MOLECULAR CHARACTERIZATION OF MUTATIONS IN THE HPRT GENE OF NORMAL HUMAN SKIN KERATINOCYTES TREATED WITH N-ETHYL-N-NITROSOUREA - INFLUENCE OF O-6-ALKYLGUANINE ALKYLTRANSFERASE

O-6-Alkylguanine-DNA alkyltransferase (AGT) is responsible for repairi ng the O-6-alkylguanine lesion in DNA. There is wide variation in the levels of AGT between organ and cell types, which appears to correlate with cell and tissue type sensitivity to the mutagenic and carcinogen ic effects of alkylating agents. In order to investigate the role of A GT in modulating the frequency and types of mutations induced in one t ype of normal human parenchymal cells, we examined the types and frequ ency of mutations in the hypoxanthine (guanine) phosphoribosyltransfer ase (hprt) gene in 1 16 mutants derived from two N-ethyl-N-nitrosourea (ENU)-treated normal human skin keratinocyte cell lines. O-6-Benzylgu anine (O-6-BZ; 5 mu M x 2 hours) was used to specifically inhibit AGT activity before ENU treatment (0 to 5 mM x 1 hour). O-6-BZ increased b oth the cytotoxic and mutagenic effects of ENU by 1.8- and 3- to 5-fol d, respectively. In both treatment groups, most of the mutations were base substitutions (72%). The proportion of GC to AT transitions in th e O-6-BZ group (14/31) was twice that in the group treated with ENU al one, consistent with the loss of AGT activity in these cells. There wa s no strand specificity of GC to AT and AT to GC transitions in both g roups. Base transversions accounted for 28% of total base substitution s. A lower than expected proportion of AT to TA transversions were obs erved in both cell lines, which decreased in the O-6-BZ pretreated gro up. A strand bios was observed for GC to TA and AT to TA transversions . Most of the G to A and G to T bose substitutions had one or more pur ines flanking 3' to the mutated deoxyguanosines. There were more delet ion mutants with the deletion of exon 1, 4, 6, and 8 in the BZ group t han in the control group. These data, characterizing the mutational sp ectra of ENU in normal human keratinocytes treated in vitro, indicate that GC to AT and AT to GC transition mutations predominate in these c ells depleted or not depleted of AGT. (C) 1997 Wiley-Liss, Inc.