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AIM:To understand the response of human REV3gene togastric cancer inducing carcinogen N-methyI-N’nitro-N-nitrosoguanidine (MNNG) and its role in humanmutagenesis.METHODS:The response of the human REV3 gene toMNNG was measured in human 293 cells and FL cells byRT-PCR.By using antisense technology,mutation analysisat HPRTIocus (on which lesion-targeted mutation usuallyoccurs) was conducted in human transgenic cell line FL-REV3-by 8-azaguanine screening,and mutation occurredon undamaged DNA template was detected by using a shuttleplasmid pZ189 as the probe in human transgenic cell lines293-REV3-and FL-REV3-.The blockage effect of REV3 wasmeasured by combination of reverse transcription-polymerase chain reaction to detect the expression ofantisense REV3 RNA and Western blotting to detect the REV3protein level.RESULTS:The human REV3 gene was significantlyactivated by MNNG treatment,as indicated by theupregulation of REV3 gene expression at the transcriptionallevel in MNNG-treated human cells,with significant increaseof REV3 expression level by 0.38 fold,0.33 fold and 0.27fold respectively at 6 h,12 h and 24 h in MNNG-treated293 cells (P<0.05);and to 0.77 fold and 0.65 fold at 12 hand 24 h respectively in MNNG-treated FL cells (P<0.05).In transgenic cell line (in which REV3 was blocked byantisense REV3 RNA),high level of antisense REV3 RNAwas detected,with a decreased level of REV3 protein.MNNG treatment significantly increased the mutationfrequencies on undamaged DNA template (untargetedmutation),and also at HPRT locus (lesion-targetedmutation).However,when REV3 gene was blocked byantisense REV3 RNA,the MNNG-induced mutationfrequency on undamaged DNA templates was significantlydecreased by 3.8 fold (P<0.05) and 5.8 fold (P<0.01)respectively both in MNNG-pretreated transgenic 293 cellsand FL cells in which REV3was blocked by antisense RNA,and almost recovered to their spontaneous mutation levels. The spontaneous HPRTmutation was disappeared in REV3-disrupted cells,and induced mutation frequency at HPRTlocus significantly decreased from 8.66×10~(-6) in FL cells to0.14×10~(-6) in transgenic cells as well (P<0.01).CONCLUSION:The expression of the human REV3 canbe upregulated at the transcriptional level in response toMNNG.The human REV3 gene plays a role not only inlesion-targeted DNA mutagenesis,but also in mutagenesison undamaged DNA templates that is called untargetedmutation.
AIM: To understand the response of human REV3gene togastric cancer inducing carcinogen N-methyI-N’nitro-N-nitrosoguanidine (MNNG) and its role in human motifnesis. METHODS: The response of the human REV3 gene toMNNG was measured in human 293 cells and FL cells by RT-PCR. Using antisense technology, mutation analysisat HPRTIocus (on which lesion-targeted mutation usuallyoccurs) was conducted in human transgenic cell line FL-REV3-by 8-azaguanine screening, and mutation originon undamaged DNA template was detected by using a shuttleplasmid pZ189 as the probe in human transgenic cell lines 293-REV3-and FL-REV3-The blockage effect of REV3 wasmeasured by combination of reverse transcription-polymerase chain reaction to detect the expression of isisense REV3 RNA and Western blotting to detect the REV3 protein level .RESULTS: The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptionallevel in MNNG-treated human cell s, with a significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27 fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P <0.05); and 0.77 fold and 0.65 fold at 12 hand 24 h respectively, in MNNG-treated FL cells (P <0.05) .In transgenic cell line (in which REV3 was blocked byantisense 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 (untargetedmutation), and also at HPRT locus (lesion-targeted motif). When, the REV3 gene was blocked byantisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly created by 3.8 fold (P <0.05) and Both spontaneous HPRTmutation was disappeared in REV3-disrupted cells, and in (P <0.01). Both spontaneous HPRTmutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRTlocus significantly decreased from 8.66 × 10 ~ (-6) in FL cells to0.14 × 10 ~ (-6) in transgenic cells as well (P <0.01) .CONCLUSION: The expression of the human REV3 canbe upregulated at the transcriptional level in response toMNNG.The human REV3 gene plays a role not only inlesion-targeted DNA mutagenesis, but also in mutagenesison undamaged DNA templates that is called untargetedmutation.