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.
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.
Authors: A Umar; M Koi; J I Risinger; W E Glaab; K R Tindall; R D Kolodner; C R Boland; J C Barrett; T A Kunkel Journal: Cancer Res Date: 1997-09-15 Impact factor: 12.701
Authors: T W Davis; C Wilson-Van Patten; M Meyers; K A Kunugi; S Cuthill; C Reznikoff; C Garces; C R Boland; T J Kinsella; R Fishel; D A Boothman Journal: Cancer Res Date: 1998-02-15 Impact factor: 12.701
Authors: A Sobrero; D Kerr; B Glimelius; E Van Cutsem; G Milano; D M Pritchard; P Rougier; M Aapro Journal: Eur J Cancer Date: 2000-03 Impact factor: 9.162
Authors: L Lo Muzio; P Nocini; M D Mignogna; G Pannone; S Staibano; M Procaccini; C Rubini; M Fioroni; S Fanali; A Piattelli Journal: Anticancer Res Date: 2000 Mar-Apr Impact factor: 2.480
Authors: V Abkevich; A Zharkikh; A M Deffenbaugh; D Frank; Y Chen; D Shattuck; M H Skolnick; A Gutin; S V Tavtigian Journal: J Med Genet Date: 2004-07 Impact factor: 6.318
Authors: Abhijit Rath; Akriti Mishra; Victoria Duque Ferreira; Chaoran Hu; Gregory Omerza; Kevin Kelly; Andrew Hesse; Honey V Reddi; James P Grady; Christopher D Heinen Journal: Hum Mutat Date: 2019-08-17 Impact factor: 4.878
Authors: Lise Lotte Christensen; Reetta Kariola; Mari K Korhonen; Friedrik P Wikman; Lone Sunde; Anne-Marie Gerdes; Henrik Okkels; Carsten A Brandt; Inge Bernstein; Thomas V O Hansen; Rikke Hagemann-Madsen; Claus L Andersen; Minna Nyström; Torben F Ørntoft Journal: Fam Cancer Date: 2009-08-21 Impact factor: 2.375
Authors: Sanjeevani Arora; Peter J Huwe; Rahmat Sikder; Manali Shah; Amanda J Browne; Randy Lesh; Emmanuelle Nicolas; Sanat Deshpande; Michael J Hall; Roland L Dunbrack; Erica A Golemis Journal: Cancer Biol Ther Date: 2017-05-11 Impact factor: 4.742
Authors: M Clyne; J Offman; S Shanley; J D Virgo; M Radulovic; Y Wang; A Ardern-Jones; R Eeles; E Hoffmann; V P C C Yu Journal: Br J Cancer Date: 2009-01-13 Impact factor: 7.640