Expression of five selected human mismatch repair genes simultaneously detected in normal and cancer cell lines by a nonradioactive multiplex reverse transcription-polymerase chain reaction.
Abnormalities in at least 1 of 5 mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, hPMS2 and GTBP/hMSH6) are found in hereditary nonpolyposis colon cancer and sporadic colon cancers. We used a single-reaction multiplex reverse transcription (RT)-polymerase chain reaction (PCR), with the beta-actin gene as an internal control, to simultaneously evaluate expression of these 5 known human MMR genes in normal and tumor cell lines with known or uncharacterized mutations in MMR genes. The relative quantitation of the transcripts is demonstrated by controlling the number of PCR cycles and titrating cDNA with a dose-curve. The 13 normal cell lines tested were derived from normal lymphocytes, skin, thymus, breast, lung, colon, liver and kidney. The 26 cancer cell lines were derived from melanoma and cancers of the brain, breast, lung, colon, pancreas and prostate. All 5 MMR genes were ubiquitously expressed in all normal cell lines tested, suggesting their housekeeping roles. Aberrant MMR gene expression was only observed in the colon cancer cell lines. Two previously uncharacterized colon cancer cell lines did not express hMLH1. These data suggest that this nonradioactive multiplex RT-PCR assay for MMR gene expression may be useful for fast screening for genetic alterations that may affect gene expression and so may aid molecular analysis of MMR-related colon cancer.
Wei
Q
Q
Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. qwei@request.mdacc.tmc.edu
Guan
Y
Y
Cheng
L
L
Radinsky
R
R
Bar-Eli
M
M
Tsan
R
R
Li
L
L
Legerski
R J
RJ
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000235:genetics; D002352:Carrier Proteins; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005796:Genes / Q000235:genetics; D006801:Humans; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012313:RNA / Q000032:analysis / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C088996:PMS1 protein, human; D011518:Proto-Oncogene Proteins; D012313:RNA; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; C497172:MSH2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D045643:DNA Repair Enzymes