Mismatch repair and p53 independently affect sensitivity to N-(2-chloroethyl)-N '-cyclohexyl-N-nitrosourea

The contributions of defective mismatch repair (MR;IR) and the p53-response to cell killing by N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU) we re evaluated. MMR defects were previously shown to be associated with CCNU sensitivity (G, Aquilina et al. Cancer Res., 58: 135-141, 1998), Unexpected ly, eight MMR-deficient variants of the A2780 human ovarian carcinoma cell line were 3-fold more resistant to CCNU than the MMR-proficient parental ce lls, The variants were members of a preexisting subpopulation of drug-resis tant A2780 cells. In addition to deficient expression of the MMR protein hM LH1, an essential component of the hMutL alpha repair complex, the variants exhibited alterations in the expression of other genes that influence drug sensitivity. Although A2780 cells possess a wild-type p53 gene, all of the clones contained a heterozygous G to T tranversion at codon 172, This chan ge resulted in a Val to Phe substitution and was associated with a constitu tive production of high levels of p53, which was inactive as a transcriptio nal activator of bar and p21, The hMLH1/p53 defective variants displayed a less prominent cell cycle arrest and reduced apoptosis after CCNU treatment , In contrast, MMR-defective A2780 variants, which had a similar hMutL alph a defect but retained a wild-type p53, did exhibit the expected CCNU sensit ivity. Expression of a dominant-negative p53val135 increased CCNU resistanc e of both MMR-proficient and MMR-deficient A2780 cells. Thus, defective MMR and p53 influence CCNU sensitivity in opposite directions, Their effects a re independent, and sensitization by defective MMR does not require a Funct ional p53 response.