IL-22 has been shown to promote hepatoma cell proliferation and survival12; however, no tumor was observed in all organs, including the PLX-4720 in vivo liver, in IL-22TG mice up to 2 years of age (data not shown). Next, we examined whether IL-22TG mice were more susceptible to DEN-induced liver tumorigenesis. As illustrated in Fig. 5A,B, IL-22TG mice had a larger liver tumor size and a higher liver/body weight ratio than WT mice 9 months after DEN injection. Liver histology confirmed that the liver tumors from both WT and IL-22TG mice are hepatocarcinoma (Supporting Information Fig. 3a). The incidence of tumor development in IL-22TG

mice was 100%, whereas the WT littermate group only had 60% and 80% tumor incidence after injection of 10 μg/g and 20 μg/g, respectively (Supporting Information Fig. 3b). In addition, the maximum size of tumors and the number of tumors were greater in IL-22TG mice compared with WT mice after DEN injection (Fig. 5C,D). Western blot analysis demonstrated that pSTAT3 activation was detected

in both nontumor and tumor regions in IL-22TG mice, but was detected only in the tumor region in WT mice. In addition, cyclin D1 and Bcl-xL expression were higher in the tumors from IL-22TG mice than in those from WT mice (Fig. 5E). Real-time PCR analyses revealed that IL-22 was detected at low levels in nontumor and tumor tissues from DEN-treated WT mice, but was detected at very high levels in nontumor and tumor tissues from DEN-treated IL-22TG mice (Fig. Selleck Adriamycin 5F). The levels of IL-22 in tumor tissues were slightly lower compared with those in nontumor tissues from DEN-treated IL-22 mice (Fig. 5F). Furthermore, the number of Ki67-positive cells was higher in the tumor tissues from IL-22TG mice compared with WT mice (Supporting Information Fig. 3c). To rule out whether increased DEN-induced liver tumorigenesis in IL-22TG mice was due to an increase in DEN metabolism–associated liver injury and oxidative stress induced by a single dose of DEN injection at age 15 days, we measured serum ALT and AST, liver glutathione (an antioxidant), malondialdehyde (a marker of

lipid peroxidation), and 8-hydroxy-2′-deoxyguanosine (a major see more product of DNA oxidation). As illustrated in Fig. 6A, DEN injection induced elevation of serum ALT and AST in both WT and IL-22TG mice, with lower serum ALT in the latter group 12 hours after injection. A single dose of DEN injection induced elevation of malondialdehyde and 8-hydroxy-2′-deoxyguanosine but reduction of glutathione in WT mice. Similar changes were observed in IL-22TG mice. These findings indicate that a single injection of DEN induced similar levels of lipid peroxidation and DNA oxidation in the liver of WT and IL-22TG mice at age 15 days, suggesting the increased DEN-induced liver tumorigenesis in IL-22TG is not caused by an increase in DEN-metabolism.