Abstract
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.
Footnotes
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ABBREVIATIONS: BCNU, carmustine or 1,3-bis(2-chloroethyl)-1-nitrosourea; TMZ, temozolomide or 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide; MMR, mismatch repair; NER, nucleotide excision repair; BER, base excision repair; MGMT, O6-methylguanine-DNA methyltransferase; CCNU, lomustine or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; ACNU, nimustine or 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride; MX, methoxyamine; XTT, sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate; RT-PCR, reverse transcription-polymerase chain reaction; GST, glutathione transferase; shRNA short hairpin RNA; APE, AP endonuclease; BG, O6-benzylguanine.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received October 14, 2007.
- Accepted April 22, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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