Response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and its role in mutagenesis

Abstract

Aim: To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (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 HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3(-) 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 transcription-polymerase chain reaction to detect the expression of antisense 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 transcriptional level in MNNG-treated human cells, with 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 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 REV3 was blocked by antisense RNA, and almost recovered to their spontaneous mutation levels. The spontaneous HPRT mutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66 x 10(-6) in FL cells to 0.14 x 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.

Figure 1

The detection system of untargeted mutation occur-ring on undamaged DNA template. Cells were pretreated by MNNG, and then intact and undamaged shuttle plasmid pZ189 was transfected into cells after removing MNNG. After repli-cation for 48 h in cells, replicated pZ189 plasmid was rescued and then transformed to host bacterial MBM7070 to screen pZ189 mutants.

Figure 2

The response of the human REV3 to MNNG at different time points. The response of REV3 to MNNG was measured in human 293 cells and FL cells at the transcriptional level by using RT-PCR. ^aP < 0.05, compared with the control.

Figure 3

Detection of antisense REV3 RNA fragment expressed in 293-REV3^- cells using RT-PCR. A, 100 bp DNA ladder; B, RT-PCR result in Dex-treated 293-REV3^- cells; C, RT-PCR result in 293-REV3^- cells (Dex-free); D, a positive control.

Figure 4

Western blotting showing the loss of REV3 protein in transgenic FL-REV3^- cells. Western analysis showing the level of expression of REV3 protein in nuclear extracts from REV3 antisense-expressing transfectant FL-REV3^- and its parental strain FL. Ku-70 was used as the loading control.