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Updated: Oct 1, 2026
Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Published on: February 24, 2015
Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers
M L Veigl1, L Kasturi, J Olechnowicz
1Ireland Cancer Center, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106, USA.
Abstract:
Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI-positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism.
Insights
Epigenetic silencing of the hMLH1 gene, through promoter methylation, inactivates DNA repair in many microsatellite instability (MSI) colon cancers. This silencing can be reversed by demethylating agents, offering potential therapeutic strategies for MSI cancers.
Area of Science:
- Molecular Biology
- Cancer Genetics
- Epigenetics
Background:
- DNA mismatch repair (MMR) gene mutations, including hMLH1, are linked to microsatellite instability (MSI) in human cancers.
- While germline and somatic mutations explain some MSI cancers, a subset of sporadic MSI colon cancers have wild-type MMR genes.
- Understanding the mechanisms behind MSI in these tumors is crucial for cancer diagnosis and treatment.
Purpose of the Study:
- To investigate the molecular basis of hMLH1 inactivation in sporadic MSI colon cancers with wild-type coding sequences.
- To explore the role of epigenetic modifications, specifically promoter methylation, in hMLH1 silencing.
- To assess the potential for reversing hMLH1 silencing using demethylating agents.
Main Methods:
- Analysis of hMLH1 protein expression in MSI cancer cell lines.
- Examination of hMLH1 gene promoter methylation status.
- Treatment of cell lines with the demethylating agent 5-azacytidine to assess hMLH1 re-expression.
- Single-cell cloning experiments to study dynamic regulation of hMLH1 expression.
Main Results:
- hMLH1 protein was absent in five of six MSI cancer cell lines despite wild-type coding sequences.
- Absence of hMLH1 protein correlated with methylation of the hMLH1 gene promoter.
- Treatment with 5-azacytidine restored hMLH1 protein expression, which could be reversibly modulated.
- Epigenetic silencing affected both maternal and paternal hMLH1 alleles, and was reversible by 5-azacytidine.
Conclusions:
- A significant proportion of human MSI cancers arise from epigenetic silencing of the hMLH1 gene via promoter methylation.
- This epigenetic mechanism inactivates both hMLH1 alleles, leading to defective DNA repair and MSI.
- Reactivation of hMLH1 expression by demethylating agents demonstrates the reversibility of this silencing mechanism.
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