title: Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants.
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.
  
  
  OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Medizinische Klinik II, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Brieger
authors.forename: J
authors.initials: J
authors.lastname: Trojan
authors.forename: J
authors.initials: J
authors.lastname: Raedle
authors.forename: G
authors.initials: G
authors.lastname: Plotz
authors.forename: S
authors.initials: S
authors.lastname: Zeuzem
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D000251:Adenosine Triphosphatases / Q000235:genetics; D017209:Apoptosis; D020137:Base Pair Mismatch; D002352:Carrier Proteins; D002455:Cell Division; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics*; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C501486:MLH1 protein, human; C098790:MSH6 protein, S cerevisiae; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; C497172:MSH2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D045643:DNA Repair Enzymes