DB-1 melanoma cells were developed from lymph node biopsies from metastatic patients at Thomas Jefferson University, Philadelphia [17
]. The cells were grown in α
-minimum essential medium (MEM) complete medium in a 5% CO2
incubator at 37°C and stably express the pcDNA3 vector as previously described [12
]. SK Mel 28 (mutant for p53) and SK Mel 5 (wild type for p53) melanoma cell lines were obtained from American Tissue Culture Collections (Rockville, MD, USA). Quercetin (3, 3, 4, 5, 7-pentahydroxy flavone), α
-MEM and dimethylsulfoxide (DMSO) were purchased from Sigma, (St. Louis, MO, USA). TMZ was a kind gift from The Developmental Therapeutics Program, National Cancer Institute (Bethesda, MD, USA). Antibodies for Bax, p53 and Tyrosinase for western blotting were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for phosphorylated p53 (at ser 15, 37, 392), phosphorylated ATM (ser 1981), DNApk and PARP were obtained from Cell Signaling (Danvers, MA, USA). Antibody for GAPDH was purchased from Millipore-Chemicon (San Francisco, CA, USA). p73 antibody for western blotting and immunocytochemistry (ICC) was obtained from IMGENEX (San Diego, CA, USA). Sterile DMSO (0.1%) dissolved in α
-MEM complete medium was used as vehicle. Quercetin and TMZ were prepared in sterile filtered DMSO.
TMZ and Qct treatment
TMZ (20 mg/ml) was dissolved in DMSO and then dissolved in α-MEM complete medium and sterile filtered after adjusting the pH to 7.4. For combination treatments the cell lines were treated with TMZ 400 μM for 48 hr followed by Qct 75 μM for 24 hr.
Cell lysates were electrophoresed in 7 and 10% NUPAGE gels (Invitrogen Corp., CA, USA). Separated proteins were electrophoretically transferred to Hybond PVDF membrane (Amersham Pharmacia Biotech, UK) and the membrane was blocked for 1 hr by incubating the membrane in I-block (Tropix kit, Applied Biosystems, CA, USA). Primary antibodies were used at the dilutions which the manufacturers suggested. ALP conjugated goat anti-rabbit IgG was used at a dilution of 1:10000 for antibodies for phospho-p53, DNApk, Bax and PARP whereas, anti-mouse IgG was used for Total p53, Total p73, phospho-ATM and GAPDH at a dilution of 1:10000. Western detection was carried out using CDP star from Tropix kit, Applied Biosystems, CA, USA.
Annexin V-FITC staining
The p53 wild type and mutant cell lines were grown up to 50% confluency and were treated as mentioned above. Apoptosis was determined using Fluorescein isothiocyanate-conjugated Annexin V (Annexin V-FITC)/Propidium Iodide (PI) apoptosis detection kit (R&D systems, Minneapolis, MN, USA) as per manufacturer's instructions. Approximately 5 × 105 cells were resuspended in 100 μl of 1× binding buffer, 1 μl of Annexin V-FITC and 10 μl of propidium iodide. After 15 min incubation at room temperature in the dark, 400 μl of 1× binding buffer was added and the cells positive for Annexin V-FITC and/or PI were analyzed using a BD FACS flow cytometer.
RNA isolation and RT-PCR
Homogenization of cells and isolation of RNA were performed using QIAshredder spin columns and an RNeasy Kit as instructed by the manufacturer (Qiagen, Valencia, CA). 1 μg of RNA was reverse transcribed using a Super Script III Kit as instructed by the manufacturer (Invitrogen, Carlsbad, CA) and diluted 1:5 for subsequent analysis. The following PCR reaction mix was used: 5 ul of diluted cDNA, 1 ul of mixed forward and reverse primers (10 uM each), 12.5 ul SYBR Green (Qiagen), and nuclease-free water to a final volume of 25 ul. For non-quantitative PCR, cDNA was amplified for thirty cycles. Forty cycles of quantitative PCR were performed (95°C for 15 seconds, 54°C for 30 seconds, 72°C for 30 seconds) using an iQ5 Real-Time PCR Detection System (BioRad, Hercules, CA) and run on a 1% agarose gel. Real-time PCRs were run in triplicate for each cDNA sample using an iQ5 Real-Time PCR Detection System. Forty cycles of PCR were performed (as described above) with fluorescence detection during the 72°C step at each cycle. The data were analyzed using the 2-ΔΔCt
], and results were normalized to S15, which remains unchanged in response to treatment. Normalized values were plotted as relative fold over untreated. The following primers were purchased from Integrated DNA Technologies (Coralville, IA, USA): S15 [19
] transcriptionally active p73 (TAp73) [20
] and ΔNp73 [21
The cells were grown on cover slips in 100 mm tissue culture dishes and were treated with TMZ and Qct as mentioned above. The cover slips were placed in 6 well dishes and washed with PBS and fixed with 95% ethanol and 5% glacial acetic acid for 5 min. The slides were rinsed with PBS and were incubated with 0.5% of Triton X-100 in PBS for 10 min to permeabilize the membranes and rinsed again. After blocking the endogenous peroxidase with 3% hydrogen peroxide (H2O2) in PBS for 20 min the cover slips were processed according to staining procedure of the manufacturer's protocol for Histostain plus kits, Zymed Laboratories (Invitrogen, CA, USA). Total p73 antibody was used in the dilution of 1:250.
siRNA transfection was carried out according to manufacturer's protocol (Invitrogen, CA, USA). Cells were grown up to 50% confluence in antibiotic free medium in 100 mm dishes. Stealth RNAi for p73 at varying concentrations (1 nM-50 nM) was diluted in 1.5 ml OPTI-MEM I reduced serum. 30 μl of Lipofectamine™ 2000 was diluted in 1.5 ml OPTI-MEM I reduced serum medium, mixed gently. After 5 min incubation at RT, diluted oligomer was combined with diluted Lipofectamine™ 2000, mixed gently and incubated at room temp for 20 min. The oligomer-Lipofectamine™ 2000 complexes were added to each plate in OPTI-MEM I reduced serum medium by mixing gently and by rocking the plate back and forth. After 6 hrs incubation in a 5% CO2 incubator at 37°C, the plates were subjected to TMZ treatment for 48 hrs without removing the complexes. BLOCK-iT Fluorescent oligomer was used as positive control.
Transient transfection with Tyrosinase
DB-1 cells were transiently transfected with 6 μg of tyrosinase or pcDNA3 DNA using Lipofectamine™ 2000 in serum free OPTI-MEM medium for 5 hr followed by leaving the complex in Neomycin containing α MEM complete medium for at least 18 hrs as demonstrated previously [12