Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes.

Abstract

The ada-alkB operon protects Escherichia coli against the effects of many alkylating agents. We have subcloned it into the pSV2 mammalian expression vector to yield pSV2ada-alkB, and this plasmid has been introduced into Mer-HeLa S3 cells, which are extremely sensitive to killing and induction of sister chromatid exchange by alkylating agents. One transformant (the S3-9 cell line) has several integrated copies of pSV2ada-alkB and was found to express a very high level of the ada gene product, the 39-kDa 06. methylguanine-DNA methyltransferase. S3-9 cells were found to have become resistant to killing and induction of sister chromatid exchange by two alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine and N,N'-bis(2-chloroethyl)-N-nitrosourea. This shows that bacterial DNA alkylation-repair genes are able to suppress the alkylation-repair defects in human Mer-cells.DNA alkylation damage causes mutation and cell death in both human and bacterial cells. The probability that a particular cell will die or mutate is determined by the types of DNA damage incurred and by the cellular response to the damage. Comparisons of the repair of alkylated bases in populations of alkylation-sensitive and -resistant Escherichia coli have identified 06-methylguanine as the main mutagenic lesion that causes G-C to A'T transitions (1-3) and 3-methyladenine and 3-methylguanine as lesions that lead to cell death (4-6). It is unclear whether these lesions produce the same effects in human cells. Alkylation-sensitive (Mer-) human cell lines do exist (7,8), but their phenotypes are complex and it has been difficult to use them to determine which lesions are mutagenic in human cells and which are lethal. Here we describe an approach in which we study killing and the induction of sister chromatid exchange (SCE) in Mer-human cells that have integrated the bacterial ada and alkB genes and are producing the bacterial DNA methyltransferase.The E. coli ada gene encodes a 39-kDa DNA methyltransferase that directly transfers methyl groups from o6-methylguanine, O4-methylthymine, and methyl phosphotriester DNA adducts to cysteine residues within the enzyme itself (9-12). The methyl groups remain covalently bound to the enzyme, and its DNA methyltransferase activity is consequently inactivated; i.e., the methyltransferase is a suicide enzyme (10). The alkB gene lies in the same operon as the ada gene (13). The specific function of the 27-kDa alkB gene product is not known, but it is known to be involved in the protection of bacteria from the lethal effects of alkylation damage (13, 14). We have subcloned the ada-alkB operon into a mammalian expression vector such that the operon comes under the control of the simian virus 40 (SV40) early promoter, and we have transfected this construct into alkylation-sensitive human cells.Human 10% fetal bovine serum, streptomycin (100 ,tg/ml), penicillin (100 units/ml), and L-glutamine (0.03%) and incubated in humidified 5% CO2 at 370C. G418-resistant transformants were grown in the presence of 200 ,ug ...

Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas

“…The D30 values (the dose at which 30% of the cells survived) together with the initial ATase activities of the cells used for the survival assay are shown in Table II. Several other groups have now reported increased resistance to alkylating agents following overexpression of the ada (Samson et al, 1986;Kataoka et al, 1986;Ishizaki et al, Dose of agent (,ug ml -1) 1987) and human ATase genes (Hayakawa et al, 1990;Kaina et al, 1991;Wu et al, 1992) in mammalian cells. The relative increases in resistance of LH2 and SB cells compared to 6E (based on the D30 values) were not proportional to ATase expression in the cells for any of the drugs tested.…”

Sequential administration of varying doses of dacarbazine and fotemustine in advanced malignant melanoma

“…Experimental models using ATase-deficient cell lines or xenografts show them to be more sensitive to DTIC than lines or xenografts with high activity (Hayward & Parsons, 1984;Gibson et al, 1986;Catapano et al, 1987;D'Incalci et al, 1988;Lunn & Harris, 1988;Foster et al, 1990). The strongest evidence for the cytotoxic effects of 06-alkylguanine in DNA comes from ATase cDNA transfection experiments which show that expression of prokaryotic or eukaryotic ATase cDNA in mammalian cells protects them against the toxic effects to these agents (Brennand & Margison, 1986;Kataoka et al, 1986;Samson et al, 1986;Kaina et al, 1991).…”

Protection of committed murine haemopoietic progenitors against BCNU toxicity does not predict protection of primitive, multipotent spleen colony-forming cells – implications for chemoprotective gene therapy

“…14,15 The latter protein, which confers resistance to the biological effects of O 6 -alkylating agents comprising the chloroethylnitrosoureas (eg N,NЈ-bis(2-chloroethyl)-N-nitrosourea (BCNU); 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU); fotemustine) and related methylating agents (eg temozolomide; dimethyltriazenoimidazolecarboxamide (DTIC); streptozocin; procarbazine), has been widely studied in cells and animal systems. These investigations have shown that the expression of ATase in cells which are normally deficient for this function produced a marked increase in cellular resistance to toxicity, [16][17][18][19][20] chromosome damage, 17,20,21 mutagenesis 16,20,[22][23][24][25] and transformation [25][26][27][28][29][30][31] as a consequence of enhanced repair of O 6 -alkylguanine (O 6 -alkG). In contrast, mice carrying homozygous knockout mutations for ATase have been demonstrated to be particularly sensitive to the biological effects of O 6 -alkylating agents.…”