Truncation of the mismatch repair protein PMS2 during the neoplastic transformation of human breast epithelial cells in vitro.
Alterations in known mismatch repair (MMR) genes have been found in many cancers, such as in hereditary non-polyposis colorectal cancer syndrome (HNPCC), in addition to specific oncogenes and tumor suppressor gene abnormalities. We have reported mutations in the MMR genes hMSH2, hPMS2 and hMSH6 in human breast epithelial cell lines (HBEC). In hPMS2 we found a non-sense mutation in codon number 471 by a change of glutamine (CAG) by stop codon (TAG). We also observed that the number of alterations in the MMR genes were increased during the process of cell transformation and tumorigenesis. In the present study in order to determine the role of those mutations in the hPMS2 protein activity, we used western blotting, protein truncation test and MMR activity assay employing HBEC cells. In the transformed and tumor cell lines we found a premature hPMS2 protein truncated by western blot and also we confirmed this truncation using the protein truncation test. In the MCF-10F cells only the wild-type hPMS2 protein was detected. The functionality of the hPMS2-truncated protein was confirmed by DNA heteroduplex assay using specific mispaired bases in mutated M13mp2 phages. DNA repair activity was proficient in MCF-10F cells, partially deficient in the transformed cells, and totally deficient in the tumor cell lines. In order to test whether the MMR deficiency was due to an hPMS2 defect, we used the wild-type heterodimer hPMS2-hMLH1 in the in vitro reaction of DNA heteroduplex assay; we have found that addition of the hPMS2 wild-type increased the MMR repair efficiency by more than 40% in tumor cell lines. Our results indicate that a protein-truncating mutation in the hPMS2 gene had occurred during the process of cell transformation and tumorigenesis and that truncation is related to a lack of function of this gene.
Balogh
Gabriela A
GA
Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Russo
Irma H
IH
Russo
Jose
J
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D020137:Base Pair Mismatch / Q000235:genetics; D001940:Breast / Q000166:cytology*; D001943:Breast Neoplasms / Q000235:genetics; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D000070976:Mismatch Repair Endonuclease PMS2; D009154:Mutation; D017384:Sequence Deletion
D004268:DNA-Binding Proteins; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D045643:DNA Repair Enzymes