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) act ivity, and exhibits microsatellite instability. To determine whether the in troduction of a wild type hMSH2 into LoVo restores MMR activity and stabili zes 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 wil d-type hMSH2. Complete correlation was found between the presence of the wi ld-type hMSH2 and hMSH2 expression, an increased stability in microsatellit e loci, and competency in MMR. The hMSH2-positive LoVo hybrids also showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine. This enha nced toxicity is associated with G(2) cell-cycle arrest followed by prematu re mitosis and cell death. These results suggest that hMSH2 may be responsi ble 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 targeti ng vector containing an hMSH2 exon 4 disrupted by the zeocin-resistant gene . The results showed that the hMSH2 locus in DT40 2C was efficiently target ed by an exogeneously transfected homologous sequence, suggesting that tran sfer of a modified hMSH2 from DT40 2C to LoVo via chromosome transfer could be used to determine the function of hMSH2. Mel. Carcinog. 29:37-49 2000. dagger published 2000 Wiley-Liss, Inc.