Sensitivity to temozolomide of cell lines derived from brain tumours and melanomas: relationship to MGMT gene expression

Abstract

BackgroundWe have developed a series of low-passage melanoma and glioma cell lines and are using them to study responses to antitumour drugs. Here we have assessed responses to temozolomide, a commonly used clinical drug used for the treatment of glioblastoma and melanoma. Temozolomide acts mainly by methylation of DNA guanine and lesions can be removed by the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Low expression of MGMT is one of the main features associated with sensitivity to temozolomide and is usually associated with CpG methylation of the MGMT promoter. We wished to determine whether temozolomide sensitivity in these lines was related to MGMT promoter methylation and MGMT mRNA synthesis. Methods: 63 melanoma and 37 glioblastoma lines were derived from developed from surgical samples, using atmosphere of 5% oxygen for development and propagation in order to minimise oxygen toxicity. Temozolomide sensitivity (IC50 data) was analysed by its effect on 3H-thymidine incorporation in 5-day assays. MGMT promoter methylation status was assessed by the methylation-specific polymerase chain reaction (PCR). MGMT mRNA expression was assessed using reverse transcription and PCR. Results: 40% of the melanoma lines and 24% of the glioblastoma showed some sensitivity to temozolomide. Of the 79 cell lines analysed for MGMT mRNA expression, a good correlation was found to temozolomide resistance (p < 0.0001). Of the 100 cell lines assessed for MGMT promoter status, 12 showed methylation of both promoter sequences and were all temozolomide-sensitive; 50 lines showed no MGMT promoter methylation and 90% of these were temozolomide resistant; 30 lines showed partial promoter methylation and 53% of these were temozolomide resistant. Conclusion: Temozolomide sensitivity matched MGMT mRNA expression well but only partially matched MGMT promoter methylation. Other factors, such as loss of DNA mismatch repair capacity, may contribute to resistance.