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Carmustine-Resistant Cancer Cells Are Sensitized to Temozolomide As A Result of Enhanced Mismatch Repair during the Development of Carmustine Resistance

Department of Hematology and Oncology, University of Fukui, Matsuoka, Eiheiji, Fukui, Japan (T.Y., T.U.); and Preclinical Research Department, Schering-Plough K.K., Osaka, Japan (M.O.)

The cytotoxicity of the monofunctional alkylator temozolomide (TMZ) is mediated by mismatch repair (MMR) triggered by O⁶-alkylguanine, whereas MMR protects cells against bifunctional alkylators, including carmustine (BCNU). Therefore, TMZ may be cytotoxic to BCNU-resistant cancer cells because MMR affects sensitivity to TMZ and BCNU in a converse way. We evaluated TMZ cytotoxicity on BCNU-resistant variant (CEM-R) compared with the parental CCRF-CEM cell line (CEM-S). The mechanisms of its BCNU-resistance involved DNA repairs including nucleotide excision repair, base excision repair, alkylguanine alkyltransferase, MMR, and apoptotic and survival pathways. In particular, transcript levels of MMR-related hMLH1 and hMSH2 were enhanced in CEM-R cells. CEM-R cells were 8-fold more BCNU-resistant but surprisingly 9-fold more TMZ-sensitive than were CEM-S cells. Although TMZ-induced adducts include N-alkylated purines and O⁶-alkylguaine, DNA excision repair was enhanced in CEM-R cells, suggesting the efficient repair of N-alkylation adducts. Cotreatment with methoxyamine, a base excision repair inhibitor, did not sensitize CEM-R cells to TMZ, suggesting little or no contribution of N-alkylation to TMZ-induced cytotoxicity. Cotreatment with O⁶-benzylguanine, an alkylguanine alkyltransferase inhibitor, further sensitized CEM-R cells to TMZ, confirming the cytotoxic impact of O⁶-alkylguanine. Cotreatment with cadmium chloride, an MMR inhibitor, disrupted the sensitivity of CEM-R cells to TMZ. The sensitivity to TMZ was reversed in the CEM-R variant clone that had been established by transfecting CEM-R cells with short hairpin hRNA against hMLH1, suggesting the critical role of MMR on sensitization to TMZ. In conclusion, BCNU-resistant CEM-R cells were sensitized to TMZ as a result of enhanced MMR during the development of BCNU resistance.

Address correspondence to: Dr. Takahiro Yamauchi, Department of Hematology and Oncology, University of Fukui, 23 Shimoaizuki, Matsuoka, Eiheiji, Fukui, 910-1193, Japan. E-mail: tyamauch{at}u-fukui.ac.jp