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Updated: Aug 30, 2026
Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence
Published on: February 10, 2023
Mismatch repair gene expression defects contribute to microsatellite instability in ovarian carcinoma
John P Geisler1, Michael J Goodheart, Anil K Sood
1Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA. john-geisler@indianagynonc.com
Background:
hMLH1, the human MutL homologue, has been linked to microsatellite instability (MSI) in gastrointestinal tumors. However, to the authors' knowledge, the role of hMLH1, the other mismatch repair genes (MMR), and MSI in ovarian carcinoma has not been well defined. The purpose of the current study was to determine the relation between MSI of ovarian carcinoma and MMR gene expression, hMLH1 and hMSH2 hypermethylation, and hMLH1 and hMSH2 null mutations.
Methods:
hMLH1 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and amplification of cDNA using a housekeeping gene (glycerol 3-phosphate dehydrogenase) as a control for mRNA quality and quantity. Methylation-specific PCR (MS-PCR) was used to correlate methylation of the hMLH1 and hMSH2 CpG islands with mRNA expression status. Similar techniques were used to evaluate the concomitant expression of five other MMR: hMSH2, hMSH3, hMSH6, PMS1, and PMS2. Microsatellite instability was studied using the National Cancer Institute consensus markers (D2S123, D5S346, D17S250, BAT25, and BAT26) and NM23 as described previously.
Results:
One hundred twenty-five primary tumors were analyzed. High-frequency MSI (MSI-H) was found in 21 tumors (16.8%). hMLH1 mRNA was absent in 10 of these 21 tumors (47.6%). In each case, coordinated hypermethylation of both regions A and C of the promoter was identified. Microsatellite stable and low-frequency MSI tumors all were found to express not only hMLH1 but the other MMR genes as well (P < 0.001). Absence of expression of hMSH2 and the four other MMRs occurred in tumors with absent hMLH1 mRNA expression because of CpG island hypermethylation. No absence of expression of hMSH2, hMSH3, hMSH6, PMS1, or PMS2 was found to occur in tumors expressing hMLH1. None of the 11 MSI-H tumors without promoter hypermethylation demonstrated a null mutation in hMLH1 or hMSH2.
Conclusions:
A molecular mechanism to explain > 50% of the MSI-H phenotype in ovarian carcinoma cases was demonstrated. MSI-H may occur because of MMR defects, especially hMLH1 promoter hypermethylation. Additional mechanisms are required to explain the balance between the cases of MSI-H as well as the phenomenon of MSI-L tumors.
Insights
Microsatellite instability-high (MSI-H) ovarian cancers are often caused by defects in DNA mismatch repair (MMR) genes, particularly hMLH1 promoter hypermethylation. This study clarifies the molecular mechanisms underlying MSI in ovarian carcinoma.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The role of DNA mismatch repair (MMR) genes and microsatellite instability (MSI) in ovarian carcinoma is not well-defined.
- hMLH1, a key MMR gene, is implicated in MSI in other cancers.
- This study investigates MMR gene expression, hypermethylation, and mutations in ovarian cancer.
Purpose of the Study:
- To determine the relationship between MSI in ovarian carcinoma and MMR gene expression.
- To investigate the role of hMLH1 and hMSH2 promoter hypermethylation in MSI.
- To examine hMLH1 and hMSH2 null mutations in relation to MSI status.
Main Methods:
- hMLH1 mRNA expression was assessed using RT-PCR.
- Methylation-specific PCR (MS-PCR) was employed to detect promoter hypermethylation of hMLH1 and hMSH2.
- MMR gene expression (hMSH2, hMSH3, hMSH6, PMS1, PMS2) and MSI status were evaluated using established techniques.
Main Results:
- High-frequency MSI (MSI-H) was observed in 16.8% of 125 ovarian tumors.
- hMLH1 mRNA absence, linked to promoter hypermethylation, occurred in 47.6% of MSI-H tumors.
- Absence of other MMR gene expression correlated with hMLH1 absence due to hypermethylation; no null mutations were found in MSI-H tumors without hypermethylation.
Conclusions:
- hMLH1 promoter hypermethylation is a significant molecular mechanism driving MSI-H in ovarian carcinoma, explaining over 50% of cases.
- MMR defects, particularly hMLH1 hypermethylation, are key drivers of MSI in ovarian cancer.
- Further research is needed to elucidate mechanisms behind remaining MSI-H and MSI-L cases.
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