Liver Samples and Primary Human Hepatocytes
Tissue samples from patients undergoing liver surgery at the University Medical Center Regensburg were used, including primary hepatocellular carcinoma and adjacent nontumorous tissue. Primary human hepatocytes (PHH) were isolated and cultivated as described recently (31). Briefly, nonneoplastic tissue samples from liver resections were obtained from patients undergoing partial hepatectomy for metastatic liver tumors of colorectal cancer. PHHs were isolated using a modified two-step ethylene glycol tetraacetic acid (EGTA)/collagenase perfusion procedure and plated on collagen coated dishes. Experimental procedures were performed according to the guidelines of the charitable state controlled foundation HTCR (Human Tissue and Cell Research, Regensburg, Germany), with the informed patient’s consent approved by the local ethical committee of the University of Regensburg. All experiments involving human tissues and cells have been carried out in accordance to “WMA International Code of Medical Ethics” (30).
Human hepatoma cell lines HepG2, HepG2.2.15 (10), Huh7.5 (11) and PLC (PLC/PRF/5 ATCC CRL-8024) were cultivated in DMEM/1% MEM (Invitrogen/Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), penicillin (100 units/mL), and streptomycin (10 µg/mL) and maintained at 37°C, 5% CO2. HepG2 cells (2 × 105) were seeded and, after 24 h, cells were stimulated with 200 µmol/L tBHQ (tertbutylhydrochinone) for 24 h.
Promoter Constructs, Transfections and Luciferase Reporter Gene Assays
The 5′-flanking region of human ALR gene (gene ID 2671) residing from −733 to +240 bp (32) was amplified by PCR and inserted into pGL2-basic plasmid. HepG2 and Huh-7 cells (2 × 105) were transiently cotransfected with ALR promoter construct and caNrf2 (constitutively active Nrf2) or dnNrf2 (transdominant negative Nrf2) expression plasmids (0.5 µg each) (10), using the siPORT XP-1 method (Ambion/Life Technologies). Cells were lysed 24 h after transfection and the luciferase activity was determined. To determine the transfection efficiency, pRL-TKRenillavector was cotransfected and the promoterless vector pGL2-Basic served as negative control.
Nrf2−/− Mouse Model
Male Nrf2−/− mice in the C57BL/6 background were provided by M Yamamoto (University of Tsukuba, Japan) (33). As controls, age-matched male C57BL/6 mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice (8 to 10 wks old) were anesthetized by intraperitoneal (IP) injection of ketamine (100 mg/g bodyweight [bw])/xylazine (5 mg/g bw) and subjected to two-thirds partial hepatectomy as described previously (34). At different stages after injury, they were euthanized by CO2 inhalation, and the remaining livers were harvested. For each time point studied, at least four animals were harvested. All experiments were approved by the local authorities.
Transgenic HBV Mouse Model
The HBV transgenic mouse model used in this study is based on a 1.3-fold HBV genome and has been initially described elsewhere (35). Liver samples were derived from 10-12-wk-old transgenic mice or the corresponding sex and age matched wild-type (WT) littermates.
HCV Replicating Cells and HBV-Infected Primary Human Hepatocytes
The plasmids pJFH1/J6 and pJFH1/J6_GND were described recently (36). In vitro transcription, electroporation of HCV RNAs and infection of Huh7.5 cells with HCV were performed as described (37). The transfection efficiency was up to 65%. RNA was isolated 72 h after electroporation. Primary human hepatocytes were infected with HepAD38-derived supernatant as described (38). Cells were harvested 8 d after infection.
RNA Isolation and cDNA Synthesis
Total RNA was isolated from cultivated cells, mouse and human liver tissues using RNeasy kit (Qiagen, Hilden, Germany). Subsequently, first strand cDNA was synthesized using 1 µg of total RNA and virus-reverse transcription reaction (Promega, Madison, WI, USA). For cDNA synthesis of samples from HCV and HBV experiments, RNA isolation was performed using TRIzol (Invitrogen/Life Technologies) and 5 µg of total RNA were treated with DNase I followed by first-strand synthesis using SupercriptII reverse transcriptase (Invitrogen/ Life Technologies).
Quantification of mRNA Expression by Real-Time PCR
Transcript levels of NQO1, ALR, GSTα1 and 18S ribosomal RNA were quantified using the RT-PCR (LightCycler, Roche, Penzberg, Germany). Primers used to amplify human ALR were: forward 5′-CAC AAT GAA GTG AAC CGC AAG-3′, reverse 5′-CAC CCA ACT GAG ACA CAA CAG-3′, mouse ALR: forward 5′-CAC AGG ATC GGG AAG AAT TG-3′, reverse 5′-ATT CCT CGC AGG GGT AAA AC-3′, human NQO1: forward 5′-GCA CTG ATC GTA CTG GCT CA-3′, reverse 5′-GAA CAC TCG CTC AAA CCA G-3′, mouse NQO1: forward 5′-GGC TGC TTG GAG CAA AAT AG-3′, reverse 5′-TTC TCT GGC CGA TTC AGA G-3′, mouse GSTα1: forward 5′-CCG GAA GAT TTG GAA AAG C-3′, reverse 5′-TTT GGT GGC GAT GTA GTT GA-3′, 18S rRNA: forward 5′-GTA ACC CGT TGA ACC CCA TT-3′, reverse 5′-CCA TCC AAT CGG TAG TAG CG-3′. The PCR reaction was evaluated by melting curve analysis.
Western Blot Analysis
Total protein fractions (20 µg/ lane) were separated by 14% SDS-PAGE under reducing conditions using 100 mmol/L DTT. Proteins were transferred onto polyvinylidene fluoride membranes and incubated with anti-ALR (1:440), anti-β-actin (1:1000) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) and anti-GAPDH (1:1000) (New England Biolabs GmbH, Frankfurt, Germany) antibodies, and developed with ECL reactions (Pierce, Rockford, IL, USA).
Immunostaining was performed as described recently (39). Briefly, sections of paraffin-embedded liver samples were deparaffinized and incubated with anti-ALR antibody (0.26 µg/mL) overnight at 4°C. Immunostaining was performed by incubation with secondary mouse anti-rabbit antibody (DAKO, Hamburg, Germany) for 1 h and development using APAAP complex (DAKO) and Fast Red Chromogen (Roche). Signal intensity of staining was defined as follows: 0, no staining; 1 weak staining; 2 when moderate staining; and 3, strong staining.
Significant differences in paired and nonpaired samples were evaluated using Wilcoxon signed-rank t test and Mann-Whitney U test, respectively.