P53 GENE-MUTATIONS ARE ASSOCIATED WITH DECREASED SENSITIVITY OF HUMANLYMPHOMA-CELLS TO DNA-DAMAGING AGENTS

The present study assessed the role of the p53 tumor suppressor gene i n cell cycle arrest and apoptosis following treatment of Burkitt's lym phoma and lymphoblastoid cell lines with gamma-rays, etoposide, nitrog en mustard, and cisplatin. Cell cycle arrest was measured by flow cyto metry; p53 and p21(Waf1/Cip1) protein levels were measured by Western blotting; cell survival was measured in 72-96-h growth inhibition assa ys and by trypan blue staining, and apoptotic DNA fragmentation was as sessed by either agarose gel electrophoresis or a modified filter elut ion method. We found that gamma-rays and etoposide induced a strong G( 1) arrest in the wild-type p53 lines while nitrogen mustard and cispla tin induced relatively little G(1) arrest. All agents failed to induce G(1) arrest in cells containing mutant p53 genes. The degree of G(1) arrest observed with these agents correlated with the rate of p53 and p21(Waf1/Cip1) protein accumulation: gamma-rays and etoposide induced rapid accumulation of both p53 and p21(Waf1/Cip1); nitrogen mustard an d cisplatin induced slow accumulation of p53 and no major accumulation of the p21(Waf1/Cip1) protein. Despite differences in G(1) arrest and kinetics of p53 or p21(Waf1/Cip1) protein accumulation, all agents te nded to decrease survival to a greater extent in the wild-type p53 lin es compared to the mutant p53 lines. Cell death in the wild-tgpe p53 l ines was associated with intracellular DNA degradation into oligonucle osomal sized DNA fragments, indicative of apoptosis. We also observed an inverse sensitivity relationship between nitrogen mustard/cisplatin and etoposide in the mutant p53 lines and this was found to correlate with topoisomerase II mRNA levels in the cells. Our results suggest t hat p53 gene status is an important determinant of both radio- and che mosensitivity in lymphoid cell lines and that p53 mutations are often associated with decreased sensitivity to DNA damaging agents.