Distinct molecular mechanisms of acquired resistance to temozolomide in glioblastoma cells

J. Neurochem. (2012) 122, 444–455. Temozolomide (TMZ) is an alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma. Clinical benefit is more prominent in patients with methylation of the O6‐methyl‐guanine DNA methyltransferase (MGMT) promoter. However, all patients eventually suffer from tumor progression because their tumors become resistant to TMZ. Here, we modeled acquired TMZ resistance in glioma cells in vitro to identify underlying molecular mechanisms. To this end, the glioma cell lines LNT‐229, LN‐308, and LN‐18 were exposed repetitively to increasing concentrations of TMZ to induce a stable resistant phenotype (R) defined by clonogenic survival assays. The molecular mechanisms mediating acquired resistance were assessed by immunoblot, PCR, and flow cytometry. Rescue experiments were performed with siRNA‐mediated candidate gene silencing. We found in LN‐18 cells constitutively expressing MGMT a strong up‐regulation of MGMT levels in TMZ‐resistant cells. TMZ resistance in the MGMT‐negative cell lines LNT‐229 and LN‐308 was not associated with de novo expression of MGMT. Instead, we found a down‐regulation of several DNA mismatch‐repair proteins in resistant LNT‐229 cells. A TMZ‐resistant phenotype was also achieved by silencing selected DNA mismatch repair proteins in parental LNT‐229 cells. No obvious mechanism of resistance was identified in the third cell line, LN‐308, except for reduced methylation of LINE‐1 repetitive elements. In conclusion, we demonstrate that different molecular mechanisms may contribute to the development of acquired TMZ resistance in glioma cells, indicating the need to develop distinct strategies to overcome resistance. Identification of molecular mechanisms leading to temozolomide resistanceAcquired resistance to temozolomide is an important challenge in the setting of therapy failure in glioblastoma patients. Here, we identify distinct mechanisms by which glioma cells become resistant to the anti‐tumor effects of temozolomide. These findings highlight the importance of different therapy‐induced molecular changes, which might be exploited for novel therapeutic approaches in the future.