Overexpression of Rev1 promotes the development of carcinogen-induced intestinal adenomas via accumulation of point mutation and suppression of apoptosis proportionally to the Rev1 expression level

Abstract

Cancer development often involves mutagenic replication of damaged DNA by the error-prone translesion synthesis (TLS) pathway. Aberrant activation of this pathway plays a role in tumorigenesis by promoting genetic mutations. Rev1 controls the function of the TLS pathway, and Rev1 expression levels are associated with DNA damage induced cytotoxicity and mutagenicity. However, it remains unclear whether deregulated Rev1 expression triggers or promotes tumorigenesis in vivo. In this study, we generated a novel Rev1-overexpressing transgenic (Tg) mouse and characterized its susceptibility to tumorigenesis. Using a small intestinal tumor model induced by N-methyl-N-nitrosourea (MNU), we found that transgenic expression of Rev1 accelerated intestinal adenoma development in proportion to the Rev1 expression level; however, overexpression of Rev1 alone did not cause spontaneous development of intestinal adenomas. In Rev1 Tg mice, MNU-induced mutagenesis was elevated, whereas apoptosis was suppressed. The effects of hREV1 expression levels on the cytotoxicity and mutagenicity of MNU were confirmed in the human cancer cell line HT1080. These data indicate that dysregulation of cellular Rev1 levels leads to the accumulation of mutations and suppression of cell death, which accelerates the tumorigenic activities of DNA-damaging agents.

Figure 1.

MNU-induced small intestinal tumorigenesis in Rev1 Tg mice. (a) Rev1 expression levels of wild-type and Rev1 Tg (Hemi) male mice were measured by semi-quantitative RT-PCR in indicated tissues. Gapdh was used as a loading control. (b) Relative Rev1 expression level in small intestines. Significant differences were observed between Wt and Rev1 Tg (Hemi) mice (P = 0.046), and between Wt and Rev1 Tg (Homo) mice (P = 0.0008). (c) Survival curves of wild-type (Wt), Rev1 Tg (Hemi) and Rev1 Tg (Homo) male mice [Wt; black line, Rev1 Tg (Hemi); blue line, and Rev1 Tg (Homo); red line] after repeated intraperitoneal injection of MNU (2 × 50 mg/kg of body weight at 6 and 7 weeks of age). Significant differences were observed between Wt and Rev1 Tg (Hemi) mice (P = 0.0059), and between Wt and Rev1 Tg (Homo) mice (P = 0.0001). (d) Numbers of adenomas per mouse in Wt (gray), Hemi (blue) and Homo (red) mice. Each circle indicates the result from each mouse.

Table 1.

Comparison of tumor number, tumor size and adenocarcinoma incidence

Figure 2.

Initiation and progression of tumor formation 1 and 2 months after MNU treatment. (a) Images of intestines from Wt, Rev1 Tg (Hemi) and Rev1 Tg (Homo) mice 1 or 2 months after MNU treatment. Magnification: ×10. Arrows indicate adenomas. (b) Numbers of adenoma tumors in the intestinal tracts of Wt and Rev1 Tg mice. Each circle indicates the result from one mouse [Wt; white circle, Rev1 Tg (Hemi); gray circle, and Rev1 Tg (Homo); black circle]. P = 0.018 was calculated by the t-test. (c) Histogram of intestinal adenoma tumor size in Wt, Rev1 Tg (Hemi) and Rev1 Tg (Homo) mice after MNU treatment. In each histogram, the number of adenomas for each size interval is presented as average from the mice used. In all cases five mice were used, with the exception of Rev1 Tg (Hemi) at 2 months, where four mice were used due to technical error.

Figure 3.

Apoptotic response in the intestinal crypts of Wt and Rev1 Tg mice after MNU treatment. (a) Hematoxylin and eosin staining of Wt and Rev1 Tg (Homo) mice intestines. Asterisks represent apoptotic cells. Magnification, ×200. (b) Number of apoptotic bodies per 50 half-crypts in Wt and Rev1 Tg (Homo) mice at the indicated time points after MNU treatment (Wt, open circles; and Rev1 Tg (Homo), closed circles). Each data point represents the mean and standard deviation from three mice. The rate of apoptosis at 6 h differed significantly (P = 0.002) between Wt and Rev1 Tg (Homo) mice. (c) Prevalence of apoptotic bodies per 50 half-crypts in Wt, Rev1 Tg (Hemi) and Rev1 Tg (Homo) mice at 6 h after MNU treatment [Wt, white bars; Rev1 Tg (Hemi), gray bars; and Rev1 Tg (Homo), dark gray bars]. Each bar represents the mean and standard deviation from three mice. The rates of apoptosis differed significantly between Wt and Rev1 Tg (Hemi) mice and between Rev1 Tg (Hemi) and Rev1 Tg (Homo) mice (P = 0.017 and P = 0.002, respectively).

Figure 4.

Association between hREV1 expression levels and cytotoxicity and mutagenicity of MNU in HT1080 cell lines. (a) hREV1 expression levels in parental HT1080 cells and the HT1080-6TR, -C6 and -C7 sublines, as determined by Western blotting; histone H1 was used as an internal control. Full length plot was exhibited in Supplementary Figure 7a, available at Carcinogenesis Online. (b) Cytotoxic effects of MNU treatment in the hREV1-overexpressing HT1080-hREV1-C6, -C7 cell lines. Cell survival was determined by colony-formation assays. (c) HPRT mutation frequency in HT1080-hREV1-C6 cells after MNU treatment. (d) hREV1 expression level in HT1080-6TR cells transfected with si-Ctrl or si-hREV1. Full-length plot was exhibited in Supplementary Figure 7b, available at Carcinogenesis Online. (e) Sensitivity of HT1080-6TR cells transfected with si-Ctrl or si-hREV1 to the cytotoxic effects of MNU treatment. Cell survival was determined by colony-formation assays. (f) HPRT mutation frequency in HT1080-6TR cells transfected with si-Ctrl or si-hREV1 after MNU treatment.