NINDS, Prestwick Peakdale 1, Mixed Commercials, Antimitotic, and Enamine and Known Bioactive Compounds libraries were obtained from The Institute of Chemistry and Cell Biology (ICCB) at Harvard Medical School (MA, USA). Information about the libraries can be found at http://iccb.med.harvard.edu.
SMIPs and other chemical compounds
SMIP001 to SMIP018 were purchased from Ryan Scientific (SC, USA). SMIP0019 was purchased from ChemDiv, Inc (CA, USA). Roscovitine was purchased from Enxo Life Sciences (formerly Biomol International; NY, USA), bortezomib from LC Laboratories (MA, USA), cycloheximide (CHX) and camptothecin from Sigma (MO, USA), MG132 and epoxomicin from BostonBiochem (MA, USA). All drugs were dissolved in DMSO and kept at -80°C.
Anti-p27 antibody (610241, 1:5000 for immunoblotting and 1:1000 for immunofluorescence) was obtained from BD Biosciences (NJ, USA). Anti-SKP2 (32-3300, 1:2000 for immunoblotting.) and anti-ubiquitin (13-1600, 1:2000) was from Zymed Laboratories (CA, USA). Anti-SKP2 (sc-7164, 1:500 for immunofluorescence), anti-CDK2 (sc-163, 1:1000 for immunoblotting, 2 μg/500 mg protein for immunoprecipitation) and anti-CDK4 (sc-260, dil 1:500) from Santa Cruz Biotechnology, Inc (CA, USA). Antibodies for Cyclin E Ab-2 (clone HE12, dil 1:200) and Cyclin A Ab-6 (clone 6E6, dil 1:100) from Thermo Scientific (CA, USA). Anti-p21 (2947, 1:2000) and anti-PARP (9542, 1:1000) were from Cell Signaling Technology (MA, USA). Anti-Tubulin (T5168, 1:5000) was from Sigma Life Science. Anti-Actin (69100, 1:20000) was from MP Biomedicals, LLC (CA, USA). HRP Donkey anti-mouse IgG (715-035-150, 1:5000) and HRP Donkey anti-rabbit IgG (711-035-152, 1:5000) was obtained from Jackson ImmunoResearch Laboratories (PA, USA).
Creation of the LNCaP-S14 cell line and culture conditions
Parental LNCaP cells were stably transfected with a plasmid driving the expression of Myc epitope-tagged SKP2 or empty vector (pcDNA3). Briefly, 5 x106 cells were transfected with 20 μg of each plasmid using Lipofectamine 2000 reagent (Invitrogen, CA, USA) following the manufacturer's protocol. Cells were selected in 600 μg/mL geneticin (G418) until individual colonies were visible. Colonies were picked, passed to 24 well plates and expanded. The levels of p27 and SKP2 were analysed by immunoblotting as described below. LNCaP-S14 (positive clone for SKP2 overexpression), LNCaP-P10 (vector control cell line), LNCaP (parental cell line), PC3 and DU145 cells were maintained in RPMI supplemented with 10% fetal bovine serum and 5% penicillin/streptomycin solution. IMR90 and HeLa cells were maintained in DMEM containing 10% fetal bovine serum, 5% penicillin/streptomycin and 4 mM glutamine. Cells were grown as a monolayer in a humidified incubator at 37°C and 5% CO2.
Immunofluorescence assay on cover slips and in 384 well plates
The screen relies on the detection of differences in the levels of endogenous p27 by immunofluorescence. LNCaP-S14 cells were seeded at a density of 100,000 cells onto 15 mm glass cover slips and allowed to attach for 1 day. Cells were treated with vehicle (DMSO) or positive controls (roscovitine, MG132) for 18 h. After the incubation, cells were fixed by the addition of 10% paraformaldehyde in phosphate buffered saline (PBS; final concentration 4%) and incubated for 20 min at room temperature (RT). Cells were permeabilized by washing 3 × 5 min with PBS, 0.1% Triton X100, followed by blocking in 5% nonfat dry milk in PBS, 0.1% Triton X100 for 1 h at RT. Cells were incubated with 50 μl primary antibody (anti-p27, 1:1000 in blocking buffer) for 1 h, followed by one wash with blocking buffer. 50 μL of secondary antibody (Alexa Fluor 594 goat anti-mouse, Invitrogen A11005, 1:200 in blocking buffer) was added for 1 h. Cells were washed 3 × 5 min in PBS, 0.1% Triton X100 and stained with Hoechst dye (1 μg/mL in PBS) for 2 min. Cells were washed twice in PBS and mounted for imaging with a Nikon Type 120 inverted fluorescent microscope using 60× magnification. Compounds used as positive controls included MG132, epoxomycin and roscovitine, while DMSO was used as negative control. Cells stained with secondary antibody only were used to assess the non-specific (NS) staining background.
The above protocol was adapted to 384 well plates as follows: 4000 LNCaP-S14 cells per well were seeded into 384-well plates (black with clear bottom) in 30 μL RPMI, 10% fetal bovine serum (FBS). Nuclear p27 staining was done under the same conditions as above but with reducing the volume of solutions to 20 μL/well for 10% para-formaldehyde in PBS, 30 μL/well for blocking and wash solutions and 15 μL/well for primary and secondary antibody solutions and Hoechst dye. All liquid handling was done with an eight-channel multidrop liquid dispenser (Wellmate, OH, USA) and a wand aspirator (VP Scientific, CA, USA; VP-186L). After staining, plates were sealed and stored in the dark at 4°C until scanning.
Images acquisition and analysis
Images from 384 well plates were acquired by using the ImageXpress Micro inverted epifluorescent automated microscope (Molecular Devices, CA, USA) at dual wave length to detect Hoechst and p27. The microscope was equipped with the Photometrics CoolSNAP ES digital CCD camera and an automated objective and filter cube changers. Two images per well at a 20× magnification were obtained at each wave length.
Images were analysed with the MetaXpress cellular imaging analysis software using the cell scoring module. Cells were scored positive for Hoechst, if the integrated pixel intensity was 210-fold above local background and positive for p27 when staining was 30-fold above background (signal obtained with secondary antibody). MetaXpress processing of the raw images provided quantitative measures of the total cell number and the number of p27 positive cells in a given field. The data from both images of each well were combined to get a single number of positive cells. The percentage of positive cells was calculated relative to the total number of cells. Background correction was done by subtracting the number of p27 positive cells in wells stained with secondary antibody only.
The staining protocol was also evaluated using another imaging and software package, the Cell Lab IC100 and Cytoshop software (Beckman Coulter, CA, USA). Minor changes were introduced to the protocol: The cell number was reduced to 3000/well and the secondary Alexa Fluor 568 goat anti mouse antibody was diluted 1:500 in blocking buffer. Four images per well were taken using 10× magnification with a numerical aperture of 0.25 and a camera binning of 2 × 2. A manual threshold was established by comparison of the total nuclear intensity in the positive control and the total intensity obtained in cells stained with the secondary antibody only (unspecific staining) and/or the vehicle control (DMSO). Processing of the raw images provided quantitative measures of the total cell number and the number of p27 positive cells in a given field.
Z'-factor calculation: This parameter was used to assess the quality of the assay in the HTP optimization (HTP Company, CA, USA) . In three independent experiments, LNCaP-S14 cells were seeded in 384 well plates followed by treatment with 0.3% DMSO (192 wells, negative control) or 20 μM roscovitine (192 wells, positive control) for 18 h. The percentage of p27 positive cells was determined as described above and the Z' factor calculated from 576 replicates (each condition) as follows: Z'= 1- (3SD+ + 3SD-)/(Ave+ - Ave-), where SD+ is the standard deviation for the positive control, SD- the standard deviation for the negative control, Ave+ the average for the positive control and Ave- is the average for the negative control .
The cell-based screen was performed at the Institute of Chemistry and Cell Biology, Harvard Medical School, using libraries of uncharacterized compounds (Peakdale 1, Mixed Commercials, Antimitotic and Enamine, total of 4888 compounds) plated in 5 mg/mL stock solutions in DMSO, the NINDS library (1040 compounds, 10 mM in DMSO), the Prestwick Library (960 compounds, 2 mg/mL) and a library of 489 known bioactive compounds (plated at 5 mg/mL, 1.1 mg/mL, and 0.25 mg/mL in DMSO) for a total of 7638 compounds. LNCaP-S14 cells were seeded onto 384 well plates as described above. Compound stocks (100 nL) were pin-transferred using a robot-controlled stainless-steel pin array, resulting in a 300-fold-dilution (5 to 35 μM final screening concentration, depending on individual compound molecular weight) followed by incubation for 18 h. All compound plates were screened in duplicates. Each individual plate contained at least eight positive controls (roscovitine, 20 μM), eight negative controls (DMSO, 0.3%) and eight wells of cells treated with 0.3% DMSO and stained only with secondary antibody (non-specific staining). Staining of p27, imaging and analysis was performed as described above. Potential hits were classified according to their Z score. The Z score is a simple and widely known normalization method calculated as follows: Z = Xi - X/Sx, where Xi is the raw measurement on the ith compound, X and Sx are the mean and the standard deviation, respectively, of all measurements within each plate . Potential hits were classified as weak if their Z score was between 2 and 3, medium if the Z score was between 3 and 5 and strong if the Z score was greater than 5.
Target specificity was analysed using LNCaP cells stably expressing the homeobox transcription factor NKX3.1 fused to YFP. In brief, 4000 LNCaP-NKX3.1-YFP (clone 4A) cells were seeded in a 384 well plate and treated with SMIPs for 18 h. Proteasome inhibitors were used as positive controls. Images from 384 well plates were taken using the Cell Lab IC100 automated microscope (Beckman Coulter), and the percentage of cells positive for NKX3.1-YFP was quantified using Cytoshop software (Beckman Coulter). The percentage of NKX3.1-YFP positive cells was determined relative to DMSO treated cells.
Cell specificity was evaluated using HeLa cells stably transfected with a p27-luciferase (p27Luc) construct . Briefly, HeLa-p27Luc cells were seeded in 96 well plates and treated with 40 μM of the respective SMIPs or positive controls (proteasome inhibitors). After incubation for 18 h, cells were lysed using Cell Culture Lysis Reagent from Promega (WI, USA) and luciferase activity was determined by the Luciferase Assay System (Promega) using a Veritas microplate luminometer (Turner Biosystems, CA, USA). Protein concentration was measured in a parallel plate using Bio-Rad Protein Assay (Bio-Rad, CA, USA). Luciferase activity was normalized against protein concentration and compared to the activity recovered from DMSO-treated cells.
Total cell lysate from cells treated with vehicle (DMSO) or compounds were obtained by incubating cells in lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, protease inhibitors) for 15 min at 4°C. The supernatant was collected by centrifugation and the amount of protein was determined with a Bio-Rad protein assay (Bio-Rad) using BSA as standard. Equal amounts of protein were subjected to SDS-PAGE (4%-20% gels, Invitrogen) and transferred to PVDF membranes. The membranes were blocked with TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) containing 5% non-fat dry milk or bovine serum albumin (BSA) for 1 h at room temperature, followed by incubation in primary antibodies 4°C overnight. Membranes were washed and incubated with secondary antibodies conjugated to HRP (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Signals were visualized using the ECL Western Blotting Substrate (Pierce Biotechnology, IL, USA) according to the manufacturer's instructions. Membranes were stripped using Restore™ Western Blot Stripping Buffer (Pierce Biotechnology). Films were scanned and quantification of the bands was performed using Image J software http://rsbweb.nih.gov/ij/.
Total lysates of cells treated with vehicle (DMSO), SMIPs or positive controls were prepared by incubation in immunoprecipitation (IP) buffer (25 mM Tris-HCl, pH 7.4; 150 mM NaCl, 0.5% Triton X-100, protease inhibitors) for 15 min at 4°C. Upon centrifugation, the supernatant was collected and the amount of protein was determined with a Bio-Rad protein assay (Bio-Rad Laboratories) using BSA as standard. Five hundred micrograms of protein was adjusted to 1 mL with IP buffer and incubated with 10 μL anti-CDK2 antibody (sc-163, Santa Cruz Biotech) at 4°C for 3 h with constant rotation, followed by addition of 100 μL protein G-sepharose beads (10% slurry) for 2 h. After four washes for 5 min each with 1 mL IP buffer, beads were resuspended in 2X-SDS Laemmli buffer, followed by SDS-PAGE and immunoblotting.
In vitro kinase assay
Histone H1 kinase assays were performed as described in reference . Briefly, the total cell lysates from LNCaP-S14 cells treated with the respective SMIP (40 μM) for 24 h were prepared in IP lysis buffer supplemented with protease inhibitors followed by IP. Kinase reactions were performed by adding histone H1 (1 μg) and 7.5 μCi [γ-32P]ATP (800 Ci/mmol Perkin Elmer Life Sciences, MA, USA) in kinase buffer (20 mM MgCl2, 10 mM EGTA, 40 mM Hepes, pH 7). After incubation at 30°C for 20 min, the reaction was stopped by adding 20 μL 2× SDS gel loading buffer. Samples were separated by electrophoresis, gels were stained and dried, followed by exposure to X-ray film. DMSO was used as negative control and roscovitine (CDK2 inhibitor) as positive control.
LNCaP-S14 (10,000 cells), PC3, DU145 or IMR90 cells (5000 cells) were seeded in 96-well plates and treated with increasing concentrations of the respective SMIPs for 72 h. Cell viability was assessed using the MTT cell proliferation assay (ATCC®, Middlesex, UK) according to the manufacturer's protocol. IC50 was calculated using the BioDataFit 1.02 software http://www.changbioscience.com/stat/ec50.html.
Determination of protein half-lives by cycloheximide chase
LNCaP-S14 cells were treated with SMIPs (40 μM) for 18 h followed by the addition of 100 μg/mL cycloheximide (CHX). Cell extracts were obtained as described above at different times after CHX addition. Protein lysate was subjected to SDS-PAGE and immunoblotting for p27, p21 and SKP2. Tubulin was used as loading control. Protein half-lives were calculated using a web based calculator http://www.1728.com/halflife.htm.
RNA isolation and quantitative polymerase chain reaction (q-PCR)
Total RNA was isolated from LNCaP-S14 cells treated with 40 μM SMIPs or vehicle (18 h) using the RNeasy Mini Kit (Quiagen, CA, USA), followed by first-strand cDNA synthesis using Omniscript® Reverse Transcription (Quiagen). Real-time PCR analysis was performed on the Stratagene Mx3000p detection system (Stratagene, Agilent, CA, USA) using the SYBR Green PCR Master Mix (ABI, CA, USA) at the Microarray and q-PCR Facility of the Sanford-Burnham Medical Research Institute. The primers used were:
5'-ACTGCAGAGACATGGAAGAG-3' and 5'-ATGCGTGTCCTCAGAGTTAG-3' for p27 (to amplify a region from nucleotides 608 to 852); 5'-CGATGGAACTTCGACTTTG-3' and 5'-GGGTACAAGACAGTGACAGG-3' for p21 (to amplify a region from nucleotides 142 to 355); and 5'-TGAGCTGCTAAAGGTCTCTG-3' and 5'-ATGTGCTGTACACGAAAAGG-3' for SKP2 (to amplify a region spanning from nucleotide 527 in exon No. 2 to 739 in exon No.4). Briefly, reactions were done in a 25 μL reaction volume of q-PCR mixture containing 2 μL of cDNA (40 ng) and 250 nM of each primer. Activation of the enzyme was done at 95°C for 10 min followed by 40 cycles of amplification at 95°C for 30 s, 56°C for 1 min and 72°C for 30 s. All reactions were done in duplicates and normalized using GAPDH as control. Primers were design using Primer 3 http://frodo.wi.mit.edu/primer3/ and synthesized by Valuegene, Inc, CA, USA.
Cell cycle analysis
Cells were exposed to SMIPs (40 μM) for 24 h and 48 h. Cells were trypsinized, washed with PBS and suspended in 600 μL PBS. Cells were fixed by adding 1.4 mL cold absolute ethanol and kept at -20°C for at least 12 h. After washing once with PBS, the cells were resuspended in 250 μL PBS containing 2.5 μg RNAse A and incubated for 45 min at room temperature. Staining was done by adding 40 μg/mL of propidium iodine followed by incubation for 15 min at room temperature. DNA-bound fluorescence was read at 564 to 606 nm using FACSort and FACSCanto flow cytometers (BD Biosciences) at the Flow Cytometry Facility of the Sanford-Burnham Medical Research Institute. Distribution of cells in the different cell cycle phase was determined with ModFit LT 3.2.1 or FlowJo 8.6 software. Aggregates were excluded from the analysis manually using pulse shape or identified by automated modelling in ModFit LT.
Apoptosis was measured using the Cell Death Detection ELISA (Roche, Basel, Switzerland) following the manufacturer's instructions. Briefly, LNCaP-S14 cells (0.1 × 106) were seeded in 12-well plates and treated with SMIPs (40 μM) for 24 h. Cells were collected in medium, spun at 1000 rpm, and resuspended in 1 mL PBS at the specified time points (day 1 to day 3). The cell suspension was divided into two parts. The first half was used for determination of cytoplasmic histone-associated DNA fragments (ELISA), the second for protein determination to normalize the ELISA data to the amount of input protein.
LNCaP-S14 cells were seeded in 6 cm dishes or six-well-plates coated with poly-lysine the day before transfection. 10-20 nM of siRNA was transfected using DharmaFECT 3 (Thermo Scientific) according to the manufacturer's instructions. Briefly, siRNAs were dissolved in siRNA suspension buffer (Quiagen) at 20 μM, heated to 90°C for 1 min and incubated at 37°C for 1 h. siRNAs were added to the DharmaFECT 3 reagent diluted in Optimem media (1:100) and incubated for 20 min at room temperature. The mix was added to the cells for 24 h. Treatment with SMIPs (40 μM) or vehicle was carried on for another 24 h. Cells were obtained for FACS or lysed for immunoblotting analysis as described above. Untransfected cells as well as cells transfected with non-specific siRNA were used as controls. Silencer® Negative Control siRNA No.1 from Ambion (Applied Biosciences, CA, USA) was used as non-specific control siRNA. siRNA target sequences for p27 and p21 synthesized by IDT (Integrated DNA Technologies Inc, Iowa, USA) were as follows; p27 siRNA: AAGGUUGCAUACUGAGCCAAG, and p21 siRNA: AACAUACUGGCCUGGACUGUU .
Soft agar assay
Agar Noble (Difco, MD, USA) was suspended at 6% in water and autoclaved. A dilution 1:10 was made with RPMI culture medium and added to six-well plates (2 mL). We suspended 20,000 cells were in 0.5 mL RPMI culture medium (containing drugs), added to 0.5 mL of agar (0.6%) and poured immediately into a six-well plate containing hardened bottom agar. Cells were fed with fresh medium containing DMSO, SMIP001, SMIP004 or bortezomib every third days. Images were taken after 14 days using a Nikon Eclipse E600 Microscope.