Plasmids
Two oligonucleotide pairs (mERα_1395: 5'-gatccccgctcctgtttgctcctaacttcaagagagttaggagcaaacaggagctttttggaaa-3' and 5'-agcttttccaaaaagctcctgtttgctcctaactctcttgaagttaggagcaaacaggagcggg-3', mERα_1103: 5'-gatccccgaatagccctgccttgtcc ttcaagagaggacaaggcagggctattc tttttggaaa-3' and 5'-agc ttttccaaaaagaatagccctgccttgtcctctcttgaaggacaaggcagggctattcggg) were ordered (Eurogentec, United kingdom). The bold nucleotides correspond to nucleotides 1395–1418 and 1103–1120 of the mRNA mERα sequence (GenBank accession number NM_ 007956). The underlined nucleotides represent a BglII and a HindIII site. These oligo's were annealed and ligated between the BglII and HindIII sites of pSUPER-H1prom [2]. The pSUPER-shERα sequences were verified by restriction and sequence analysis (ABI 3700, LGTC, Leiden).
The H1prom plus or minus shERα were cloned from the pSUPER into the promoter less pAdTrack vector [5] by use of XbaI and XhoI restriction sites. The Ad.shERα_tandem construct was generated by ligation of H1prom-shERα_1103 between the NotI and KpnI sites of pTrack-H1prom- shERα_1395.
The (ERE)3TATA-Luc was cloned from pGl3-basic as a ClaI-blunt/ KpnI fragment in EcoRV- and KpnI- digested promoter less Shuttle vector (pShuttle) (He et al. 2509–14). The functionality of this construct was verified by transfection. hERα was cloned from pCMV5 (pCMV5-hERα) [17] as a BamHI fragment in the BglII digested pShuttle-CMV vector. The pcDNA3.1-mERα expression vector was provided by Larry Jameson [18] and subcloned as a EcoRI-blunt fragment in the EcoRV digested pShuttle-CMV vector.
Cell culture
The MXT+ (murine breast cancer) cell line was generously provided by Dr. Bernards. H5V (a murine endothelial cell line derived from heart), EOMA (murine hemangioma-derived micro vascular cell line) and MXT cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL) supplemented with 10% fetal calf serum, 100 units/ml Penicillin, 100 μg/ml Streptomycin and glutamax (Invitrogen) (Complete DMEM). PERC6 cells [19] were maintained in complete DMEM supplemented with 10 mM MgCl2+. For large-scale production of recombinant Ad in PERC6 cells (Crucell, Leiden, he Netherlands), complete DMEM with 2% horse serum (Gibco) was used.
Luciferase reporter assays
Transient transfections were performed in triplicate in 12-wells plates (1.105 cells per well) using Lipofectamine (Invitrogen). The effect of shERα on ERα mediated transcription regulation was determined by co-transfecting the cells with 100 ng of reporter construct (ERE)3TATA-LUC and 500 ng expression vector pSUPER-shERα or an empty pSUPER control vector together with 100 ng pCMV-LacZ. After 24 hours, the cells were stimulated with complete DMEM containing 10-9M Estrogen for an additional 24 hours. The cells were lysed with reporter lyses buffer (Promega) and after centrifugation of 2 min, supernatant was used for determining β-galactosidase normalized luciferase activity by adding 100 μl luciferyl-CoA (Promega) to 20 μl of cell extract in a monolight luminometer (BD Biosciences). β-galactosidase was measured in a 96-well microtiter plate using the β-Galactosidase Enzyme Assay System in reporter lyses buffer (Promega). Absorbance at 450 nm was determined in a microplate reader. Luciferase activities were normalized for transfection efficiency with the β-galactosidase activity and expressed as a percentage relative to expression levels induced by endogenous estrogen receptor (ER). Expression of endogenous ERα in those cells was verified by real time PCR.
Western blot analysis
Immunoblotting procedures were as described previously [20]. H5V cells seeded in triplicate in 12-wells plate were co-transfected with 20 ng pCMV-mERα and 500 ng expression vector pSUPER-shERα or an empty pSUPER control vector as described above. 28 hours post-transfection, the cells were lysed in 200 μl of RIPA buffer (1% NP40, 0.5% DOC, 0.1% SDS, 50 mM Tris pH 8.0, 150 mM NaCl, 2.5 mM EDTA) containing protease inhibitor (40 ul/ml, Roche). Extracts were cleared by centrifugation (4°C, 14 000 g, 5 min), and protein content was determined using the BCA kit (Pierce). Protein samples were denaturated (5 min, 90°C) and separated on SDS/PAGE by use of 8% gradient gels and were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). Blots were stained with Ponceau S before blocking to verify equal loading and appropriate protein transfer. Membranes were blocked for 90 min in PBS, pH 7.4, containing 0.05% Tween 20 and 10% milk powder. Thereafter, membranes were incubated for 16 h at 4°C with ab MC20, 1:1000 (mERα rabbit polyclonal antibody, Santa Cruz Biotechnology, CA). After extensive washing with blocking buffer without milk powder or BSA, membranes were incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit IgG, 1:5000 (Promega). Membranes were again extensively washed and bound peroxidase conjugates were visualized by enhanced chemiluminescence (ECL, Amersham) on a LumiImager workstation. Additionally, filters were stripped by a 30 min incubation in 100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.8 at 50°C, to proceed with the whole procedure as described above. However, now membranes were incubated for 16 h at 4°C with p-38 ab, 1:1000 (N-20, cs-728, rabbit polyclonal antibody, Santa Cruz Biotechnology, CA). Immunoblots were quantified using LUMIANALYST software on a LumiImager (Boehringer-Mannheim).
Adenoviral vectors
Recombinant adenoviral plasmids were generated by homologous recombination of pAdtrack or pShuttle vectors with pAdEasy1 in BJ5183 cells as described previously [5]. Correct clones were propagated in DH5α cells (Life Technologies). For the generation of the Ad.shERα vectors, Ad.Empty and Ad.ERE-Luc, PERC6 cells were transfected with 4 μg Pac-I-linearized adenoviral construct using LipofectAMINE PLUS (Life Technologies). After 16 hours transfection medium was replaced by growth medium. Transfected cells were harvested at day seven post-transfection and after three freeze-thaw cycles the lysate was used for large-scale production of Ad vectors in PERC6 cells. Virus was purified by double CsCl centrifugation and subsequently dialysed as described previously [21]. Final yields as assessed by plaque assays on 911 cells were approximately 2 × 1010 plaque forming units (pfu)/ml. The control virus (Ad.Empty) carries the green fluorescent protein (GFP) under control of cytomegalovirus promoter (CMV) and contained the H1prom. Ad.shERα_1395 and Ad.shERα_1103 carry GFP under control of CMV and shERα_1395 or shERα_1103 under control of H1prom. Ad. shERα_tandem carries both shERα_1395 and shERα_1103 under control of their own H1prom. Ad.ERE-Luc does not contain CMV-GFP and its functionality was verified in vitro and in vivo.
Infection cells
24 hours before transfection, 1.105 cells per well were seeded into12 wells-plate. Cells were transiently transfected by use of lipofectamine with a total of 450 ng of DNA per well (150 ng of reporter plasmid (ERE)3TATA-LUC and 300 ng pCMV-LacZ). After 4 hours cells were infected with either Ad.shERα or control Ad.Empty (MOI 5.000). Additionally, they received 10-9M estrogen for 24 hours. Cells were lysed in 300 μl reporter lyses buffer. β-galactosidase and luciferase activity was determined as described above.
Animals and Ad Injection
The Ethics Committee for Animal Experiments of the Leiden University approved all animal work and the experimental protocols complied with the national guidelines for use of experimental animals. Male C57Bl/6JIco (Charles river, The Netherlands) were given a standard m diet Chow (Hope Farms, Woerden, NL) and housed under standard conditions in conventional cages with free access to water and food.
Recombinant Ad, with a maximum of 4 × 109 pfu in 200 μl of PBS, were administered by injection into the tail vein of mice at the age of 14 weeks. Within five days post-infusion, mice were sacrificed; liver pieces were removed and immediately deep-frozen in liquid nitrogen and stored at -80°C.
Pharmacological treatment
The experiment was carried out in 12-wks old C57BL/6 male mice. To prevent sequestration of low doses of Ad.ERE-Luc by liver Kupffer cells, mice were pre-injected with Ad.LacZ (5 × 108 pfu) 4 hours before administration of 8 × 108 pfu Ad.ERE-Luc. 17β-estradiol (Sigma, E8875) was dissolved in sesame oil (Sigma). In the dose-response experiment, five days post-injection of Ad.ERE-Luc, 0, 5, 25 and 50 μg/kg 17β-estradiol was injected for 6 hours. Then liver pieces were rapidly dissected and immediately deep-frozen in liquid nitrogen and stored at -80°C for further analysis.
Bioluminescent reporter imaging
The experiment was carried out in 12 wks old C57BL/6 male mice co-injected with Ad.ERELuc (5 × 108 pfu) plus either Ad.Empty or Ad.shERα (3 × 109 pfu). Bioluminescent signals (BLS) were performed at time 0 and at several days after 6 and 24 hours s.c injections of 5 μg/kg 17 β-estradiol with the Xenogen IVIS imaging system (IVIS 100). The living mice were intraperitoneal (ip) injected with the luciferase substrate, luciferin, at a dose of 150 mg/kg body weight approximately 5 minutes before imaging. The mice were anaesthetized with isoflurane/oxygen and placed on the imaging stage. Total photon emission of each animal was acquired for 1 minute. Captured images were then quantified by using the Living Image software (Xenogen Corp, Almeda, CA) and the IGOR software (WaveMetrics Corp, Lake Oswego, OR). BLS from the region of interest (ROI) was expressed using the pseudo colour scale (Red most intense and Blue least intense luminescence) and the data were presented as the cumulative photon counts collected within each ROI. Because layers of tissue may limit photon emission from inner organs, the experiment was repeated. Of these mice the livers were rapidly dissected at day 5, 6 hours after 17β-estradiol administration, verifying the results from the bioluminescent reporter imaging by determining the luciferase activity in liver lysates
Luciferase enzymatic assay
The liver extracts were prepared by homogenisation with the minibead beater in reporter lyses buffer (Promega), two cycles of freeze-thawing and 2 min. of centrifugation at maximum speed. Supernatants were used for determining protein-normalized luciferase activity by adding 100 μl luciferyl-CoA (Promega) to 20 μl of liver extract in a monolight luminometer (BD Biosciences). Protein content was measured in a 96-well microtiter plate using the BCA protein assay kit (Pierce). Absorbance at 562 nm was determined in a microplate reader.
Real time quantitative PCR analysis
Total RNA was extracted from liver using TRIzol reagent (Life technologies). Purified RNA was treated with RQ1 RNase-free DNase (Promega, 1 units/2 μg of total RNA) and reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. Quantitative gene expression analysis was performed on an ABI prism7700 Sequence Detection System (Applied Biosystems) using SYBR Green as described earlier (Hoekstra et al. 25448–53). PCR primer sets (Cyclophilline, Fw: AAAAGGAAGACGACGGAGCC Rev: TCGGAGCGCAATATGAAGGT and mERα, Fw: CTAGCAGATAGGGAGCTGGTTCA, Rev: GGAGATTCAAGTCCCCAAAGC) were designed via Primer Express 1.7 software with the manufacturer's default settings (Applied Biosystems) and were validated for amplification efficiency. The absence of genomic DNA contamination in the RNA preparations was confirmed in a separate PCR reaction on total RNA samples that were not reverse transcribed. Cyclophilline was used as a control.
Data Analysis – The significance of differences in relative gene expression numbers Ct (Ct((Cyclo)-Ct(target gene)) measured by real time quantitative PCR was calculated using a two-tailed Student's t test. Probability values less than 0.05 were considered significant.