AIM: To understand the response of human REV3gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) and its role in human mutagenesis.METHODS: The response of the human REV3 gene to ...AIM: To understand the response of human REV3gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) and its role in human mutagenesis.METHODS: The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRTlocus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FLREV3- by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3- and FL-REV3-. The blockage effect of REV3 was measured by combination of reverse transcriptionpolymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3protein level.RESULTS: The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P＜0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P＜0.05).In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein.MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P＜0.05) and 5.8 fold (P＜0.01)respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3was blocked by antisense RNA,and almost recovered to their spontaneous mutation levels.The spontaneous HPRT mutation was disappeared in REV3disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66×10-6 in FL cells to 0.14×10-6 in transgenic cells as well (P＜0.01).CONCLUSION: The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.展开更多
基金the National Natural Science Foundation of China for Key Program,No.39830210a grant from the National Natural Science Foundation of China,No.39960067a grant from National Key Basic Research and Development Program,No.2002CB512904
文摘AIM: To understand the response of human REV3gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) and its role in human mutagenesis.METHODS: The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRTlocus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FLREV3- by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3- and FL-REV3-. The blockage effect of REV3 was measured by combination of reverse transcriptionpolymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3protein level.RESULTS: The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P＜0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P＜0.05).In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein.MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P＜0.05) and 5.8 fold (P＜0.01)respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3was blocked by antisense RNA,and almost recovered to their spontaneous mutation levels.The spontaneous HPRT mutation was disappeared in REV3disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66×10-6 in FL cells to 0.14×10-6 in transgenic cells as well (P＜0.01).CONCLUSION: The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.
