Using a mouse model of diethylnitrosamine (DEN)-induced HCC, we found that TLR4 mutant (TLR4mut) mice shows an increase in the initiation and progression of HCC and a decrease in the animal survival compared to wild-type (WT) littermates. Our studies indicate that TLR4-controlled immunity supporting the senescent induction and the expression of DNA repair proteins plays an integrated defense role against genotoxic carcinogenesis and tumor progression in the liver. Ectopic expression of DNA damage repairing protein Ku70 attenuates the DEN-induced

HCC in TLR4mut mice, MG-132 order suggesting that Ku70 may act as a tumor suppressor by restoring immunity, senescent response, and autophagy flux in TLR4mut liver. All animals received care according to the Guide for the Care and Use of Laboratory Animals. TLR4mut mice (C3H/He background) were originally obtained from The Jackson Laboratory (Bar Harbor, ME). Fifteen-day-old WT and TLR4mut mice were injected intraperitoneally with or without DEN (25 mg/kg) (Sigma-Aldrich, St. Louis, MO).18 The mice were fed normal chow and

sacrificed on months 1, 3, 6, and 18 after DEN injection to observe tumor development and animal survival. For adenovirus infection experiments, the mice were infected intramuscularly with 1 × 105 viral particles (V.P.) of Ku70 adenovirus or green fluorescent protein (GFP) adenovirus per mouse on day 1, 7, and 14 after DEN injection, and were sacrificed at day 30 and month CAL-101 price 6 after DEN injection. For assessing HCC, external visible tumors (>0.5 mm) were counted and measured by stereomicroscopy.19 The largest liver lobes were fixed in 4% formalin, paraffin-embedded, and sliced into sections. Sections were stained with hematoxylin and eosin and the tumor areas were measured as described.20

Liver function was monitored by measuring serum alanine aminotransferase 上海皓元 activities. Western blot assays of liver tissue were performed with commercial antibodies as described,21 using β-actin as loading controls. Detergent-soluble and insoluble fractions of livers were performed as described.22 Immunohistochemistry and immunofluorescence assays were performed as described.23 To detect total contents of reactive oxygen species (ROS), frozen liver sections or single cell suspensions were prepared as described21 and incubated with 2′,7′-dichlorofluorescein diacetate (Sigma-Aldrich, St. Louis, MO) as described.23 Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed with detection kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. Data are expressed as the mean ± SE. Groups were compared via one-way analysis of variance followed by a Tukey-Kramer or Dunnett multiple comparisons test. Comparisons between two groups were performed via an unpaired Student t test. The survival rates were analyzed using the Kaplan-Meier method. P < 0.05 was considered significant.