Carmustine-Resistant Cancer Cells Are Sensitized to Temozolomide As A Result of 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 O 6 -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 -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.

Leukemia cells are sensitized to temozolomide, carmustine and melphalan by the inhibition of O6-methylguanine-DNA methyltransferase

“…Our previous study evaluated TMZ cytotoxicity in a BCNU-resistant variant leukemia cell line, in comparison with a BCNU-sensitive cell line (17). The study identified that the BCNU-resistant cells were more sensitive to TMZ compared with the BCNU-sensitive cells (17). In addition, the BCNU-resistant cells possessed increased hMLH1 and hMSH2 transcript levels (17).…”

“…Intact MMR is required for the exertion of the cytotoxicity of TMZ, while interstrand crosslinks formed by bifunctional agents are, in part, repaired by MMR (21,27). Our previous study evaluated TMZ cytotoxicity in a BCNU-resistant variant leukemia cell line, in comparison with a BCNU-sensitive cell line (17). The study identified that the BCNU-resistant cells were more sensitive to TMZ compared with the BCNU-sensitive cells (17).…”

“…Our previous study demonstrated the important role of MMR in the exertion of the cytotoxicity of monofunctional agent temozolomide (17). Restored MMR sensitized the cancer cells to temozolomide.…”

“…RT-qPCR was performed according to the method of our previous study (17). ERCC1 is responsible for incision of the damaged DNA strand in the NER pathway, while hMLH1 and hMSH2 are two key factors in the MMR response.…”

“…To evaluate the growth-inhibitory effect of each agent on the two cell lines, the trypan blue exclusion assay was performed (17,18). Briefly, the cells were incubated with various concentrations of TMZ, BCNU or MEL (10 nM, 100 nM, 1 µM or 10 µM), alone or in combination with BG (10 µM), for 72 h. Subsequently, the samples were stained with trypan blue (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the viable cells, which exhibited negative staining, were counted.…”