title: Functional effects of the MLH1-93G>A polymorphism on MLH1/EPM2AIP1 promoter activity.
abstract: Defective mismatch repair leads to the microsatellite instability (MSI) phenotype of colorectal cancer (CRC). We previously showed that the MLH1-93G>A promoter polymorphism is strongly associated with MSI tumours, suggesting a modifier role for this polymorphism in CRC. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. In order to evaluate the functional effects of this polymorphism, we transfected a panel of CRC, endometrial cancer and non-tumourigenic cell lines with MLH1 luciferase promoter constructs. We used constructs in reverse orientation to assess the effect of this polymorphism on EPM2AIP1. The luciferase activities were compared using a two-sided Student's t-test. Electrophoretic mobility shift assays (EMSAs) were used to evaluate whether differential protein binding was responsible for the differences in promoter activity. We observed a higher level of activity with the -93G allele in all the cell lines observed; including the CRC cell line, HCT116 (P=0.002), the endometrial cancer cell line, HEC-1-A (P<0.001) and the normal colonic cell line, CCD-841-CoTr (P=0.002). This polymorphism also affected EPM2AIP1 transcription with the -93A allele demonstrating higher promoter activity in the HCT116 (P=0.007) and HEC-1-A (P=0.004) cells. The EMSA results suggest that this polymorphism alters the affinity of nuclear factors that bind to this region. Our findings indicate that the -93G>A polymorphism modifies the efficiency of MLH1/EPM2AIP1 transcription.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 1 King's College Circle, Toronto, ON, Canada.
authors.forename: Sheron
authors.initials: S
authors.lastname: Perera
authors.forename: Miralem
authors.initials: M
authors.lastname: Mrkonjic
authors.forename: James B
authors.initials: JB
authors.lastname: Rawson
authors.forename: Bharati
authors.initials: B
authors.lastname: Bapat
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000502:physiology; D002277:Carcinoma / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D002478:Cells, Cultured; D015179:Colorectal Neoplasms / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D053842:Microsatellite Instability; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology; D020641:Polymorphism, Single Nucleotide / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C483189:EPM2AIP1 protein, human; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1