Molecular events after antisense inhibition of hMSH2 in a HeLa cell line.
To establish a cause-effect relationship between the human mismatch repair pathway deficiency and the observed phenotypes, a hMSH2 deficient HeLa cell line (HeLa-MSH2-) was established by transfecting the HeLa cells with an antisense RNA expression plasmid. The expression plasmid was constructed by inserting an 851 bp fragment of hMSH2 cDNA into the polyclonal site of the vector pREP9 in a reversed orientation. The production of the mismatch binding protein, hMSH2, was inhibited in HeLa-MSH2- cells, as demonstrated by Western blotting and band shift assay of its whole cell extract. The growth rate of this cell line was not different from the parental HeLa cells soon after transfection. However, the rate was faster after 10 subcultures. The spontaneous mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus increased markedly, but no N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) tolerance appeared in this cell line. Our results clearly demonstrated several molecular events happened after the inhibition of a major mismatch recognition protein, hMSH2, in the mismatch repair pathway, mimicking carcinogenesis processes.
Department of Pathophysiology and Laboratory of Medical Molecular Biology, Zhejiang Medical University, Hangzhou, Zhejiang 310031, China.
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Qian
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Yu
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Cheng
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Luo
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Xie
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Shen
D020137:Base Pair Mismatch / Q000235:genetics; D015153:Blotting, Western; D002273:Carcinogens / Q000737:chemistry; D002455:Cell Division / Q000235:genetics; D002850:Chromatography, Gel; D004260:DNA Repair / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008769:Methylnitronitrosoguanidine / Q000737:chemistry; D051718:MutS Homolog 2 Protein; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000502:physiology; D012313:RNA / Q000737:chemistry; D012326:RNA Splicing; D016372:RNA, Antisense / Q000737:chemistry* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D002273:Carcinogens; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016372:RNA, Antisense; C039919:RNA, recombinant; D008769:Methylnitronitrosoguanidine; D012313:RNA; D007041:Hypoxanthine Phosphoribosyltransferase; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein