Tissue Specimens and Tissue Microarrays
Paraffin-embedded sections of prostate samples from 482 patients with prostate cancer were obtained as archival specimens from the Department of Pathology at the University Hospital in Malmö. Hematoxylin–eosin (H&E)–stained slides of prostate biopsies were examined by a national board–certified pathologist (L. Helczynski). Selected areas of prostate cancer and corresponding benign samples were applied in duplicate to tissue microarray constructs of 1.0 mm in diameter. The study was approved by the Ethics Committee, Lund University, and the Helsinki Declaration of Human Rights was strictly observed.
Immunohistochemistry and Quantification of Angiogenesis
Immunohistochemistry on tumor tissue arrays was performed as previously described (21
). The slides were incubated with the following primary antibodies: anti–cyclin A1, 1:50 (Pharmingen, San Diego, CA); anti-VEGF, 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA); anti-MMP9, 1:100 (Chemicon, Temecula, CA); anti-MMP2, 1:100 (Lab Vision, Fremont, CA); and anti-AR, 1:200 (Dako, Glostrup, Denmark A/S). Anti-rabbit or anti-mouse peroxidase–conjugated secondary antibodies were applied. Diaminobenzidine colorimetric reagent solution from Dako was used. Slides were counterstained with hematoxylin (Sigma, St Louis, MO). The specimens were viewed with an Olympus BX51 microscope at magnification of ×20 or ×40. The staining intensity was scored as 0 (negative), 1 (weakly positive or positive), 2 (moderate positive), 3 (strongly positive), or 4 (very strong positive) using an arbitrary semiquantitative scale as previously described (21
). The scores are representative for at least 60%–70% of the area of tissue analyzed, and the highest score for both cytoplasmic and nuclear compartments was considered in the statistical analyses. For analysis of tumor angiogenesis, tumor sections were stained with an antibody against CD31 (BD Pharmingen, San Diego, CA, and Dako). Regions of high vascular density within the tumors were examined. The number of CD31-positive pixels per microscopic field was recorded. At least two sections per tumor and three views per section were determined.
Cell Culture and Androgen R1881 Treatment
Human prostatic cell lines LNCaP and PC3 were purchased from the American Type Culture Collection (Manassas, VA), and treatment with the synthetic androgen R1881 (Dupont/NEN, Boston, MA) was performed as previously described (22
). Time-resolved analyses of cyclin A1–GFP fusion protein in living cells in response to R1881 treatment were performed using a Fluorescence Zeiss Apoptome Axiovert microscope at ×40 magnification.
Retroviral- and Adenoviral-Mediated Transduction
Micro-RNA–adapted short hairpin RNA (shRNAmir) against human cyclin A1 in pSM2 vector was obtained from Open Biosystems (Huntsville, AL). The shRNAmirCyclin A1 sequence was subcloned to MSCV-LTRmiR30-PIG (LMP) vector (Open Biosystems) with Xho
I and Eco
RI restriction enzymes. Clones were verified by restriction site analysis and sequencing. For production of retrovirus, HEK 293 packaging cells were transiently transfected with shRNAmir-cyclinA1-1α-LMP, VSVG, and Gag-Pol plasmids by use of Lipofectamine 2000 (Invitrogen, Inc, Carlsbad, CA). Media containing viruses were collected every 12 hours, and supernatants were added to the culture medium containing PC3 cells followed by further incubation for 14 hours. PC3 cells were then selected with 5 μg/mL of puromycin (Sigma) for 1 week, and the knockdown of cyclin A1 was tested by immunoblotting and reverse transcription–polymerase chain reaction (RT-PCR). Adenoviruses containing cyclin A1 were prepared by standard protocols as previously described (23
). Adenovirus-cyclin A1 vector was also generated by cloning full-length cyclin A1 cDNA into the Xba
I and Sal
I sites of adenoviral vector pShuttle-PGK-EGFP vector kindlly provided by Dr. Xiaolong Fan (Department of Molecular Medicine and Gene Therapy, Lund University). This vector contains Ad5-PGK1-IRES-EGFP-B-globin intron-poly A and pShuttle backbone. Cyclin A1 cDNA was cloned between PGK1 and IRES. This adenoviral vector carrying cyclin A1 cDNA was designated as Ad5-Cyclin A1. The control vector was the empty adenoviral vector expressing EGFP and was designated as Ad5-Control. These vectors were used for the induction of long-term expression of cyclin A1 in prostate cancer cell lines.
Mouse Models of Tumor Growth and Metastasis
Three sets of mouse experiments were performed. All animal experiments met the requirements of Lund University Animal Care Facility and the National Institutes of Health guidelines. To analyze of the effects of cyclin A1 overexpression in subcutaneous tumor xenografts, 40 athymic nude mice aged 8–12 weeks (Harlan Nederland, Horst, The Netherlands) were used. For measurement of tumor growth, 10 of these mice were injected subcutaneously with PC3 cells infected with adenoviruses expressing cyclin A1–EGFP and 10 mice were injected with PC3 cells infected with adenoviruses expressing control-EGFP (4 × 106 cells per mouse). Tumor diameters were measured twice weekly using calipers, and volumes were calculated using the equation a(b2/2), where a and b represent the larger and smaller diameters, respectively. Mice were killed 6 weeks after inoculation of tumor cells. Tumor invasion and metastasis was examined in the remaining 20 mice (10 injected with PC3 cells expressing cyclin A1–EGFP and 10 with PC3 cells expressing control-EGFP) that were killed 12–14 weeks after injection. For assessment of cyclin A1 knockdown in subcutaneous tumor xenografts, 20 mice were injected with PC3 cells infected with retroviruses containing cyclin A1 shRNA-EGFP or control shRNA-EGFP (4 × 106 per mouse, 10 mice per group). For assessment of cyclin A1–overexpressing tumors in orthotopic tumor xenografts, eight mice were injected in their prostates with PC3 cells (1 × 106 cells per mouse) infected with adenovirus expressing cyclin A1–EGFP or control-EGFP (four mice for each tumor type). Mice were killed by CO2 asphyxia or by cervical dislocation. Prostates, lymph node, liver, lung, brain, spleen, and femurs were removed from each mouse. (These tissues have been taken from all the mice used in this study.) Half of the tissues were used for histology and immunohistochemical analysis. For histology analysis, tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were stained with H&E and were subjected to analysis under microscopy. The other half of the tissues were snap frozen in liquid nitrogen for use in protein analyses.
For immunohistochemistry of mouse organs, tissues were fixed for 24 hours in 4% paraformaldehyde and embedded in paraffin. The following antibodies were used: anti–cyclin A1, 1:50 (US Biological) and anti–cyclin A1, 1:100 (Santa Cruz Biotechnology); anti–cytokeratin-5, 1:200 (CK5) and anti-CD31, 1:200 (BD Pharmingen and Dako); and anti-VEGF, 1:100 (Santa Cruz Biotechnology).
Detection of VEGF Expression by Enzyme-Linked Immunosorbent Assay
The enzyme-linked immunosorbent assay (ELISA) for the detection of human VEGF in tissue lysates was performed as previously described (21
). Briefly, tissue homogenate was diluted to the final concentration of 1 mg/mL and 100 μL of the lysate was used for measuring human VEGF using the VEGF ELISA kit (R&D Systems, Minneapolis, MN).
Cell Synchronization and Cell Cycle Analysis
To obtain cells in G0 cell cycle phase, LNCaP cells were starved in RPMI-1640 containing 0.1% fetal bovine serum for 48 hours as previously described (24
). Cells were then fed with medium containing 10% FBS for 6 hours (G1 phase), followed by growth in medium containing 200 μM mimosine dissolved in ethanol (Sigma) for additional 24 hours (late G1 phase). To obtain G1/S cells, 2 μg/mL aphidicolin in dimethyl sulfoxide (Sigma) was added to the cells; after 24 hours, cells were washed and cultured in medium containing 10% FBS for additional 6 hours (S phase). Nocodazole (0.1 μg/mL) in DMSO (Sigma) was then added into the medium, and cells were grown for 30 hours (G2/M phase). Cells were harvested at different time points. A total of 1 × 106
cells were fixed and permeabilized. Cells were then washed twice with phosphate-buffered saline (PBS) containing 10% fetal calf serum and resuspended and incubated in PBS containing 5 μg/mL propidium iodide (Sigma), 100 mM sodium citrate, pH 7.3, and 0.05 mg RNase A (Sigma) for 20 minutes at 4°C. The cell fluorescence was measured in a FACSCalibur cytofluorometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed using CELL Quest software (Becton Dickinson).
BrdU Proliferation Assay
The effect of cyclin A1 expression on cell proliferation was determined using the nonradioactive BrdU–based cell proliferation assay (Roche, Basel, Switzerland) according to the manufacturer’s protocol. Twenty-four hours after transfection, PC3 cells (3 × 103 cells per well) were incubated for 24 hours in a 96-well plastic plate that contained complete growth medium (triplicate cultures). BrdU was then added, and cells were cultured for another 8 hours. BrdU incorporation into the DNA was determined by measuring the absorbance at both 450 and 690 nm on an ELISA plate reader.
The invasion of PC3 cells was measured using Boyden transwell chambers (Chemicon) according to the manufacturer's protocol. Briefly, the cells were seeded onto the membrane of the upper chamber at a concentration of 3 × 104 in 400 μL medium. RPMI-1640 medium containing 10% FBS was then added to the lower chamber. Cells that passed through the Matrigel-coated membrane to the lower chamber were stained with crystal violet and were then dissolved in 10% acetic acid. The absorbance of the stained cells was measured on an ELISA plate reader.
uPA Activity Assay and Zymography
uPA activities in conditioned medium of cells transfected with small interfering RNA (siRNA) targeting uPA and control siRNA was measured with a colorimetric uPA activity assay kit (Millipore, Billerica, MA) following the manufacturer's protocol. Zymography was performed using Novex Zymogram Gels (Invitrogen) following the manufacturer's protocol. Briefly, PC3 cells were transfected with pCMS-EGFP-Cyclin A1 or control pCMS-EGFP vector (see below), or cells were stably transduced with shRNA against cyclin A1; 24 hours after transfection or seeding the stably transduced cells, culture supernatants were collected, and equal amount of volumes were subjected to gelatin zymography experiment (Invitrogen).
Preparation of Nuclear and Cytoplasmic Extracts
Nuclear and cytoplasmic fractions from control LNCaP cells or LNCaP cells treated with R1881 were prepared using the subcellular fractionation kit from Pierce (Rockford, IL) following the manufacturer's protocol.
Plasmids, Transfection, siRNA, and Luciferase Assay
The pCMS-EGFP-Cyclin A1 and pCMS-EGFP plasmids have been described previously (21
). Human cyclin A1 cDNA (1.8 kb) was cloned into pCMS-EGFP plasmid at the Xho
I and Sal
I restriction sites. These vectors were used in experiments assessing the effects of induction of transient expression of cyclin A1 in prostate cancer cell lines. AR-pcDNA3.1 was kindly provided by Dr Steven Balk (Beth Israel Deaconess Medical Center, Harvard Medical School). pcDNA3.1 was purchased from Invitrogen. PSA-luc and control constructs were kindly provided by Dr Hans Lilja (Memorial Sloan-Kettering Cancer Center, New York, NY). VEGF promoter constructs were kindly provided by Dr Dieter Marme (Tumor Biology Center, Freiburg, Germany) (25
). The fragment designated as 2068 contains the full-length promoter; fragment 1340 consists of two activator protein-1 (Ap-1) sites; fragment 840 contains only one Ap-1 site; and fragments 102, 105, 316, and 415 lack the Ap-1 motifs. Transient transfection of pCMS-EGFP-Cyclin A1, pCMS-EGFP, AR-pcDNA3.1, or luc vectors containing different fragments into prostate cancer cell lines was performed using the electroporation system with Nucleofection kit (AmaxaBiosystems, Gaithersburg, MD) according to the manufacturer's instructions. In luciferase assay, 100–200 ng of luc plasmids were used. Luciferase activity was measured using the Luciferase Assay kit (Promega, Madison, WI) following the manufacturer's protocol. siRNAs against MMP2, uPA, and VEGF and scrambled siRNA were purchased from Ambion, Austin, TX. siRNA (50 nM) in 3.5 μL of nuclease-free water was transfected into 1 × 104
PC3 cells in a total volume 250 μL of Optimem medium using 7 μL of Lipofectamine 2000 (Invitrogen). Cells were used 24 hours later for invasion assays.
Immunoblotting and Immunoprecipitation Assays
Immunoblot analysis was performed as previously described (21
). Co-immunoprecipitation of cyclin A1 and AR was performed as previously described (23
). Protein extracts were separated on 12% sodium dodecyl sulfate (SDS)–polyacrylamide gels and were subsequently transferred to nitrocellulose membrane Hybond ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were probed with appropriate primary antibodies, including: anti–cyclin A1, 1:200 (Pharmingen and US Biological, Swampscott, MA) and anti–cyclin A1, 1:500 (Santa Cruz Biotechnology); anti-AR, 1:1000 (Dako); anti–β-actin, 1:1000 and anti–lamin B, 1:1000 (Santa Cruz Biotechnology); anti-uPA, 1:500 (American Diagnostics, Stamford, CT); and anti–prostate-specific antigen (PSA), 1:1000 (code 2E9) (26
). Membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies (Amersham Life Sciences, Alesbury, UK) at 1:5000 dilution and visualized using the Enhanced ChemiLuminescence detection system (ECL) and ECL films (Amersham Pharmacia Biotech).
RNA Preparation, RT-PCR Analysis, and cDNA Microarray Analysis
RNA was isolated from PC3 cells as previously described (21
). cDNA was synthesized from 2 μg of RNA (Invitrogen) and was used for semiquantitative RT-PCR or real-time RT-PCR. β-Actin was used as an internal control. Primers for human VEGF were previously described (27
). The other primers were as follows: cyclin A1: 5′-CTCCTCTCCCAGTCTGAAGA-3′ and 5′-CAGGAAGTTGACAGCCAGAT-3′; MMP2: 5′-CGGCCGCAGTGACGGAAA-3′ and 5′-CATCCTGGGACAGACGGAAG-3′; MMP9: 5′-GACGCAGACATCGTCATCCAGTTT-3′ and 5′-GCCGCGCCATCTGCGTTT-3′; MMP11: 5′-CGATGTGACGCCACTCACCTTT-3′ and 5′-GGCCAGGGCTGGCCATATA-3′; MT1MMP: 5′-CCAGGGTCTCAAATGGCAACATAATGAAA-3′ and 5′-CCATGGAAGCCCTCGGCAAA-3′; GAPDH: 5′-AACAGCGACACCCACTCCTC-3′ and 5′-GGAGGGGAGATTCAGTGTGGT-3′; and β-actin: 5′-CGCGAGAAGATGACCCAGATC-3′, 5′-TCACCGGAGTCCATCACGA-3′. For cDNA microarray analysis, the experimental procedures have been described in Varambally et al. (28
). We used expression profiling data that were available in the Gene Expression Omnibus (GEO) under accession number GSE3325
(available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3325
). We used the data from benign prostate tissues (n = 6, N1–N4, NX1, NX2, GEO Profiles numbers: GSM74875–GSM74880), clinically localized primary prostate cancer (n = 7, P1–P5, PX1-2-tissue pools, GEO Profiles numbers: GSM74881–GSM74887), and metastatic prostate cancer (n = 6, W1–W4, WX1-2-tissue pools, GEO Profiles numbers: GSM74888–GSM74893).
LNCaP cells were incubated with 0.1 nM R1881 for 1 hour and then rinsed with PBS. Protein–DNA complexes were then cross-linked with 5% formaldehyde in serum-free medium for 5 minutes. The samples were precleared with 40 μL of protein A/G beads, and the supernatant was collected. Immunoprecipitation was performed on 0.5–1 mg protein with antibody against cyclin A1 or IgG (negative control) by rocking samples with 40 μL G beads and 200 μg/mL salmon sperm DNA at 4°C in total volume of 500 μL for 3 hours. After incubation, beads were extensively washed with low-salt buffer (RIPA) and DNA–cyclin A1 complexes were eluted with 200 μL elution buffer (1% SDS, 1× Tris-Acetate Buffer) by rocking the beads at room temperature for 15 minutes. To dissociate histone–DNA complexes, 20 μL of 5 M NaCl was added and incubated for 4 hours at 65°C. To release DNA fragments, proteinase K was added in incubation buffer (10-μL 0.5 M EDTA, 20 μL 1 M Tris–HCl, pH 6.5) and incubated for 1 hour at 45°C. DNA was purified by phenol–chloroform extraction. For PCR the following primers were used: human VEGF promoter: 5′-CTGGCCTGCAGCAGACATCAAAGTGAG-3′ and 5′-CTTCCCGTTCTCAGCTCCACAAAC-3′ (amplicon: −913 to −783 bp in the promoter); and human MMP2 promoter: 5′-TGTTCCCTAAAACATTCCCC-3′ and 5′- GTCTCTGAGGAATGTCTTCT-3′ (amplicon: −1340 to −1120 bp in the promoter).
Sequence-Based Structure Prediction
The secondary structure of cyclin A1 was predicted by using the SABLE server (29
Possible pairwise correlations between the groups were analyzed using the Spearman rank correlation test. The statistical significance of differences between groups was evaluated by paired Wilcoxon rank sum test and Student t test or Mann–Whitney tests (comparison of means), and the Fisher exact test was used for comparison of incidence, as indicated. For measurement of tumor growth, two-way repeated analysis of variance (ANOVA) was performed using ANOVA test. All outcome variables are presented as the mean values with 95% confidence intervals (CIs). All data presented are representative of at least three independent experiments. All statistical tests were two-sided, and P values less than .05 were considered to be statistically significant.