Multiple DNA repair mechanisms and alkylator resistance in the human medulloblastoma cell line D-283 Med (4-HCR)

Purpose: We have previously reported preferential repair of DNA interstrand crosslinks in the 4-hydroperoxycyclophosphamide-resistant human medullobla stoma cell line D-283 Med (4-HCR). We now report further studies that explo red the potential mechanisms underlying this repair. Methods: Limiting dilu tion assays and Western, Southern, and Northern blots were used to compare specific differences between D-283 Med (4-HCR) and its parental line D-283 Med. Results: D-283 Med (4-HCR) was cross-resistant to melphalan and 1,3-bi s(2-chloroethyl)-1-nitrosourea (BCNU), with O-6-alkylguanine-DNA alkyltrans ferase (AGT) levels of 466 +/- 164 fmol/mg protein; AGT levels in the paren tal line, D-283 Med, were 76 +/- 96 fmol/mg. The increase in AGT activity w as not a result of gene amplification. Depleting AGT with O-6-benzylguanine partially restored sensitivity to BCNU. Both cell lines were deficient in the human mismatch protein MutL alpha. ERCC4 mRNA and poly(ADP-ribose) poly merase levels were similar in both cell lines, and ERCC1 mRNA levels were 2 - to 2.5-fold lower in D-283 Med (4-HCR). Topoisomerase I levels were 2- to 2.5-fold higher in D-283 Med compared with D-283 Med (4-HCR). Conclusion: These results, while illustrating the multiple differences between D-283 Ms d and D-283 Med (4-HCR), do not explain the enhanced DNA interstrand crossl ink repair seen in D-283,Med (4-HCR).