The overall goal of this proposal is to enhance the chemotherapeutic usefulness of chloroethylnitrosoureas and other chloroethylating agents. This goal will be accomplished by utilizing O6-alkylguanines to specifically deplete tumor cells of the DNA repair protein, O6- alkylguanine-DNA alkyltransferase (AGT) prior to administration of chloroethylating agents. As a prerequisite to these studies, a highly sensitive assay to determine AGT activity will be further developed. The sensitive assay utilizes short chain oligodeoxynucleotides containing O6- methylguanine labeled at the 5' end with 32p as substrates for the AGT protein. Upon reaction with AGT, which removes the methyl group form O6- methylguanine, the demethylated oligomer will be separated from its parent by chromatographic or immunological techniques. Conditions for this assay will be optimized through a systematic study of the reaction kinetics of AGT with oligomers of differing length and sequence. This assay will allow for determination of AGT levels in mammalian tumors and non-neoplastic tissue when limited activity or only a small amount of tissue is present. Enhancement of chloroethylating agents by exposure to O6-alkylguanines will be studied in two animal tumor models: the well differentiated Dunning PAP rat model of prostatic carcinoma and human brain tumor xenografts grown in athymic nude mice. In order to achieve maximal AGT depletion in the tumor and minimal bone marrow toxicity, the optimal route of administration and treatment schedule for O6-methylguanine and O6-n-butylguanine will be determined. The newly proposed assay will allow for precise determination of levels of AGT in tumor, liver and bone marrow after O6-alkylguanine treatment. The effects of chemotherapeutic chloroethylating agents subsequent to AGT suppression by treatment with O6-alkylguanines will be evaluated in terms of inhibition of tumor growth and animal toxicity. This method of therapy may have clinical implications in terms of sensitizing human cancers to chloroethylating agents.
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