Expression Array Analysis
Total RNA from stable PC-3(NKX3.1) and PC-3(pcDNA3.1) cells was harvested using the RNeasy Miniprep kit (Qiagen, Valencia, CA). First strand cDNA synthesis from total RNA was carried out using the GeneChip® T7-Oligo(dT) primer kit (Affymetrix, Santa Clara, CA). Second strand cDNA synthesis was performed using the SuperScript™ Choice System (Invitrogen, Carlsbad, CA). The cDNA was then processed using the GeneChip® Sample Cleanup Module (Affymetrix). Amplification and biotin labeling of antisense cRNA was carried out using the BioArray High Efficiency RNA Transcript Labeling system (Affymetrix). Finally, the GeneChip® Sample Cleanup Module (Affymetrix) was utilized to cleanup the biotinylated cRNA before it was sent out for analysis on an Affymetrix U-133 array system.
Cell Culture and Reagents
The prostate cancer cell lines PC-3 and LNCaP, and the A172 human glioblastoma cell line were obtained from the American Type Culture Collection, Rockville, MD. PC-3 and A172 cell lines are grown in Modified IMEM (Invitrogen, Carlsbad, CA) containing 10% FBS. LNCaP cells are grown in Modified IMEM with phenol red (Invitrogen) containing 10% fetal bovine serum (FBS). The PC-3 cells stably expressing the NKX3.1 expression vector are continuously grown in Modified IMEM (Invitrogen) containing 10% FBS and 1.2mg/ml G418 (Invitrogen). LNCaP cells were serum starved overnight in IMEM supplemented with 5% charcoal charcoal-stripped calf serum (CCS) and treated with 10nM R1881 for 48hr before harvesting.
Plasmids and Transfection
Full length NKX3.1 was cloned into the mammalian expression vector, pcDNA3.1 (Invitrogen) as previously described (19
). The PTEN expression vector, cloned into pcDNA3.1 was a kind gift from Charles Sawyers, Memorial Sloan-Kettering Cancer Center, NY, NY (20
). Transient and stable transfections were carried out in 75 cm2
cell culture flasks (Corning Inc., Corning, NY). Briefly, PC-3 and LNCaP prostate cancer cells were grown to 40–60% confluence and 4μg of plasmid DNA was transfected into the cell lines using Lipofectamine and Plus reagents (Invitrogen) in Opti-MEM (Invitrogen). After a 4hr incubation, the media was replaced with IMEM containing 10% FBS for an additional 24 hours. The PC-3 clones that stably express NKX3.1 were derived by transfection. After 4hr incubation in transfection reagent, PC-3 cells were trypsinized and seeded at a 1:30 density in Falcon Integrid 20mm grid tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ) in modified IMEM containing 10% FBS and 1.2mg/ml G418 (Invitrogen). The media was replaced every 4 days until colonies derived from a single cell could be seen with a light microscope. Single clone colonies were isolated with sterile cloning disks (Scienceware, Pequannock, NJ) soaked in 0.25% Trypsin-EDTA (Invitrogen) and grown to confluence in 6-well tissue culture dishes (Corning) for further study.
Western Blot Analysis
Cells were grown to 60–80% confluence and media was aspirated from the tissues culture dish. Immediately following media aspiration, lysis buffer was pipetted directly onto the cell monolayer and cells were scraped from the tissue culture flask. Cells were lysed with radio-immunoprecipitation assay (RIPA) buffer containing complete mini protease inhibitors (Roche, Nutley, NJ) and/or phosphatase inhibitors (Cell Signaling, Danvers, MA) followed by brief sonication to complete lysis. Sixty to ninety μg of total cell lysate was boiled in Novex® 2X Tris-glycine SDS sample buffer (Invitrogen) containing β-mercaptoethanol for six minutes and resolved on a 10–20% Tris-glycine SDS-PAGE gel (Invitrogen) Protein was then transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA) and probed with primary antibody concentrations, as follows; β-Actin (Sigma, St. Louis, MO) 1:10,000; NKX3.1 (2) 1:2000; IGFBP-3 (sc-9028, Santa Cruz Biotechnology, Santa Cruz, CA) 1:8,000, AKT (#9272, Cell Signaling) 1:7500, phospho-AKT Thr308 (#9275, Cell Signaling) 1:7500, at 4°C overnight, followed by three washes in PBST. HRP conjugated Goat-anti-Rabbit and Goat-anti-Mouse (ImmunoPure® antibodies, Pierce Biotechnology, Rockford, IL) secondary antibodies in 1% milk or 1% BSA were applied for 1 hour at room temperature. Signal detection was performed with Super-Signal West Pico Chemiluminescent Substrate (Pierce Biotechnology).
Reverse Transcriptase PCR analysis
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and cells were homogenized using the Qiashredder (Qiagen) method. 125–250ng of RNA was added to the RT-PCR master mix from One-step RT-PCR kit (Qiagen) (includes 5x buffer, DNTPs, and Taq polymerase). The following primers were used in the RT-PCR reactions: β-Actin (Fwd 5′-GGC CAC GGC TGC TTC-3′ and Rev 5′-GTT GGC GTA CAG GTC TTT GC-3′); NKX3.1 (Fwd 5′-GCC GCA CGA GCA GCC AGA GAC A-3′ and Rev 5′-TTC AGG GCC GGC AAA GAG GAG TG-3′); IGFBP-3 (Fwd 5′-CGC CAG CTC CAG GAA ATG-3′ and Rev 5′-GCA TGC CCT TTC TTG ATG ATG-3′); IGFBP-4 (Fwd 5′-TTA GCC CAA GAG GTC TGA GC-3′ and Rev 5′-CTG TGC TTC AAG TCT TCC TTT G-3′); Lamin A/C (Fwd 5′-AAC TTC AGG ATG AGA TGC TGC G-3′ and Rev 5′-GTC CAG AAG CTC CTG GTA CTC GT-3′). RT-PCR was performed in a Techne Techgene PCR machine; 30 min. at 50°; 15 min. at 94°; 22–30 cycles of 30s–1 min. at 94°, 30s–1 min. at melting temperatures of 55°–65°, and 30s–1 min. at 72°; followed by 15 min. at 72°. Samples were stored on ice until and mixed with 10x Blue Juice gel loading buffer (Invitrogen) and run on a 1.5% agarose gel containing 0.1μg/ml ethidium bromide in TAE buffer. Gels were imaged on a luminometer and recorded using a Kodak 1D digital camera.
Real-time RT-PCR analysis of murine prostate RNA
Frozen anterior prostates from three individual mice of each of the following genotypes: Nkx3.1+/+
, and Nkx
of animals both 4 and 12 months of age, were generously provided by Cory Abate-Shen, Columbia University, New York, NY (6
). mRNA was extracted using Qiagen RNeasy mini kit (Qiagen Inc.). The Real-Time Quantitative PCR TaqMan Assays were performed on the ABI PRISM 7700 Sequence Detection System (SDS) equipment (Applied Biosystems, Foster City, California). The primers and probe were selected for igfbp-3
using Primer Express software (Applied Biosystems). The primer sequences were: forward 5′-GCAGGCAGCCTAAGCACC-3′, reverse 5′-CCTCCTCGGACTCACTGAT-3′. The probe sequence (TCCCCTCCCAACCTGCTCCAGG) was labeled at the 5′ end with the reporter molecule 6-carboxyfluorescein (FAM) and at the 3′ with the quencher BHQ-1. Amplification of commercially available endogenous VIC labeled control, Rodent-GAPDH (Applied Biosystems), was used to standardize the amount of sample DNA added. Dilutions of DNA from the cell line (LNCaP) were used to construct standard curves for the target gene and endogenous control. TaqMan Universal PCR Master Mix was combined with 100ng of sample DNA, 900nM final concentration of primers and 100nM final concentration of the probe. All samples were analyzed as replicates of 4 wells. Relative quantitation of the data from 7700 SDS was performed using SDS 2.1 Software (Applied Biosystems).
IGF-IR Activation and Signaling
Cells were plated in 100mm culture dishes and washed twice with 1x phosphate buffered saline (PBS) before being serum starved for 14–16 hours in Modified IMEM containing 1.2mg/ml G418 (Invitrogen). Cells were then washed once with PBS and treated for 3 minutes with 100pM IGF-I (a gift from Dr. J. Toretsky, Georgetown University, Washington, DC) or Long-R3-IGF-I (GroPep) in IMEM at 37°C. The media was immediately aspirated and cells were scraped from the flask and suspended in 2X Cell Lysis Buffer (Cell Signaling) containing phosphatase inhibitors and protease inhibitors by using Complete Mini tablets (Roche). Western blot analysis was completed as described above using anti-IGF-I Receptor β (#3027, Cell Signaling), anti-Phospho-IGF-I Receptor (Tyr1131) (#3021, Cell Signaling), anti-IRS-1 (06-248, Upstate), anti-IRS-1[pY612] (44-816G, Biosource), anti-PI3K p85 (#4257, Cell Signaling), and anti-phospho-PI3K [pY458] (#4228, Cell Signaling) primary antibodies. Bands were quantified by Scion Imager software and p-values were assessed from triplicate experiments by t-test analysis using Prism Graphpad software. [* indicates p-value of <0.05, ** indicates p-value of <0.005, *** indicates p-value of <0.001].
Cell Proliferation Assay
PC-3, PC-3(pcDNA3.1), and PC-3(NKX3.1) cells were seeded in triplicate in 96-well plates at a concentration of 4000 cells per well in IMEM containing 10% FBS (PC-3) or 10% FBS plus 1.2mg/ml G418 [PC-3(pcDNA3.1) and PC-3(NKX3.1)] and incubated for 24 hr at 37°C. At 24, 48, 72, and 96 hr after seeding, wells were trypsinized, suspended in IMEM, and immediately counted in a Beckman Coulter Z1 cell counter. Doubling times were calculated using Microsoft Excel and p-values were calculated by ANOVA.
Animal studies were carried out under the approved protocol AAAA-7422 as per Columbia University’s Institutional Animal Care and Use Committee guidelines and approval. Cell lines were grown to 80% confluence in IMEM + 10% FBS + 1.2mg/ml G418 in a Hyperflask (Corning) and trypsinized with 0.25% Trypsin-EDTA (Invitrogen). Cells were resuspended in IMEM containing 10% FBS to deactivate trypsin and washed twice with PBS. Cells were then counted and resuspended in PBS at a concentration of 3×107cells/ml. Cell suspensions (150μl) were injected into 5-week old female NCr-Nude mice (Taconic Farms, Hudson, NY) on their ventral surface and tumors were measured in two dimensions once a week. We performed 20 inoculations per cell line. All measurements were performed by one observer (E.M.). Once the tumors reached 500mm3 or if illness was observed, mice were sacrificed and tumors were dissected and stored in 10% buffered formalin for paraffin embedding or in RNAlater (Qiagen) at −80°C for western blot analysis.
Cells grown under tissue culture conditions were embedded in 1% agarose before sectioning and staining. Paraffin-embedding and sectioning of the tumor xenografts and agarose cell plugs was performed by the Molecular Pathology Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University. Slides were microwaved for 5 minutes and immersed in two washes of xylene, followed by successive washes in 100%, 90%, and 70% ethanol and followed by a 5 minute wash in PBS. Slides were immersed in 10mM citrate buffer at pH 6.0 and steamed in a Black and Decker vegetable steamer for 40min. Slides were fully cooled to room temperature and washed once with PBS before the blocking step, horse serum in PBS (1:66, Pierce) for 30 min. at room temperature. NKX3.1 primary antibody (1:500, Zymed) was applied for 1 hour at room temperature followed by biotin-anti-mouse secondary antibody for 30 min. (1:200, Vector Laboratories, Burlingame, CA). This was followed by application of Vectastain Elite ABC kit (Vector Laboratories) and Vector VIP substrate kit (Vector Laboratories). Methyl green (Vector Laboratories) was used as a nuclear counter stain.
siRNA knockdown of IGFBP-3
All siRNA oligonucleotides were purchased from Dharmacon, derived from sequences published by Stewart et al
). An siRNA duplex directed against nucleotides 603-623 of IGFBP-3 mRNA (reference sequence NM_000598) (5′-AAU CAU CAU
CAA GAA AGG GCA -3′), a mismatch siRNA sequence that differs from the IGFBP-3 siRNA oligonucleotide by one base pair (5′-AAU CAU CUA CAA GAA AGG GCA -3′), and the lamin A/C positive control sequence from Dharmacon (5′-GGU GGU GAC GAU GUG GGC U -3′). This single siRNA sequence was the only one of eleven siRNA sequences tested. The ten IGFBP-3
siRNA sequences that had no effect are shown in Supplementary Table 1
cells were plated, in triplicate, in a 6-well plate in IMEM + 10%FBS + 1.2mg/ml G418 the night before the exposure to the oligonucleotides. Cells were transfected with 20uM of siRNA oligonucleotide using Lipofectamine (Invitrogen) in Opti-MEM (Invitrogen). The media was changed back to IMEM + 10%FBS + 1.2mg/ml G418 3.5 hr post transfection and knockdown of IGFBP-3 mRNA was assayed at 24 and 96 hr by RT-PCR. In the cell proliferation assay, knockdown was allowed to proceed for 24 hr before the initial cell count. Assessment of IGF-IR activation and downstream signaling 1×106
cells were plated in IMEM + 10%FBS + 1.2mg/ml G418 the night before exposure to the oligonucleotides. Cells pretreated with oligonucleotide for up to 16 hr were washed twice with PBS and serum starved for 14–16 hours. Cells were further washed with PBS and treated with 100pM IGF-I for 3 minutes at 37°C.