Related Experiment Videos
Complementation of an hMSH2 defect in human colorectal carcinoma cells by human chromosome 2 transfer
Y Watanabe1, A Haugen-Strano, A Umar
1Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Abstract:
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.
Insights
Restoring the hMSH2 gene in LoVo colorectal cancer cells corrected mismatch repair (MMR) activity and microsatellite instability. This correction also increased sensitivity to a DNA-damaging agent, highlighting hMSH2
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- The LoVo colorectal tumor cell line exhibits microsatellite instability due to a homozygous deletion in the hMSH2 gene.
- This deficiency impairs DNA mismatch repair (MMR) activity.
Purpose of the Study:
- To investigate if introducing wild-type hMSH2 into LoVo cells restores MMR activity and microsatellite stability.
- To assess the functional consequences of hMSH2 restoration, including drug sensitivity.
Main Methods:
- Chromosome transfer of hMSH2 into LoVo cells using chicken DT40/human hybrid and mouse A9 hybrid cells.
- Analysis of MMR competency, microsatellite stability, and drug sensitivity (N-methyl-N'-nitro-N-nitrosoguanidine).
- Homologous recombination targeting of hMSH2 in DT40 2C cells.
Main Results:
- Successful transfer of wild-type hMSH2 into LoVo cells restored MMR activity and microsatellite stability.
- hMSH2-positive LoVo hybrids showed increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine, linked to G2 cell-cycle arrest and cell death.
- Efficient homologous recombination targeting of hMSH2 in DT40 2C cells was demonstrated.
Conclusions:
- Restoration of hMSH2 complements mutator and drug-resistant phenotypes in LoVo cells.
- Chromosome transfer is a viable method for introducing functional or modified hMSH2 into cancer cells.
- Further studies using modified hMSH2 from DT40 2C could elucidate specific gene functions.