DNA DAMAGE BY ALKYLATING AGENTS AND THEIR REPAIR IN HUMAN TUMOR CELLS

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D. Yarosh, R. S. Day,III, and others have shown that approximately 20% of human tumor lines and viral transformed lines are hypersensitive to alkylating agents due to an apparent absence of O6-alkylguanine DNA alkyltransferase (O6AT); this phenotype has been designated mer-. This enzyme removes alkylation damage at the 0-6 position of guanine but not at other sites in DNA. Dr. D. Yarosh in collaboration with our unit has been able to partially purify the enzyme from human liver and raise polyclonal antibodies to this protein. Studies have been initiated to develop monoclonal antibodies to this protein. Recently, a cDNA clone for a human O6AT has been isolated on the basis that it protects O6AT-deficient bacteria from certain alkylating agents (Tano, K, Shiota, S, Collier, J, Foote, RS, and Mitra, S. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine. Proc. Natl. Acad. Sci. USA 1990;87:686-690). Using oligonucleotides based on this sequence, we have isolated a full-length O6AT cDNA clone from a human liver library which we had constructed. Using this clone as a probe, we have found that O6AT mRNA was markedly reduced in all the mer- tumor cells lines (8) examined. The level of O6AT mRNA varied by 10-fold in mer+ cell lines and correlated with known levels of the protein in particular cells. Our findings may have important implications in cancer therapy where agents such as BCNU are used. D. Yarosh has preliminary evidence that some brain tumors have no detectable O6AT activity. Determining which tumors are mer- and which mer+ tumors have low levels of O6AT mRNA and protein would probably be useful in planning chemotherapy. Efforts are underway to develop approaches to measure O6AT mRNA and protein levels in vivo.