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Intracellular Localization of p53 Tumor Suppressor Protein in -irradiated Cells Is Cell Cycle Regulated and Determined by the Nucleus¹

Department of Molecular Genetics, University of Illinois, Chicago, Illinois 60607 [E. A. K., M. Z., A. V. G.]; Advanced Cellular Diagnostics, Elmhurst, Illinois 60126 [C. R. Z., D. C., S. S. B.]; and Department and School of Medicine, University of California at San Diego, La Jolla, California 92093-0648 [A. S. G]

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^waf1 after a single dose of -irradiation. Quantitation of DNA content in p53-positive and -negative nuclei 4–6 h after 10 Gy of -irradiation of human breast carcinoma MCF7 cells, fibrosarcoma HT1080 cells, and diploid skin fibroblasts showed that p53 and p21^waf1 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^waf1-negative. This suggests that there is a cell cycle window during which p53 can accumulate in the nucleus and activate expression of p21^waf1. 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^waf1 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.

¹ Supported by the National Cancer Institute Grants CA60730 and CA75179 (to A. V. G. and E. A. K.) and United States Army Breast Cancer Research Fellowship (to M. Z.).

² To whom requests for reprints should be addressed, at Department of Genetics (M/C 669), University of Illinois, 900 South Ashland Avenue, Chicago, IL 60607-7170. Phone: (312) 413-0349; Fax: (312) 996-0683; E-mail: gudkov@uic.edu.

Received 8/ 8/97. Accepted 10/17/97.

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