Clinical samples, ethics statement and protein extraction
Prostate tissue samples were obtained from the University Medical Center Hamburg Eppendorf after informed consent. The study was approved by the local ethics committee of the University Hospital Eppendorf, Hamburg. For expression profiling whole prostates were collected after radical prostatectomy from patients with elevated PSA values and preoperative pathological examination performed at Martini Clinics, Hamburg, Germany. Patients received no preoperative therapy. After radical prostatectomy samples were frozen in liquid nitrogen until use. Tumor and benign areas were marked on the sections. We employed manual micro dissection method to obtain pathologically characterized materials for gene and protein expression profiling. The corresponding areas on the remaining blocks were sliced out with sharp knife, embedded in Tissue-tek® and stored at -80°C until use for total protein extraction. Protein preparation has been described previously .
RNA and DNA extraction from tissue sections
After surgical removal of the prostate, tissue samples were immediately taken with a 6 mm punch biopsy instrument (Stiefel, Wächtersburg, Germany) from areas that were suspected to contain tumor foci based on information obtained from the preoperative systematic biopsies. Tissue biopsy was immediately immersed in RNAlater (Qiagen, Hilden, Germany), stored overnight at ambient temperature and frozen at -20°C until use. For nucleic acid isolation, the specimen was thawed at room temperature and immediately washed two times each of 5 minutes in 10 ml ice-cold sterile PBS-buffer to elute most of the RNAlater from the tissue. Cryo sections were taken by fixing the tissue using Tissue-Tek® (Sakura, Netherlands) followed by freezing in a cryo-microtome and stained with haematoxylin and eosin (H&E) and analyzed by pathologists. Tissues were only enrolled into the study if at least 70% of cells were epithelial prostate tumor cells. Then, 10-15 subsequent unstained sections were transferred to a cryo tube for RNA and DNA isolation and the final section was again H&E stained and analyzed by pathologists. In parallel, normal prostate tissues were collected from tumor free areas and processed in the same way as tumor samples. Adjacent cryo sections were used for DNA and RNA extraction from the same prostate tissue specimens. Total RNA and DNA were extracted using the All Prep DNA/RNA Mini kit (Qiagen) according to the manufacturer's instructions. The quantity of the DNA and total RNA was checked using the Nanodrop and RNA quality by Bioanalyzer. Samples with low RNA quality (RIN < 6) were excluded from further analysis.
The PCa cell lines LNCaP and DU145 were purchased from DSMZ (Braunschweig, Germany) and cultivated in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/mL) penicillin and streptomycin as recommended by suppliers Phoenix amphotrophic packaging cells were grown in DMEM with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were regularly tested for mycoplasma contamination using the MycoAlert Kit (Cambrex Bio Science Rockland, Inc., Rockland, ME, USA).
Bisulfite treatment and methylation specific PCR (MSP)
DNA isolation from prostate tissues was performed using Qiagen all prep kit according to supplier's protocol. The DNA concentration was measured by nanodrop spectrophotometer (Peqlab, Germany). Approximately 1 μg DNA was sodium bisulfite-modified and subjected to MSP with primers specifically recognizing the unmethylated or the methylated sequence of UCHL1. MSP primers for the UCHL1 gene were adapted from previous publication . Primers used for MSP are mentioned in additional files (Additional file 1). The PCR was run for 35 cycles with an annealing temperature of 56°C. Normal DNA from peripheral blood was treated in vitro with Sss I methyltransferase (New England Biolabs, Beverly, MA) in order to generate in vitro methylated DNA (IVD) that served as a positive control for methylated alleles. PCR products were separated on 2.5% agarose gels and visualized by ethidium bromide staining.
For quantitative analysis of regional DNA methylation, pyrosequencing was used. Following PCR amplification of bisulfite-converted DNA using primer sequences (see Additional file 1) the final biotin-labeled PCR products were captured by Streptavidin Sepharose HP (Amersham Biosciences). PCR products bound on the beads were purified and made single-stranded in a Pyrosequencing Vacuum Prep Tool (Pyrosequencing Inc.). The forward sequencing primers were annealed to single-stranded PCR products and pyrosequencing was performed using the PSQ HS 96 Pyrosequencing system (Biotage AB). Quantification of cytosine methylation was performed using the PSQ HS96A 1.2 software package.
RNA isolation and quantitative real time PCR
Quantitative real time PCR for analysis of transcriptional levels of UCHL1 was performed in 48 benign and 45 tumour samples using SYBR Green. RNA isolation and cDNA synthesis carried out according to standard protocols. Quanti Tect primers for UCHL1, p53, MDM2 and p27Kip1 and GAPDH (housekeeping gene) were purchased directly from Qiagen, Germany. Quantitative real time PCR was performed in thermal cycler (Stratagene, Germany) using Dynamo Flash SYBR Green qPCR kit (Finnzymes, Finland) under optimized cycling conditions. PCRs for the target and housekeeping genes were performed in triplicates and mean relative expression levels were reported. To obtain statistical significance data obtained were analyzed by unpaired student t-test and p value < 0.05 was considered as significant. For semiquantitative UCHL1 RTPCR we used the cloning primers and for RPLP0 primers refer additional files (Additional file 1).
Cloning strategy for UCHL1 overexpression
An UCHL1 protein expressing recombinant vector was generated by cloning the coding region of the human UCHL1 (Accession number NM_004181) cDNA derived from prostate tissue into the pMSCV-Puro vector (Clontech, Palo Alto, CA, USA). UCHL1 coding sequence (CDS) was amplified from total cDNA by PCR using Phusion DNA Polymerase (Finnzymes Oy, Finland). After digestion of the PCR product and pMSCV vector with BglII/XhoI enzymes (Fermentas GmbH, Germany), ligation of the PCR product with linear vector resulted in recombinant pMSCV-UCHL1 construct. The sequence of the cloned PCR fragment was confirmed by DNA sequencing (MWG Operon).
Virus production and infection of target cells
Phoenix amphotrophic packaging cells were transfected with either empty or pMSCV-UCHL1 vector using CaCl2 transfection method. The transfection mixture was prepared by mixing 15 μg of plasmid DNA and 125 mM Cacl2 in 1 ml of HBS. The DNA precipitate was added drop wise into the cell culture medium containing 25 μm of chloroquine. After 12 h of transfection, medium was replaced with fresh medium and further incubated for 12 h. The virus containing medium was collected, filtered by 0.45 μm sterile filters directly on the target cells at around 50% of confluence. The cells were fed with fresh medium to continue another round of virus collection. Both target and packaging cells were continued to grow. 12 h later, two more infection cycles were repeated. After three cycles of infection, the target cells were grown in normal cultivation medium for 24 hrs and selected for integration of the target gene and/or puromycin at a concentration of 2 μg/ml until all cells died in control dishes. The colonies appeared with resistance to puromycin were propagated further and verified for overexpression of UCHL1 in LNCaP cells.
Cell proliferation assay
For cell proliferation assays, cells were plated at a density of 1.5 × 105 cells/well in 6-well format in complete growth medium. Cells were allowed to grow under optimal culture conditions over a period of 0 to 8 days. Cells were harvested by trypsinization and counted using Vi-Cell Cell counter (Beckaman Coulter GmbH, Germany). The growth rate was shown by plotting the mean total number of cells from triplicate experiments vs. growth time in days. Each experiment was performed in triplicate wells and repeated 3 times. The significance of difference in growth between UCHL1 positive and mock LNCaP cells was calculated using student t-test.
Detection for SA-ß-galactosidase was performed as described elsewhere . Briefly, LNCaP cells were harvested at sub confluent density and fixed with 2% PFA and 0.25% glutaraldehyde in PBS supplemented with 1 mM MgCl2 (pH 6.0) and incubated in a staining solution containing potassium cyanide/X-gal in PBS/MgCl2 (pH 6.0) at 37°C over night. Slides were analyzed using an Axioplan microscope (10 × magnifications) (Carl Zeiss AG, Germany). 200 cells/triplicate were analysed for a positive staining.
Colony formation assay
Effect of UCHL1 expression on anchorage-independent LNCaP cell growth was analyzed by soft agar assays. In soft agar assay, bottom agar was prepared by mixing 1% of agarose (Bacto Agar: Becton, Dickinson, Sparks, MD) with 2 × RPMI 1640 with 10% FBS in 6-well plates at 37°C to achieve final concentration of 0.5% of agar. After solidifying the bottom agar, 1 × 104 cells were mixed with cultivation medium and agar solution to obtain a final concentration of 0.35% agar. The mixture was spread on the surface of pre prepared base agar plates immediately. The culture medium was replenished every 3 days with fresh medium. After 14 days of incubation, plates were stained with 0.005% crystal violet solution until colonies turned purple color. After washing excess stain solution colonies were photographed and counted under a light microscope. Each experiment was performed in triplicates and repeated 3 times.
Proteasomal activity assay
Cells were lysed in M-PER buffer with complete protease inhibitor cocktail without EDTA (Roche, Germany). Protein concentration was measured by BCA method (Thermo Fischer Scientific, Germany) according to supplier's protocol. For proteasomal activity assay, 10 μg of total protein diluted to final volume of 50 μl with incubation buffer (5 mM DTT, 0.5 mMEDTA, 20 mM HEPES, 0.1 mg/ml ovalbumin in ddH2O, pH 7.8). Protein lysate prepared in incubation buffer were pre incubated for 2 h at 4°C. For blank control, incubation buffer alone was included in each assay. After incubation, the substrate suc-LLVY-AMC (Calbiochem) was added to the incubation mixtures to achieve final of concentration of 60 μM in final volume of 100 μl. The assay plate was incubated at 37°C for 1 h in the dark before measuring the proteasomal activity in fluorescent spectrophotometer (Mitras LB 940, Berchthold Technology, TN, US) at 355 and 460 nm. Each experiment was performed in triplicates and repeated 3 times.
Commercially available antibodies against protein targets of interest were purchased. Protein extracts prepared in M-PER (Pierce) with protease and phosphatase inhibitors were separated by 4-12% Bis-Tris-NuPAGE in Nupage running buffer and electrophoretically transferred onto PVDF membrane (Millipore). Blocking was carried out in 1 × Rotiblock solution (Roth Chemicals) followed by incubating the membrane with primary antibodies purchased all from cell signaling except anti UCHL1 from Millipore diluted at 1:1000 in 3% BSA in TBST overnight at 4°C. Excess antibodies were removed by washing with NaCl-Tris-Tween 20. Incubation with secondary antibody conjugated to horseradish peroxidase [anti-(mouse IgG) or anti-(rabbit IgG) from cell signaling, diluted 1:5000 in 1 × Rotiblock] was performed for 1 h at room temperature. After three washes, the reaction was developed by the addition of LumiGLO substrate (Thermo). The emitted light was captured on X-ray film (GE Healthcare).
Cell culture experiments were carried out in triplicates and repeated three times. Data points were expressed as mean of triplicates and median of repeated experiments. Graph Pad PRISM Version 5.0 statistics program was used to test significance of the results with Mann-Whitney test or t-test and p values less than 0.05 with 95% confidence interval were considered as significant.