Hypersensitivity of human tumor xenografts lacking O6-alkylguanine-DNA alkyltransferase to the anti-tumor agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitros ourea.

Human tumor cell strains having different activities of O6-alkylguanine-DNA alkyltransferase (ATR) were transplanted into nude mice and chemotherapeutic responses of tumor xenografts were compared after intraperitoneal injection of the anti-tumor drug 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). The tumor strains used were four Mer+ strains possessing high ATR activity and three Mer- strains lacking this activity. Included in these Mer+ strains was a clone 5'dD which expresses the Escherichia coli ATR in Mer- HeLa cells and thus shows the Mer+ phenotype. All the Mer- tumor xenografts were much more sensitive than tumors of Mer+ strains, including the clone 5'dD; after the highest ACNU dose (three injections of 50 mg/kg), some Mer- tumors disappeared completely and the growth of other tumors was severely retarded, whereas all Mer+ tumors continued to grow. These results demonstrate that ATR activity in tumor cells is a major determinant of tumor response to ACNU, and further suggest that measurement of ATR activity in biopsy specimens may provide a useful guide to predict the response to chemotherapy.

O6-methylguanine methyltransferase activity and sensitivity of Japanese tumor cell strains to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)- 3-nitrosourea hydrochloride.

Using 40 tumor cell strains derived from various organs of Japanese tumor patients and also 12 normal cell strains, we have measured the activity of O6-methylguanine-DNA methyltransferase (MT), which can repair O6-methylguanine produced in DNA by alkylating agents. Then, the lethal sensitivities of the strains to the anti-tumor drug 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-)2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) were measured. The MT activity was assayed by measuring the 3H radioactivity transferred from the substrate DNA containing [methyl-3H]-O6-methylguanine to acceptor molecules in the cell extracts. Extracts from the majority of tumor strains and all normal cell strains contained substantial MT activity of varying degree, while the extracts of 6 tumor strains showed virtually undetectable MT activity. Hence these 6 strains were assigned as Mer-, the phenotype which is characterized by the inability to repair O6-methylguanine in DNA due to the lack of MT. The Mer- tumor strains were much more sensitive to ACNU than the rest of Mer+ strains, as measured by colony-forming ability. Furthermore, with all the tumor and normal strains tested, a good correlation was observed between MT activity and cellular resistance to ACNU. These results indicate that the frequency of Mer- strain is about 15% among Japanese tumor cell strains so far analyzed, and further suggest that MT may be the only system able to repair lethal DNA damage induced by ACNU.

Expression of the truncated E. coli O6-methylguanine methyltransferase gene in repair-deficient human cells and restoration of cellular resistance to alkylating agents.

We have constructed a truncated E. coli O6-methylguanine methyltransferase (MT) gene (ada gene) to express the MT activity for O6-methylguanine and O4-methylthymine but not for methylphosphotriester in human cells and transferred it into Mer- HeLa MR cells. The transfectant cells expressed the truncated E. coli MT were resistant to alkylating agents as same as the transfectant cells with the intact ada gene in cell killing, sister-chromatid exchange induction and host-cell reactivation of adenovirus 5. These results strongly suggest that methylphosphotriester may not contribute to the biological effect of alkylating agents in human cells.

Comparative analysis of O6-methylguanine methyltransferase activity and cellular sensitivity to alkylating agents in cell strains derived from a variety of animal species.

Using 26 cultured cell lines derived from 17 different animal species, we have measured both the activity of O6-methylguanine (O6-MeG) methyltransferase (MT) in cell extracts and the sensitivity of the strains to the lethal effects of the alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). The MT activity was assayed by measuring the amount of 3H radioactivity transferred from methyl-[3H]-labeled O6-MeG in DNA to acceptor protein molecules in the extracts. In all the 21 mammalian cell strains, lethal sensitivity to ACNU as measured by colony-forming ability correlated well with cellular MT activity, indicating that the major lethal ACNU damage is reparable by the MT. On the other hand, MNNG sensitivity did not necessarily correlate with the MT activity.

Transfer of the E. coli O6-methylguanine methyltransferase gene into repair-deficient human cells and restoration of cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine.

We have constructed a plasmid on which the E. coli O6-methylguanine-DNA methyltransferase (MT) gene (ada gene) was linked with an SV40 promoter sequence and a poly(A) site. After transferring this plasmid into Mer- HeLa MR cells by DNA transfection, effective expression of E. coli MT was observed. Isolated stable transformant clones showed higher resistance to N-methyl-N'-nitro-N-nitrosoguanidine in colony formation and sister-chromatid exchange induction than HeLa MR cells.