Intracellular localization of p53 tumor suppressor protein in γ- irradiated cells is cell cycle regulated and determined by the nucleus

DNA damage leads to the stabilization of p53 protein and its translocation to the nucleus, resulting in activation or suppression of p53- responsive genes. However, a significant proportion of cell nuclei remain negative for p53 and p53-inducible cyclin-dependent kinase inhibitor p21(wafl) after a single dose of γ-irradiation. Quantitation of DNA content in p53-positive and -negative nuclei 4-6 h after 10 Gy of γ-irradiation of human breast carcinoma MCF7 cells, fibrosarcoma HT1080 cells, and diploid skin fibroblasts showed that p53 and p21(wafl) nuclear accumulation occurs predominantly in the G₁ phase and at the beginning of the S phase of the cell cycle. The majority of the nuclei in late S phase and in G₂-M phase remained p53- and p21(wafl)-negative. This suggests that there is a cell cycle window during which p53 can accumulate in the nucleus and activate expression of p21(wafl). To determine whether cell cycle-dependent distribution of p53 is caused by cytoplasmic modifications of p53 protein or by properties of the nucleus, p53 localization was analyzed in multinucleated cells obtained by polyethylene glycol-mediated cell fusion. Dramatic differences in p53 accumulation were found among the nuclei in individual multinucleated cells. Distribution of p53-positive and -negative nuclei among the phases of the cell cycle was similar to that observed in a regular cell population. These results suggest that the observed differences in p53 accumulation in the nuclei of irradiated cells are determined by cell cycle- dependent nuclear functions. In contrast to p53, p21(wafl) was equally distributed among the nuclei of multinucleated cells regardless of the stage of the cell cycle, indicating that the observed phenomenon is specific for p53.