Abstract
The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) strongly influences the effectiveness of cancer treatment with chemotherapeutic alkylating agents, and MGMT status in cancer cells could potentially contribute to tailored therapies for individual patients. However, the promoter methylation and immunohistochemical assays presently used for measuring MGMT in clinical samples are indirect, cumbersome and sometimes do not accurately report MGMT activity. Here we directly compare the accuracy of 6 analytical methods, including two fluorescent reporter assays, against the in vitro MGMT activity assay that is considered the gold standard for measuring MGMT DNA repair capacity. We discuss the relative advantages of each method. Our data indicate that two recently developed fluorescence-based assays measure MGMT activity accurately and efficiently, and could provide a functional dimension to clinical efforts to identify patients who are likely to benefit from alkylating chemotherapy.
Highlights
O6-methylguanine DNA Methyltransferase (MGMT; known as alkylguanine alkyltransferase, AGT) repairs DNA damage induced by endogenous, environmental, and therapeutic alkylating agents, and is the most important pathway for repairing O6-alkylguanine adducts in human cells [1, 2]
Clinical studies have demonstrated that glioblastoma patients with MGMT deficient tumors exhibit longer overall survival following treatment with temozolomide [4], and are more likely to respond to radiotherapy [5], highlighting the potential for personalized cancer therapy based on MGMT status in cancer cells
A panel of 24 lymphoblastoid cell lines derived from apparently healthy individuals from diverse genetic backgrounds [26], Coriell #1–24, has been characterized previously for MGMT levels using transcriptional profiling and MGMT activity using a fluorescence based multiplex host cell reactivation (FM-HCR) assay [17]
Summary
O6-methylguanine DNA Methyltransferase (MGMT; known as alkylguanine alkyltransferase, AGT) repairs DNA damage induced by endogenous, environmental, and therapeutic alkylating agents, and is the most important pathway for repairing O6-alkylguanine adducts in human cells [1, 2]. MGMT prevents cell killing by repairing O6-methylguanine (O6-MeG) cytotoxic DNA lesions induced by the cancer chemotherapy agents Temozolomide and Dacarbazine, and cytotoxic O6-chloroethylguanine lesions induced by cross-linking agents such as BCNU [3]. These chemotherapeutic agents are used to treat a variety of cancers including. Over 10-fold inter-individual variation in MGMT activity has been observed in normal tissues [6, 7], and lower MGMT activity is associated with therapy related leukemia [8], myelotoxicity in patients receiving temozolomide [9], and lung cancer risk [10]. Studies investigating the relationships between MGMT activity and disease risk and cancer therapy outcomes have been limited by cumbersome and indirect assays that may not accurately predict MGMT activity
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