Received for publication October 15, 2007.
Revised April 22, 2008.
Accepted for publication April 22, 2008.

Carmustine-resistant cancer cells are sensitized to temozolomide due to enhanced mismatch repair during the development of carmustine resistance

Abstract

The cytotoxicity of the monofunctional alkylator temozolomide (TMZ) is mediated by mismatch repair (MMR) triggered by O⁶-alkylguanine, while 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, and MMR, and apoptotic and survival pathways. Especially, 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. Co-treatment with methoxyamine, a base excision repair inhibitor, did not sensitize CEM-R cells to TMZ, suggesting no or little contribution of N-alkylation to TMZ-induced cytotoxicity. Co-treatment with O⁶-benzylguanine, an alkylguanine alkyltransferase inhibitor, further sensitized CEM-R cells to TMZ, confirming the cytotoxic impact of O⁶-alkylguanine. Co-treatment 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 shRNA against hMLH1, suggesting the critical role of MMR on sensitization to TMZ. In conclusion, BCNU-resistant CEM-R cells were sensitized to TMZ due to enhanced MMR during the development of BCNU resistance.

Key words: Mechanisms of cell killing/apoptosis, Resistance