Gene Mutations Are Associated with Decreased Sensitivity of Human Lymphoma Cells to DNA Damaging Agents

The present study assessed the role ofthep53 tumor suppressor gene in cell cycle arrest and apoptosis following treatment of Burldtt's lymphoma and lymphoblastoid cell lines with †̃y-rays, etoposide, nitrogen mustard, and cisplatin. Cell cycle arrest was measured by flow cytometry; p5.3 and @ protein levels were measured by Western blotting; cell sur vival was measured in 72-96-h growth inhibition assays and by trypan blue staining, and apoptotic DNA fragmentation was assessed by either agarose gel electrophoresis or a modified ifiter elution method. We found that y-rays and etoposide induced a strong G1 arrest in the wild-typeps3 lines while nitrogen mustard and cisplatin induced relatively little G1 arrest. All agents failed to induce G1 arrest in cells containing mutantp53 genes. The degree of C1 arrest observed with these agents correlated with the rate ofp53 and p2JW41fI/C4PI protein accumulation: †̃y-rays and etopo side induced rapid accumulation of both p5.3 and p21@'@―1;nitrogen mustard and cisplatin induced slow accumulation of p5.3 and no major accumulation of the @2jWafI/C1@1 protein. Despite differences in G1 arrest and kinetics ofp53 orp21@―@ protein accumulation, all agents tended to decrease survival to a greater extent in the wild-type p5.3 lines com pared to the mutant p5.3 lines. Cell death in the wild-type p53 lines was associated with intracellular DNA degradation into oligonucleosomal sized DNA fragments, indicative ofapoptosis. We also observed an inverse sensitivity relationship between nitrogen mustard/cisplatin and etoposide in the mutantp53 lines and this was found to correlate with topoisomerase II mRNA levels in the cells. Our results suggest thatp53 gene status is an important determinant of both radioand chemosensitivity in lymphoid cell lines and that p53 mutations are often associated with decreased sensitivity to DNA damaging agents. that imposed by ionizing radiation (11), and lack ofp53 can increase the likelihood of genomic instability (12). A potential mechanism by which p53 induces G1 arrest has recently been proposed based on the identification of a p53 regulated gene product, Wafi/Cipi, which is a potent inhibitor of cyclin dependent kinases (8, 9). The Wafi/Cipi gene product is identical to the p21 protein first identified by Xiong et a!. (13) and the senescent cell derived inhibitor (Sdil) identified by Noda et a!. (14). Accumulation 0f@2JWafl@i@@1 following DNA damage correlates with G1 arrest and inhibition of cyclin E/Cdk2 kinase activity in cells with wild-type p5.3 (15). Cells with mutant p13 genes, cells from patients with ataxia telangiectasia or cells infected with human papilloma virus E6 fail to arrest in G@and fail to inhibit cyclin E/Cdk2 following †̃y-irradiation (15, 16). Several studies have shown that p53 mutationsreduce radiosensitivity (17—21),although this has not been observed in all cases (22), possibly because of the different cell types studied and their susceptibility to apoptosis. Activation of the p53 control system can therefore have two consequences for a cell: stable G1 arrest; and perhaps enhanced DNA repair or deletion of cells with DNA damage through an apoptotic pathway. In the present study we assessed the role of the p53 tumor suppres sor gene in cell cycle arrest and apoptosis following treatment of Burkitt's lymphoma and lymphoblastoid cell lines with †̃y-rays, eto poside, nitrogen mustard, and cisplatin. Our findings indicated that p53 gene status is an important determinant of both radioand chemosensitivity and that p53 mutations are often associated with decreased sensitivity to DNA damaging agents.