12 hours. Clones of REFs with inducible p21 plasmids were screened for satisfactory induction of p21 following removal of tetracycline, and clones with a >4x increase in p21 were used for cell cycle analysis. The effect of p21 induction upon G2 arrest was seen in the REF clone with inactivated p53, where the G2 population 9 hours following exposure to 8 Gy of X-rays increased from 30% to 60% with induced p21. In the REF clone with wild-type p53, the G2 population at 9 hours was 35% with or without induction of p21. This suggests that p21 can affect the G2/M transition. H1 kinase assays are in progress to confirm that p21(+/+) cells maintain kinase inhibition in the face of caffeine treatment, whereas p21(-/-) cells do not, and to show that induction of p21 can independently inhibit kinase activity in synchronized cells. Conclusions: We conclude that p53 mediated transactivation of p21 can influence the G2/M transition in response to DNA damage. The effect is most easily observed by the effect of caffeine revealing a differential sensitivity of p21(-/-) and p53(-/-) cells. These data suggest a model in which caffeine inhibits the DNA damage signal pathway involving tyrosine phosphorylation of CDC2, and a separate p53-p21 pathway can also inhibit CDC2/cyclin B kinase activity in vivo