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Expression of O6-methylguanine-DNA methyltransferase in malignant human glioma cell lines
L E Ostrowski1, M A von Wronski, S H Bigner
1Department of Pathology, Duke University Medical Center, Durham, NC 27710.
Abstract:
When animals are treated with carcinogenic agents that alkylate O6-guanine residues, the incidence of tumors in specific tissues often relates inversely to the level of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) present in the tissue. Similarly, the hypersensitivity to anticancer chloroethylnitrosoureas of some human tumor cell lines is believed to result from their deficiency in MGMT. We have undertaken a comprehensive investigation of MGMT expression in a panel of nine characterized human glioma cell lines. Methyltransferase activity determined by incubating protein extracts of these glioma lines with [3H]methylated DNA ranged from undetectable in six lines (the Mer- phenotype) to greater than 0.8 pmol/mg in two lines (U-373 MG and D-392 MG). MGMT protein was undetectable in Western blots of the Mer- cell extracts probed with specific anti-MGMT monoclonal antibodies. Consistent with these results, steady-state levels of MGMT mRNA, determined by Northern blot analysis, were detectable only in the three Mer+ glioma lines (U-373 MG, D-392 MG, D-263 MG). Southern analysis of EcoRI-digested DNA probed with MGMT cDNA revealed no amplification, rearrangement or deletions of the MGMT gene in any of the glioma cell lines. This is the first report that examines MGMT expression at the biochemical, molecular and genetic levels in a particular tumor type. These studies suggest that transcriptional regulation is the basis of the Mer- phenotype in these malignant human glioma cell lines, since no gross structural or quantitative abnormalities of the MGMT gene were seen in the phenotypically Mer- lines.
Insights
The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is deficient in many human glioma cell lines, leading to a Mer- phenotype. This deficiency is due to transcriptional regulation, not gene abnormalities.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme crucial for protecting against DNA alkylation damage.
- MGMT deficiency is linked to increased tumor incidence and hypersensitivity to certain anticancer drugs like chloroethylnitrosoureas.
- Understanding MGMT expression in human gliomas is vital for cancer therapy and prognosis.
Purpose of the Study:
- To comprehensively investigate O6-methylguanine-DNA methyltransferase (MGMT) expression at biochemical, molecular, and genetic levels in human glioma cell lines.
- To determine the basis of the Mer- phenotype (MGMT deficiency) in these cell lines.
- To explore the role of MGMT in glioma pathogenesis and its implications for treatment.
Main Methods:
- Biochemical assays to measure methyltransferase activity in protein extracts.
- Western blot analysis to detect MGMT protein levels.
- Northern blot analysis to assess MGMT mRNA expression.
- Southern blot analysis to examine the MGMT gene structure and copy number.
Main Results:
- Six out of nine human glioma cell lines exhibited undetectable MGMT activity (Mer- phenotype).
- MGMT protein and mRNA were undetectable in Mer- cell lines, but present in Mer+ lines.
- No gene amplification, rearrangement, or deletions of the MGMT gene were observed in any of the glioma cell lines.
- MGMT expression varied significantly across the glioma cell panel.
Conclusions:
- The Mer- phenotype in human glioma cell lines is primarily attributed to transcriptional dysregulation of the MGMT gene.
- Gross structural or quantitative abnormalities of the MGMT gene do not explain its absence in phenotypically Mer- glioma cells.
- These findings highlight the importance of transcriptional control in MGMT expression and its potential as a therapeutic target in gliomas.