array scanning protocol
After visual check and background subtraction, endpoint signal intensities for each spot, i.e. for each kinase peptide substrate per array, were calculated by BioNavigator (PamGene International B.V.).
nucleic acid hybridization to array protocol
The kinase substrate array technology (PamGene International, The Netherlands) allows functional comparison of biological samples without prior knowledge of which signaling pathways are activated. Tyrosine Kinase PamChip®12 Array contains 12 arrays, with each array containing 144 peptide substrates. These target peptides consist of 13 -14 amino acids with tyrosine residues for phosphorylation, representing 100 different proteins. The sample reaction mixtures are actively pumped through the arrays to allow contact with the reactive surface for enzymatic reaction with the peptide substrates. Substrate phosphorylation levels are determined by measuring fluorescence of bound labeled anti-phosphotyrosine antibody. Spot images are recorded after every fifth completed pumping cycle by a CCD-camera until the reactions are terminated. Images were inspected visually, image analysis (gridding and spot quantitication) were performed using Bionavigator software (PamGene International, The Netherlands).
nucleic acid labeling protocol
Microarrays were blocked with 25 ul of 2 % bovine serum albumin (BSA) solution each, and washed thrice with 1x protein kinase (PK) reaction buffer (diluted from 10x concentration in Ambion ultrapure water (Applied Biosystems, Oslo, Norway)). The sample reaction mixture consisted of 15 µg of total protein from each sample’s lysate, 4 ul of 10x PK buffer, 0.4 ul of 100x BSA solution, 0.4 ul of 1 mM dithiothreitol (DTT), 0.3 ul of monoclonal fluorescein isothiocyanate (FITC)-conjugated antibody PY20 (1 µg/ul in Tris buffer; PamGene International B.V., ‘s-Hertogenbosch, The Netherlands), 4 ul of 4 mM ATP, and ultrapure water adjusted to a total volume of 40 ul per array. Following loading of the sample reaction mixture, a manual kinetic protocol of 60 cycles of 60 seconds each was commenced, where the mixtures were pumped up and down through the porous arrays, enabling kinetic phosphorylations of the kinase peptide substrates by the samples to be recorded. For every fifth cycle, a charge-coupled device camera imaged the FITC fluorescence using a 50 ms exposure time. The fluorescence signal intensity per spot corresponds to the phosphorylation level of each kinase peptide substrate. During imaging, a wedge separates the unbound solution from the array, allowing imaging of bound solution only, i.e. of the phosphorylated kinase peptide substrates. After completing all the cycles, the arrays were washed twice with 1x PK buffer before end-level images with five different exposure times (10, 20, 50, 100, and 200 ms) were recorded.
Xenografts representing ADT-naïve disease were recruited when their shortest diameter reached 8 mm. Second, ADT was performed when the shortest xenograft diameter reached 12 mm, by surgical castration and simultaneous discontinuation of exposure to the testosterone pellet. Androgen-deprived (AD) xenografts were recruited when their diameter was reduced to 8 mm. Third, long-term AD (ADL) xenografts were recruited when their diameter was reduced to 4. Last, CR xenografts with renewed growth were included when their diameter reached 8 mm. Xenograft diameters were measured by a caliper twice weekly from implantation until castration and once weekly after castration until the end of the experiment. Castration was performed under anesthesia, using s.c. injections of zoletil mixture (2.4 mg/ml tiletamine, 2.4 mg/ml zolazepam (Zoletil vet, Virbac Laboratories, Carros, France), 3.8 mg/ml xylazine (Narcoxyl vet, Roche, Basel, Switzerland), and 0.1 mg/ml butorphanol (Torbugesic, Fort Dodge Laboratories, Fort Dodge, IA)), diluted 1:5 in sterile water, in a dose of 7.5 ?l/g. Analgesia was given to castrated animals as s.c. injections of buprenorphine (Temgesic, Schering-Plough, Brussels, Belgium), in a dose of 0.1 mg/kg. To investigate changes in kinase activity induced by hypoxia, exponentially growing cells at ~ 60% confluence, with fresh medium, were transferred to a hypoxia chamber for 24 hours (Invivo2 200; Ruskinn Technologies, Leeds, UK). Moderate hypoxia (1% O2) was achieved by 5% CO2 and residual N2. Severe hypoxia (anoxia) was obtained by 5% CO2 and a mixture of 10% H2 and 90% N2 in combination with a catalyst that catalyzed the conversion of H2 with any O2 left to H2O.
nucleic acid extraction protocol
Xenografts were first evaluated for tumor cell content, after being sectioned using a cryostat microtome and stained with hematoxylin and eosin. Any normal tissue present in tumor samples was roughly removed by dissection guided by the stains. All tumor specimens used had tumor cell content higher than 80%. The specimens were subsequently cut in 10 µm sections, continuously kept frozen and immediately re-stored at -80 degrees C. A volume of ~ 3 mm3 was cut of all xenograft samples, the required number of sections to cut calculated after measuring the samples’ width and length. By identical procedures, ~ 5 mm3 was sectioned of each prostatectomy biopsy (normal and tumor) specimen. Protein lysates were prepared by adding 100 ul of lysis buffer (M-PER (Mammalian Protein Extraction Reagent) containing Halt phosphatase inhibitor cocktail and EDTA-free Halt protease inhibitor cocktail (Fisher Scientific, Oslo, Norway) and mixed by pipetting and vortexing before 25 minutes of incubation on ice, followed by 15 minutes of centrifugation (15000 rpm, 4 degrees C). The resulting supernatant was filtered (Millex-GP, 0.22 µm; Millipore) before aliquoted into five vials, including one separate vial to protein concentration quantification, and immediately put on dry ice before being stored at -80 degrees C. Protein concentrations (of all samples simultaneously) were measured using a Micro BCA protein assay kit (Fisher Scientific). To generate lysates of normoxic and hypoxic prostate carcinoma cells, the medium was removed before cells were washed with 10 ml ice-cold PBS twice. Then, after adding 4 ml ice-cold PBS, the cells were loosened by scraping and divided into four 1.5 ml vials. Cell pellets were obtained by centrifugation (10 minutes, 2500 rpm, 4 degrees C). The supernatant was removed and identical lysis buffer (150 ul/vial) as for the xenografts was added to each pellet. Following pipetting and vortexing, the cells were lysed for 15 minutes on ice. After centrifugation (15 minutes, 15000 rpm, 4 degrees C), resulting supernatants from individual samples were mixed in one vial before aliqouted and immediately frozen at -80 degrees C. Protein concentrations were measured using a Micro BCA protein assay kit (Fisher Scientific).