Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.

Alkylating agents and compounds converted metabolically to alkylating agents form an important class of carcinogens and mutagens that includes a number of environmentally important /V-nitrosocompounds. A number of alkylating agents are also useful antitumor drugs. There are many factors determining the sensitivity to the toxic, mutagenic, and carcinogenic poten tial of these compounds (1-4).2 One of these is the capacity to

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Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents.

Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials

Mechanisms of human DNA repair: an update.

MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents.

The role of O6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis

The ability of extracts of human tumor cells to demethylate O6-methylguanine (O6-MeG) in DNA was assayed using the synthetic DNA polymer poly(dC,dG,m6dG). Cell strains proficient in repair of O6-MeG in vivo (Mer+ phenotype) contained a methyltransferase activity while repair deficient cells (Mer- phenotype) had little or no activity. Mixing extracts of different Mer- strains did not result in the appearance of the activity. Extracts of Mer- cells did not inhibit the activity in extracts of Mer+ cells. Both Mer+ and Mer- strains contained methylnitrosourea-damage-specific endonuclease activity. The data suggest that the Mer- strains are deficient in methyltransferase and that this is the fundamental reason for their hypersensitivity to the cytotoxic effects of DNA alkylation. The activity was partially purified from a Mer+ colon carcinoma cell strain. Its kinetics parallel the repair of O6-MeG in DNA in vivo and suggest that the activity is inactivated during repair of DNA.

Repair of O6-methylguanine in DNA by demethylation is lacking in Mer- human tumor cell strains.

Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylguanine-DNA methyltransferase were incubated with mM concentrations of the free base O6-methylguanine for up to 24 h. This treatment depleted the cells of their transferase activity, and sensitized the cells to killing by the antineoplastic drug 1-[2-chloroethyl]-1-nitrosourea. Cells constitutively lacking the methyltransferase were not sensitized to cell killing. Cell free extracts incubated with O6-methylguanine also lost methyltransferase activity. Other alkylpurines, such as O6-methylguanosine, S6-methylthioguanine, O6-ethylguanine, and 3-methyladenine, did not have this effect on extracts of human tumor cells, while O6-methylguanosine and O6-methylguanine inactivated purified methyltransferase from Escherichia coli. The data suggest that the free base O6-methylguanine is probably a substrate for the methyltransferase. Calculation of the second order rate constants for free base versus O6-methylguanine in DNA, and experiments in which the free base was mixed with DNA containing O6-methylguanine before reaction with methyltransferase, indicated that the base in DNA is about 4 X 10(7) better as a substrate than is the free base. These results demonstrate that DNA repair capacity of tumor cells can be diminished without DNA damage, and suggest a method for increasing the efficiency of chemotherapy.

https://cancerres.aacrjournals.org/content/canres/46/4_Part_1/1663.full.pdf

Inactivation of O6-methylguanine-DNA methyltransferase and sensitization of human tumor cells to killing by chloroethylnitrosourea by O6-methylguanine as a free base.

SUMMARY O 6 -methylguanine ( O 6 mG) produced in DNA by such SN1 methylating agents as N -methyl- N -nitrososurea and N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) has been suggested by some to be the lesion that leads to certain biological endpoints in mammalian cells: cell killing, sister chromatid exchange (SCE) production, mutagenesis and cellular transformation. Other evidence is interpreted as inconsistent with this point of view. The finding of Karran & Williams (1985) that O 6 mG delivered to cells in culture resulted in the depletion of the activity of the protein responsible for repair of O 6 mG in DNA ( O 6 mG-DNA methyltransferase, O 6 MT) provided a tool for the assessment of the role of O 6 mG in producing biological endpoints. In this paper we review much of the literature on human cells pertinent to this question. In addition we present our survival data obtained using the depletion technique of Karran & Williams as well as data supporting a model invoking a mismatch and excision response to O 6 mG proposed by Sklar & Strauss (1980). Although data linking O 6 mG to causation are inconclusive, it is premature to conclude that O 6 mG is not a lesion lethal to certain cultured cells.

The Role of O6-Methylguanine in Human Cell Killing, Sister Chromatid Exchange Induction and Mutagenesis: A Review

O6-Methylguanine-DNA methyltransferase activity was measured in extracts of human tumor cells and was partially purified from human placenta. Repair of O6-methylguanine in DNA inactivated the methyltransferase, and treatment of cells with MNNG, which produces this alkylated base in DNA, depleted the cells of active methyltransferase. RNA and protein synthesis were required for restoration of methyltransferase activity, which transiently exceeded the original levels by 50% 48 h after treatment. One species of methyltransferase of Mr = 22 kd was present in human tumor cells and human placenta.

Daniel B. Yarosh

Papers

The role of O6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis

Repair of O6-methylguanine in DNA by demethylation is lacking in Mer- human tumor cell strains.

Inactivation of O6-methylguanine-DNA methyltransferase and sensitization of human tumor cells to killing by chloroethylnitrosourea by O6-methylguanine as a free base.

The Role of O6-Methylguanine in Human Cell Killing, Sister Chromatid Exchange Induction and Mutagenesis: A Review

O6-Methylguanine-DNA methyltransferase in human cells