The role of O⁶-alkylguanine in cell killing and mutagenesis in Chinese hamster ovary cells

Abstract

Chinese hamster ovary cells with no detectable (less than 200 molecules/cell) O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) were transfected with human cell DNA and pSV2neo plasmid by electroporation. Two stable transformant clones, GC-1 and GC-2, containing 4 X 10(4) and 4-6 X 10(3) methyltransferase molecules/cell respectively were isolated by successive screening in the presence of G418 and 2-chloroethyl-N-nitrosourea (CNU). Only three or four copies of pSV2neo DNA and no repetitive human DNA sequence were detected in these isolates. Secondary transfection of parent cells with GC-1 DNA yielded several clones containing 2-10 X 10(3) methyltransferase molecules/cell. The rate of removal of O6-methylguanine in GC-1, GC-2 and parent cells in vivo reflected their methyltransferase levels, while the N-methylpurines were removed at similar rates in all three cell lines. The differential sensitivity of these cells to several alkylating agents, namely CNU, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and methyl-methane sulfonate (MMS), known to yield different proportions of O6-alkylguanine among the alkyl adducts in DNA, varied widely. The largest and smallest differences in toxic response were observed with CNU and MMS respectively. These cell lines showed no difference in sensitivity to the DNA cross-linking agent psoralen. These data strongly suggest that alkylating agents produce two classes of lethal lesions, one of which is O6-alkylguanine. Induction of mutations at the hypoxanthine-phosphoribosyltransferase locus in these cells lines suggests that, regardless of its relative yield, O6-methylguanine is the major mutagenic lesion for all alkylating agents.

Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations.

“…It seems reasonable that this partial blockage may be related to the lethality ascribed to m6G in vivo (6)(7)(8)(9)(10)(11)(12).…”

“…However, m6G is also capable of pairing with cytosine, in which case no mutation would result (1). In addition, there is evidence that m6G is a lethal lesion in eukaryotic and prokaryotic cells (6)(7)(8)(9)(10)(11)(12). These data raise the question of how this simple modification ofguanine can generate such different biological end points.…”

Self-destruction and tolerance in resistance of mammalian cells to alkylation damage

“…Whatever the explanation for its mechanism, there clearly exists a means of amplifying the DNA synthesis inhibitory effects of relatively minor amounts of DNA damage. Models of methylation tolerance It has been suggested that tolerance may result from an enhanced ability to repair methylation lesions in DNA other than 06-meGua (24,62,87). While the apparent recessive nature of the tolerant phenotype would indicate otherwise, and the circumstantial evidence linking 06-meGua to cytotoxicity is strong, a definite answer to this question awaits a molecular characterization of tolerance.…”

Regulation of O6-methylguanine-DNA methyltransferase by methionine in human tumour cells

“…Such adducts are major contributors to the toxicity of alkylating anti-tumour agents, such as procarbazine, temozolomide, carmustine and related nitrosoureas commonly used for the treatment of several tumours (Schold et al, 1989;Dunn et al, 1991;Egyhazi et al, 1991;Brent et al, 1993;Marathi et al, 1993).…”