Antisense inhibition of hMLH1 is not sufficient for loss of DNA mismatch repair function in the HCT116+chromosome 3 cell line.
We have reported that transfer of chromosome 3 (Chr3) containing a single wild-type copy of the hMLH1 gene into HCT116 colon cancer cells, a cell line deficient in DNA mismatch repair (MMR) activity attributable to inactivating hMLH1 mutations, corrects all of the aspects of the MMR repair-deficient phenotype. We inhibited the expression of the wild-type hMLH1 gene using antisense RNA in HCT116+Chr3 cells to determine if this would result in reversion to the MMR-deficient phenotype. Despite profound inhibition of hMLH1 expression, DNA MMR activity and alkylation sensitivity were not impaired in the antisense-transfected HCT116+Chr3 cells. Additionally, arrest of the cell cycle at the G2 phase with alkylation damage occurs in these cells, a phenotype associated with MMR proficiency. These results indicate that even with a reduction in the expression of hMLH1 protein below the limits of detection by Western blotting, DNA MMR activity remained fully functional (by direct DNA MMR activity assay). We would speculate that hMLH1 is expressed in substantially greater abundance than would be minimally necessary for DNA MMR and that minor reductions in the expression of this protein would not be sufficient to permit DNA MMR dysfunction. Alternatively, Chr3 may contain a second hMLH1 homologue that might overlap with the function of hMLH1.
Chauhan
D P
DP
Department of Medicine and Cancer Center, The University of California San Diego, La Jolla 92093-0688, USA. dchauhan@ucsd.edu
Yang
Q
Q
Carethers
J M
JM
Marra
G
G
Chang
C L
CL
Chamberlain
S M
SM
Boland
C R
CR
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch; D015153:Blotting, Western; D002352:Carrier Proteins; D002453:Cell Cycle / Q000187:drug effects; D002893:Chromosomes, Human, Pair 3 / Q000235:genetics*; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D012313:RNA; D004247:DNA; D000070957:MutL Protein Homolog 1