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Radiation-induced G₁ Arrest Is Selectively Mediated by the p53-WAF1/Cip1 Pathway in Human Thyroid Cells¹

Department of Cell Physiology [H. N., T. H., T. T., S. Y.], Atomic Disease Institute and First Department of Internal Medicine [K. M., S. N.], Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki, 852 and Ito Hospital, 150, Tokyo [N. I., K. I.], Japan

We investigated the sensitivity and sequential events that take place in thyroid epithelial cells after irradiation. Cell survival ratios at a dose of 2 Gy were 18 ± 2.5%, 58 ± 1.0%, 59 ± 1.5%, and 98 ± 1.8% in primary thyroid cells, papillary thyroid carcinoma cells, follicular thyroid carcinoma cells, and anaplastic thyroid carcinoma cells, respectively. Thyroid carcinoma cell lines carrying mutations in the p53 gene were resistant to ionizing radiation. Although irradiated cells were accumulated at G₁ in primary thyroid cells even after low-dose irradiation (0.2 Gy), this phenomenon was not observed in the thyroid carcinoma cell lines. Wild-type p53 expression in primary thyroid cell was increased following irradiation, but mutated p53 in the thyroid carcinoma cell lines was unchanged. To clarify the signal transduction in the G₁ arrest following irradiation, levels of expression of the p53 putative downstream effectors GADD45 and WAF1/Cip1 were examined. Despite the consistent level of GADD45 mRNA, the level of WAF1/Cip1 transcripts was increased in a radiation dose-dependent manner in primary thyroid cells. This increase in the WAF1/Cip1 mRNA level was observed 30 min after irradiation and continued for at least 48 h. A mobility shift assay performed using the sequence of the putative p53 DNA binding site on the WAF1/Cip1 and GADD45 genes as a probe showed that nuclear protein extracted from primary thyroid cells, anti-p53 antibody, and probe oligonucleotide-bound complex was clearly shifted. An increase in binding activity of the p53/antibody/DNA complex was observed following irradiation. In contrast, the nuclear extract from thyroid carcinoma cells could not bind the specific DNA site, suggesting that mutant p53 has lost its binding ability. Actinomycin D inhibited WAF1/Cip1 and GADD45 mRNA levels and cycloheximide stimulated up-regulation of both basal mRNA levels, but an additional increase of the mRNA expression following irradiation was observed only in the WAF1/Cip1 gene. These data suggest that p53 in postradiation acts at a transcriptional level on WAF1/Cip1 gene expression and that de novo protein synthesis is not required for this effect.

These results suggest that the p53-WAF1/Cip1 pathway may play a central role in induction of G₁ arrest following irradiation in human thyroid epithelial cells.

¹ This work was supported by Grants-in-Aid 06671039 (H. N.) and 06454340 (S. Y.) from the Ministry of Education, Culture, and Science of Japan.

² To whom requests for reprints should be addressed.

Received 11/29/94. Accepted 3/20/95.

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