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Complementation of an hMSH2 defect in human colorectal carcinoma cells by human chromosome 2 transfer
Author(s) -
Watanabe Yoh,
HaugenStrano Astrid,
Umar Asad,
Yamada Kanae,
Hemmi Hiromichi,
Kikuchi Yoshinori,
Takano Shoichi,
Shibata Yoichiro,
Barrett J. Carl,
Kunkel Thomas A.,
Koi Minoru
Publication year - 2000
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/1098-2744(200009)29:1<37::aid-mc5>3.0.co;2-2
Subject(s) - biology , homologous recombination , dna mismatch repair , microbiology and biotechnology , genetics , chromosome , locus (genetics) , gene , dna repair
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 . Mol. Carcinog. 29:37–49, 2000. Published 2000 Wiley‐Liss, Inc.