Chemical reagents and antibodies
Copper chloride (CuCl2) at a cell culture tested grade was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3Vo4, 1 μg/ml leupeptin) was from Cell Signaling Technology (Danvers, MA, USA). Anti-ATOX1, anti-Cox17 (cytochrome C-17), anti β-actin mouse monoclonal antibodies, and horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG secondary antibody were all purchased from Novus Biologicals (Littleton, CO, USA). Anti-CCS antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell lines and cell culture
Human non-small cell lung carcinoma (NSCLC) cell lines (A549, H460, H1299, H1355, H1703 and SKLU-1) were purchased from ATCC (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco-BR, Grand Island, NY, USA), with 10% fetal bovine serum purchased from Atlanta Biologicals (Lawrenceville, GA, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin purchased from Biosource International (Camarillo, CA, USA). Antibiotic G418 was purchased from Sigma. For copper-stimulated cell proliferation assay, cells were cultured in DMEM medium containing 2% fetal bovine serum (FBS) supplemented with various doses of CuCl2.
Construction of plasmid vectors encoding ATOX1 shRNA
To prepare plasmid vectors encoding shRNA specific for ATOX1 (pATOX1-shRNA), ATOX1 shRNA sequences were designed and cloned downstream of the SP3 promoter in a plasmid vector from SABiosciences, Inc. (Frederick, MD, USA), using a method specified by the manufacturer. Scramble RNA sequence with no homology to any human mRNA by BLAST search (National Center for Biotechnology Information, NIH, Bethesda, MD, USA) was used for construction of a control plasmid vector (pSCR-shRNA). The nucleotide sequence of ATOX1-shRNA and SCR-shRNA in the plasmid vector was confirmed by sequencing prior to use for testing the knockdown of ATOX1 in NSCLC cells. Efficacy of ATOX1 shRNA for knockdown of ATOX1 expression was evaluated by immunoblot assay of cell lysates of SKLU-1 NSCLC cells (ATCC) stably transfected with the pATOX1-shRNA plasmid vector DNA after selection of G418-resistant stable transfected cells.
Construction of lentiviral vectors encoding AOTX1 shRNA
For constant suppression of ATOX1 expression in NSCLC cells, a lentiviral vector encoding ATOX1 shRNA (Lenti-ATOX1-shRNA) was constructed for knockdown of ATOX1, in a method similar to that previously described (23
). Briefly, the ATOX1 shRNA was designed, and the efficacy of ATOX1 shRNA for knocking down the expression of ATOX1 was determined by western blot analysis, using cell lysates of the NSCLC cells transfected with the plasmid vectors containing different ATOX1 shRNA sequences. Subsequently, pLenti-ATOX1-shRNA vector was prepared by cloning of an ATOX1 shRNA sequence with confirmed efficacy for knocking down expression of ATOX1 at the Bam
H1 and Eco
R1 site of a pGreenPuro lenti-shRNA expression vector from System Biosciences (Mountain View, CA, USA). To prepare the lentivirus encoding ATOX1 shRNA, 293 cells were transfected with a mixture of pLenti-ATOX1-shRNA and lentiviral packaging plasmid DNA. At 72 h post-transfection, the supernatant containing lentivirus particles was harvested, and the titers (pfu/ml) of the Lenti-ATOX1-shRNA virus were determined using the UltraRapid Lentiviral Titer kit from System Biosciences. A stock solution of the Lenti-ATOX1-shRNA virus was stored at −80°C for use within 6 months post-preparation.
Western immunoblot assay
NSCLC cells were infected with the Lenti-ATOX1-shRNA virus and subjected to puromycin selection to obtain the cells in which expression of ATOX1 was constantly suppressed. Western immunoblot assay was performed to examine the expression of ATOX1 in the NSCLC cells infected with Lenti-ATOX1-shRNA, in a method similar to that previously described (25
). Briefly, cell lysates were prepared using cell lysis buffer (Cell Signaling Technology), and the total protein concentration of the supernatant was determined using the Bio-Rad BCA protein assay kit (Hercules, CA, USA). Following separation of cellular proteins (30–50 μg) by SDS-PAGE (12%) and transfer onto a PVDF membrane, the blots were blocked in 5% de-fatted milk powder in PBST (130 mM NaCl, 10 mM NaH2
, pH 7.4, 0.1% Tween-20) at 4°C overnight. The blocked blots were incubated with mouse anti-ATOX1 monoclonal antibody (1:1,000 dilution) at 37°C for 1 h, followed by washing with PBST and incubation with HRP-conjugated rabbit anti-mouse secondary antibody (Novus) in 5% milk/PBST at room temperature for 1 h. Immunoreactivity on the blots was visualized by chemiluminescence using a Supersignal Western Blot Enhancer kit (Pierce, Rockford, IL, USA). Following stripping of the blots using a blot stripping buffer from Pierce, the blots were reprobed with anti-β actin antibody (1:10,000) for visualization of the sample loading control. Semi-quantitative analysis of the western immunoblot was conducted by densitometry using AlphaEase FC™ software (Alpha Innotech, Santa Clara, CA, USA), in a method modified from that previously described (26
). The integrated density value (IDV) measured from ATOX1 and β-actin bands on blots was used for calculation of the percentage of ATOX1 suppression or silencing in the cells transfected with the plasmid DNA encoding shRNA targeting ATOX1 or the cells transduced by the lentiviral vector encoding shRNA targeting ATOX1, relative to ATOX1 expression in wild-type cells. Briefly, IDV of the β-actin band for a test sample was normalized to IDV of the β-actin band of the wild-type cell sample to minimize variation in the protein loading controls. A ratio of ATOX1 expression in the cells transduced with ATOX1 shRNA vectors relative to the ATOX1 expression in wild-type cells was calculated by dividing IDV of the ATOX1 band from the cells transduced with ATOX1 shRNA vectors with IDV of the ATOX1 band from the wild-type cell lysates. Subsequently, the normalized ratio of ATOX1 expression was obtained following normalization of the ATOX1 expression ratio against the β-actin loading control. Finally, the percentage of ATOX1 suppression or silencing was calculated as (1 − normalized ATOX1 expression ratio × 100%).
Cell proliferation assay
Cell proliferation assay was conducted using an MTT cell proliferation assay kit purchased from ATCC (Manassas, VA, USA), using the protocol specified by the manufacturer based on a previously described method (25
). Briefly, cells were inoculated into a 96-well plate (1×103
cells/well) and cultured in DMEM without serum under serum starvation conditions for 24 h. Subsequently, the cells were incubated with DMEM supplemented with CuCl2
dissolved in PBS (10 μM) at 37°C for up to 72 h. At 24 h post inoculation, MTT agent solution was added, and the optical density (OD) was measured at a wavelength of 570 nm with a reference wavelength of 630 nm. The cell numbers at the time of the MTT assay were recorded as percentages of the cell numbers initially inoculated. The experiment was conducted in triplicate for each time point and repeated three times.
Immunohistochemical (IHC) analysis of copper chaperone expression in tumor tissue samples was conducted using a a previously described method (12
), according to a protocol approved by the Institutional Review Board, University of Texas Southwestern Medical University at Dallas, Texas. Briefly, formalin fixed, paraffin-embedded, archived tissue samples from the tissue bank were subjected to de-paraffining and antigen retrieval by steam cooking for 5 min in citrate at pH 8.0. After quenching of the endogenous peroxidase and blocking with 5% BSA for 30 min, sections were incubated with mouse monoclonal anti-ATOX1 antibody (1:200), anti-Cox17 antibody (1:200), or anti-CCS antibody (1:200), at room temperature for 2 h, followed by visualization of immunoreactivity with HRP-labeled rabbit anti-mouse IgG secondary antibody (ABC method). Immunoreactivity of copper chaperones (ATOX1, CCS or Cox17) was examined under a microscope. Semi-quantitative analysis of ATOX1 expression in tissue samples was conducted by visual assessment of the intensity of immunoreactivity on tissue samples, assuming that the intensity of immunoreactivity was correlated with the level of expression. Intensity of immunoreactivity was scored visually and recorded as following: 3 + for strong, 2+ for moderate, 1+ for weak immunoreactivity, and 0 for negative immunoreactivity.
The percentages of cells determined from the MTT assay are expressed as means ± SD. In order to determine whether growth of the cells of two groups (Lenti-ATOX1-shRNA vs. Lenti-SCR-shRNA cells) differed between the three time-points (24, 48 and 72 h), a (2 × 3) mixed design ANOVA was applied, where the between-subject factor was the group (with 2 levels) and the within-subject factor was time (with 3 levels). These tests were applied for MTT assays in the presence and absence of CuCl2 at the indicated concentration. If a significant (time × group) interaction was determined for each of the comparisons, it was followed up with an independent sample t-test for each of the 3 time points (24, 48 and 72 h). A P-value of <0.05 was considered to indicate a statistically significant result.