MOLECULAR EVENTS AFTER ANTISENSE INHIBITION OF HMSH2 IN A HELA-CELL LINE

To establish a cause-effect relationship between the human mismatch re pair pathway deficiency and the observed phenotypes, a hMSH2 deficient HeLa cell line (HeLa-MSH2(-)) was established by transfecting the HeL a cells with an antisense RNA expression plasmid. The expression plasm id 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 H eLa-MSH2- cells, as demonstrated by Western blotting and band shift as say of its whole cell extract. The growth rate of this cell line was n ot different from the parental HeLa cells soon after transfection. How ever, the rate was faster after IO subcultures. The spontaneous mutati on frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locu s increased markedly, but no N-methyl-N'-nitro-N-nitrosoguanidine (MNN G) tolerance appeared in this cell line. Our results clearly demonstra ted several molecular events happened after the inhibition of a major mismatch recognition protein, kMSH2, in the mismatch repair pathway, m imicking carcinogenesis processes. (C) 1998 Elsevier Science B.V. All rights reserved.