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Departments of ¹ Surgery, ² Pediatrics, ³ Pathology, ⁴ Medicine, and ⁵ Biochemistry and ⁶ Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina; ⁷ Department of Cellular and Molecular Physiology, College of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania; ⁸ Department of Medicine, University of Chicago, Chicago, Illinois; and ⁹ Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland

Requests for reprints: Henry S. Friedman, Department of Surgery, Duke University Medical Center, Box 3624, Durham, NC 27710. Phone: 919-684-5301; Fax: 919-684-8918. E-mail: fried003{at}mc.duke.edu

The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O⁶-benzylguanine (O⁶-BG). The reaction of O⁶-BG with O⁶-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O⁶-chloroethyl lesions caused by BCNU. In clinics, the combination of O⁶-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O⁶-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O⁶-BG and BCNU [O⁶-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O⁶-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O⁶-BG plus an alkylator may produce resistance to this intervention in clinical settings.

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¹⁰ Unpublished results.

Received 3/10/04; revised 6/ 3/04; accepted 7/ 6/04.