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 the same tumor. The first cell line, HEC-1-A, contains a nonsense mutation in the hPMS2 gene, which results in premature termination and a truncated hPMS2 protein. In addition, HEC-1-A cells carry a splice mutation in the hMSH6 gene and lack wild-type hMSH6 protein. The second cell line, HEC-1-B, possesses the same defective hMSH6 locus. However, HEC-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 cells also contain a wild-type hPMS2 gene. Initial recognition of mismatches 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 via the hMSH2/hMSH3 heterodimer. The data presented here in HEC-1-B cells illustrate (i) the reduction of instability at microsatellite sequences, (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 reduction in base substitution mutation rate at HPRT in HEC-1-B cells (hMSH6-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/hMSH3 heterodimer in maintaining genomic stability in the absence of a wild-type hMSH2/hMSH6 heterodimer.
Glaab
W E
WE
Laboratory of Environmental Carcinogenesis and Mutagenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. warren_glaab@merck.com
Risinger
J I
JI
Umar
A
A
Kunkel
T A
TA
Barrett
J C
JC
Tindall
K R
KR
D020137:Base Pair Mismatch; D001483:Base Sequence; D017931:DNA Primers; D004260:DNA Repair; D019281:Dimerization; D016889:Endometrial Neoplasms / Q000235:genetics / Q000473:pathology*; D005260:Female; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D018895:Microsatellite Repeats; D009363:Neoplasm Proteins / Q000235:genetics; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D009363:Neoplasm Proteins; D007041:Hypoxanthine Phosphoribosyltransferase