Effect of hMSH6 cDNA expression on the phenotype of mismatch repair-deficient colon cancer cell line HCT15.
Mismatch recognition in human cells is mediated primarily by a heterodimer of hMSH2 and hMSH6. Cells mutated in both alleles of the hMSH6 gene are deficient in the correction of base/base mispairs and insertion/deletion loops of one nucleotide and thus exhibit a strong mutator phenotype, evidenced by elevated mutation rates and microsatellite instability, as well as by tolerance to methylating agents. The decrease in replication fidelity associated with a loss of mismatch correction implies that with each division, these cells are likely to acquire new mutations throughout their genomes. Should such secondary mutations occur in genes linked to replication fidelity or involved in the maintenance of genomic stability, they might contribute to the observed mutator phenotype. The human colon tumour line HCT15 represents one such case. Although it carries inactivating mutations in both hMSH6 alleles, it has also been shown to contain a missense mutation in the coding sequence of the proofreading domain of the polymerase-delta gene. In an attempt to find out whether the phenotype of HCT15 cells was indeed brought about solely by the lack of hMSH6, we stably transfected them with a vector carrying the wild-type hMSH6 cDNA. Our results show that although the levels of transgenic hMSH6 were low, expression of the wild-type protein resulted in a substantial restoration of mismatch binding, mismatch repair capacity and the stability of mononucleotide repeats, as well as in the reduction of mutation rates. Although methylation tolerance of the hMSH6-expressing cells was not markedly affected, the G2 cell cycle checkpoint, absent in N-methyl-N'-nitro-N-nitrosoguanidine-treated control cells, was restored.
Lettieri
T
T
Institute of Medical Radiobiology, Zürich, Switzerland.
Marra
G
G
Aquilina
G
G
Bignami
M
M
Crompton
N E
NE
Palombo
F
F
Jiricny
J
J
D020137:Base Pair Mismatch; D001483:Base Sequence; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D017931:DNA Primers; D004260:DNA Repair; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics*; D016195:G2 Phase; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008770:Methylnitrosourea / Q000494:pharmacology; D018895:Microsatellite Repeats; D009154:Mutation; D010641:Phenotype; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D008769:Methylnitronitrosoguanidine; D006147:Guanine; D008770:Methylnitrosourea; C008449:O-(6)-methylguanine