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• W2074970823 abstract "The BRAF gene, encoding a mitogen-activated protein kinase kinase kinase, is mutated in several human cancers, with the highest incidence occurring in cutaneous melanoma. The activating V599E mutation accounted for 80% of all mutations detected in cutaneous melanoma cell lines. Reconstitution experiments have shown that this mutation increases ectopically expressed B-Raf kinase activity and induces NIH3T3 cell transformation. Here we used tumor-derived cell lines to characterize the activity of endogenous mutated B-Raf protein and assess its specific role in transformation. We show that three cell lines (OCM-1, MKT-BR, and SP-6.5) derived from human choroidal melanoma, the most frequent primary ocular neoplasm in humans, express B-Raf containing the V599E mutation. These melanoma cells showed a 10-fold increase in endogenous B-RafV599E kinase activity and a constitutive activation of the MEK/ERK pathway that is independent of Ras. This, as well as melanoma cell proliferation, was strongly diminished by siRNA-mediated depletion of the mutant B-Raf protein. Moreover, blocking B-RafV599E-induced ERK activation by different experimental approaches significantly reduced cell proliferation and anchorage-independent growth of melanoma cells. Finally, quantitative immunoblot analysis allowed us to identify signaling and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Although the expression of signaling molecules was not sensitive to U0126 in melanoma cells, the expression of a cluster of cell cycle proteins remained regulated by the B-RafV599E/MEK/ERK pathway. Our results pinpoint this pathway as an important component in choroidal melanoma cell lines. The BRAF gene, encoding a mitogen-activated protein kinase kinase kinase, is mutated in several human cancers, with the highest incidence occurring in cutaneous melanoma. The activating V599E mutation accounted for 80% of all mutations detected in cutaneous melanoma cell lines. Reconstitution experiments have shown that this mutation increases ectopically expressed B-Raf kinase activity and induces NIH3T3 cell transformation. Here we used tumor-derived cell lines to characterize the activity of endogenous mutated B-Raf protein and assess its specific role in transformation. We show that three cell lines (OCM-1, MKT-BR, and SP-6.5) derived from human choroidal melanoma, the most frequent primary ocular neoplasm in humans, express B-Raf containing the V599E mutation. These melanoma cells showed a 10-fold increase in endogenous B-RafV599E kinase activity and a constitutive activation of the MEK/ERK pathway that is independent of Ras. This, as well as melanoma cell proliferation, was strongly diminished by siRNA-mediated depletion of the mutant B-Raf protein. Moreover, blocking B-RafV599E-induced ERK activation by different experimental approaches significantly reduced cell proliferation and anchorage-independent growth of melanoma cells. Finally, quantitative immunoblot analysis allowed us to identify signaling and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Although the expression of signaling molecules was not sensitive to U0126 in melanoma cells, the expression of a cluster of cell cycle proteins remained regulated by the B-RafV599E/MEK/ERK pathway. Our results pinpoint this pathway as an important component in choroidal melanoma cell lines. One of the main hallmarks of cancer is the acquisition by transformed cells of self-sufficiency in growth signals. In contrast, normal cells require mitogenic growth signals transmitted by transmembrane receptors to enter the cell cycle (1Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22397) Google Scholar). The Raf/MEK 1The abbreviations used are: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; FCS, fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; KSR, kinase suppressor of Ras; EGFP, enhanced green fluorescent protein; NCM, normal human choroidal melanocyte; GST, glutathione S-transferase; Cdk, cyclin-dependent kinase. (MAPK or ERK kinase)/ERK (extracellular signal-regulated kinase) module of the mitogen-activated protein kinase cascade is a major intracellular mediator of mitogenic signaling that regulates numerous biological processes (2Peyssonnaux C. Eychène A. Biol. Cell. 2001; 93: 53-62Crossref PubMed Scopus (615) Google Scholar, 3Pearson G. Robinson F. Beers Gibson T. Xu B-E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3547) Google Scholar). Activation of this cascade downstream of membrane-bound receptors occurs through the interaction of Raf proteins with the small GTPase Ras upon its conversion to the GTP loaded, activated form. Mutations in genes encoding components of the Ras/Raf/MEK/ERK signaling pathway contribute to the development of many human cancers. Point mutations in HRAS, KRAS, and NRAS that lead to the constitutive activation of their protein products are found in ∼30% of all human cancers (4Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar), suggesting that the deregulation of downstream effectors of Ras is involved in oncogenesis. Although constitutive activation of ERK1/2 was detected in 36% of 50 studied human tumor cell lines (5Hoshino R. Chatani Y. Yamori T. Tsururo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Hohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar), no mutations have been found in either ERK1/2 or MEK1/2 in human tumors. However, mutations in the BRAF gene were detected recently in ∼20% of 530 human tumoral cell lines (6Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix- Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8325) Google Scholar). B-Raf, the major MEK activator, has a higher affinity for and is a more efficient activator of MEK1 and MEK2 than Raf-1 (7Catling A.D. Reuter C.W. Cox M.E. Parson S.J. Weber M.J. J. Biol. Chem. 1994; 269: 30014-30021Abstract Full Text PDF PubMed Google Scholar, 8Pritchard C.A. Samuels M.L. Bosh E. McMahon M. Mol. Cell. Biol. 1995; 15: 6430-6442Crossref PubMed Scopus (197) Google Scholar, 9Marais R. Light Y. Paterson H.F. Mason C.S. Marshall C.J. J. Biol. Chem. 1997; 272: 4378-4383Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 10Papin C. Denouel A. Calothy G. Eychène A. Oncogene. 1996; 12: 2213-2221PubMed Google Scholar, 11Papin C. Denouel-Galy A. Laugier D. Calothy G. Eychène A. J. Biol. Chem. 1998; 273: 24939-24947Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Two classes of BRAF gene mutation have been detected in the kinase domain of B-Raf; both of them lead to a constitutive increase in its kinase activity (6Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix- Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8325) Google Scholar). One consists of single amino acid substitutions within the glycine-rich loop located in exon 11 of BRAF. These mutations are frequently associated with a RAS gene mutation. The other consists of single amino acid substitutions within the activation loop (exon 15 of BRAF). This class is never associated with a RAS gene mutation (6Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix- Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8325) Google Scholar). B-Raf is more strongly activated by the second class of mutations, which may explain why the first class is always found associated with a RAS mutation. The highest incidence of BRAF mutations was found in cutaneous melanomas. Sixty-six percent of malignant cutaneous melanomas contain somatic missense mutations in BRAF, and the V599E single substitution in exon 15 of BRAF accounts for ∼80% of all mutations in this cancer (6Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix- Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8325) Google Scholar). In contrast to cutaneous melanomas, which have been extensively studied, little is known about the molecular pathogenesis of choroidal melanomas. Choroidal melanoma is the most frequent primary intraocular neoplasm in adult humans in western countries. Cutaneous and choroidal melanomas share a common embryological origin, the neural crest, and similar histological features, but they differ in epidemiological and cytogenetic aspects. No RAS mutations have been detected in choroidal melanoma (12Mooy C.M. Van der Helm M.J. Van der Kwast T.H. De Jong P.T. Ruiter D.J. Zwarthoff E.C. Br. J. Cancer. 1991; 64: 411-413Crossref PubMed Scopus (32) Google Scholar, 13Soparker C.N. O'Brien J.M. Albert D.M. Investig. Ophthalmol. Vis. Sci. 1993; 34: 2203-2209PubMed Google Scholar), and no data concerning BRAF mutations in choroidal melanoma were reported by Davies et al. (6Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix- Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8325) Google Scholar), leaving open the question of the involvement of the Ras/Raf/MEK/ERK signaling pathway in this cancer. In addition, despite the identification of mutations in the BRAF gene, the specific role of endogenous mutated B-Raf in tumoral progression has not been documented. For instance, the kinase activity of endogenous mutated B-Raf protein in cancer cells has not been investigated thus far. Likewise, the contribution of B-RafV599E-mediated constitutive activation of the MEK/ERK pathway in the establishment of the transformed phenotype has yet to be directly assessed in tumor-derived cell lines. In this study, we report the presence of the V599E B-Raf mutation in three short-term choroidal melanoma cell cultures. We have investigated the consequences of this mutation on the basal kinase activity of endogenous B-Raf protein and the downstream activation of the MEK/ERK module relative to normal human choroidal melanocytes. Using small interfering RNA (siRNA)-mediated inhibition of B-Raf expression in melanoma cells, we demonstrate that both constitutive activation of the MEK/ERK module and cell proliferation are linked directly to the presence of endogenous mutated B-Raf protein. We also show that one hallmark of transformation, anchorage-independent growth in soft agar, is strongly dependent on the B-Raf/MEK/ERK pathway in the choroid melanoma cell lines. Finally, we identify several signaling molecules and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Strikingly, only the expression of a subset of cell cycle proteins proved to be regulated by the B-RafV599E/MEK/ERK pathway in melanoma cells. Cell Cultures and Treatments—Normal choroidal melanocytes were isolated from post-mortem human enucleated eyes (generously provided by Dr. C.-A. Maurage, CHRU Lille, France, and Prof. P. Gain, EA 3063, Saint-Etienne, France) as described previously (14Hu D.N. McCormick S.A. Ritch R. Pelton-Henrion K. Investig. Ophthalmol. Vis. Sci. 1993; 34: 2210-2219PubMed Google Scholar). They were cultured in FIC medium (F12 Ham's medium supplemented with 20% fetal calf serum (FCS), 2.5 μg/ml fungizone/amphotericin B, 50 μg/ml gentamycin, 2 mm l-glutamine, 10 ng/ml cholera toxin, 0.1 mm isobutyl methylxanthine, and 10 ng/ml FGF2 (obtained from Dr. H. Prats (INSERM U397, Toulouse, France) (14Hu D.N. McCormick S.A. Ritch R. Pelton-Henrion K. Investig. Ophthalmol. Vis. Sci. 1993; 34: 2210-2219PubMed Google Scholar)). OCM-1 (ocular choroidal melanoma-1), MKT-BR (kindly provided by Dr. M. Jager, University of Leiden, The Netherlands), and SP6.5 cells (kindly provided by Dr. F. Malecaze, Ophthalmology Dept., CHU Toulouse, France) were grown in RPMI 1640 medium supplemented with 5% FCS, 2.5 μg/ml fungizone/amphotericin B, 50 μg/ml gentamycin, and 2 mml-glutamine (complete medium) (15Kan-Mitchell J. Mitchell M.S. Rao N. Liggett P.E. Investig. Ophthalmol. Vis. Sci. 1989; 30: 829-834PubMed Google Scholar). Normal melanocytes and melanoma cells were cultured at 37 °C in a humidified air/CO2 (19:1) atmosphere. Stock solutions of pharmacological inhibitors were made by dilutions in dimethyl sulfoxide (Me2SO) such that the final Me2SO concentration in the culture media did not exceed 0.1%. Cells were seeded in triplicate in 24-well plates at a density of 5 × 104 cells/well for normal melanocytes and 2.5 × 104 cells/well for melanoma cells. The plates were incubated for 3 days at 37 °C in a humidified air/CO2 (19:1) atmosphere. Inhibitors of Ras (FPT III inhibitor and farnesylthiosalicylic acid) and MEK1/2 (U0126) (VWR International) were added 2 h before induction of cell proliferation, on the first day of stimulation and on day 3 of the cell proliferation assay. Cell proliferation was assessed daily by two methods: 1) counting the cells remaining in the culture dish after staining with trypan blue and 2) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method (16Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46492) Google Scholar). The number of trypan blue-positive cells never exceeded 3% of the total cell population. Cells were examined by phase-contrast microscopy before being treated and before the MTT assay, to assess the degree of cell death. The percentage of growth inhibition was calculated by comparison with control Me2SO-treated cells. Plasmid Construction and Cell Transfection—The CA5 domain of murine Ksr-1 (amino acid sequence from Gly-450 to the stop codon) was cloned from pGBT9/Ksr-CA5 (17Denouel-Galy A. Douville E.M. Warne P.H. Papin C. Laugier D. Calothy G. Downward J. Eychène A. Curr. Biol. 1998; 8: 46-55Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) into the pEGFP-C1 vector (Clontech Laboratories, Inc.) such that the KSR (kinase suppressor of Ras) domain was fused to enhanced green fluorescent protein (EGFP). pcDNA3/HA1-RasN17 was described previously (18Buscà R. Abbe P. Mantoux F. Aberdam E. Peyssonnaux C. Eychène A. Ortonne J.P. Ballotti R. EMBO J. 2000; 19: 2900-2910Crossref PubMed Scopus (293) Google Scholar). Melanoma cells were seeded at a density of 6 × 104 cells/well in a 6-well plate in RPMI culture medium supplemented with 5% FCS. After 24 h cells were transfected with 300 ng of pEGFP/KSR-CA5, or pEGFP-C1 empty vector and pcDNA3/HA1-RasN17, or pcDNA3 empty vector, in the presence of 2 μg/ml LipofectAMINE 2000 reagent (Invitrogen) for 5 h. The culture medium was then replaced, and cells were cultured in fresh growth medium supplemented with 600 ng/ml G418 for 15 days. The culture medium was replaced every 3 days. Western Blot Analysis—Normal melanocytes and melanoma cells were washed twice in phosphate-buffered saline, lysed in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1% Nonidet P-40, 1% deoxycholate, 50 mm β-glycerophosphate, 0.2 mm sodium orthovanadate, 50 mm sodium fluoride, 1 μg/ml leupeptin, 5 μm pepstatin, 20 trypsin inhibitor units/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride), and centrifuged at 4 °C for 10 min at 10,000 × g. Protein concentrations were determined with the Bio-Rad protein assay reagent according to the Bradford method. Cell lysates were mixed with 3× Laemmli buffer and heated for 5 min at 95 °C. They were then resolved by SDS-PAGE (12-15% polyacrylamide gel), transferred onto polyvinylidene difluoride membranes (Immobilon 228, Millipore), and probed with polyclonal antibodies directed against MEK1/2 (dilution 1:1000, Cell Signaling Technology), ERK2 (dilution 1:1000, BD Pharmingen), ERK1/2 (dilution 1:1000, Cell Signaling Technology), or cyclin D1 (dilution 1:400, NeoMarkers). ERK1/2 and MEK1/2 activation was analyzed with polyclonal antibodies directed against phospho-MEK1/2 (Ser-217/Ser-221) and phospho-ERK1/2 (Thr-202/Tyr-204) (dilution 1:1000, Cell Signaling Technology). Horseradish peroxidase-conjugated anti-rabbit antibodies were used as secondary antibodies, and proteins were visualized by ECL (Amersham Biosciences). Quantitative Immunoblot Analysis—Protein extracts from normal human choroidal melanocytes (NCM) and OCM-1 cells were processed by the PowerBlot facility at BD Biosciences. The expression level of 120 signal transduction and cell cycle proteins was measured using a combination of SDS-PAGE (5-15% gradient) immunoblotting with specific monoclonal antibodies revealed by a secondary goat anti-mouse horseradish peroxidase antibody, acquisition of chemiluminescence with a CCD camera, and computer-aided processing of densitometric data in triplicate after normalization on the mean protein expressions. All blots were loaded with protein standard mixtures containing exportin-1, nucleoportin p62, α-tubulin, actin, KNP-1, NTF2, p190, HipR, transportin, calreticulin. Changes ≥2 were well above background variations observed in this assay. Analysis of B-Raf Expression and B-Raf Kinase Assay—Protein extracts from NCM and OCM-1 cells were immunoprecipitated with 2.5 μl of IS11 B-Raf polyclonal antibody, which recognizes all B-Raf splicing variants, as described previously (11Papin C. Denouel-Galy A. Laugier D. Calothy G. Eychène A. J. Biol. Chem. 1998; 273: 24939-24947Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). B-Raf expression was analyzed by Western blotting using a monoclonal anti-B-Raf antibody (1:2000, Santa Cruz Biotechnology), a secondary horseradish peroxidase-conjugated sheep anti-mouse antibody (1:10000 dilution, Amersham Biosciences), and ECL substrate. B-Raf expression was analyzed using a CCD camera and GeneSnap software (GeneGenome Bioimaging, Syngene). For the B-Raf kinase assay, NCM and OCM-1 cells were cultured in serum-depleted medium for 18 h. B-Raf was immunoprecipitated as described above with the monoclonal anti-B-Raf antibody using a lysis buffer supplemented with 25 mm sodium pyrophosphate. Test kinase buffer containing 25 mm sodium pyrophosphate, 200 μm ATP, and 80 ng of recombinant GST-MEK-wt (Upstate Biotechnology) was then added. After 30 min at 4 °C, 10 μCi of [32P]ATP and 1 μg of recombinant GST-ERK2 (K52R) (Cell Signaling Technology) were added. The enzymatic reaction was stopped after 15 min by adding 2× Laemmli buffer. The proteins were subjected to 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The radioactive signal was detected using a PhosphorImager (Storm 820, Amersham Biosciences) and quantified using ImageQuant software (Amersham Biosciences). To determine the relative B-Raf kinase activity, B-Raf protein amount on the membrane was then quantified by Western blotting analysis as described above using the GeneTools software (GeneGnome Bioimaging, Syngene). Inhibition of B-RafV599E Expression Using Small Interfering RNA (siRNA)—19 nucleotides of siRNA for B-RafV599E (forward, 5′-GAG-AAA-UCU-CGA-UGG-AGU-G-dTdT-3′; and reverse, 5′-CAC-UCC-AUC-GAG-AUU-UCU-C-dTdT-3′) targeting 1795 nucleotides downstream of the start codon were synthesized (Eurogentec). As a control, a nonspecific siRNA duplex containing the same nucleotides but in irregular sequence (scrambled) was used (forward, 5′-AAA-UGG-GUG-GAG-CUC-UUG-A-dTdT-3′; and reverse, 5′-UCA-AGA-GCU-CCA-CCC-AUU-U-dTdT-3′). Transfection of siRNA duplex (300 ng) was performed using LipofectAMINE 2000 reagent as recommended by the manufacturer (Invitrogen) for 5 h. Then, the transfection medium was replaced by the culture medium. Genomic DNA Purification and Mutation Screening—Genomic DNA from normal melanocytes and melanoma cell lines was prepared according to standard procedures, checked in an 0.8% agarose gel, and quantified by spectrophotometry. Polymerase chain reaction (PCR) primers were designed to amplify exons 11 and 15 of the human BRAF gene. The following primers were used: Exon 11, sense, 5′-AA-ACA-CTT-GGT-AGA-CGG-GAC-3′, and antisense, 5′-GTC-TAC-AAG-GGA-AAG-TGG-CAT-G-3′; exon 15, sense, CTT-CAT-GAA-GAC-CTC-ACA-GT and antisense, 5′-CT-GGA-TCC-ATT-TTG-TGG-ATG-3′. PCR was performed with 250 ng of genomic DNA using the Expand High Fidelity PCR kit (Roche Molecular Biochemicals). PCR products were precipitated and separated in 3% agarose gels. The 117-bp (exon 11) and 111-bp (exon 15) PCR fragments were purified and sequenced (MWG Biotech). Transformation Assay—Cell transformation was analyzed in a clonogenic assay by the ability of cells to form colonies in soft agar under anchorage-independent conditions. Melanoma cells were suspended in 0.3% agar in complete medium and plated on a layer of 0.7% agar in complete medium in 6-well culture plates (3 × 105 cells/well). U0126 was added 2 h before plating cells and then every 3 days during the culture period. Statistics—Two-tailed Student's t tests (normal distributions with equal variances) and Mann-Whitney tests (nonparametric tests) were used for statistical analysis. Constitutive Activation of the MEK/ERK Module in Choroidal Melanoma Cells—When cultured in the presence of 20% FCS, 10 ng/ml FGF2, 10 ng/ml cholera toxin, and 0.1 mm isobutyl methylxanthine, normal human choroidal melanocytes grew slowly, with a doubling time of ∼72 h (Fig. 1A). In contrast, the human choroidal melanoma cell lines OCM-1, MKT-BR, and SP-6.5 grew rapidly in medium supplemented only with 5% FCS, with a doubling time of 18-24 h (Fig. 1A). These three cell lines also proliferated in the presence of 0.5% FCS, unlike normal melanocytes despite the addition of FGF2, isobutyl methylxanthine, and cholera toxin (Fig. 1B). This suggested that a major mitogenic signaling pathway is constitutively activated in melanoma cells. We concentrated our attention on the Ras/Raf/MEK/ERK signaling cascade, a major mediator of proliferation in many cell types. Initially, we compared the activation levels of ERK1/2 following a 24-h period of serum stimulation in the three melanoma cell lines versus normal melanocytes. In the absence of serum, high basal levels of ERK1/2 phosphorylation/activation were observed in the three melanoma cell lines only (Fig. 2A). 24 h of serum stimulation slightly increased ERK1/2 phosphorylation in normal melanocytes; however, the level of phosphorylated ERK1/2 never reached that observed in the three melanoma cell lines (Fig. 2A). Kinetic analysis at different times of serum stimulation confirmed these observations (Fig. 2B). Consistent with this finding, the phosphorylation/activation of MEK1/2, the immediate upstream activators of ERK1/2, was strong and sustained in all three melanoma cell lines relative to normal cells (Fig. 2C). Interestingly, the level of MEK1/2 expression is lower in normal melanocytes than in melanoma cells (Fig. 2C; see below and Fig. 8). These data clearly show that the basal level of MEK/ERK module activation is much higher in melanoma cells than in normal choroidal melanocytes.Fig. 8Effects of MEK1/2 inhibition on signal transduction and cell cycle proteins in melanoma cells. A, effect of U0126 on cyclin D1 expression in choroidal melanoma cells. OCM-1, MKT-BR, and SP-6.5 cells were cultured in the presence (+) or absence (-) of 20 μm U0126 for 2 h. Equal amount of protein extracts were analyzed by Western blotting with an anti-cyclin D1 antibody. Similar results were obtained in three independent experiments. B, comparison of signal transduction and cell cycle proteins expression between normal melanocytes and OCM-1 melanoma cells. Protein extracts from serum-starved cells were processed as described under “Materials and Methods.” Only signal transduction and cell cycle proteins with expression levels that changed by a factor ≥2, as determined by quantitative immunoblots (X ± S.E., n = 3) in OCM-1 cells relative to normal melanocytes, are presented. C, effects of MEK1/2 inhibition on protein expression in melanoma cells. OCM-1 cells were cultured for 3 days in the presence or absence of U0126 (20 μm) for 24 h and then processed as described in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Constitutive Activation of the MEK/ERK Module Is Ras-independent in Choroidal Melanoma Cells—The small GTPase Ras is the major upstream activator of the MEK/ERK signaling pathway in response to proliferative signals. Although no Ras mutations have been detected in choroidal melanoma (12Mooy C.M. Van der Helm M.J. Van der Kwast T.H. De Jong P.T. Ruiter D.J. Zwarthoff E.C. Br. J. Cancer. 1991; 64: 411-413Crossref PubMed Scopus (32) Google Scholar, 13Soparker C.N. O'Brien J.M. Albert D.M. Investig. Ophthalmol. Vis. Sci. 1993; 34: 2203-2209PubMed Google Scholar), we nevertheless investigated whether Ras is involved in the constitutive activation of the MEK/ERK module and proliferation in choroidal melanoma cell lines. Farnesylation, a post-translational modification required for Ras membrane anchoring and activation (19Adjei A. J. Natl. Cancer Inst. 2001; 93: 1062-1074Crossref PubMed Scopus (746) Google Scholar), is prevented by treatment with FPT III inhibitor (20Wang D. Yu X. Brecher P. J. Biol. Chem. 1998; 273: 33027-33034Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Thus, we used this pharmacological compound to investigate the role of Ras in melanoma cell proliferation. Treatment of OCM-1, MKT-BR, and SP-6.5 cells with 25 μm FPT III inhibitor reduced their proliferation by 35, 38, and 29%, respectively, after 6 days of culture (Fig. 3A). A similar inhibitory effect (41Keyomarsi K. Pardee A.B. Proc. Natl. Acad. Sci. 1993; 90: 1112-1116Crossref PubMed Scopus (506) Google Scholar, 39Ewanowich C. Brynes R.K. Medeiros L. McCourty A. Lai R. Arch. Pathol. Lab. Med. 2001; 125: 208-210Crossref PubMed Google Scholar, and 32% in OCM-1, MKT-BR, and SP-6.5, respectively) was observed in melanoma cells treated with 50 μm farnesylthiosalicylic acid, a Ras antagonist that dislodges Ras from its membrane anchoring sites (Fig. 3A). To confirm the role of Ras in melanoma cell proliferation, we transfected cells with an expression vector for a dominant negative mutant of Ras, RasN17. RasN17 overexpression reduced cell proliferation in OCM-1, MKT-BR, and SP-6.5 by 38, 34, and 61%, respectively, 6 days after cell transfection (Fig. 3A). These data suggested that Ras plays a significant role in choroidal melanoma cell proliferation. Therefore, we investigated whether the inhibition of Ras, either through treatment with FPT III inhibitor or overexpression of RasN17, affected the activation of the MEK/ERK module in this context. Interestingly, ERK1/2 phosphorylation was not diminished by either inhibitory strategy in any of the three melanoma cell lines (Fig. 3, B, FPT III inhibitor, and C, Ras N17). These data suggested that the role of Ras on melanoma cell proliferation is not mediated through the activation of the MEK/ERK module and that constitutive activation of this pathway is independent of Ras activity. A Somatic Mutation in the BRAF Gene Induces High Basal Kinase Activity of Endogenous B-Raf Protein in Choroidal Melanoma Cells—The results presented above strongly suggested that constitutive activation of the MEK/ERK pathway in choroidal melanoma cells occu" @default.
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