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Transient transfection permits analysis of mismatch repair genes

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How variants of mismatch repair genes affect development of colorectal cancer can now be studied much more easily, thanks to a method tested for this purpose by German researchers.

They used a transient transfection model between wild type (wt) and two mutant DNA constructs of the major human mismatch repair (MMR) genes hMLH1 and hMSH2 and two cell lines, each deficient in one or other gene. These cell lines were derived from patients with hereditary non-polyposis colorectal cancer (HNPCC), and their gene mutations were matched by the constructs.

For mismatch repair to occur hMLH1 and hMSH2 proteins must interact with other specific proteins—hMLH1 with hPMS2 and hMSH2 with hMSH6. Transient transfection of cell line HCT-116 (hMLH1 deficient) with wt but not mutant hMLH1 increased expression of hPMS2 protein. Similarly, in the LOVO cell line (hMSH2 deficient) expression of hMSH6 depended on transfection with wt hMSH2. Synthesis of hMSH6 mRNA in LOVO cells depended on transfection with wt hMSH2 but that of hPMS2 was the same with wt and mutant hMLH1, suggesting post transcriptional control.

Transfections to establish the role of the MMR genes in apoptosis and cell proliferation were also investigated. They showed that the wt hMSH2 gene controlled apoptosis and that wt hMLH1 gene but not wt hMSH2 gene regulated cell proliferation.

Germline mutations in the MMR genes account for most cases of HNPCC. A role in controlling apoptosis or cell proliferation has been suggested for these genes, but previous results are thought to be unreliable because of confounding factors.