Culture of B16-F10 melanoma cells
Murine B16-F10 melanoma cells (from the ATCC, Rockville, MD) were cultured in serum-free Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY), pH 7.4, supplemented with 10 mM HEPES, 40 mM NaHCO3, 100 U/ml penicillin and 100 μg/ml streptomycin . Cells were harvested by incubation for 5 min with 0.05% (w/v) trypsin (Sigma, St. Louis, MO) in PBS (10 mM sodium phosphate, 4 mM KCl, 137 mM NaCl), pH 7.4, containing 0.3 mM EDTA, followed by the addition of 10% calf serum to inactivate the trypsin. Cell numbers were determined using a Coulter Counter (Coulter Electronic Inc., Miami, FL). Cell integrity was assessed by trypan blue exclusion and leakage of lactate dehydrogenase activity .
Syngenic male C57BL/6J mice (12 weeks old) from Charles River Laboratories (Barcelona, Spain) were fed ad libitum on a standard diet (Letica, Barcelona, Spain). Mice were kept on a 12-h light/12-h dark cycle with the room temperature maintained at 22°C. Procedures involving animals were in compliance with international laws and policies (EEC Directive 86/609, OJ L 358. 1, December 12, 1987; and NIH Guide for the Care and Use of Laboratory Animals, NIH Publ. No. 85-23, 1985). Experimental research on mice was performed with the approval of the ethics committee on animal research of the University of Valencia (Spain).
Transfection of red fluorescent protein
The pDsRed-2 vector (Clontech Laboratories Inc., Palo Alto, CA) was used to engineer B16-F10 melanoma clones stably expressing red fluorescent protein (RFP). Cultured B16-F10 cells were transfected as previously described . High-Performance Cell Sorting (DAKO, Copenhagen, Denmark) was used to select geneticin-resistant B16-F10 clones expressing the RFP (B16-F10-RFP) and showing high fluorescence emission. These cells were seeded in 96 wells plates, and their growth was followed by immune-fluorescence microscopy to select clones showing stable fluorescence emission.
Hepatic or lung metastases were produced by i.v. injection (portal vein or tail vein, respectively) into anesthetized mice (Nembutal, 50 mg/kg i.p.) of 105 viable B16-F10-RFP suspended in 0.2 ml DMEM. Mice were cervically dislocated 10 days after tumor cell inoculation. Livers and lungs were fixed with 4% formaldehyde in PBS (pH 7.4) for 24 hours at 4°C and then parafin-embedded. Metastases volume (mean % of organ volume occupied by metastases) was determined as earlier described .
Isolation of B16-F10 melanoma cells from metastatic foci
Isolation of B16-F10 melanoma cells from metastatic foci was performed as previously described . Briefly, tissues containing tumor cells were obtained by surgical means. Cell dispersion was carried out in minced tissue by trypsinization and collagenase digestion. Cells were washed three times in PBS and resuspended in 1 ml of ice-cold PBS, filtered through a 44-μm pore mesh and analyzed using a MoFlo High-Performance Cell Sorter (DAKO). Fluorescent B16-F10-RFP cells were separately gated for cell sorting and collected into individual tissue culture chambered slides (Nalge Nunc International Corp., Naperville, IL). Then the sorted tumor cells were harvested and plated in 25-cm2 polystyrene flasks (Falcon Labware).
Measurement of adrenocorticotropin hormone, corticosterone, and norepinephrine levels
Plasma levels of ACTH (Calbiotech, Inc., Spring Valley, CA), corticosterone (Kamiyama Biomedical Co., Seattle, WA), and NORA (IBL, Hamburg, Germany) were quantified by ELISA according to the instructions of the suppliers.
CRH expression in the brain (in situ hybridization)
Sections of 10 μm of the paraventricular nucleus (PVN) were cut according to a mouse brain atlas (Allen Insttute for brain science, http://www.brain-map.org) on a cryostat, mounted on polysine microscope slides (Menzel-Gläzer, Braunschweig, Germany), and stored at -80°C for 24 h. Then sections were fixed in 4% paraformaldehyde, further permeabilized by proteinase K treatment, acetylated twice with 0.25% acetic anhydride in 0.1 M triethanolamine, and dehydrated in a graded ethanol series.
Hybridization, carried out as described before , was performed using specific 48-mer, 35S-labeled oligonucleotide probes for murine CRH mRNA (5′-GGC CCG CGG CGC TCC AGA GAC GGA TCC CCT GCT CAG CAG GGC CCT GCA-3′) . Hybridized slices were exposed to BioMax MR film (Kodak, Rochester, NY). The mRNA expression of CRH in the PVN was quantified as gray density minus background in digitized images using the NIH ImageJ 1.6 program (http://rsb.info.nih.gov/ij). Bilateral measures were taken from two to four PVN sections for each mouse, which were pooled to provide individual means per mouse. For tissue background, the optical density of a nonhybridized region outside the PVN was measured.
Measurement of IL-6 levels
Blood samples were centrifuged at 14,000 rpm for 10 min at 4°C to separate the serum. Concentration of IL-6 in the serum was determined using commercially available mouse cytokine ELISA kits from Innovative Research (Novi, MI).
DNA binding activity of NF-κB, CREB, AP-1, and NF-IL-6
Nuclear extracts were prepared with a nuclear extraction kit (Millipore, Billerica, MA). The DNA binding activity of nuclear factor-κB (NF-κB, p65/p50) and activator protein-1 (AP-1, c-Jun/c-Fos) in nuclear extract was determined by the NF-κB or AP-1 EZ-TFA transcription factor assay kits (Millipore) according to the manufacturer’s protocols; whereas the DNA binding activity of cAMP response element-binding protein (CREB) and nuclear factor for IL-6 expression (NF-IL-6) were determined by specific ELISA-based TransAm™ (Active Motif North America, Carlsbad, CA) assay kits following manufacturer’s procedures.
Transfection of anti-IL-6 small interfering RNA
IL-6 silencing was performed as previously described in detail . The PSilencer 3.1-H1 linear vector from Ambion Inc. (Austin, TX) was used to obtain long term gene silencing. The siRNA molecules targeting IL-6 mRNA were purchased from Ambion. The RNA duplex against IL-6 had the sequence 5′-GGA CAU GAC AAC UCA UCU CTT-3′ (sense) and 5′-GAG AUG AGU UGU CAU GUC CTG-3′ (antisense). The negative control vector that expresses a hairpin siRNA with limited homology to any known sequences in mice was provided by the vector kit (Ambion). Stably transfected clones were selected in medium containing 0.5 mg/ml Geneticin (Invitrogen). Established clones were grown in medium supplemented with 10% FCS and 0.5 mg/ml Geneticin. Silencing was confirmed by immunoblotting.
GSH was determined, following procedures previously described , by liquid chromatography-mass spectrometry using a Quattro micro triple-quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with a Shimadzu LC-10ADVP pump and SCL-10AVP controller system with an SIL-10ADVP autoinjector (Shimadzu Corporation, Kyoto, Japan). Cell processing was performed according to published methodology, where rapid N-ethylmaleimide derivatization was used to prevent GSH auto-oxidation .
Cell death and cell cycle analysis
Apoptotic and necrotic cell death were distinguished by using fluorescence microscopy. For this purpose, isolated cells were incubated with Hoescht 33342 (10 mM; which stains all nuclei) and propidium iodide (10 mM; which stains nuclei of cells with a disrupted plasma membrane), for 3 min, and analyzed using a Diaphot 300 fluorescence microscope (Nikon, Tokyo, Japan) with excitation at 360 nm. Nuclei of viable, necrotic, and apoptotic cells were observed as blue round nuclei, pink round nuclei, and fragmented blue or pink nuclei, respectively. About 1,000 cells were counted each time. DNA strand breaks in apoptotic cells were assayed by using a direct TUNEL labeling assay (Boehringer, Mannheim, Germany) and fluorescence microscopy following manufacturer’s methodology. Quantitative determination of mitochondrial membrane potential, measurement of H2O2, flow cytometry determination of O2
.- generation, and measurements of cytochrome c release and caspase 3 activity, were performed as previously described . Cell-cycle phase distribution was determined by analytical DNA flow cytometry as previously described .
Compartmentation of B16-F10 cells
Cultured cells were harvested (see above), washed twice in DMEM, and resuspended in ice-cold Krebs-Henseleit bicarbonate medium (pH 7.4). Rapid separation of cytosolic and mitochondrial compartments, and calculation of mitochondrial volume, were performed as previously described .
Transient plasma membrane permeabilization was obtained using an electroporation unit for eukaryotic cells (BioRad, Hercules, CA). The field strength applied to each sample was of 1.0 kV/cm with a time constant of 50 ms.
RT-PCR and detection of mRNA
Total RNA was isolated using the TRIzol kit from Invitrogen and following the manufacturer’s instructions. cDNA was obtained using a random hexamer primer and a MultiScribe Reverse Transcriptase kit, as described by the manufacturer (TaqMan RT Reagents, Applied Biosystems, Foster City, CA). APCR master mix and AmpliTaq Gold DNA polymerase (Applied Biosystems) were then added containing the specific primers (Sigma-Genosys) previously reported  for IL-6 and glyceraldehyde-3P-dehydrogenase (GAPDH). Real-time quantitation of the mRNA relative to GAPDH was performed with a SYBR Green I assay, and a iCycler detection system (Biorad, Hercules, CA). Target cDNA was amplified as follows: 10 min at 95°C, then 40 cycles of amplification (denaturation at 95°C for 30 sec and annealing and extension at 60°C for 1 min per cycle). The increase in fluorescence was measured in real time during the extension step. The threshold cycle (CT) was determined, and then the relative gene expression was expressed as: fold change= 2–Δ(Δ
) , where Δ CT = CT target – CT GAPDH, and Δ (Δ CT) = Δ CT treated - Δ CT control.
Expression of results and statistical analyses
Data are presented as the means + S.D. for the indicated number of different experiments. Statistical analyses were performed using Student’s t test, and P values < 0.05 were considered significant.