alexa Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants.
Bioinformatics & Systems Biology

Bioinformatics & Systems Biology

Journal of Proteomics & Bioinformatics

Author(s): Brieger A, Trojan J, Raedle J, Plotz G, Zeuzem S

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Abstract BACKGROUND: Germline mutations in the mismatch repair (MMR) genes hMLH1 and hMSH2 can cause hereditary non-polyposis colorectal cancer (HNPCC). However, the functional in vitro analysis of hMLH1 and hMSH2 mutations remains difficult. AIMS: To establish an in vitro method for the functional characterisation of hMLH1 and hMSH2 mutations. METHODS: hMLH1 and hMSH2 wild type (wt) genes and several mutated subclones were transiently transfected in mismatch repair deficient cell lines (HCT-116 and LOVO). Apoptosis, proliferation, and regulation of mRNA expression and protein expression of interacting proteins were analysed by Hoechst staining, AlamarBlue staining, real time polymerase chain reaction, and western blotting, respectively. RESULTS: The protein expression of hMLH1 and hMSH2 mutants was significantly decreased after transfection compared with wild type transfections. The hMLH1 and hMSH2 interacting proteins hPMS2 and hMSH6 became detectable only after transfection of the respective wild type genes. In parallel, hMSH6 mRNA levels were increased in hMSH2 wt transfected cells. However, hPMS2 mRNA levels were independent of the mutation status of its interacting partner hMLH1, indicating a post-transcriptional regulating pathway. In the hMLH1 deficient HCT-116 cell line apoptosis was not affected by transfection of any mismatch repair gene, whereas complementation of hMSH2 deficiency in LOVO cells increased apoptosis. Conversely, proliferative activity of HCT-116 was decreased by complementation with hMLH1wt and unaffected in hMSH2 deficient LOVO cells. CONCLUSION: These data show that the cellular role of the MMR genes and its mutations are assessable in a simple transient transfection system and show the influence of MMR gene regulation on major cell growth regulating mechanisms. This method is applicable for the functional definition of mutations in hMLH1 and hMSH2 genes observed in patients with suspected HNPCC.
This article was published in Gut and referenced in Journal of Proteomics & Bioinformatics

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