Complementation of an hMSH2 defect in human colorectal carcinoma cells by human chromosome 2 transfer.
The human colorectal tumor cell line LoVo has a homozygous deletion in the hMSH2 gene from exon 3 to exon 8, is deficient in mismatch repair (MMR) activity, and exhibits microsatellite instability. To determine whether the introduction of a wild type hMSH2 into LoVo restores MMR activity and stabilizes microsatellite loci, we transferred a chromosome 2 fragment containing hMSH2 into a homologous recombination-proficient chicken DT40/human hybrid (DT40 2C) and a human chromosome 2 in a mouse A9 hybrid to LoVo. Transfers of these chromosomes into LoVo resulted in LoVo both with and without a wild-type hMSH2. Complete correlation was found between the presence of the wild-type hMSH2 and hMSH2 expression, an increased stability in microsatellite loci, and competency in MMR. The hMSH2-positive LoVo hybrids also showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine. This enhanced toxicity is associated with G(2) cell-cycle arrest followed by premature mitosis and cell death. These results suggest that hMSH2 may be responsible for complementing mutator and drug-resistant phenotypes in chromosome 2-transferred LoVo cells. To test whether the hMSH2 in DT40 2C cells can be modified by homologous recombination, we transfected DT40 2C with a targeting vector containing an hMSH2 exon 4 disrupted by the zeocin-resistant gene. The results showed that the hMSH2 locus in DT40 2C was efficiently targeted by an exogeneously transfected homologous sequence, suggesting that transfer of a modified hMSH2 from DT40 2C to LoVo via chromosome transfer could be used to determine the function of hMSH2.
Watanabe
Y
Y
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Haugen-Strano
A
A
Umar
A
A
Yamada
K
K
Hemmi
H
H
Kikuchi
Y
Y
Takano
S
S
Shibata
Y
Y
Barrett
J C
JC
Kunkel
T A
TA
Koi
M
M
D000818:Animals; D001483:Base Sequence; D016923:Cell Death / Q000187:drug effects; D002889:Chromosomes, Human, Pair 2; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D017931:DNA Primers; D004268:DNA-Binding Proteins; D016195:G2 Phase / Q000187:drug effects; D018390:Gene Targeting; D005816:Genetic Complementation Test; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D051379:Mice; D018895:Microsatellite Repeats / Q000235:genetics; D051718:MutS Homolog 2 Protein; D011518:Proto-Oncogene Proteins / Q000235:genetics*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D008769:Methylnitronitrosoguanidine; C497172:MSH2 protein, human; C497173:Msh2 protein, mouse; D051718:MutS Homolog 2 Protein