Plasmid constructs
Vector pCMVTNT-EPHX1 used for in vitro translation of microsomal epoxide hydrolase (mEH) was constructed by inserting of EPHX1 gene (GeneBank Accession No. NM_000120) cDNA into pCMVTNT (Promega, Madison, WI, USA) between the Xho I and Kpn I sites, EPHX1 gene cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the total RNA isolated from Huh-7 hepatoma cells. The primers used were: forward primer, 5′- CCGCTCGAGGCCACCATGTGGCTAGAAATCCTCCTCACT-3′; reverse primer, 5′-CGGGG TACCTCATTGCCGCTCCAGCAC-3′. pACT-EPHX1353–455 coding for 353–455 amino acids of mEH was generated by an in-frame insertion of PCR amplified fragment using screened cDNA library prey plasmid as a template into pACT between the Sal I and Not I sites (encodes a herpes simplex virus type 1 VP16 protein, Promega). The primers used were: forward primer: 5′-ACGCGTCGACTTGACCTGCTGACCAAC-3′; reverse primer: 5′-ATAAGAATGCGGCCGC TCATTGCCGCTCCAGCAC-3′. pGEX-HBSP coding for GST-HBSP protein, pBIND-HBSP, pBIND-HBSP1-47 and pBIND-HBSP48-111, which respectively codes for GAL4 DNA-binding domain fused full length, N terminal 47 amino acids and C terminal 64 amino acids of HBSP, were described previously [21].
GST pull-down assay
E. coli Rosetta (DE3) (Novagen, Madison, Wisconsin, USA) was transformed with pGEX-HBSP, and the expression of GST-fused HBSP was induced with 0.5 mM isopropy l-β-D-thiogalactopyranoside (IPTG, Merck, Darmstadt, Germany) for 3 h at 28°C. The cells were harvested and suspended in phosphatate-buffered saline (PBS) containing 5 mM DTT and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The cells were then disrupted by sonication. After centrifugation, the glutathione-Sepharose 4B beads (GE Healthcare, Uppsala, Sweden) were added to the supernatants and incubated overnight at 4°C. Then the glutathione-Sepharose 4B beads were washed three times with PBS containing 5 mM DTT and protease inhibitor cocktail (Calbiochem, La Jolla, CA). 35S-Labeled mEH protein was prepared using the TNT T7 Coupled Reticulocyte Lysate System (Promega) by adding 2 μg of pCMVTNT-EPHX1 and 50 μCi of 35S-methionine (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For GST pull-down assay, 35S-labeled mEH was added to the GST recombinant proteins and glutathione-Sepharose 4B beads, and incubated overnight at 4°C. Beads were washed three times with 1% Triton X-100 in PBS, re-suspended and subjected to 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of 35S-mEH was detected by autoradiography.
Co-immunoprecipitation (Co-IP) assay
A total of 4 × 106 Huh-7 hepatoma cells in a 10 cm dish were transfected with 12 μg each of the constructs pBIND-HBSP, pBIND-HBSP1–47, pBIND-HBSP48–111 or pBIND. Forty-eight hours after transfection, the cells were washed three times with PBS and lysed using RIPA lysis buffer (Pierce, Rockford, IL, USA) containing a proteinase inhibitor cocktail (Roche Diagnsotics). The soluble proteins were pre-cleared with 100 μL of a 50% slurry of protein A agarose (Invitrogen, Carlsbad, CA, USA), and then the clear lysates were mixed with 2 μg of goat polyclonal anti-mEH IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 100 μL of a 50% slurry of protein A agarose. The immunoprecipitated complexes were washed with lysis buffer and then analyzed by 12% SDS-PAGE and western blot, using specific antibodies including anti-GAL4BD monoclonal antibody (1:4000 dilution, Clontech, Palo Alto, CA, USA) and anti-mEH antibody (1:200 dilution, Santa Cruz Boitechnology).
Mammalian two-hybrid assay
CheckMate Mammalian Two-Hybrid System (Promega) was used following the manufacturer’s instructions. Briefly, Huh-7 hepatoma cells (106 cells per 6 cm dish) were co-transfected with pACT-EPHX1353–455 and pBIND-HBSP, pBIND-HBSP1–47 or pBIND-HBSP48–111 vector with the pG5luc as a reporter plasmid, using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Paired empty plasmids pBIND and pACT were used as negative controls. At 48 h post-transfection, the cells were harvested and renilla-normalized firefly luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. The relative luciferase activities were obtained by comparison to the negative controls (pBIND and pACT), which were set to 1 in each experiment. Each transfection was performed three times, and each time in duplicate. The relative luciferase activities are presented as mean ± SD.
Recombinant adenoviruses preparation
Recombinant adenoviruses were generated by using AdEasy XL System (Stratagene, La Jolla, CA, USA) following the manufacturer’s instruction. Briefly, plasmid pShuttle-IRES-hrGFP-1-HBSP was generated by inserting HBSP gene into the vector pShuttle-IRES-hrGFP-1. HBSP-expressing (pAdHBSP) or control (pAdControl) recombinant adenoviral vectors were then generated by homologous recombination of pAdEasy-1 and pShuttle-IRES-hrGFP-1-HBSP or pShuttle-IRES-hrGFP-1 in E. coli BJ5183-AD-1. The colonies obtained were screened for appropriate recombination events by Pac I restriction endonuclease analyses. The pAdHBSP and pAdControl were then digested with Pme I and used to transfect the 293A packaging cell line (Invitrogen) to produce HBSP-expressing (HBSP-Ad) and control (GFP-Ad) recombinant adenoviruses. Exponentially growing Huh-7 hepatoma cells were infected with these recombinant adenoviruses at a multiplicity of infection (MOI) of 100. This dose of virus was sufficient to give 100% infectivity as determined by GFP expression under fluorescence microscopy.
Analysis of effects of HBSP on styrene oxide (STO) hydrolysis
4 μg of mouse liver microsomal protein (Sigma, St. Louis, MO, USA) was incubated with 40 or 100 μg of bacterially expressed NUS-StrepII-tagged HBSP, HBSP1–47 (N terminal 47 amino acids of HBSP), HBSP48–111 (C terminal 64 amino acids of HBSP) or Nus-StrepII (negative control) [21] for 20 min at 37°C. After incubation, the reaction was initiated by adding 400 μL of 0.1 M potassium phosphate buffer (pH7.4) and 1 mM of STO (Sigma). Then the mixture was incubated for 20 min at 37°C. The reaction was terminated by adding 1 mL of cold ethyl acetate. The mixtures were measured by high performance liquid chromatography (HPLC) as described before [22]. The STO was identified by comparison of their retention times with co-injected authentic standards of STO, and quantified by integrating the areas under the peaks. The assay was performed three times, and the results were expressed as mean ± SD.
Analysis of effects of HBSP on B[alpha]P metabolism
4 μg of mouse liver microsomal protein was incubated with 40 or 100 μg of StrepII-tagged HBSP, HBSP1–47, HBSP48-111or NUS-StrepII (negative control) for 20 min at 37°C. After incubation, the reaction was initiated by adding 200 μL of 0.1 M potassium phosphate buffer (pH7.4), 10 μM NADPH (Sigma) and 0.5 μM of B[alpha]P (Sigma). Then the mixtures were incubated for 20 min at 37°C. The reaction was terminated by adding 1 mL of cold ethyl acetate. The mixtures were measured by HPLC as previously described [23]. The B[alpha]P metabolites were identified by comparing their retention times with co-injected authentic standards of B[alpha]P and B[alpha]P-7,8-diol-9,10-epoxide (BPDE) and were quantified by integrating the areas under the peaks. B[alpha]P overall metablic turnover was expressed as percentage of initial substrate concentration. The analyses were performed three times, and the results were expressed as mean ± SD.
Cell culture and B[alpha]P exposure
Huh-7 hepatoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). 5 × 105 Huh-7 hepatoma cells were infected with 100 MOI of recombinant adenoviruses GFP-Ad and HBSP-Ad, respectively. 72 hours after the infection, the cells were treated with 1 μM of B[alpha]P for 24 h, and used for detection of cellular BPDE-DNA by immunocytochemistry. Cells used for assay of proliferation, cell cycle, transformation and tumorigenicity were prepared by viral infection and continuously treated with 1 μM of B[alpha]P for 4 weeks (10 passages), then the cells were harvested and kept at -80°C until use. These cells were designated as Huh-7/HBSP/B[alpha]P (Huh-7 hepatoma cells infected with HBSP-Ad and treated with B[alpha]P), Huh-7/GFP/B[alpha]P (Huh-7 hepatoma cells infected with GFP-Ad and treated with B[alpha]P), Huh-7/HBSP (Huh-7 hepatoma cells infected with HBSP-Ad and without treating by B[alpha]P) or Huh-7/GFP (Huh-7 hepatoma cells infected with GFP-Ad and without treating by B[alpha]P).
RNA interference assay
100 pmol of either small interfering RNA targeting mEH (mEH siRNA, Santa Cruz Biotechnology) or negative control was used for transfection by lipofectamine RNAiMAX reagent (Invitrogen) in accordance with the manufacturer’s instructions. The cells were collected using RIPA lysis buffer (Pierce) 48 h after transfection. The protein concentration was measured with a BCA Protein Quant Kit (Bio-Rad, Hercules, CA, USA).
Western blotting analysis
A total of 30 μg protein was subjected to 12% SDS-PAGE, and then electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Protein blots were incubated separately with a panel of specific antibodies such as anti-mEH (1:500 dilution, Santa Cruz Biotechnology) and anti-β-actin (1:4000 dilution, Sigma). An alkaline phosphatase (AP)-conjugated goat anti-mouse IgG was used as a secondary antibody. CDP-Star reagent (Roche Diagnostics) was used for color development.
Immunocytochemistry assay of BPDE-DNA
The procedure was performed as previously described [24]. Briefly, when the cells on sterilized glass coverslips reached 70-80% confluence, they were fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS. After incubation with anti-BPDE-DNA (1:50 dilution, Santa Cruz), the cells were incubated with a biotinylated secondary antibody followed by streptavidin conjugated with horseradish peroxidase (HRP) for 10 min. The immunoreaction was visualized using 3-3′ diaminobenzidine tetrachloride (DAB, Santa Cruz). The slides were mounted with Eukitt (Sigma) and observed with an Olympus BX60 microscope. Images were captured with Image-Pro Express 6.0 (IPE6.0) software.
Bromodeoxyuridine cell proliferation assay
Huh-7/HBSP/B[alpha]P, Huh-7/GFP/B[alpha]P, Huh-7/HBSP or Huh-7/GFP were seeded triplicate in 96-well plates at 2 × 103/well. Cell proliferation was determined daily for 9 days by bromodeoxyuridine (BrDU) assay according to the manufacturer’s instructions. Briefly, after the cells were plated and serum-starved for 24 hours, BrDU was added to each well at a dilution of 1:2000 and the cells incubated for an additional 24 h. The BrDU incorporation (a measure of DNA synthesis and growth) was measured by using the BrDU Cell Proliferation assay kit (CalBiochem, San Diego, CA, USA) at 450 nm using a microplate reader. The assay was performed three times, and the results were expressed as mean ± SD.
Cell cycle analysis
Huh-7/HBSP/B[alpha]P or Huh-7/GFP/B[alpha]P were seeded in 6-well plates at 1 × 104/well and serum-starved with 0.5% FBS DMEM for 24 h. Cells were harvested after serum starvation at day 2, 4, and 6 days. Cells were fixed with ice-cold 70% ethanol (pre-chilled at -20°C), washed with PBS (pH 7.2), incubated with 0.05 mg/mL propidium iodide (PI, Sigma) and 1 μg/mL RNase A at 37°C for 30 min in dark. The DNA content of 10,000 cells was analyzed by flow cytometry (Beckman Coulter, Miami, Florida, USA) and CXP 2.2. software. The percentage of each phase of the cell cycle was determined.
Anchorage-independent growth in soft agar
The assay was performed as previously described [25]. Briefly, 2 × 103 Huh-7/HBSP/B[alpha]P or Huh-7/GFP/B[alpha]P were suspended in 0.3% agarose in DMEM supplemented with 10% FBS, and plated in 60 mm dishes over a basal layer of 0.6% agarose in the same medium. All dishes were incubated at 37°C in a 5% CO2 humidified atmosphere, and were examined microscopically for colony formation after a 2-week incubation.
Tumor xerograft
All the procedures involving animals were approved by Experimental Animal Ethics Committee, Fujian Medical University. The method was used as previously described [26] with minor changes. Briefly, the right flanks of BALB/c nude mice (nu/nu) (male, 4 weeks old) were inoculated subcutaneously with pooled 2 × 106 of Huh-7/HBSP/B[alpha]P or Huh-7/GFP/B[alpha]P in 0.2 mL PBS per animal. Tumor size was measured every 3 days, and the tumor volume was calculated by using the following formula: (length × width2)/2. After 21 days of inoculation, all mice were sacrificed, tumors were dissected out, weighted, and processed for immunohistochemistry.
Immunohistochemistry
Tissues were fixed in 4% neutral buffered formalin, processed, then embedded in paraffin and cut into 5 μm sections. Tissues sections were deparaffinized and rehydrated. Endogenous peroxidase were blocked with 10 min incubation in 3% H2O2 in PBS. After blocking of non-specific sites with 1.5% blocking serum in PBS for 1 h at room temperature (RT), tissue sections were incubated 1 h at RT with the anti-BPDE-DNA. After a 30 min reaction with a biotinylated secondary antibody, slides were washed with PBS and incubated with streptavidin conjugated with HRP for 10 min. The reaction was then revealed with DAB. Then the slides were mounted with Eukitt and observed with an Olympus BX60 microscope. Images were captured with IPE6.0 software.
Availability of supporting data
The data sets supporting the results of this article are available in the Addgene plasmid repository, IDs 53113 and 53114, http://www.addgene.org/depositing/70984/