title: Exposure of normal human melanocytes to a tumor promoting phorbol ester reverses growth suppression by transforming growth factor beta.
abstract: Transforming growth factor-beta (TGF-beta), a potent inhibitor of normal melanocyte growth, does not significantly suppress growth of melanoma cells. The mechanism of melanocyte desensitization to TGF-beta in the transformation process remains largerly unknown. We investigated whether the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) may induce melanocyte resistance to TGF-beta. Cell proliferation and DNA synthesis of normal human melanocytes were strongly inhibited by TGF-beta, whereas in the presence of TPA remained largerly unaffected. The inactive phorbol ester 4alpha-phorbol 12,13 didecanoate did not modify the TGF-beta antiproliferative effect, whereas the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol counteracted TGF-beta effects. Protein kinase C (PKC) is the major cellular receptor of tumor promoting phorbol esters. PKC-alpha expression and phosphorylation were almost completely downregulated under combined treatment with TGF-beta + TPA at 24 and 72 h, as shown by immunoblots. Confocal microscopy demonstrated that TGF-beta-induced nuclear accumulation of PKC-alpha was abolished in the presence of TPA at the same time points. The selective PKC inhibitor Ro-31-8220 weakened the TGF-beta antiproliferative effect. Smads are central mediators for TGF-beta signal transduction. Smad-dependent transcriptional activity was suppressed in TGF-beta-treated melanocytes in the presence of TPA, as well as in ALK5 (constitutively active type I TGF-beta receptor)- or Smad3 + Smad4-transfected melanocytes in the presence of Ro-31-8220. In addition, an antisense oligodeoxynucleotide against PKC-alpha abolished TGF-beta-driven Smad-mediated transcription. These findings show that tumor promoting phorbol esters induce melanocyte resistance to TGF-beta, associated with downregulation of PKC-alpha and suppression of Smad-dependent transcription. This may represent an important mechanism for expansion of melanocytes exposed to PKC-targeting tumor promoters.
authors.affiliation: Department of Dermatology, Faculty of Medicine, University of Crete, Heraklion, Greece.
authors.forename: Melanthia
authors.initials: M
authors.lastname: Stavroulaki
authors.forename: Dimitris
authors.initials: D
authors.lastname: Kardassis
authors.forename: Ekaterini
authors.initials: E
authors.lastname: Chatzaki
authors.forename: George
authors.initials: G
authors.lastname: Sakellaris
authors.forename: Carsten
authors.initials: C
authors.lastname: Lindschau
authors.forename: Hermann
authors.initials: H
authors.lastname: Haller
authors.forename: Androniki
authors.initials: A
authors.lastname: Tosca
authors.forename: Konstantin
authors.initials: K
authors.lastname: Krasagakis
mesh_terms: D051017:Apoptosis Regulatory Proteins; D002273:Carcinogens / Q000494:pharmacology*; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008297:Male; D008544:Melanocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D012867:Skin / Q000166:cytology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D051017:Apoptosis Regulatory Proteins; D002273:Carcinogens; D016895:Culture Media, Serum-Free; C412403:DIABLO protein, human; D004791:Enzyme Inhibitors; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D053773:Transforming Growth Factor beta1; D008156:Luciferases; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate; C064758:Ro 31-8220