Cell lines and Growth Conditions
The Wistar Melanoma (WM) cell lines were kindly provided by Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA) and have been described in detail elsewhere [13].
The MeWo cell line was derived from a lymph node metastasis [14]. The cell lines FEMX-I and LOX were established from metastatic lymph node biopsies obtained from melanoma patients treated at the Rikshospitalet-Radiumhospitalet Medical Center [15]. The cells were routinely cultured in RPMI 1640 medium (BioWhittaker Europe, Verviers, Belgium) supplemented with 5% fetal calf serum (FCS) (Biochrom, KG, Berlin, Germany). Phorbol-12-myristate-13-acetate (PMA) was from Sigma-Aldrich (St. Louis, MO), whereas the MEK1 inhibitor, PD98059, was from Cell Signaling Technology (Beverly, MA). Multi-cellular aggregates (spheroids) were prepared as previously described [16]. Briefly, 24-well plates were coated with 1% Seaplaque agarose (BioWhittaker Molecular Application, Rockland, ME) and tumor cells (2 × 105 cells in 1 ml complete medium) were plated on top of the solidified agarose. For thymidine incorporation assay, 5000 cells per well were plated in 96-well polyhema (Sigma-Aldrich)-coated U-bottom plates. For treatment of spheroid cultures, PMA was added when plating in suspension, whereas the inhibitors in combination experiments were added 45 min prior to plating as spheroids.
Gene expression analysis
WM35 cells were grown as spheroids for 24 hrs in the presence of PMA and PD98059, alone and in combination. Total RNA was extracted using the TRIZOL reagent (Invitrogen, Carlsbad, CA). Gene expression profiling was performed using Affymetrix U133 Plus 2.0 arrays (Affymetrix, Santa Clara, CA). For microarray hybridization, the protocol described in the Affymetrix GeneChip eukaryotic one-cycle target preparation protocol, using 5 μg of total RNA, was followed. Analysis of the data was performed by Genolyze Ltd. (Turku, Finland) using statistical software R version 2.3.0. and package collection Bioconductor version 1.8. Statistical significance was assessed using p-value from two-tailed two sample t-test. P-values are replaced with q-values to control the False Discovery Rate.
Quantitative real time RT-PCR analysis
The high capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA) was used to reverse-transcribe total RNA (0.8 μg) in a 20 μl reaction mixture using random primers. The real-time PCR analyses were performed using TaqMan Fast Universal PCR Master Mix (2×) and TaqMan Gene Expression Assay (HS00361426-ml FABP7, HS99999908-ml GUS, Applied Biosystems). A total of 0.5 μl cDNA was used in 25 μl PCR mixtures with 900 nM of each primer and 250 nM TaqMan probe. The reactions were carried out in a 7900 HT Fast Real Time PCR system (Applied Biosystems) with the following program: 95°C for 20 sec. followed by 40 cycles of 95°C for 1 sec., 60°C for 20 sec. Each sample was run in triplicate. The FABP7 relative mRNA expression level was normalized with respect to the beta-glucuronidase (GUS) gene, which had stable transcript levels under these experimental conditions. The mean from three independent experiments was calculated.
Immunoblotting
Cells were lysed in ice-cold NP-40 lysis buffer (1% NP-40, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 100 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.02 mg/ml each of aprotinin, leupeptin, and pepstatin, and 10 μl/ml phosphatase inhibitor cocktail I and II (Sigma-Aldrich)). Protein quantitation was done by Bradford analysis and 25 μg protein/lane was resolved by SDS polyacrylamide gel electrophoresis. Transfer and hybridization were as described in [17]. To ensure even loading, filters were stained with naphthol-blue black (Sigma-Aldrich) and re-stained with α-tubulin. The antibodies against FABP7 and α-tubulin were from R&D Systems (Minneapolis, MN) and Calbiochem (San Diego, CA), respectively. HRP-conjugated anti-mouse IgG secondary antibody was from Promega (Madison, WI) and HRP-conjugated anti-goat secondary antibody was from DAKO A/S (Glostrup, Denmark).
Small interfering RNA transfection
Fifty thousand cells per well were seeded in 24-well plates for 24 hrs prior to transfection with 50 nM siRNA targeting FABP7 (OligioID: HSS103516; Catalog# 1299003) or negative control siRNA duplexes (Catalog#12935-300) using Lipofectamine™ RNAiMAX transfection reagent (all reagents and siRNA were from Invitrogen). Cells were detached 48 hours after transfection and plated into agarose-coated 24-well plates as spheroids for an additional 72 hrs for assessment of apoptosis, seeded into 96-well polyhema-coated U-bottom plates for the proliferation assay and plated in BioCoat Matrigel invasion chambers.
Proliferation assay
Five thousand cells per well were seeded in 96-well polyhema-coated U-bottom plates for spheroids and in 96-well flat-bottom plates for monolayer cells and cultured for 72 hrs, the last 24 hrs with the addition of 3.7 × 104 Bq [3H]Thymidine (ARC, St.Louis, MO) Thereafter, the cells were harvested using a Filtermate Harvester (Packard Instrument Co. Meriden, CT). [3H]Thymidine incorporation was assessed in a Packard Microplate Scintillation Counter. Proliferation assays were measured in triplicate. The experiment was repeated at least three times.
Flow cytometric analysis of apoptosis
The adherent cells were harvested by Trypsin and together with detached cells fixated in 100% cold methanol. Fixed cells were washed with PBS, incubated for 30 min at 37°C in 50 μl terminal transferase (TdT) solution containing 5 units TdT (Roche, Basel, Switzerland), 10 μl 5× reaction buffer (supplied with TdT), 1.5 mM CoCl2, 0.5 nmol labeled biotin-16-dUTP, 0.1 mM dithiothreitol and distilled water. The cells were subsequently washed once in PBS containing 0.1% Triton X-100 and incubated in 50 μl 1:50 streptavidin-FITC (Amersham, Buckinghamshire, UK) in PBS (0.1% Triton X-100) and 3% skimmed dry milk for 45 min at room temperature. After washing in PBS (0.1% Triton X-100) the pellet was resuspended in PBS (0.1% Triton X-100) containing 2 μg/ml Hoechst 33258 to a final concentration of 1 × 106 cells/ml and incubated for 30 min at 4°C. Data acquisition and analysis were performed on Becton Dickinson LARII (Becton Dickinson immunocytometry systems, San Jose, CA) using Multifit software (FACSDiVa House inc., Tonsham, ME).
Matrigel invasion assay
WM35 and WM239 cells were plated in BioCoat Matrigel invasion chambers (BD Biosciences, San Jose, CA) at a cell density of 3 × 104 per chamber in RPMI 1640 supplemented with 5% fetal bovine serum (inner chamber) 48 hrs post-transfection. Self-supplied fibroblast conditioned medium was used as chemoattractant in the outer chamber. The conditioned medium was obtained from fibroblasts isolated as described by Costea et al[18] cultivated in DMEM supplemented with 10% fetal bovine serum. The medium was collected when the cells were 70% confluent. After 48 hrs incubation at 37°C and 5% CO2, non-invading cells remaining on the top surface of the chamber were removed by scrubbing with a cotton-tipped swab, and the invading cells that had adhered to the bottom surface of the chamber membranes were fixed, stained with hematoxylin and counted.
Clinical melanoma specimens
Formalin-fixed, paraffin-embedded tissue from 149 primary and 68 metastatic melanomas, as well as 11 benign nevi, was examined for expression of FABP7 protein. Of the primary tumors, 93 were classified as superficial spreading (SSM) and 56 as nodular melanomas (NM). Clinical follow-up was available for all patients. The study was approved by the Regional Committee for Medical Research Ethics in Norway.
Immunohistochemical analysis
Sections of formalin-fixed, paraffin-embedded tissue were immunostained using the two-step EnVision system (DAKO EnVision™, DAKO A/S). Deparafinized sections were microwaved in low pH buffer (pH 6.0) (DAKO) at 750 W for 5 minutes and then at 500 W for 15 minutes to unmask the epitopes. After treatment with 1% hydrogen peroxide for 5 minutes to block endogenous peroxidase, the sections were incubated with polyclonal rabbit anti-human FABP7 antibody (R&D Systems) for 30 minutes at room temperature followed by 30 minutes incubation with mouse anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The sections were then incubated with HRP-labeled secondary antibody for 30 minutes followed by 5 minutes incubation at RT with DAB substrate (DAKO A/S). All series included positive controls. Four semiquantitative classes were used to describe the number of stained cells: negative, ≤ 5%, 6–50% and >50%. Both nuclear and cytoplasmic staining was scored. Staining was evaluated by a surgical pathologist (BD). A subset of the cases (n = 50) was additionally scored by another author (AS).
Statistical analysis
Statistical analysis was performed using the SPSS program version 13.0 (Chicago, IL). The differences between FABP7 expression in benign nevi, primary melanomas and metastases were analyzed using the Chi-square test. The relationship between FABP7 expression and mean tumor thickness was evaluated nonparametrically using the Mann-Whitney two sample test. The association between expression of FABP7 and cell cycle markers was performed using the Fischer's exact test. Kaplan-Meyer estimates and the log-rank test were used for survival analysis. P < 0.05 was considered statistically significant.