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[Effects of Rad9 mutants with impaired DNA mismatch repair function on tumorigenesis of colorectal cancer].
OBJECTIVE: The aim of this study was to investigate the effects of Rad9 mutants with impaired DNA mismatch repair (MMR) function on the tumorigenesis of colorectal cancer.
METHODS: The colorectal cancer tumor samples were collected from 100 patients. The mutation profiles of human Rad9 (hRad9) gene in these samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. The plasmid of pFLAG-hRad9 (L101M) was constructed following the QuickChange mutagenesis procedure and transfected into mRad9-deleted mouse cells (mRad9(-/-) cells). The expression of hRad9 protein was measured by western blot analysis. The MMR activity in live cells was detected by flow cytometry using the reporter plasmid for MMR function.
RESULTS: Mutation from Leu to Met at the residue 101 (L101M) of hRad9 gene was detected in 7 of the 100 samples. The mismatch repair efficiency of mRad9(-/-)+ L101M cells (mRad9-deleted mouse cells with ectopic expression of L101M hRad9 gene) was (34.0±5.6)%, which was significantly lower than that in the mRad9(-/-)+ hRad9 cells [mRad9-deleted mouse cells with ectopic expression of hRad9 gene, (48.0±7.5)%, P<0.05]. After N-nitroso-N-methylurea (MNU) treatment, the survival rate of mRad9(-/-)+ L101M cells was (33.7±5.9)%, which was significantly higher than that in the mRad9(-/-)+ hRad9 cells [(21.3±4.7)%, P<0.05]. Thus, ectopic expression of L101M hRad9 gene resulted in significantly reduced MMR activity and increased resistance to MNU. Furthermore, ectopic expression of hRad9 gene with mutation at the target residues of post-translational modification in mRad9(-/-) cells also led to a reduced MMR activity.
CONCLUSION: Rad9 mutants with impaired DNA mismatch repair function may promote tumorigenesis of colorectal cancer.
METHODS: The colorectal cancer tumor samples were collected from 100 patients. The mutation profiles of human Rad9 (hRad9) gene in these samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. The plasmid of pFLAG-hRad9 (L101M) was constructed following the QuickChange mutagenesis procedure and transfected into mRad9-deleted mouse cells (mRad9(-/-) cells). The expression of hRad9 protein was measured by western blot analysis. The MMR activity in live cells was detected by flow cytometry using the reporter plasmid for MMR function.
RESULTS: Mutation from Leu to Met at the residue 101 (L101M) of hRad9 gene was detected in 7 of the 100 samples. The mismatch repair efficiency of mRad9(-/-)+ L101M cells (mRad9-deleted mouse cells with ectopic expression of L101M hRad9 gene) was (34.0±5.6)%, which was significantly lower than that in the mRad9(-/-)+ hRad9 cells [mRad9-deleted mouse cells with ectopic expression of hRad9 gene, (48.0±7.5)%, P<0.05]. After N-nitroso-N-methylurea (MNU) treatment, the survival rate of mRad9(-/-)+ L101M cells was (33.7±5.9)%, which was significantly higher than that in the mRad9(-/-)+ hRad9 cells [(21.3±4.7)%, P<0.05]. Thus, ectopic expression of L101M hRad9 gene resulted in significantly reduced MMR activity and increased resistance to MNU. Furthermore, ectopic expression of hRad9 gene with mutation at the target residues of post-translational modification in mRad9(-/-) cells also led to a reduced MMR activity.
CONCLUSION: Rad9 mutants with impaired DNA mismatch repair function may promote tumorigenesis of colorectal cancer.
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