CHARACTERIZATION OF DISTINCT HUMAN ENDOMETRIAL CARCINOMA CELL-LINES DEFICIENT IN MISMATCH REPAIR THAT ORIGINATED FROM A SINGLE TUMOR

The role of specific mismatch repair (MMR) gene products was examined by observing several phenotypic end points in two MMR-deficient human endometrial carcinoma cell lines that were originally isolated from th e same tumor. The first cell Line, HEC-1-A, contains a nonsense mutati on ire the hPMS2 gene, which results in premature termination and a tr uncated hPMS2 protein. In addition, REC-1-A cells carry a splice mutat ion in the hMSH6 gene and lack wild-type hMSH6 protein, The second cel l line, HEC-1-B, possesses thee same defective hMSH6 locus, However, H EC-1-B cells are heterozygous at the hPMS2 locus; that is, along with carrying the same nonsense mutation in hPMS2 as in HEC-1-A, HEC-1-B ce lls also contain a wild-type hPMS2 gene. initial recognition of mismat ches in DNA requires either the hMSH2/hMSH6 or hMSH2/hMSH3 heterodimer , with hPMS2 functioning downstream of damage recognition. Therefore, cells defective in hPMS2 should completely lack MMR (HEC-1-A), whereas cells mutant in hMSH6 only (HEC-1-B) can potentially repair damage vi a the hMSH2/hMSH3 heterodimer. The data presented here in HEC-1-B cell s illustrate (i) the reduction of instability at microsatellite sequen ces, (ii) a significant decrease in frameshift mutation rate at HPRT, and (iii) the in vitro repair of looped substrates, relative to HEC-1- A cells, illustrating the repair of frameshift intermediates by hMSH2/ hMSH3 heterodimer. Furthermore, the role of hMSH2/hMSH3 heterodimer in the repair of base:base mismatches is supported by observing the redu ction in base substitution mutation rate at HPRT in HEC-1-B cells (hMS H6-defective but possessing wild-type hPMS2), as compared with HEC-1-A (hMSH6/hPMS2-defective) cells, These data support a critical role for hPMS2 in human MMR, while further defining the role of the hMSH2/hMSH 3 heterodimer in maintaining genomic stability in the absence of a wil d-type hMSH2/hMSH6 heterodimer.