Suppression of tumor suppressor Tsc2 and DNA repair glycosylase Nth1 during spontaneous liver tumorigenesis in Long-Evans Cinnamon rats

Abstract

Chronic inflammation and oxidative stress are arguably associated with an increased risk of cancer. Certain diseases that are characterized by oxyradical overload, such as Wilson's disease (WD), have also been associated with a higher risk of liver cancer. The Long-Evans Cinnamon (LEC) rat, an animal model for WD, is genetically predisposed to the spontaneous development of liver cancer and has been shown to be very useful for studying the mechanisms of inflammation-mediated spontaneous carcinogenesis. Endonuclease III (Nth1) plays a significant role in the removal of oxidative DNA damage. Nth1 and a tumor suppressor gene Tuberous sclerosis 2 (Tsc2) are bi-directionally regulated in humans, mice, and rats by a common minimal promoter containing two Ets-binding sites (EBSs). In this study, we examined the expression of Nth1 and Tsc2 genes during disease progression in the LEC rat liver. During the period of acute hepatitis (16-17 weeks), we observed decreased Nth1 and Tsc2 mRNA levels and a continued decrease of the Tsc2 gene in 24 weeks in LEC rats, while the effect was minimal in Long-Evans Agouti (LEA) rats. This reduction in the mRNA levels was due to the reduced binding of EBSs in the Nth1/Tsc2 promoter. Increase in protein oxidation (carbonyl content) during the same time period (16-24 weeks) may have an effect on the promoter binding of regulatory proteins and consequent decrease in Nth1 and Tsc2 gene expressions during tumorigenesis.

Fig. 1

a Stages of hepatitis and hepatocellular carcinoma (HCC) in LEC rat. b mRNA expression profile of Nth1 and Tsc2 in livers from LEA and LEC. The data presented are the mean ± SD values of three to five independent measurements from two rats of each strain. The arrows denote the important time points for development and progression of hepatitis and HCC in LEC rats

Fig. 2

a Sequence of oligonucleotides used for gel shift assay. b Gel shift analysis of protein binding to the Nth1 promoter. All lanes contained 10 μg of nuclear extracts from livers of LEC and LEA rats and 1 ng of radiolabeled oligonucleotide containing two ETS-binding sites (EBS) denoted by I and II. Arrowheads indicate DNA bound with ETS family protein. Competition experiments were performed using non-radiolabeled 40-fold molar excess of oligonucleotide containing wild-type EBSs (lanes indicated by “W”) or the mutant (lanes indicated by “M”) EBSs. The β-actin expression is shown as loading control

Fig. 3

Protein oxidation profile in LEC and LEA liver tissues. Carbonyl content as an indicator of protein oxidation was measured by DNPH-based colorimetric analysis. The details are described in “Materials and methods” section. The data presented are the mean ± SD values of three to five independent measurements from two rats of each strain