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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA036888-07
Application #
3174491
Study Section
Special Emphasis Panel (SSS (08))
Project Start
1984-12-01
Project End
1992-11-30
Budget Start
1990-12-01
Budget End
1991-11-30
Support Year
7
Fiscal Year
1991
Total Cost
Indirect Cost
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
067717892
City
Memphis
State
TN
Country
United States
Zip Code
38105
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Natarajan, A T; Vermeulen, S; Darroudi, F et al. (1992) Chromosomal localization of human O6-methylguanine-DNA methyltransferase (MGMT) gene by in situ hybridization. Mutagenesis 7:83-5
von Wronski, M A; Harris, L C; Tano, K et al. (1992) Cytosine methylation and suppression of O6-methylguanine-DNA methyltransferase expression in human rhabdomyosarcoma cell lines and xenografts. Oncol Res 4:167-74
Gonzaga, P E; Potter, P M; Niu, T Q et al. (1992) Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA. Cancer Res 52:6052-8
Harris, L C; Potter, P M; Tano, K et al. (1991) Characterization of the promoter region of the human O6-methylguanine-DNA methyltransferase gene. Nucleic Acids Res 19:6163-7
Tano, K; Shiota, S; Remack, J S et al. (1991) The origin of O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells transfected with human DNA. Mutat Res 255:175-82
Brent, T P; von Wronski, M; Pegram, C N et al. (1990) Immunoaffinity purification of human O6-alkylguanine-DNA alkyltransferase using newly developed monoclonal antibodies. Cancer Res 50:58-61
Gonzaga, P E; Harris, L; Margison, G P et al. (1990) Evidence that covalent complex formation between BCNU-treated oligonucleotides and E. coli alkyltransferases requires the O6-alkylguanine function. Nucleic Acids Res 18:3961-6
Gonzaga, P E; Brent, T P (1989) Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide. Nucleic Acids Res 17:6581-90
Smith, D G; Brent, T P (1989) Response of cultured human cell lines from rhabdomyosarcoma xenografts to treatment with chloroethylnitrosoureas. Cancer Res 49:883-6

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