MGMT methylation status and expression level do not correlate with sensitivity to CCNU in short-term cultures derived from malignant astrocytoma

Abstract

Adjuvant chemotherapy using DNA-damaging agents has largely failed to make a significant impact on the outcome of patients with malignant astrocytoma. One of the primary mechanisms of resistance to nitrosureas such as CCNU is mediated through O6-methylguanine-DNA methyltrans-ferase (MGMT). This DNA repair enzyme removes the cytotoxic alkyl adducts from O6-guanine, and hence the level of MGMT activity in tumor cells is related to their sensitivity to nitroureas. It has been proposed that functional inactivation of MGMT through hypermethylation of the gene promotor region could be predictive of chemosensitivity. We have previously reported differential sensitivity to CCNU in a panel of 17 short-term cultures derived from malignant astrocytoma. In this study, we determined the methylation status of MGMT using methylation-specific PCR in these 17 cultures. We also assessed the amounts of MGMT mRNA and protein present in each culture using real-time quantitative PCR and immunohistochemistry with a commercial antibody against MGMT. There was good correlation between MGMT promotor methylation and presence of MGMT mRNA and protein in all but 2 cases. In both these cultures, mRNA and protein were not detected even though the MGMT promotor was unmethylated. However, there was no correlation between sensitivity to CCNU and MGMT status. In the 2 most resistant cultures, the MGMT gene was methylated and was not expressed. Similarly, in 4/5 of the most sensitive cultures, MGMT was unmethylated, and in 2 of these cases, there was commensurate MGMT expression. However, in the remaining 2 cultures, MGMT expression was not detected, indicating that an alternative mechanism to gene methylation is responsible for MGMT inactivation. This study highlights that the resistance of malignant astrocytoma to nitroureas may be more complex than simple reliance on MGMT activity and prediction of response to such agents by MGMT methylation status should be used with caution.