A MUTATION IN THE P53 TUMOR-SUPPRESSOR GENE OF AHH-1 TK(+ -) HUMAN LYMPHOBLASTOID-CELLS/

Loss-of-function mutations in the p53 tumor suppressor gene result in an altered response to DNA-damaging agents. Included in the mutant p53 phenotype are the loss of the G(1) checkpoint and delayed apoptotic c ell death, characteristics we have consistently observed in the the AH H-1 tk(+/-) cell line following exposure to DNA-damaging agents. In or der to determine the functional status of p53 in the AHH-1 tk(+/-) cel l line, molecular analysis (single-strand conformational polymorphism [SSCP] and sequence analysis) was performed on exons 5-9 of the p53 ge ne. In addition, the status of the p53 gene in the closely related lym phoblast line, MCL-5, which, in our hands, has a much higher spontaneo us rate of apoptosis than AHH-1 tk(+/-), was also determined by molecu lar analysis. Initial SSCP analysis of AHH-1 tk(+/-) revealed an abnor mal migration pattern of exon 8 when compared to a wild-type control. Subsequent sequence analysis indicated that a base-pair substitution ( (C) under bar GG --> (T) under bar GG) mutation had occurred at codon 282, a reported 'hot spot' for 5-methylcytosine mutations in the human p53 gene. Neither SSCP nor sequence analysis of exons 5-9 of MCL-5 in dicated any differences from wild-type DNA. These results suggest that the lack of a G(1) arrest and the delayed entrance into apoptosis obs erved in chemically-exposed AHH-1 tk(+/-) cells are, at least partiall y, accounted for by a loss-of-function mutation in the p53 gene.