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[Cancer Research 54, 5824-5830, November 15, 1994]
© 1994 American Association for Cancer Research
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p53 Gene Mutations Are Associated with Decreased Sensitivity of Human Lymphoma Cells to DNA Damaging Agents

Saijun Fan, Wafik S. El-Deiry, Insoo Bae, Jim Freeman, Daniel Jondle, Kishor Bhatia, Albert J. Fornace, Jr., Ian Magrath, Kurt W. Kohn and Patrick M. O'Connor1

Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment [S. F., I. B., J. F., D. J., A. J. F., K. W. K., P. M. O.], and Lymphoma Biology Section, Pediatric Branch [K. B., I. M.], National Cancer Institute, NIH, Bethesda, Maryland 20892, and the Oncology Center and Program in Human Genetics and Molecular Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231 [W. S. E.]

The present study assessed the role of the p53 tumor suppressor gene in cell cycle arrest and apoptosis following treatment of Burkitt's lymphoma and lymphoblastoid cell lines with {gamma}-rays, etoposide, nitrogen mustard, and cisplatin. Cell cycle arrest was measured by flow cytometry; p53 and p21Waf1/Cip1 protein levels were measured by Western blotting; cell survival 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 filter elution method. We found that {gamma}-rays and etoposide induced a strong G1 arrest in the wild-type p53 lines while nitrogen mustard and cisplatin induced relatively little G1 arrest. All agents failed to induce G1 arrest in cells containing mutant p53 genes. The degree of G1 arrest observed with these agents correlated with the rate of p53 and p21Waf1/Cip1 protein accumulation: {gamma}-rays and etoposide induced rapid accumulation of both p53 and p21Waf1/Cip1; nitrogen mustard and cisplatin induced slow accumulation of p53 and no major accumulation of the p21Waf1/Cip1 protein. Despite differences in G1 arrest and kinetics of p53 or p21Waf1/Cip1 protein accumulation, all agents tended to decrease survival to a greater extent in the wild-type p53 lines compared to the mutant p53 lines. Cell death in the wild-type p53 lines was associated with intracellular DNA degradation into oligonucleosomal 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 that p53 gene status is an important determinant of both radio- and chemosensitivity in lymphoid cell lines and that p53 mutations are often associated with decreased sensitivity to DNA damaging agents.

1 To whom requests for reprints should be addressed.

Received 8/19/94. Accepted 10/ 5/94.




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