Expression of O<sup>6</sup>-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

Abstract

The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions, depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included (a) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, (b) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and (c) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumor-selective reduction in MGMT activity exist in human tissue.

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

“…Therefore, in order to check the clinical applicability of the developed DNA sensor, the implemented methodology was applied to analyze the endogenous MGMT status in the reference cell line U87. Figure 5b shows that a larger amperometric response was measured when 100 ng genomic DNA extracted from these cells were analyzed, in comparison with the currents measured for both in the absence of target DNA and genomic DNA extracted from HeLa cells (non-methylated MGMT gene promoter cells used as control) 52 , which is consistent with the reported specific hypermethylation of the MGMT gene of U87 cell line 53 , 54 .…”

Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response

“…Current methods for measuring MGMT activity in cellular samples require several steps involving lengthy workups [ 26 ]. Hence, we next tested whether FAM probe 1 could simplify the assay of MGMT activity in such samples.…”

Influence of Glutathione and Glutathione S-transferases on DNA Interstrand Cross-Link Formation by 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the Active Anticancer Moiety Generated by Laromustine

“…MGMT plays a major role in resistance to the cytotoxicity of CNUs and 90CE and its prodrugs. 16 − 20 This resistance is believed to result from the quenching/repair of the O 6 -(2-chloroethyl)guanine and N 1 , O 6 -ethanoguanine cross-link precursors which are generated by both these classes of agents. 16 − 20 In contrast to GSH/GST, MGMT had the ability to rapidly quench 90CE generated cross-link precursors within DNA and blocked DNA cross-linking after the primary alkylation phase was completed.…”

“… 16 G-C ethane cross-link formation appears to be responsible for the vast majority of the cytotoxicity in responsive tumor models treated with 90CE prodrugs and for a large proportion of the cytotoxicity of BCNU and other chloroethylnitrosoureas (CNUs). 16 − 20 Selectivity for tumor cells arises predominately from differentials between normal and tumor cells in (1) their O 6 -alkylguanine-DNA alkyltransferase (MGMT) content, 16 − 20 the protein responsible for the repair of DNA O-6 guanine alkyl lesions, 18 and (2) in their ability to repair the cross-links formed from lesions that escape MGMT repair. 21 Additional resistance may arise from the interception of (or decreased yields of) chloroethylating species by other mechanisms.…”