An Assay Of Quantitating Mismatch Repair Activity In Live Cells And The Effect Of Estrogen On Mismatch Repair Activity In Colonic Cells

Background and AimsThe DNA mismatch repair (MMR) system plays a key role in maintaining genomic stability.Patients with germline mutations of any of the 4 major MMR genes (hMSH2, hMLH1, hMSH6,or hPMS2) have Lynch syndrome (or hereditary nonpolyposis colorectal cancer [HNPCC]), themost common familial form of colorectal cancer. Another 10%-15% of sporadic colorectalcancers are caused by epigenetic silencing of hMLH1 gene. MMR dysfunction causesmicrosatellite instability (MSI). It has been reported that in HNPCC families the incidence ofcolorectal cancer (CRC) is higher in male mutation carriers than in female, which was foundsimilar in Chinese HNPCC families. The sex-specific difference in incidence of HNPCC hasbeen attributed to estrogen. It has been reported that post-menopausal estrogen replacementtherapy can reduce the risk of MSI-positive colon cancer. These findings suggest that estrogenmight prevent CRC caused by MMR dysfunction. In fact, estrogen has been reported to have aprotective effect on colon cancer in several large-scale population studies; however theunderlying mechanism is unclear. These above studies provide foundation for the hypothesis thatestrogen may prevent carcinogenesis by altering the expression and function of DNA MMRgene(s) in colonic cells. In the next study,we found that estrogen can up-regulate the expressionof MMR and we have already certified that estrogen can suppress the propagation of normalclone cells. Therefore, at the base of former study,we design this study to construct themeasurement of DNA mismatch repair activity in live cells and study the effect of estrogen tothe activity of mismatch repair genes.Materials and MethodsAT-G mismatch was introduced into the ATG start codon of the enhanced greenfluorescent protein (EGFP) gene. Repair of the T–G mismatch to T–A in the heteroduplex plasmid generates a functional EGFP gene expression. Colonic cancer cell line SW480 wasconfected with this mismatched plasmid and different concentrational estrodiol was added intothe medium. We can observe the intension of the green flow from the Flow cytometry and thenstudy the effect of estrogen to the activity of mismatch repair genes.ResultResultsThe homoduplex and heteroduplex plasmids were constructed and we devised the assay ofthe measurement of DNA mismatch repair activity in live cells; the intension of the green flowand the number of green flow cells improved after the infection of estrogen. Estrodiol canup-regulate the activity of MMR of SW480. Estrogen can up-regulate the function of MMR.ConclusionAT–G mismatch was introduced into the ATG start codon of the enhanced greenfluorescent protein (EGFP) gene. The homoduplex and heteroduplex plasmids we got can beused in the measurement of DNA mismatch repair activity in live cells. Estrodiol can improvethe relative EGFP expression of SW480. Estrogen can up-regulate the function of MMR.