Cell lines and culture conditions
PC-3 and DU-145 cell lines were obtained from ATCC (Rockville, MD). Cells were cultured in RPMI 1640 (Bio-Whittaker, Walkersville, MD) medium supplemented with 10% FBS (Hyclone, Logan, UT), gentamicin (50 mg/l), sodium pyruvate (1 mM) and non-essential amino acids (0.1mM) under conditions indicated in the figure legends. RWPE-1 and RWPE-2 cells were maintained in Keratinocyte-Serum Free medium supplemented with 5 ng/ml of human recombinant EGF and 0.05 mg/ml of bovine pituitary extract.
Antibodies and Reagents
Antibodies to p50, IκBα TOPO I and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary horseradish peroxidase-conjugated donkey anti-rabbit antibodies were purchased from Amersham (Arlington Hts., IL). TPEN was obtained from Sigma (St. Louis, MO). IκBα dominant mutant (IκBαM) adenovirus was purchased from Imgenex (San Diego, CA).
Atomic Absorption Spectroscopy
Harvested cells were rinsed three times in PBS, digested in 2% SDS and boiled for 10 minutes. Protein concentrations were measured with BCA protein assay (Pierce, Rockford, IL). Zn levels were measured by flame mode using a Shimadzu AA-6300 atomic absorption spectrophotometer.
Stable Transfection of PC-3 cells with hZIP1
To create the hZip1 C-end FLAG-tagged expression vector, hZip1 ORF was obtained by PCR with 5′-atcttg aagctt gcc acc atg gga ccg tgg gga gag cca gag ctc ctg gtg-3′ (forward) and 5′-atcttg tctaga tta att aat cta ctt atc gtc gtc atc ctt gta atc gat ttg gat gaa gag cag gcc-3′ (reverse) primers using pRC-CMV-hZip1vector [20
] (a kind gift from Dr. R. Franklin (University of Maryland)) as a template and then cloned into the HindIII/Xba1 restriction sites of the pRC-CMV plasmid. PC-3 cells were transfected with either the hZIP1 expression vector or the CMV control vector using the TransIT-Prostate transfection kit (Mirus Bio, Madison, WI). Selection was performed using G418 (1.5 mg/mL; Invitrogen/Life Technologies), and screening of clones was based on Western Blot analysis with anti-hZIP1 antibody to determine hZIP1 expression. Stable transfectants were maintained in medium containing G418 (500 μg/mL).
Colony formation assay in soft agar
A single cell suspension of 1.5×103 PC-3 cells in 0.3% low melting point agar was plated onto a base layer of hardened 0.6% agarose. Cultures were allowed to grow for 18 days with or without various concentrations of zinc in the form of ZnSO4, colonies were stained with Thiazolyl Blue Tetrazolium Bromide (Sigma, St. Louis, MO) and analyzed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Each experimental and control group consisted of three wells.
Western Blot Analysis
Nuclear and cytoplasmic extracts and whole cell lysates were prepared as described previously [21
]. Protein concentrations were measured with BCA protein assay reagents (Pierce, Rockford, IL). Equivalent amounts of proteins (20 μg) were mixed with an equal volume of 2X Laemmli sample buffer, boiled and resolved by electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The proteins were transferred from the gel to a nitrocellulose membrane using an electroblotting apparatus (Bio-Rad) (15 V, 3 mA/cm2
for 24 minutes). Membranes were incubated in blocking solution containing 5% nonfat dry milk overnight to inhibit nonspecific binding. The membranes were then incubated with specific antibody (1–3 μg/ml) for 2 hours. After washing in TRIS/0.1% Tween 20 for 30 minutes the membranes were incubated for another 30 minutes with horseradish peroxidase-conjugated secondary antibody. The membranes were then washed and developed with enhanced chemiluminescence (ECL Western Blotting Kit, Amersham, Arlington Heights, IL).
Cells were pre-incubated with various concentrations of TPEN for 2 hours. DNA binding activity of NF-κB (p50) was examined in nuclear extracts according to the protocols provided with the TransAM kit (Active Motif, Carlsbad, CA).
Luciferase reporter assay
Cells were transfected with pNF-κB-luc (Stratagene, La Jolla, CA) and pRL-TK (Promega, Madison, WI) plasmids. Twenty-four hours after transfection, cells were treated with various concentrations of TPEN for 4 hours in fresh medium. Samples were assayed for firefly and renilla luciferase activities using Dual-Glo Luciferase assay System (Promega) and normalized as instructed by the manufacturer.
Measurement of VEGF, IL-6 and IL-8 proteins
Cells were treated in the presence or absence of IκBαM adenovirus (1:500 dilution) for 24 hours and then incubated with TPEN (6μM) for 18 hours. IL-6, IL-8 and VEGF levels in cell culture supernatants were determined by ELISA kits (R&D Systems, Minneapolis, MN).