Supplementary Methods for Majeed N et al.
Generation of transgenic mice. TRAMP mice heterozygous for the rat probasin gene fused to the SV40 T antigen (PB-Tag) transgene (Greenberg et al., 1995) were purchased from Jackson Laboratory (Ann Harbor, MI) and then bred and maintained in a pure C57BL/6 background by mating transgenic females to non-transgenic males. The lit/lit and lit/+ mice purchased from the Jackson Laboratory were bred by mating homozygous females to heterozygous males. The TRAMP/lit/lit hybrids were generated by mating heterozygous TRAMP males with lit/+ females and then backcrossing the F1 progeny again to lit/+ mice in order to obtain pups homozygous for the recessive lit mutation. All mice were maintained in a climate-controlled environment with conditions of 14 h light: 10 h darkness, a temperature of 22 ± 2 ºC, and a relative humidity of 30-60%. Mice were weaned and ear tagged for identification at 3 weeks of age. Isolation of mouse-tail DNA followed by PCR-based screening assays to identify transgenic mice were performed as previously described (Greenberg et al., 1995). TRAMP and TRAMP/lit/lit males were included in experiments and monitored throughout the 35-week period. Animal care and treatments were conducted in accordance with established guidelines and protocols approved by McGill University’s Animal Ethics Committee.
Tissue Processing and Histopathology. Mice were anaesthetized at 34-36 weeks of age and blood was collected by cardiac puncture. Serum was separated from whole blood and stored at –80oC. At necropsy, all mice were weighed and examined for gross organ abnormalities. The genito-urinary (GU) tract was excised and weighed. The 4 lobes of the prostate gland and the seminal vesicles were dissected, fixed in 4% paraformaldehyde, and transferred to 70% ethanol. Tissues were embedded in paraffin and 4-µm thick sections were cut from the paraffin-embedded tissue blocks, mounted on slides, stained with hematoxylin and eosin as described previously (Kaplan-Lefko et al., 2003), and evaluated for histopathology in a blinded fashion. The composition of the prostate epithelial tissue and seminal vesicle tissue was classified quantitatively using the following categories: normal, PIN (prostatic intraepithelial neoplasia with epithelial stratification and occasional mitotic figures or cribiform pattern), invasive cancer (ranging from well-differentiated to poorly differentiated adenocarcinoma), and phylloides-like (cells consistent with reactive stroma, stromal oedema or obstruction) (Kaplan-Lefko et al., 2003).
Ki-67 Immunostaining. Four-mm thick sections of paraffin-embedded prostate tissue from TRAMP and TRAMP/lit/lit mice were stained for Ki-67 using anti-Ki-67 rabbit monoclonal antibody, clone SP6 (LAB VISION, Fremont, CA). Immunstains were carried out using the BenchMark IHC/ISH Staining Module with the treatment standard cc1 protocol. The primary antibody was diluted at 1:200 and incubated for 20 minutes before counterstained with hematoxylin. Protein expression was evaluated in a blinded manner by a urological pathologist by measuring the proliferation index of benign, PIN and adenocarcinoma areas.
Serum IGF-I Assay. IGF-I serum levels were measured using a rat IGF-I enzyme immunoassay (EIA) (Diagnostic Systems Laboratories, Webster, TX) based upon the competitive binding of unlabeled goat anti-rat IGF-I antiserum and biotin-labeled rat IGF-I in microtitration wells coated with rabbit anti-goat gamma globulin. Ten μl of serum was processed in the assay according to manufacturer’s protocol, with the endpoint absorbance measured at 450 nm with a 620 nm reference filter. IGF-I concentration values were extrapolated from the standard curve generated using rat IGF-I as provided in the EIA kit.
Western Ligand Blotting. Serum IGFBP-3 levels were examined by Western ligand blot. Two μl of serum was subjected to 10% SDS-polyacrylamide gel electrophoresis under non-reducing conditions, and proteins were transferred to nitrocellulose membranes. The membranes were blocked and probed with 125I-IGF-I (Perkin-Elmer, Boston, MA) and exposed to X-ray film for 96 hours as previously described (Zi et al., 2000).
RNA Extraction and GHRH-R Quantitative RT-PCR. Total cellular RNA was isolated from C57BL/6, TRAMP and TRAMP/lit/lit mouse prostate tissue using the Qiagen Rneasy mini-kit (Mississauga, ONT, Canada) and from C57BL/6 mouse pituitary gland with Trizol (Invitrogen/Canada life Technologies, Burlington, ONT, Canada), according to manufacturer’s protocols. Total RNA from mouse prostates (20-320 ng) and anterior pituitaries (2.5 ng) was subjected to one-step RT-PCR using reagents and protocol of the Titan One Tube RT-PCR System kit (Roche Diagnostics, Laval, QC, Canada). RT-PCR reaction was performed in a final volume of 25 ml containing 1.5 ml of enzyme mix, 20 pmoles of mouse GHRH-R sense (nt 438-459) (5’ATCTCCATTGTAGCCCTCTGCG 3’) and antisense (nt 1013-992) (5’TGACTTGGAAAGCCGCCAGTAC 3’) primers (Peng et al., 2001) and 300 pmoles mouse GAPDH sense (nt 101-121) (5’AGGGCTGCCATTTGCAGTGGC 3’) and antisense (nt 937-917) (5’ CGGCATCGAAGGTGGAAGAGT 3’) primers (Aleppo et al., 1997). Reagents were added to final concentration of 1X RT-PCR buffer, 5 mM DTT, 1.5 mM MgCl2, and 12 U RNAse inhibitor (Invitrogen Life Technologies), 0.4 mM dNTPs and 0.5 mCi [a32P] dCTP Redivue (Amersham Biosciences, Baie d’Urfé, QC, Canada) diluted in sterile picopure water. RT-PCR reaction was performed using a Biometra T Gradient PCR (Montreal Biotech Inc., Kirkland, QC, Canada), with the following cycle profile: 30 min at 50.0°C; denaturation at 95.0°C for 3 min, annealing at 66.0°C for 70 sec, elongation at 72.0°C for 60 sec followed by 39 cycles at 95.0°C for 60 sec, at 66.0°C for 70 sec, at 72.0°C for 60 sec, and a final cycle at 95.0°C for 70 sec, 66.0°C for 60 sec, and a 5-min elongation step at 72.0°C. GHRH-R and GAPDH PCR products were analyzed by gel electrophoresis on 4.5% non-denaturating polyacrylamide gel. The molecular weight standard (Invitrogen Life Technologies), was radiolabelled with T4 polynucleotide kinase in the presence of [g32P] ATP Redivue (Amersham Biosciences) according to the 5’ DNA Terminus Labeling Protocol (Phosphate Exchange Reaction) of Gibco BRL (Invitrogen Life Technologies). Autoradiograms were generated using a Biomax MS-1 film (Kodak) with an intensifying screen and analyzed by densitometry, using an IS1000 Digital imaging system (Alpha Innotech Corp./Canberra Packard, Mississauga, ONT, Canada).
Statistical Analysis. Data are expressed as mean ± SEM. Differences between the values were evaluated with two-tailed Student’s t test. The difference in survival was evaluated using the log rank test. P < 0.05 was considered significant.
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