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Departments of Surgery [H.S.F., M.D.B., S.P.J., N.S.B.], Pediatrics [H.S.F.], Pathology [H.S.F., D.D.B.], Medicine [H.S.F., O.M.C.], and Biochemistry [P.M.] and The Howard Hughes Medical Institute [P.M.], Duke University Medical Center, Durham, NC 27710, and Department of Neurosurgery, MD Anderson Cancer Center, Houston, Texas 77030 [F.A-O.]

Medulloblastoma (D-341 MED) and rhabdomyosarcoma (TE-671) cell lines, which are resistant to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or the combination of BCNU and O⁶-benzylguanine (O⁶-BG), were generated by serial escalation of BCNU. The activities of O⁶-alkylguanine-DNA alkyltransferase (AGT), glutathione-S-transferase (GST), and total glutathione (GSH) of the parental, BCNU-resistant (BR), and BCNU + O⁶-BG-resistant (OBR) cells were measured. No significant differences in GST activity or total GSH were seen between the parental, BR, and OBR cells of both TE-671 and D-341 MED. The AGT activities of D-341 MED (BR) and TE-671 (BR) were twice those of D-341 MED and TE-671, respectively, confirming the importance of this enzyme for BCNU resistance. The D-341 MED (OBR) cells did not exhibit any AGT activity, suggesting that another mechanism must play a role in the drug resistance. Fewer DNA interstrand cross-links (ICLs) were observed in D-341 MED (OBR) than in D-341 MED after 8 h BCNU (100–400 µM) treatment. However, the amounts of DNA ICLs observed in D-341 MED and D-341 MED (OBR) were stable after 24 h. Microarray analysis showed the increased expressions of several metallothionein genes and down-regulation of several proapoptotic genes. The AGT activity of TE-671 (OBR) was 223 fmol/mg when the cells were grown in 10 µM O⁶-BG and decreased to about half this value when the O⁶-BG concentration was increased 60 µM. The AGT cDNA of TE-671 (OBR) cells was cloned and found to contain a G-to-T transversion at codon 156, resulting in conversion of glycine to cysteine (G156C). In vitro mutagenesis has shown that the G156C AGT mutant is resistant to inactivation by O⁶-BG. Thus, the selection of a mutant AGT with decreased sensitivity to O⁶-BG is a significant contributing factor to BCNU + O⁶-BG resistance.

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