Expression of O6-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.

Keywords: Laromustine (Onrigin, Cloretazine, VNP40101M, 101M); Methylation-Specific PCR (MSP); O6-Methylguanine-DNA Methyltransferase (MGMT, O6-Alkylguanine-DNA Alkyltransferase, AGT); Temozolomide; [Benzene-3H]O6-Benzylguanine.

Figure 1

The CpG island of the human MGMT gene promoter and the interrogated region in the MSP analysis. A, A vertical bar represents a CpG dinucleotide. The transcriptional start site (+1) corresponds to 46816 of AL355531 in the GenBank database. B, The DNA sequence of the primer region prior to bisulfite conversion, the oligomers (U-93-F and U-93-R) for the unmethylated product, and the oligomers (M-81-F and M-81-R) for the methylated product, are shown. The CpG sequence and base difference resulting from bisulfite conversion are underlined. Note that the primers are designed on the single strand, because double strands are no longer complementary after bisulfite conversion.

Figure 2

MGMT expression examined by alkyl-transfer assays, MSP and western blots in human cell lines. A, Cell lines with graded levels of MGMT activity were subjected to MSP analysis. U and M represent unmethylated (93 bp) and methylated (81 bp) PCR products, respectively. The percent of methylation is based on the formula [(M/U+M) × 100]. B, Cells were directly solubilized in 2X Laemmli’s sample buffer. The extracts (20 μl/5 × 10⁴ cells/lane) were resolved on 10%-PAGE and probed with monoclonal anti-human MGMT antibody MT3.1. The blot was reprobed with anti-HSC 70 antibody to show that each lane was loaded with a cell extract. C, The two-way scatter plots of the variables are shown with the Pearson’s correlation coefficient (r).

Figure 3

MGMT activity determined by alkyl-transfer assays in tumors and matched normal tissue from the colon, kidney, lung and liver. A, MGMT activity (fmol/mg protein) in normal (n) and malignant (m) tissue from the colon (C), kidney (K), Lung (Lg) and Liver (Lv) was determined using ³H-BG. The standard deviation of the triplicate determinations was less than 5% of the mean and not shown. The pairs with tumor selective reductions in MGMT activity (K7, K8 and Lv2) are squared and marked with *. B, The data are presented according to the MGMT activity in normal tissue in a column format.

Figure 4

Promoter methylation of the MGMT gene analyzed by MSP in tumors and matched normal tissue. A, U and M are 93 bp unmethylated and 81 bp methylated PCR products, respectively. The two numbers in parenthesis under the gel image are MGMT activity in fmol/mg protein on the left and % methylated signal on the right. B, The percent of promoter methylation exceeding 5 is considered as positive. C, The data from a total of 66 tissue specimens are analyzed for correlation between MGMT activity and promoter methylation.

Figure 5

Heterogeneity of the MGMT protein in human tissue revealed by western immunoblots. A, Tissue homogenates (80 μg of protein/lane) from alkyl-transfer assays and HL-60 extract (5 × 10⁴ cells/lane) were resolved on 10% PAGE and subjected to western analysis using mouse monoclonal anti-MGMT antibody (clone MT 3.1). B, Cell line homogenates (HL-60 and DU145, 40 μg/lane) and tissue homogenates (K1-n and Lv1-m, 80 μg/lane) were resolved on 12.5% PAGE. The three identical blots were prepared, and probed with MT3.1 (raised against purified recombinant human MGMT), rabbit polyclonal anti-MGMT antibody (raised against a synthetic peptide derived from the internal region of human MGMT), and goat anti-MGMT antibody (raised against purified recombinant human MGMT). Stripping and reprobing were not involved. C, Cell line homogenates (HL-60 and DU145, 80 μg/lane) and tissue homogenates (K1-n and Lv1-m, 80 μg/lane) were resolved on 12.5% PAGE and probed with rabbit polyclonal anti-ubiquitinin antibody.

Figure 6

Relationship between the MGMT protein and MGMT activity in tumors and matched normal tissue. A to F, Tissue homogenates (80 μg of protein/lane) from alkyl-transfer assays, HL-60 cell extract (in A) or HL-60 cell homogenate (in F) were resolved on 10% PAGE and probed with mouse monoclonal anti-MGMT antibody (clone MT 3.1). The MGMT content of HL-60 cells is 172 fmol/mg protein (17,000 MGMT molecules/cell). Only signals between the 20 and 30 kDa markers are shown for quantitation. The number above the tissue name is MGMT activity in fmol/mg protein and the number below the western image is signal intensity in arbitrary units. The sets with tumor-selective reduction in MGMT activity (K7, K8 and Lv2) were squared and marked with *. The scatter plots were made for each blot (A to F, bottom).