Experiments with human tissues were approved by Institutional Review Board, Hadassah-Hebrew University Medical Center. Due to the retrospective nature of this study and according to the declaration of Helsinki, participants were not obtained constantly informed. In addition, our IRB waived the need for written informed consent. All mice experiments were approved by the IACUC.
Dominant Negative β-TrCP (WD)
Dominant negative β-TrCP (WD) was cloned using E3RS excluded from pCDNA3-EE-hE3RS plasmids with the primers 5′ to 3′ forward: GCGGCCGCTATGGACCC-GGCCGAG (with NotI site in its 5′); reverse: TTATCTGGAGATGTAGGTGT; the product was cloned into TA vector (Invitrogen). The last vector was cut with AvrII/ASP718, filled in and blunt ligated. The relevant fragment was cut and inserted into pFLAG-CMV™-2 expression vector (Sigma-Aldrich) with NotI/BamHI. This procedure produced a WD construct lacking part of the F-box and conjugated to FLAG. The WD-FLAG was inserted under bidirectional teracycline promoter expressing GFP. The resulting plasmid was transfected into AT2.1 Rat prostate cancer cells expressing the tetracycline trans-activator, using FUGENE reagent (Roche Applied Science). The transfected cells were selected using hygromycin and neomycin (Sigma-Aldrich) containing media to produce a stable clone which expresses a dominant negative β-TrCP upon addition of tetracycline or its derivate doxycycline.
Inducible β-TrCP and AhR shRNA
The human shRNA 5′- GUGGAAUUUGUGGAACAUC 3′
targeted against β-TrCP1 and β-TrCP2 was constructed into pTER plasmid and inserted into a modified pRRL.sin.PPT.tetO7.MCS.PRE lentiviral vector. The vector consists of an HI promoter, tet operator, the shRNA coding sequence and eF1α promoter driving the tet repressor fused to eGFP. Virus production and infection were carried out as previously described 
. To target the AhR we used the following shRNA sequences: 1. 5′ CAGCUGAAUUAAAUAACAU 3′
; 2. 5′ CAGACAGUAGUCUGUUAUA 3′
. Both of which proved to be efficient in knocking down the receptor (the presented data represent those obtain with the latter sequence). shRNA expression is induced only after Tetracycline or Doxycycline (Sigma-Aldrich) administration. The vector alone was used as control. Double knocked down LNCaP cells were designed by co-infecting shβ-TrCP cells with lentiviral vectors carrying either of the above described sequence.
DMEM, RPMI 1640, Trypsin EDTA, Penicillin-Streptomycin solution, L-glutamine, fetal calf serum (FCS), Charcoal Stripped Serum (CSS) were purchased from Biological Industries, Kibbutz Beit Haemek, Israel. LAPC4 cells were kindly provided by Prof. Zelig Eschar (The Weizmann Institute of Science, Rehovot, Israel); AT2.1, LNCaP and 293T were furnished to us by Dr. Rachel Bar-Shavit (Department of Oncology, Hadassah Medical Center, Jerusalem, Israel). All cell lines were incubated at 37°C 5% CO2 in appropriate medium containing 10% FCS or CSS as indicated.100 pM Methyltrienolone (R1881, Perkin-Elmer, New England Nuclear) was added to LAPC4 full media.
Cell Proliferation Assay
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide) and XTT (2,3-Bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide) was used as the protocol indicates (Biological Industries, Kibbutz Beit Haemek, Israel). All experiments were carried out in 96 well plates with eight repeats of at least 3 independent infections.
Whole-cell lysates were prepared from transfected or infected cells by extraction in lysis buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 1% NP-40, 0.1% SDS, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin and 1 mM dithiothreitol. Proteins were resolved by 10% SDS-PAGE, transferred onto nitrocellulose membranes, probed with appropriate antibodies, incubated with Peroxidase-conjugated Goat anti mouse or Rabbit IgG (Jackson Laboratories) and developed using the ECL kit (Pierce). Primary antibodies used were: anti-Flag (Sigma); anti-IkB, anti-phpspho-IkB (Cell Signaling); Aryl Hydrocarbon Receptor, CYP1A1 (Santa-Cruz); phospho-β-catenin (BD Biosciences).
AT2.1 or LNCaP cells were harvested washed and reconstituted in PBS. 106
51B cells per 200 µl volume were injected subcutaneously to 6–7 weeks old atymic (Nude
) male mice. 106
LNCaP cells were injected to 6–7 weeks old rag1−/−
male mice together with Matrigel (BD bioscience). Tumors were measured in two dimensions with caliper, and tumor volume (mm3
) was calculated with the formula V
)/2. Half of the mice received Doxycycline (0.2 mg/ml, AT2.1) or tetracycline (1.5 mg/ml, LNCaP) supplemented with 5% sucrose in their drinking water. Half of the mice were surgically castrated: mice were anesthetized using Ketamine/2% Xylazine at 5.7
1 ratio (0.1 ml per 25–30 gram mouse). Surgical castration was performed via a midline scrotal incision allowing bilateral access to the hemiscrotal contents. After exposing each testicle, a 3-0 Vicryl suture was used to ligate the spermatic cord and then remove the testicle. Mice were treated with Carprofen (Rimadyl) as analgesics after surgery. Two hours before sacrifice, mice were injected with BrdU intraperitoneally 100 µl per 10 grams of body weight (RPN201, Amersham Pharmacia Biotech Inc). For AT2.1 xenografts, NUDE
mice were treated pre-injection and tumors were weighted at the end of the experiment. LNCaP xenografted mice were treated 30 days post injection when measurable tumors were established. Tumor relative growth was calculated individually for each mouse, comparing each week's measurement to the treatments' day 0 (30 days post injection). All mice experiments were approved by the IACUC.
mouse tumor specimens were fixed in 4% neutral-buffered formalin and embedded in paraffin. Patients paraffin embedded samples were collected from the archives of the Department of Pathology at the Hadassah-Hebrew University Medical Center. Experiments with human tissues were approved by the institutional review board. 5 µM sections were dewaxed and hydrated through graded ethanol dilutions, then cooked in appropriate buffer (pH 7.4) in a pressure cooker at 115°C for 3 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide followed by washing. The sections were then incubated with the indicated antibodies: anti-p65 (Neomarkers; 1
100), anti-β-catenin (Santa Cruz; 1
300) and anti-AhR (Santa Cruz; 1
200). All sections were counterstained with hematoxylin.
RNA, cDNA Micrroarray and Real-Time PCR
Total RNA was extracted from LAPC4 or LNCaP cells infected with β-TrCP shRNA lentiviral vector with TRI Reagent (Sigma). For cDNA microarray RNA was extracted from LAPC4 infected cells using TRIzol® (Invitrogen). cRNA preparation and hybridization was performed using standard manufacture protocol; Biotin-labeled target synthesis reactions were performed using standard protocols supplied by the manufacturer (Affymetrix, Santa Clara CA, USA). From each RNA sample, 5 µg were converted into double-stranded cDNA by reverse transcription with SuperScript™ II Reverse Transcriptase (Life Technologies, Helgerman CT, USA), using T7-oligo-dT as a primer. Expression value (signal) was calculated using Affymetrix Genechip software MicroArray Suite 5.0. Only probe sets that had at least an intensity of 20 and a present call at one of the microarrays were selected. Next quantile normalization was applied to the log2 transformed expression values (Bolstad BM Bioinformatics 19: 185–193). For β-TrCP knockdown determination and validation studies 2 µg of RNA were used as template for synthesis of cDNA using SuperScript™ II Reverse Transcriptase. The cDNA was subsequently used as Real Time PCR template. All Real Time PCR reactions were carried out using Absolute Blue QPCR SYBR Green Low ROX Mix (ABgene) with the following primers (5′ to 3′): β-TrCP1 Forward: ATCGGATTCCACGGTCAGAG, Reverse: AATCAACGTGTTTAGCATT-TCACCT; β-TrCP2 Forward: CCATCAAAGTCTGGAGCACGA, Reverse: CGCT-TGTGCCCATTGAGAGTA; AhR Forward: ACATCACCTACGCCAGTCG, Reverse: CTCTATGCCGCTTGGAAGGAT; CYP1A1 forward: TGAATGCCTTCAAGGAC-CTG, Reverse: TCAGGCTGTCTGTGATGTCC.
All microarray data is MIAME compliant. The raw data has been deposited in GEO (accession number GSE19141).