There is compelling evidence that the response of human malignant tumors to chemotherapy with bifunctional alkylating agents is related to the extent of formation or rate of removal of DNA interstrand crosslinks. The hypothesis to be tested in the proposed research is that reduction of crosslinking is mediated by enzymic DNA repair processes. The goal is to identify, isolate and characterize the human-cell enzymes that may repair DNA crosslinks or their precursors Immediate aims focus on crosslink prevention by 06-alkylguanine-DNA alkyltransferase (GATase) in DNA treated with the chloroethylnitrosoureas (CENUs). GATase, purified further from cultured human lymphoblasts or human liver, will be used as a probe to define the molecular mechanism for crosslink formation in DNA treated with CENUs and other classes of drug that produce adducts at 06-guanine in DNA (e.g., clomesome, mitozolomide, procarbazine, mitomycin C and cisplatin): We shall determine GATase activity in a variety of human tumor lines, in- cluding tumors of the central nervous system, to establish the relationship between cellular GATase levels and drug resistance. Development of GATase-specific antibodies and cDNA probes should eventually permit GATase levels to be readily determined in clinical biopsy specimens. The in vitro methodologies that we have developed for studying the CENUs and related drugs will be applied to investigation of crosslink formation and enzymic repair induced by other bifunctional alkylating agents (e.g., cyclophosphamide, ifosfamide, mitomycin C and cisplatin). Where necessary activated derivatives will be used. DNA interstrand crosslinking will be determined in vitro by measuring the renaturability of isolated DNA with fluorometric, filter- binding or electrophoretic methods. Both purified human DNA repair enzymes, such as alkylpurine-DNA glycosylase or GATase, and cruder extracts from human cells will be assayed for activities that 1) repair monoadduct precursors of crosslinks and 2) cleave existing crosslinks. The ultimate goal of this research is to establish correlations of repair enzyme activity, crosslink formation and crosslink persistence with cytotoxicity and clinical response to chemotherapy. Such information should enable prediction of thera- peutic response based on direct biochemical assay of repair enzyme levels in tumor and normal tissues. Intervention in these repair processes by enzyme inhibition could provide a strategy for overcoming drug resistance in cancer patients.
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