We have previously shown that melanoma cells express TRH, as well, and that TRH induces proliferation of these cells (1). Melanomas also strongly indicated the leptin receptor, whereas nevi indicated this receptor to a much lesser degree. We conclude that leptin is definitely a melanoma growth factor and that a leptin autocrine-loop may contribute to the uncontrolled proliferation of these cells. (obese) gene, exhibits a wide range of physiological functions, with tasks in energy balance, reproduction, immunity, and swelling. Best understood is the secretion of leptin by adipocytes like a satiety transmission to the hypothalamus, where neurons of the paraventricular nucleus communicate leptin receptors (3). Multiple forms of the leptin receptor (ObR), resulting from alternate mRNA splicing, have been described and are generally classified as the long form (ObRb), several short forms (ObRa, c, d, and f), and the soluble form (ObRe) (4). ObRb is definitely thought to function as the major signaling isoform and dominates in the hypothalamus where binding of leptin initiates production of TRH. TRH induces TSH production and secretion from the pituitary gland, the first step in the initiation of metabolic processes involving energy costs. Leptin receptors have been described in various types of tumor cells, including breast, prostate, colon, endometrium, while others, and leptin has been implicated as a growth element for these cancers (5C9). Based on these reports and our earlier data concerning the endocrine nature of melanoma, we hypothesized that melanoma cells communicate practical leptin receptors. Here, we statement that melanoma cells do indeed communicate these receptors which, in the presence of leptin, transmission through the MAPK pathway and induce proliferation. Furthermore, we demonstrate that melanomas also communicate leptin, providing a potential autocrine growth pathway. Materials and methods Patient material Sections of melanomas and nevi were from the M. D. Anderson Malignancy Center (MDACC) Melanoma Tumor Standard bank. Procurement of all patient materials was carried out with IRB authorization and in accordance with HIPAA recommendations. Antibodies and reagents Antibodies to human being leptin and ObR were from Santa Cruz Biotechnology (Santa Cruz, CA) for western blotting, and from R&D Systems (Minneapolis, MN) for immunohistochemistry and IF. Antibodies to ERK, phosphorylated ERK, Stat3, and phosphorylated Stat3 were purchased from Cell Signaling Technology (Beverly, MA). Recombinant human being leptin was from Sigma (St. Louis, MO) and adipocyte mRNA from Clontech (Mountain Look at, CA). Cell lines The human being melanoma L-Glutamic acid monosodium salt cell lines WM793 and WM35 were generous gifts of Dr. Robert Kerbel (Sunnybrook Health Science Center, Toronto, ON, Canada). The TXM18 melanoma cell collection was kindly provided by Dr. Janet Price (MDACC) and A375 melanoma cells were purchased from your American Type Tradition Collection (Manassas, VA). Cells were cultivated in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS). Main melanocytes were derived from neonatal foreskin and were managed as previously explained (10). Whole cell components and western blotting Cells were washed with chilly PBS and harvested into PBS with 1 mM phenylmethylsulfonyl fluoride. Cell pellets were then treated with lysis buffer (140 mM NaCl, 25 mM Tris HCl pH 7.4, and 1% NP-40) with freshly added protease inhibitor cocktail (BD Biosciences, San Jose, CA). The supernatants were collected after a 20-minute incubation and protein concentrations were measured. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and clogged for 1 hour in 5% nonfat dry milk in PBS. Main antibody was diluted in 5% nonfat dry milk/PBS/0.1% Tween and incubated overnight at 4C, followed by 45 minutes incubation with horseradish peroxidase labeled secondary antibody, again diluted in 5% L-Glutamic acid monosodium salt nonfat dry milk/PBS/0.1% Tween. Membrane development was accomplished with enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Indirect immunofluorescence Cells cultivated on chamber slides were fixed with 2% paraformaldehyde on snow for 30 minutes, clogged with 5% serum in PBS for 30 minutes at space temp, and incubated with main antibody diluted in obstructing remedy for 2 hours at 4C. This was followed by incubation with FITC-labeled secondary antibody for 1 hour at space temp. Staining was observed and imaged having a Nikon Eclipse TE 2000-U microscope equipped with a Nikon digital DXM 1200F video camera. RT-PCR RT-PCR was carried out using the GeneAmp RNA PCR Kit (Applied Biosystems, Foster City, CA) and oligonucleotide primers prepared by Sigma Genosys (The Woodlands, TX). Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription to cDNA, carried UVO out using 2 ug of total RNA, took place at 42C for 30 minutes. PCR primers and conditions are as follows: L-Glutamic acid monosodium salt Leptin:Forward: 5-CACCAAAACCCTCATCAAGACA-3Reverse: 5-TAGAGAAGGCCAGCACGTGAA-3Conditions: 95C 45 mere seconds; 58C 45 mere seconds; 72C 45 mere seconds, magnesium chloride 3.0 mM, 38 cycles.ObRb:Forward: 5-CCAGAAACGTTTGAGCATCT-3Reverse: 5-CAAAAGCACACCACTCTCTC-3Conditions: 94C 60 mere seconds; L-Glutamic acid monosodium salt 58C 60 mere seconds; 72C 60 mere seconds, magnesium chloride 5.0 mM, 35 cycles. DNA sequencing Sequencing of PCR products was performed from the MDACC DNA Core Facility using an ABI Prism.