June 2008
Volume 49, Issue 6
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Biochemistry and Molecular Biology  |   June 2008
17-AAG and 17-DMAG–Induced Inhibition of Cell Proliferation through B-Raf Downregulation in WTB-Raf–Expressing Uveal Melanoma Cell Lines
Author Affiliations
  • Narjes Babchia
    From the Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France;
    Université Paris Descartes, Paris, France;
    INSERM, Paris, France; and
  • Armelle Calipel
    From the Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France;
    Université Paris Descartes, Paris, France;
    INSERM, Paris, France; and
    Service Universitaire d’Ophtalmologie, CHRU, Caen, France.
  • Frédéric Mouriaux
    From the Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France;
    Université Paris Descartes, Paris, France;
    INSERM, Paris, France; and
    Service Universitaire d’Ophtalmologie, CHRU, Caen, France.
  • Anne-Marie Faussat
    From the Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France;
  • Frédéric Mascarelli
    From the Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France;
    Université Paris Descartes, Paris, France;
    INSERM, Paris, France; and
Investigative Ophthalmology & Visual Science June 2008, Vol.49, 2348-2356. doi:10.1167/iovs.07-1305
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      Narjes Babchia, Armelle Calipel, Frédéric Mouriaux, Anne-Marie Faussat, Frédéric Mascarelli; 17-AAG and 17-DMAG–Induced Inhibition of Cell Proliferation through B-Raf Downregulation in WTB-Raf–Expressing Uveal Melanoma Cell Lines. Invest. Ophthalmol. Vis. Sci. 2008;49(6):2348-2356. doi: 10.1167/iovs.07-1305.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) has been shown to have promising results in antitumor activity through the degradation of the activated V600E mutant of B-Raf (V600EB-Raf) in cutaneous melanoma cell lines. It has different effects, however, on the wild-type form of B-Raf (WTB-Raf), according to the WTB-Raf activation levels in the tumor cells. Uveal melanoma cells express WTB-Raf and only rarely express V600EB-Raf. This study was conducted to investigate the effects of HSP90 inhibition on uveal melanoma cell lines.

methods. Human uveal melanoma cell lines were treated with the HSP90 inhibitors 17-AAG and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG). Cell proliferation was assessed by MTT staining, and apoptosis was quantified by flow cytometry. Analysis of the expression of HSP90 and activation of the MEK/ERK downstream signaling of B-Raf was performed by Western blot. Effects of the downregulation of the HSP90 cochaperone, cdc37, on cell proliferation and activation of MEK/ERK was investigated by siRNA strategy.

results. The inhibition of HSP90 downregulated B-Raf, decreased cell proliferation, and reduced activation of MEK/ERK in uveal melanoma cell lines expressing WTB-Raf. HSP90 inhibition also reduced the expression of Akt, but the inhibition of Akt had no effect on cell proliferation, ruling out a role of Akt in the 17-AAG–induced inhibition of cell proliferation. The downregulation of cdc37 did not affect MEK/ERK signaling and cell proliferation, demonstrating that the cochaperone was not required for HSP90-controlled stability of B-Raf. c-Kit was also downregulated after HSP90 inhibition. The combination of 17-DMAG with imatinib mesylate, the inhibitor of c-kit, had synergistic inhibitory effects on cell proliferation in WTB-Raf uveal melanoma cell lines.

conclusions. These results suggest that targeting HSP90 in tandem with c-Kit inhibition may be a promising therapeutic approach to uveal melanoma.

Uveal melanoma is the most common primary ocular neoplasm in adults in developed countries. Unlike cutaneous melanoma, which has been extensively studied, little is known about the molecular pathogenesis of uveal melanoma. Cutaneous and uveal melanomas share similar histologic features. However, mutations in genes encoding major signaling pathway proteins that control cell proliferation, including those affected in cutaneous melanoma, are infrequent in uveal melanoma. 1 2 3 4 5 6 7 8 Our previous study demonstrated the role of the B-Raf/MEK/ERK signaling pathway in the rare uveal melanoma cell lines expressing the activating V600E mutation in B-Raf (V600EB-Raf), indicating that oncogenic V600EB-Raf activates a similar signaling pathway for tumorigenesis in uveal and cutaneous melanomas. 1 No B-Raf kinase activity and no constitutive activation of ERK1/2 have been detected in cutaneous melanoma cells expressing the wild-type form of B-Raf (WTB-Raf). 9 In contrast, constitutive ERK1/2 activation has been shown in primary tumors and cell lines of uveal melanoma expressing WTB-Raf, highlighting a major difference between cutaneous and uveal melanomas. 10 11 12 More recently, we demonstrated WTB-Raf–mediated control of ERK1/2 activation for cell proliferation and transformation in uveal melanoma cell lines. 10 In fact, uveal melanoma cell lines expressing WTB-Raf grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as did melanoma cell lines expressing V600EB-Raf. The receptor c-Kit autocrine loop participated in the cell proliferation and transformation of WTB-Raf–expressing uveal melanoma cell lines through the activation of ERK1/2. 13 These data together suggest that inhibiting B-Raf/ERK signaling may be a powerful method for treating uveal melanoma. Unfortunately, no validated therapy specifically inhibits B-Raf function in patients. 14 15  
The 90-kDa heat shock protein (HSP90) is a protein chaperone required for folding, activation, and assembly of many proteins associated with the initiation and development of cancer. HSP90 client proteins include the receptor c-Kit, the signaling proteins MEK, Raf-1, and Akt, and the cell cycle regulator cyclin D1, which are deregulated in cancers. 16 17 18 19 HSP90 inhibition results in the proteasome degradation of the HSP90 client proteins and leads to potent antitumor activity. 17 20 Overexpression of HSP90 has been observed in uveal melanomas, and we detected an overexpression of its cochaperone protein, cdc37, in the OCM-1 uveal melanoma cell line. 1 21 Uveal melanoma cells may, therefore, be particularly dependent on HSP90 for their growth, and HSP90 client proteins may be potential targets for therapeutic strategies against uveal melanomas. The chemical HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), characterized by its ability to disrupt the function of HSP90, has been shown to have promising results in antitumor activity 22 and is undergoing clinical tests in patients with advanced-stage cancer. 17 20 Recent findings that endogenous WTB-Raf is weakly or not at all sensitive to 17-AAG-induced HSP90 inhibition, whereas endogenous V600EB-Raf is rapidly degraded after 17–1AAG treatment in cutaneous melanoma cells, indicate that HSP90 is required for V600EB-Raf but not for WTB-Raf stability. 23 24  
However, the role of HSP90 and the effects of 17-AAG have never been studied in uveal melanoma. For this reason, we studied the effects and mechanisms of action of 17-AAG and its more soluble derivative, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), on human uveal melanoma cell lines. We investigated the effects of HSP90 inhibition on the expression levels of members of the B-Raf/MEK/ERK signaling pathway, the activation levels of the MEK/ERK module, and the expression levels of proteins downstream of MEK/ERK. We showed that the B-Raf/MEK/ERK signaling pathway is targeted by 17-AAG and 17-DMAG for cell proliferation through the control of B-Raf expression, the subsequent activation of the MEK/ERK module in WTB-Raf uveal melanoma cell lines. Moreover, the combination of 17-DMAG with imatinib mesylate, the inhibitor of c-kit, had synergistic inhibitory effects on cell proliferation in WTB-Raf uveal melanoma cell lines. Therefore, targeting HSP90, alone or in combination with c-kit inhibitor, may provide an interesting strategy against uveal melanoma. 
Methods
Cell Cultures
Cell lines 92.1, Mel270 (provided by Martine Jager, University of Leiden, The Netherlands), OCM-1 (provided by François Malecaze, Ophthalmology Department, CHU Toulouse, France), and TP31 (provided by Sylvain Guerin, Centre Hospitalo-Universitaire de Quebec, Canada) were grown in RPMI 1640 medium supplemented with 5% FCS, 2.5 μg/mL fungizone/amphotericin B, 50 μg/mL gentamicin, and 2 mM l-glutamine (Gibco/BRL, Invitrogen, Carlsbad, CA), as previously described. 1 13 Cells were cultured at 37°C in a humidified air/CO2 (19:1) atmosphere. 
Cell Proliferation Assay
We investigated cell proliferation by treating cells with specific pharmacologic inhibitors of signaling pathways. Stock solutions were made up in dimethyl sulfoxide (DMSO), with a final concentration of DMSO in the culture media not exceeding 0.1%; this concentration has been shown to have no effect on melanoma cell proliferation. Cells were seeded in triplicate in 24-well plates at a density of 1.5 × 104 cells/well. The plates were incubated for 3 days and then treated with the MEK inhibitor UO126, the Akt inhibitors Akt inhibitor II and Akt inhibitor V/triciribine (VWR; Calbiochem, La Jolla, CA), the c-Kit tyrosine kinase inhibitor imatinib mesylate (Glivec/STI571; Novartis Pharma AG, Basel, Switzerland), and the HSP90 inhibitors 17-AAG and 17-DMAG. Inhibitors were added 1 hour before the induction of cell proliferation and at induction, and the activator was then added at the indicated times. The number of viable cells was determined by using the MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric method after 3 days of culture in low (0.5% FCS) or normal (5% FCS) serum conditions. The percentage of growth inhibition was calculated with respect to control DMSO-treated cells. 
Cell Cycle Progression Analysis
We analyzed cell cycle progression by determining the cell DNA content with propidium iodide. 25 Cells were washed in PBS and fixed in ice-cold 70% ethanol by incubation at 4°C for at least 2 hours. The cells were rehydrated in cold PBS, treated with 1 mg/mL RNase A (Boehringer Mannheim, Mannheim, Germany), and stained with 50 μg/mL propidium iodide by incubation at 4°C for at least 15 minutes. Stained cells were analyzed by flow cytometry (Epics ALTRA; Beckman Coulter, Fullerton, CA). 
Transformation Assay
We analyzed cell transformation, with the use of a clonogenic assay, by determining the ability of cells to form colonies in soft agar under anchorage-independent conditions. Melanoma cells were suspended in complete medium containing 0.3% agar and either pharmacologic inhibitors or vehicle. The cells were then plated on a layer of 0.7% agar in complete medium in six-well culture plates at a density of 3 × 105 cells/well and were incubated at 37°C for 2 weeks. Pharmacologic inhibitors or vehicle were added every 3 days during the culture period. Colonies in three randomly chosen 9-cm2 areas were counted on day 10 of the culture period. 
Western Blot Analysis
Cells were washed twice in PBS, lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% NP-40, 1% deoxycholate, 50 mM β-glycerophosphate, 0.2 mM sodium orthovanadate, 50 mM sodium fluoride, 1 μg/mL leupeptin, 5 μM pepstatin, 20 kIU/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride), and centrifuged for 10 minutes at 10,000g at 4°C. Protein concentrations were determined using a Bio-Rad (Hercules, CA) kit. Cell lysates were mixed with 3× Laemmli buffer and heated for 5 minutes at 95°C. They were then resolved by SDS-PAGE (10% or 15% polyacrylamide gels), electroblotted onto polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore, Billerica, MA), and probed with polyclonal antibodies directed against ERK1/2 (dilution 1:1000; Cell Signaling Technology, Beverly, MA), MEK1/2 (dilution 1:1000; Cell Signaling Technology), B-Raf (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), Raf-1 (dilution 1:1000; Santa Cruz Biotechnology), HSP90 (dilution 1:10 000; Santa Cruz Biotechnology), and CDC37 (dilution 1:10,000; Santa Cruz Biotechnology). We used a polyclonal antibody directed against phospho-ERK1/2 (T202/Y204; dilution 1:1000; Cell Signaling Technology) to analyze the activation of these kinases during melanoma cell proliferation. Membranes were probed with a rat monoclonal antibody directed against α-tubulin (dilution 1:4000; Serotec, Oxford, UK) or with a goat antibody directed against actin (dilution 1:1000; Santa Cruz Biotechnology) to control for equal loading. Primary antibodies were tagged with specific secondary horseradish peroxidase-conjugated antibodies. Antibody complexes were detected by enhanced chemiluminescence (ECL; Amersham, Amersham, UK), and the membrane was placed against Kodak film (BioMax Light-1; Eastman Kodak, Rochester, NY). Quantification was carried out using a Kodak image station (2000 MM) and Kodak software (1D3.6). 
ERK Phosphorylation Assay
ERK activation was determined with a fast-activated, cell-based, enzyme-linked immunosorbent assay kit (FACE; Active Motif Inc., Carlsbad, CA) according to the manufacturer’s instructions. Briefly, cultured cells placed in 96-well plates were further cultured for 3 days and then treated with or without UO126 and 17-AAG. The inhibitors were added 1 hour before stimulation with culture medium and at stimulation. At the indicated times, the cells were fixed with 4% formaldehyde for 20 minutes, extensively washed, and incubated with a specific anti-phosphorylated ERK1/2 antibody, followed by incubation with a secondary horseradish peroxidase-conjugated antibody. Phosphorylated ERK1/2 levels were quantified with a colorimetric readout and are expressed as the absorbance at 450 nm measured in each well. 
Gene Silencing
Uveal melanoma cells were plated at 50% to 60% confluence in complete melanoma cell culture medium in 12-well plates and incubated for 24 hours. Cells were then transiently transfected in medium (Optimem I; Invitrogen) with reagent (Lipofectamine 2000; Invitrogen) and siRNA for cdc37 (siRNA gene silencers; Santa Cruz Biotechnology). We used a nonspecific siRNA duplex containing the same nucleotides but in an irregular sequence (scrambled) as a control. We replaced the medium (Optimem I; Invitrogen) with complete culture medium after 5 hours. With the use of an MTT assay and Western blotting, as previously described, 1 we assessed the effects of cdc37 downregulation on cell proliferation and B-Raf and Raf-1 expression and on ERK1/2 activation and expression 72 hours after transfection. 
Statistical Analysis
The two-tailed Student’s t-test (normal distribution with equal variance) and the Mann–Whitney U test (nonparametric) were used for statistical analysis. 
Results
Inhibition of Cell Proliferation, Arrest in G1 Cell Cycle, and Loss of B-Raf Protein Expression in 17-AAG–Treated B-Raf Uveal Melanoma Cell Lines
HSP90 inhibition with 17-AAG has no effect on WTB-Raf, whereas it induces V600EB-Raf degradation in cutaneous melanoma cell lines. 23 Therefore, we hypothesized that the exposure of WTB-Raf uveal melanoma cell lines to 17-AAG may have no significant effect on cell proliferation, whereas 17-AAG should reduce the proliferation of V600EB-Raf uveal melanoma cell lines. In the first set of experiments, we compared the effects of 17-AGG on cell proliferation in the WTB-Raf and V600EB-Raf uveal melanoma cell lines. Cells were treated with various concentrations of 17-AAG in the presence of serum, and cell proliferation was analyzed using the MTT method. Surprisingly, cell treatment with 17-AAG reduced proliferation in a concentration-dependent manner in WTB-Raf uveal melanoma cell lines (Fig. 1A) . In control experiments, we showed that 17-AAG also reduced the proliferation of V600EB-Raf uveal melanoma cell lines, confirming the effects of the HSP90 inhibitor on the V600EB-Raf–dependent cell proliferation (Fig. 1A) . Maximum inhibition of cell proliferation (73%–81% inhibition) was observed with 3000 nM 17-AAG, with no significant difference between WTB-Raf and V600EB-Raf uveal melanoma cell lines (Fig. 1A) . We quantified the efficacy of 17AAG by determining the concentration of the HSP90 inhibitor needed to inhibit cell proliferation by 50% (IC50). IC50 values were similar in the WTB-Raf and V600EB-Raf melanoma cell lines after culturing for 72 hours (WTB-Raf cells, 111–172 nM; V600EB-Raf cells, 105–123 nM; Fig. 1B ). This is in contrast to the data showing that 17-AAG affects cutaneous melanoma cell lines differently, depending on the expression of WTB-Raf or V600EB-Raf. 23 Therefore, we conducted similar experiments in serum-free conditions to avoid the potential effect of serum on 17-AAG. Serum-free conditions resulted in similar results. IC50 values showed that WTB-Raf uveal melanoma cell lines were as sensitive to 17-AAG as V600EB-Raf uveal melanoma cell lines (WTB-Raf cells, 60–97 nM; V600EB-Raf cells, 65–77 nM; Fig. 1B ). This discounts the presence of serum as a factor, ruling out this explanation for the absence of a difference of sensitivity of WTB-Raf and V600EB-Raf uveal melanoma cell lines to 17-AAG. Similar expression levels of B-Raf, MEK1/2, ERK1/2, and HSP90, and of its cochaperone, cdc37, were observed in the four melanoma cell lines, suggesting that alteration in the expression levels of these proteins does not account for the sensitivity of WTB-Raf melanoma cell lines to 17-AAG (see 2 Fig. 3A ). Cell transformation is also controlled by B-Raf in uveal melanoma cells. Therefore, we investigated whether 17-AAG affects cell transformation in the WTB-Raf uveal melanoma cell lines by examining the ability of cells to grow under anchorage-independent conditions. Treatment of 92–1 cells with 17-AAG markedly inhibited colony formation, and the efficiency of 17-AAG to inhibit WTB-Raf uveal melanoma cell transformation was similar to that observed in the OCM-1 cells (V600EB-Raf cells; Fig. 1C ). 
We then investigated more precisely the mode of action of 17-AAG in uveal melanoma cell lines by analyzing cell cycle and apoptosis. Flow cytometry analysis showed that in WTB-Raf melanoma cell lines, the cell cycle stopped at G1 (82% [G1 phase of the cell cycle in 17-AAG–treated cells] vs. 70% [untreated cells]) after 48-hour treatment with 17-AAG (Fig. 2A) . We observed similar results in V600EB-Raf melanoma cell lines (83.5% [G1 phase of the cell cycle in 17-AAG–treated cells] vs. 59.7% [untreated cells]; Fig. 2A ). Moreover, cells underwent apoptosis after 48 hours of 17-AAG treatment, with cell death rates increasing by factors of 5.6 and 7.8 in WTB-Raf and V600EB-Raf melanoma cell lines, respectively (Fig. 2B) . Apoptosis was accompanied by the activation of caspase 9, detected by cleavage of the caspase (Fig. 2C) . By contrast, no activation of caspase 3 was detected in 17-AAG–treated uveal melanoma cell lines (Fig. 2D) . Together these data show that 17-AAG inhibits proliferation and transformation and induces G1 cell-cycle arrest and apoptosis similarly in WTB-Raf and V600EB-Raf uveal melanoma cell lines. 
Raf-1 is a client protein of HSP90, and 17-AAG–induced disruption of the Raf-1/HSP90 complex results in the degradation of Raf-1. 26 27 We investigated the effects of 300 nM 17-AAG on Raf-1 expression, a concentration that reduced cell proliferation by 70% to 80%. Cell treatment with 17-AAG induced large, similar decreases in Raf-1 expression levels in WTB-Raf and V600EB-Raf uveal melanoma cell lines over 72 hours (reductions by 77% in WTB-Raf and 85% in V600EB-Raf on day 3; Figs. 3B 3C ). Therefore, WTB-Raf and V600EB-Raf cells were similarly sensitive to 17-AAG. In contrast to cutaneous melanoma cell lines, the similar effects observed of 17-AAG on the inhibition of cell proliferation in the WTB-Raf and V600EB-Raf uveal melanoma cell lines cannot be explained by the effects of 17-AAG on Raf-1 expression levels in WTB-Raf uveal melanoma cell lines because Raf-1 was not involved in cell proliferation in these cells. 10 Therefore, we investigated the role of B-Raf in the mechanism of action of 17-AAG in uveal melanoma cell lines. We suggested that, in contrast to cutaneous melanoma cell lines, 17-AAG induces a decrease in B-Raf expression in WTB-Raf uveal melanoma cell lines. Cell treatment with 300 nM 17-AAG decreased the B-Raf expression levels (reduction by 53% on day 3) in WTB-Raf melanoma cell lines over the 72-hour culture period (Figs. 3B 3C) . In control experiments, we show that cell treatment with 300 nM 17-AAG also decreased the B-Raf expression levels (reduction by 85% on day 3) in V600EB-Raf melanoma cell lines (Figs. 3B 3C) . Similar B-Raf expression levels were observed in the four untreated melanoma cell lines, suggesting that B-Raf overexpression does not account for the lower sensitivity of WTB-Raf melanoma cell lines to 17-AAG (Fig. 3A) . The kinetics of B-Raf downregulation with various concentrations of 17-AAG (30–1000 nM) confirmed that WTB-Raf is a target of 17-AAG (Figs. 4A 4B)
Inhibition of Constitutive Activation of MEK/ERK and Subsequent Decrease in Cyclin D1 and Increase in p27 Expression 17-AAG–Treated B-Raf Uveal Melanoma Cell Lines
In contrast to WTB-Raf cutaneous melanoma cell lines, B-Raf–induced activation of the MEK/ERK module controls cell proliferation of WTB-Raf uveal melanoma cell lines. 10 Therefore, we analyzed the effects of 17-AAG on the activation levels of the MEK/ERK module. 17-AAG induced a rapid, large downregulation of ERK1/2 phosphorylation/activation over the 72-hour culture period in WTB-Raf uveal melanoma cell lines (Fig. 5A) . As expected, cell treatment with 17-AAG also induced a rapid, large decrease in ERK1/2 phosphorylation in V600EB-Raf uveal melanoma cell lines over the 72-hour culture period (Fig. 5A) . Cell treatment with 17-AAG had no effect on ERK1/2 expression levels in WTB-Raf or V600EB-Raf uveal melanoma cell lines. Thus, ERK1/2 expression levels are not involved in the downregulation of ERK1/2 phosphorylation/activation. We used FACE (Active Motif Inc.)-based ELISA to quantify ERK1/2 phosphorylation relative to total ERK1/2 after cell treatment with 17-AAG in uveal melanoma cell lines. 17-AAG induced similar decreases in ERK1/2 activation in WTB-Raf and V600EB-Raf uveal melanoma cell lines (Fig. 5B)
MEK1, the direct upstream activator of ERK1/2, is a client protein for HSP90 and a target of 17-AAG in various cell lines. 16 Therefore, we investigated whether the ERK1/2 deactivation-mediated inhibition of cell proliferation might have resulted from MEK1 inhibition or from the downregulation of MEK1 expression in 17-AAG-treated melanoma cell lines. We infer these facts—by showing that the levels of MEK1 expression were similar in 17-AAG–treated and untreated cells in WTB-Raf and V600EB-Raf uveal melanoma cell lines over the 72-hour culture period—using Western blot analysis (Fig. 6A) . MEK1/2 phosphorylation levels decreased in a manner and with kinetics similar to those of ERK1/2 phosphorylation in 17-AAG-treated WTB-Raf and 17-AAG-treated V600EB-Raf uveal melanoma cell lines (Fig. 6A)
siRNA-mediated downregulation of cyclin D1 expression reduced the proliferation of uveal melanoma cell lines. 10 In cutaneous melanoma cell lines, treatment with 17-AAG induces the downregulation of cyclin D1, 28 which may also be true for uveal melanoma cell lines. 17-AAG similarly decreased cyclin D1 expression levels in WTB-Raf and V600EB-Raf uveal melanoma cell lines (Fig. 6B) . Inhibition of uveal melanoma cell proliferation was accompanied by p27 overexpression. 29 We observed a significant increase in the expression levels of p27 after treatment with 17-AAG in WTB-Raf and V600EB-Raf melanoma cell lines (Fig. 6B)
No Role of Akt in 17-AAG–Induced Inhibition of Cell Proliferation in Uveal Melanoma Cell Lines
Phosphoinositiol-3 kinase (PI3K) inhibition with a high concentration of LY294002 greatly reduced the proliferation of uveal melanoma cell lines. 25 Moreover, the various forms of Akt, downstream targets of PI3K, are also targets of 17-AAG and are involved in the 17-AAG–induced inhibition of cell proliferation and transformation. 30 31 This may also be the case in uveal melanoma cells. We observed no significant difference in Akt expression and activation/phosphorylation levels in WTB-Raf and V600EB-Raf uveal melanoma cell lines (Fig. 7A) . Cell treatment with 17-AAG completely decreased the expression levels and the subsequent activation of Akt in WTB-Raf uveal melanoma cell lines (Fig. 7A) . However, cell treatment with the Akt inhibitor, triciribine/Akt inhibitor V, which inhibits the activation of the three Akt forms, had no inhibitory effect on the proliferation of the four uveal melanoma cell lines (Fig. 7B) . Similar results were obtained with Akt inhibitor II (Fig. 7B) . Akt inhibition had no effects on the activation levels of ERK1/2 (data not shown). These results rule out a role for Akt in the 17-AAG–mediated inhibition of cell proliferation in uveal melanoma cell lines. 
Treatment with 17-AAG has been shown to downregulate the expression levels and the kinase activity of c-Kit. 30 c-Kit autocrine loop-induced activation plays a major role in the constitutive ERK1/2 activation-mediated cell proliferation of WTB-Raf uveal melanoma cell lines. 13 We investigated the effects of 17-AAG on c-Kit expression levels. Interestingly, cell treatment with 17-AAG completely reduced c-Kit expression levels in the Mel270 WTB-Raf uveal melanoma cells, indicating that 17-AAG targets c-Kit in addition to B-Raf for the control of cell proliferation in c-Kit-positive WTB-Raf uveal melanoma cell lines (Fig. 7C)
No Role of cdc37 in the HSP90-Mediated Control of the B-Raf/MEK/ERK Signaling Pathway for Cell Proliferation in 17-AAG–Treated B-Raf Uveal Melanoma Cell Lines
We investigated the role of cdc37 in uveal melanomas, given that it is an HSP90 cochaperone and that it is overexpressed in uveal melanoma cell lines and binds to WTB-Raf and V600EB-Raf in cutaneous melanoma cell lines. 1 23 32 We investigated the role of cdc37 in the control of the B-Raf/MEK/ERK signaling pathway and cell proliferation using an siRNA-based approach to inhibit cdc37 protein expression. Uveal melanoma cell lines treated with lipofectamine had no effects on cdc37 expression, Raf-1 and B-Raf expression, or ERK1/2 phosphorylation (Fig. 8A) . By contrast, transfection with cdc37-specific siRNA downregulated cdc37 protein expression substantially but did not affect Raf-1 or B-Raf expression in V600EB-Raf and WTB-Raf melanoma cell lines (Fig. 8A) . Therefore, cdc37 was not necessary for HSP90-mediated downregulation of B-Raf in uveal melanoma cell lines, and the phosphorylation of ERK1/2 was not affected by cdc37 downregulation (Fig. 8A) . Consequently, siRNA-mediated cdc37 downregulation did not affect the proliferation of WTB-Raf and V600EB-Raf uveal melanoma cell lines (Fig. 8B) . Cell treatment with a combination of 17-AAG and cdc37-specific siRNA had similar effects on the inhibition of cell proliferation, as did cell treatment with 17-AAG alone (Fig. 8B) . Thus, cdc37 is not necessary for HSP90-mediated control of the B-Raf/MEK/ERK signaling pathway for cell proliferation in uveal melanoma cell lines. 
Additive Inhibitory Effects of 17-DMAG with Imatinib Mesylate, the c-Kit Inhibitor, on the Proliferation of Uveal Melanoma Cell Lines
Uveal melanoma cell lines were treated with 17-DMAG, a recently developed and more soluble analogue of 17-AAGs. 33 Cell treatment with 17-DMAG reduced proliferation in a concentration-dependent manner in WTB-Raf and V600EB-Raf uveal melanoma cell lines (Fig. 9A) . Maximum inhibition of cell proliferation with 17-DMAG (82%–94% decrease in cell proliferation) was higher than with 17-AAG in WTB-Raf uveal melanoma cell lines (compare Figs. 1A and 9A ). Moreover, WTB-Raf melanoma cell lines were as sensitive as V600EB-Raf melanoma cell lines to treatment with 17-DMAG, as indicated by IC50 values (WTB-Raf cells, 17–130 nM; V600EB-Raf cells, 45–200 nM; Fig. 9A ). Treatment with 17-DMAG induced a rapid, large decrease in B-Raf level that remained low over a 72-hour period in WTB-Raf melanoma cell lines (Fig. 9B) . We observed a pronounced downregulation of B-Raf in 17-DMAG–exposed WTB-Raf cell lines (Fig. 9C) . Consequent to B-Raf downregulation, we also observed a large, decreased activation of ERK1/2 without modulation of ERK1/2 expression levels (Fig. 9B) . B-Raf levels decreased with similar kinetics in 17-DMAG–treated WTB-Raf and 17-DMAG-treated V600EB-Raf melanoma cell lines, showing a similar efficiency of 17-DMAG in WTB-Raf and V600EB-Raf uveal melanoma cell lines (Figs. 9B 9C)
Tumor cells that have become resistant to imatinib mesylate remain sensitive to 17-AAG, suggesting that 17-AAG could be beneficial to patients. 34 Therefore, we determined whether 17-DMAG sensitizes uveal melanoma cell lines to imatinib mesylate. Cell treatment with 17-DMAG, with a concentration corresponding to its IC50, sensitized the two c-Kit–positive WTB-Raf uveal melanoma cell lines to micromolar concentrations of imatinib mesylate (Fig. 10) . This shows a significant decrease in the concentration required for maximal inhibition of cell proliferation. Interestingly, 17-DMAG also enhanced the effects of imatinib mesylate in the c-Kit-positive V600EB-Raf melanoma cell line TP31 (Fig. 10) . As expected, no additive effects of 17-DMAG with imatinib mesylate were observed in the c-Kit–negative V600EB-Raf melanoma cell line OCM-1 (Fig. 10)
Discussion
Difference in the HSP90-Mediated Control of WTB-Raf Protein Expression between Uveal and Cutaneous Melanomas
In this study, we reported that the HSP90 inhibitor 17-AAG and, to a greater extent, its more soluble analogue 17-DMAG are very potent inhibitors of cell proliferation in WTB-Raf uveal melanoma cell lines. HSP90 inhibition induces the downregulation of WTB-Raf protein. This downregulation leads to the inactivation of the MEK/ERK module and the decrease in cyclin D1, which is necessary for the proliferation of uveal melanoma cell lines. 10 It has been reported recently that B-Raf is unaffected by 17-AAG in WTB-Raf cutaneous melanoma cell lines. 23 This shows a marked difference between cutaneous and uveal melanomas. Interestingly, 17-AAG also causes the downregulation of V600D, G465V, and G468A B-Raf expression in cutaneous melanoma cell lines. 23 These mutants had levels of B-Raf kinase activity as high as those observed in cutaneous melanoma cell lines expressing V600EB-Raf. Therefore, it appears that 17-AAG exclusively caused the downregulation of activated B-Raf. Thus, 17-AAG cannot downregulate WTB-Raf in cutaneous melanoma cell lines because the tyrosine kinase activity of WTB-Raf is not detectable in these cells. Marais et al. 24 confirmed this hypothesis in cutaneous melanoma cell lines. Moreover, they showed that when WTB-Raf is activated by coexpression with G12VRAS in COS cells, 17-AAG treatment greatly reduces its protein levels. 24 Conversely, WTB-Raf is activated in the absence of a mutation in RAS in uveal melanoma cell lines. 10 These data explain why WTB-Raf is as sensitive to 17-AAG as V600EB-Raf in uveal melanoma cell lines; these cells have B-Raf kinase activity levels similar to those of cells expressing V600EB-Raf. 10 Our data are therefore consistent with those showing that activated WTB-Raf is more sensitive to 17-AAG than nonactivated WTB-Raf. 24 HSP90 and V600EB-Raf associate with each other, whereas HSP90 and nonactivated WTB-Raf do not. This suggests that the association of HSP90 and B-Raf is the key process in the specificity of WTB-Raf and V600EB-Raf cell responses to 17-AAG. 23 24 Surprisingly, not all high-activity B-Raf mutants are sensitive to 17-AAG, whereas some impaired-activity mutants are. 24 Moreover, HSP90 association with Raf-1 cannot be detected in WTB-Raf or V600EB-Raf cutaneous melanoma cell lines, though 17-AAG downregulates Raf-1 expression. 23 24 Therefore, the mechanism of action of 17-AAG seems to be more subtle than previously expected. The type of conformation (active or not) may also determine 17-AAG sensitivity, rather than only the level of B-Raf kinase activity or the type of cancer cells. 24  
Expression levels of Raf-1 and Akt decrease in 17-AAG-treated V600EB-Raf–expressing cutaneous melanoma cell lines, indicating that some of these HSP90 client proteins may also be involved in cutaneous melanoma tumorigenesis. 23 24 28 Treatment of uveal melanoma cell lines with 17-AAG also downregulates Raf-1. However, this does not account for the 17-AAG–induced reduction of cell proliferation in uveal melanoma cell lines because siRNA downregulation of Raf-1 expression did not affect cell proliferation in WTB-Raf or V600EB-Raf uveal melanoma cell lines. 10 We also rule out the role of 17-AAG–mediated downregulation and deactivation of Akt in uveal melanoma cell lines because inhibition of the three forms of Akt did not affect cell proliferation in these cells. However, this does not exclude a role for Akt in the survival of uveal melanoma cells treated with cytotoxic agents. We previously showed that the WTB-Raf–ERK1/2 pathway plays a role in controlling cyclin D1 expression for cell proliferation in uveal melanoma cell lines. 10 17-AAG downregulated cyclin D1 in these cell lines. The 17-AAG–mediated inhibition of cell proliferation may, therefore, be attributed to a direct effect on cyclin D1 stability or to an indirect effect through the downregulation of B-Raf and the inhibition of ERK1/2. 10  
HSP90 Inhibitors—Potential Therapeutic Agents for Uveal Melanoma?
Our data suggest that inhibition of the B-Raf/MEK/ERK signaling pathway by HSP90 inhibitors may be an important therapeutic strategy for uveal melanoma. Maximal growth inhibition was achieved with submicromolar concentrations of 17-AAG in cutaneous melanoma cells. 35 The higher concentrations of 17-AAG/17-DMAG needed to achieve complete inhibition of cell proliferation in the four uveal melanoma cell lines we tested (IC90, 1–7 μM) limited the clinical efficacy of these HSP90 inhibitors in this disease. Another approach may be to target HSP90 accessory proteins, such as the cochaperone cdc37. Interestingly, the quantity of cdc37 associated with V600EB-Raf is five times greater than that associated with WTB-Raf in cutaneous melanoma cell lines. 23 This may play a role in the differential sensitivity to 17-AAG of V600EB-Raf and WTB-Raf. The recent finding that the inhibition of cdc37 phosphorylation suppresses HSP90 association with target kinases suggests that drugs targeting the HSP90 cochaperone may be used in inhibiting a subset of HSP90 functions. 36 We ruled out a role of cdc37 in the mechanism of action of HSP90-mediated control of uveal melanoma cell proliferation because siRNA-induced cdc37 downregulation had no effect on B-Raf expression levels, MEK/ERK activation, or cell proliferation. 
As part of combination therapy, 17-AAG markedly sensitizes cancer cells to a variety of agents, often with a reduction in the concentration required for each agent. The tyrosine kinase receptor c-Kit is a client protein for HSP90 and a target of HSP90 inhibitors. Our previous study on the effects of imatinib mesylate demonstrated the role of the SCF/c-Kit autocrine loop for cell proliferation. 13 A clinical study on the effects of imatinib mesylate in patients with uveal melanoma is under way. Cell treatment with 17-DMAG in conjunction with imatinib mesylate was more efficient than treatment with each compound separately in c-Kit–positive uveal melanoma cells, showing a synergistic effect of HSP90 inhibition and imatinib mesylate. Therefore, the method of combining 17-DMAG with imatinib mesylate would have the advantage of lowering doses of the two compounds that may reduce systemic drug exposure and may perhaps achieve greater tumor specificity. 37 Therefore, our study suggests that a therapeutic approach may lie in targeting HSP90 in tandem with c-Kit inhibition in uveal melanoma. 
 
Figure 1.
 
Effects of 17-AAG on proliferation and transformation in uveal melanoma cell lines. WTB-Raf cells (Mel270 and 92.1) and V600EB-Raf cells (OCM-1 and TP31) were cultured for 3 days in complete growth medium, as described in Experimental Procedures. Then cells were treated with 17-AAG at the indicated concentrations in 5% FCS culture medium (A) or 0.5% FCS culture medium (B). Cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. (C) WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days and were resuspended in 0.3% agar in complete medium and either 300 nM 17-AAG or vehicle. Plates were incubated at 37°C for 2 weeks. Macroscopic colonies were counted on day 12. Similar results were obtained in five (A, B) and three (C) independent experiments.
Figure 1.
 
Effects of 17-AAG on proliferation and transformation in uveal melanoma cell lines. WTB-Raf cells (Mel270 and 92.1) and V600EB-Raf cells (OCM-1 and TP31) were cultured for 3 days in complete growth medium, as described in Experimental Procedures. Then cells were treated with 17-AAG at the indicated concentrations in 5% FCS culture medium (A) or 0.5% FCS culture medium (B). Cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. (C) WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days and were resuspended in 0.3% agar in complete medium and either 300 nM 17-AAG or vehicle. Plates were incubated at 37°C for 2 weeks. Macroscopic colonies were counted on day 12. Similar results were obtained in five (A, B) and three (C) independent experiments.
Figure 2.
 
Effects of 17-AAG on cell cycle, apoptosis, and caspase activation in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were treated with 300 nM 17-AAG in complete culture medium. Cell cycle (A) and cell death (B) were analyzed by FACS after 48 hours of culture. (C, D) Western blot analysis of cleaved caspase 9 and 3 in WTB-Raf cells and V600EB-Raf cells. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 2.
 
Effects of 17-AAG on cell cycle, apoptosis, and caspase activation in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were treated with 300 nM 17-AAG in complete culture medium. Cell cycle (A) and cell death (B) were analyzed by FACS after 48 hours of culture. (C, D) Western blot analysis of cleaved caspase 9 and 3 in WTB-Raf cells and V600EB-Raf cells. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 3.
 
Kinetics of 17-AAG–mediated Raf-1 and B-Raf downexpression in uveal melanoma cell lines. WTB-Raf cells (92.1 [AC], Mel270 [A]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A]) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG (B, C) or remained untreated (A) in 5% FCS culture medium and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting with anti-HSP90 (A), anti-cdc37 (A), MEK1 (A), anti-ERK1/2 (A), anti–Raf-1 (A, B) and anti–B-Raf (A, B) antibodies. (B) Effects of 17-AAG on the expression levels of Raf-1 and B-Raf were compared in WTB-Raf cells and V600EB-Raf cells by Western blotting with anti–Raf-1 and anti–B-Raf antibodies. (C) Protein expression levels were quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in four independent experiments.
Figure 3.
 
Kinetics of 17-AAG–mediated Raf-1 and B-Raf downexpression in uveal melanoma cell lines. WTB-Raf cells (92.1 [AC], Mel270 [A]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A]) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG (B, C) or remained untreated (A) in 5% FCS culture medium and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting with anti-HSP90 (A), anti-cdc37 (A), MEK1 (A), anti-ERK1/2 (A), anti–Raf-1 (A, B) and anti–B-Raf (A, B) antibodies. (B) Effects of 17-AAG on the expression levels of Raf-1 and B-Raf were compared in WTB-Raf cells and V600EB-Raf cells by Western blotting with anti–Raf-1 and anti–B-Raf antibodies. (C) Protein expression levels were quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in four independent experiments.
Figure 4.
 
Time and dose effects of 17-AAG on B-Raf expression in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 17-AAG at the indicated concentration (A) and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and were analyzed by Western blotting with an anti–B-Raf antibody. (B) Expression levels of B-Raf quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 4.
 
Time and dose effects of 17-AAG on B-Raf expression in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 17-AAG at the indicated concentration (A) and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and were analyzed by Western blotting with an anti–B-Raf antibody. (B) Expression levels of B-Raf quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 5.
 
Effects of 17-AAG on the expression and activation levels of ERK1/2 in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium and lysed at various times after serum stimulation. (A) Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting. Analysis of the expression and phosphorylation of ERK1/2 was performed with anti–phospho-ERK1/2 (P-ERK1 and P-ERK2) and anti-ERK1/2 (ERK1 and ERK2) antibodies. (B) ERK1/2 activation was quantified using an ELISA kit. The effects of the MEK1/2 inhibitor UO126 (10 μM) on ERK1/2 activation was measured in parallel as a control. Results for both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in five (A) and two (B) independent experiments.
Figure 5.
 
Effects of 17-AAG on the expression and activation levels of ERK1/2 in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium and lysed at various times after serum stimulation. (A) Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting. Analysis of the expression and phosphorylation of ERK1/2 was performed with anti–phospho-ERK1/2 (P-ERK1 and P-ERK2) and anti-ERK1/2 (ERK1 and ERK2) antibodies. (B) ERK1/2 activation was quantified using an ELISA kit. The effects of the MEK1/2 inhibitor UO126 (10 μM) on ERK1/2 activation was measured in parallel as a control. Results for both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in five (A) and two (B) independent experiments.
Figure 6.
 
Effects of 17-AAG on the expression and activation levels of MEK1/2 and on the expression levels of cyclin D1 and p27 in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium or they remained untreated and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and were analyzed by Western blotting. (A) Analysis of the expression and phosphorylation of MEK1/2 was performed with anti–phospho-MEK1/2 (P-MEK1/2) and anti-MEK1 (MEK1) antibodies. (B) Analysis of the expression of cyclin D1 and p27 by Western blotting. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 6.
 
Effects of 17-AAG on the expression and activation levels of MEK1/2 and on the expression levels of cyclin D1 and p27 in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium or they remained untreated and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and were analyzed by Western blotting. (A) Analysis of the expression and phosphorylation of MEK1/2 was performed with anti–phospho-MEK1/2 (P-MEK1/2) and anti-MEK1 (MEK1) antibodies. (B) Analysis of the expression of cyclin D1 and p27 by Western blotting. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 7.
 
Effects of 17-AAG on the expression and activation levels of Akt and c-Kit. Effects of the inhibition of Akt kinase activity on uveal melanoma cell lines. WTB-Raf cells (92.1 [AC], Mel270 [A, left]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A, left]) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium, or they remained untreated and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting. (A) Western blotting analysis of Akt expression and phosphorylation in untreated cells over a 48-hour culture period and effects of 17-AAG–treated cells over a 72-hour culture period. (B) Melanoma cells were treated with the two Akt inhibitors, triciribine (10 μM) and Akt inhibitor II (10 μM), and cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. Effects of the MEK1/2 inhibitor UO126 (10 μM) on cell proliferation were measured in parallel. The percentage of growth inhibition was calculated with respect to control cells. (C) Analysis of the expression of c-Kit was performed with an anti–c-Kit (c-Kit) antibody in 17-AAG–treated and untreated cells. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 7.
 
Effects of 17-AAG on the expression and activation levels of Akt and c-Kit. Effects of the inhibition of Akt kinase activity on uveal melanoma cell lines. WTB-Raf cells (92.1 [AC], Mel270 [A, left]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A, left]) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium, or they remained untreated and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting. (A) Western blotting analysis of Akt expression and phosphorylation in untreated cells over a 48-hour culture period and effects of 17-AAG–treated cells over a 72-hour culture period. (B) Melanoma cells were treated with the two Akt inhibitors, triciribine (10 μM) and Akt inhibitor II (10 μM), and cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. Effects of the MEK1/2 inhibitor UO126 (10 μM) on cell proliferation were measured in parallel. The percentage of growth inhibition was calculated with respect to control cells. (C) Analysis of the expression of c-Kit was performed with an anti–c-Kit (c-Kit) antibody in 17-AAG–treated and untreated cells. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 8.
 
Effect of cdc37 depletion on ERK1/2 activation, proliferation, and cell cycle in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured in complete medium. Twenty-four hours later, cells were treated with lipofectamine (Ct siRNA) or with lipofectamine and cdc37-specific siRNA (Cdc37 siRNA), or they remained untreated (siRNA), as described in Experimental Procedures. (A) Expression of cdc37, Raf-1, B-Raf, phospho-ERK1/2 (P-(ERK1/2), and total ERK1/2 was analyzed by Western blotting 3 days after cell transfection. (B) Effects of 17-AAG, alone or in combination with cdc37 depletion on cell proliferation, was measured by the MTT colorimetric assay 3 days after cell transfection. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 8.
 
Effect of cdc37 depletion on ERK1/2 activation, proliferation, and cell cycle in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured in complete medium. Twenty-four hours later, cells were treated with lipofectamine (Ct siRNA) or with lipofectamine and cdc37-specific siRNA (Cdc37 siRNA), or they remained untreated (siRNA), as described in Experimental Procedures. (A) Expression of cdc37, Raf-1, B-Raf, phospho-ERK1/2 (P-(ERK1/2), and total ERK1/2 was analyzed by Western blotting 3 days after cell transfection. (B) Effects of 17-AAG, alone or in combination with cdc37 depletion on cell proliferation, was measured by the MTT colorimetric assay 3 days after cell transfection. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 9.
 
Effects of 17-DMAG on cell proliferation, expression levels of B-Raf and ERK1/2, and ERK1/2 activation. WTB-Raf cells (92.1 [AC], Mel270 [A]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A]) were cultured for 3 days in complete growth medium, and then cells were treated with 17-DMAG at the indicated concentrations in 5% FCS culture medium. (A) Cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. (B) Equal amounts of protein extract were separated on SDS-PAGE and analyzed by Western blotting with anti–Raf-1, anti–B-Raf, anti–phospho-ERK1/2 (P-ERK1 and P-ERK2), and anti–ERK1/2 (ERK1 and ERK2) antibodies. (C) Comparison of the effects of 17-AAG and 17-DMAG on B-Raf expression levels in WTB-Raf cells and V600EB-Raf cells. Expression levels of B-Raf were quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in five (A) and three (B, C) independent experiments.
Figure 9.
 
Effects of 17-DMAG on cell proliferation, expression levels of B-Raf and ERK1/2, and ERK1/2 activation. WTB-Raf cells (92.1 [AC], Mel270 [A]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A]) were cultured for 3 days in complete growth medium, and then cells were treated with 17-DMAG at the indicated concentrations in 5% FCS culture medium. (A) Cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. (B) Equal amounts of protein extract were separated on SDS-PAGE and analyzed by Western blotting with anti–Raf-1, anti–B-Raf, anti–phospho-ERK1/2 (P-ERK1 and P-ERK2), and anti–ERK1/2 (ERK1 and ERK2) antibodies. (C) Comparison of the effects of 17-AAG and 17-DMAG on B-Raf expression levels in WTB-Raf cells and V600EB-Raf cells. Expression levels of B-Raf were quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in five (A) and three (B, C) independent experiments.
Figure 10.
 
17-DMAG sensitizes uveal melanoma cell lines to imatinib mesylate. WTB-Raf (92.1 and Mel270) and V600EB-Raf cells (OCM-1 and TP31) were cultured for 3 days in complete growth medium. Cells were treated with 17-DMAG alone at the respective concentrations corresponding to the IC50 or in combination with 1 μM imatinib mesylate, and cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. Similar results were obtained in three independent experiments.
Figure 10.
 
17-DMAG sensitizes uveal melanoma cell lines to imatinib mesylate. WTB-Raf (92.1 and Mel270) and V600EB-Raf cells (OCM-1 and TP31) were cultured for 3 days in complete growth medium. Cells were treated with 17-DMAG alone at the respective concentrations corresponding to the IC50 or in combination with 1 μM imatinib mesylate, and cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. Similar results were obtained in three independent experiments.
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Figure 1.
 
Effects of 17-AAG on proliferation and transformation in uveal melanoma cell lines. WTB-Raf cells (Mel270 and 92.1) and V600EB-Raf cells (OCM-1 and TP31) were cultured for 3 days in complete growth medium, as described in Experimental Procedures. Then cells were treated with 17-AAG at the indicated concentrations in 5% FCS culture medium (A) or 0.5% FCS culture medium (B). Cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. (C) WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days and were resuspended in 0.3% agar in complete medium and either 300 nM 17-AAG or vehicle. Plates were incubated at 37°C for 2 weeks. Macroscopic colonies were counted on day 12. Similar results were obtained in five (A, B) and three (C) independent experiments.
Figure 1.
 
Effects of 17-AAG on proliferation and transformation in uveal melanoma cell lines. WTB-Raf cells (Mel270 and 92.1) and V600EB-Raf cells (OCM-1 and TP31) were cultured for 3 days in complete growth medium, as described in Experimental Procedures. Then cells were treated with 17-AAG at the indicated concentrations in 5% FCS culture medium (A) or 0.5% FCS culture medium (B). Cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. (C) WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days and were resuspended in 0.3% agar in complete medium and either 300 nM 17-AAG or vehicle. Plates were incubated at 37°C for 2 weeks. Macroscopic colonies were counted on day 12. Similar results were obtained in five (A, B) and three (C) independent experiments.
Figure 2.
 
Effects of 17-AAG on cell cycle, apoptosis, and caspase activation in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were treated with 300 nM 17-AAG in complete culture medium. Cell cycle (A) and cell death (B) were analyzed by FACS after 48 hours of culture. (C, D) Western blot analysis of cleaved caspase 9 and 3 in WTB-Raf cells and V600EB-Raf cells. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 2.
 
Effects of 17-AAG on cell cycle, apoptosis, and caspase activation in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were treated with 300 nM 17-AAG in complete culture medium. Cell cycle (A) and cell death (B) were analyzed by FACS after 48 hours of culture. (C, D) Western blot analysis of cleaved caspase 9 and 3 in WTB-Raf cells and V600EB-Raf cells. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 3.
 
Kinetics of 17-AAG–mediated Raf-1 and B-Raf downexpression in uveal melanoma cell lines. WTB-Raf cells (92.1 [AC], Mel270 [A]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A]) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG (B, C) or remained untreated (A) in 5% FCS culture medium and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting with anti-HSP90 (A), anti-cdc37 (A), MEK1 (A), anti-ERK1/2 (A), anti–Raf-1 (A, B) and anti–B-Raf (A, B) antibodies. (B) Effects of 17-AAG on the expression levels of Raf-1 and B-Raf were compared in WTB-Raf cells and V600EB-Raf cells by Western blotting with anti–Raf-1 and anti–B-Raf antibodies. (C) Protein expression levels were quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in four independent experiments.
Figure 3.
 
Kinetics of 17-AAG–mediated Raf-1 and B-Raf downexpression in uveal melanoma cell lines. WTB-Raf cells (92.1 [AC], Mel270 [A]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A]) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG (B, C) or remained untreated (A) in 5% FCS culture medium and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting with anti-HSP90 (A), anti-cdc37 (A), MEK1 (A), anti-ERK1/2 (A), anti–Raf-1 (A, B) and anti–B-Raf (A, B) antibodies. (B) Effects of 17-AAG on the expression levels of Raf-1 and B-Raf were compared in WTB-Raf cells and V600EB-Raf cells by Western blotting with anti–Raf-1 and anti–B-Raf antibodies. (C) Protein expression levels were quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in four independent experiments.
Figure 4.
 
Time and dose effects of 17-AAG on B-Raf expression in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 17-AAG at the indicated concentration (A) and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and were analyzed by Western blotting with an anti–B-Raf antibody. (B) Expression levels of B-Raf quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 4.
 
Time and dose effects of 17-AAG on B-Raf expression in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 17-AAG at the indicated concentration (A) and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and were analyzed by Western blotting with an anti–B-Raf antibody. (B) Expression levels of B-Raf quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 5.
 
Effects of 17-AAG on the expression and activation levels of ERK1/2 in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium and lysed at various times after serum stimulation. (A) Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting. Analysis of the expression and phosphorylation of ERK1/2 was performed with anti–phospho-ERK1/2 (P-ERK1 and P-ERK2) and anti-ERK1/2 (ERK1 and ERK2) antibodies. (B) ERK1/2 activation was quantified using an ELISA kit. The effects of the MEK1/2 inhibitor UO126 (10 μM) on ERK1/2 activation was measured in parallel as a control. Results for both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in five (A) and two (B) independent experiments.
Figure 5.
 
Effects of 17-AAG on the expression and activation levels of ERK1/2 in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium and lysed at various times after serum stimulation. (A) Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting. Analysis of the expression and phosphorylation of ERK1/2 was performed with anti–phospho-ERK1/2 (P-ERK1 and P-ERK2) and anti-ERK1/2 (ERK1 and ERK2) antibodies. (B) ERK1/2 activation was quantified using an ELISA kit. The effects of the MEK1/2 inhibitor UO126 (10 μM) on ERK1/2 activation was measured in parallel as a control. Results for both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in five (A) and two (B) independent experiments.
Figure 6.
 
Effects of 17-AAG on the expression and activation levels of MEK1/2 and on the expression levels of cyclin D1 and p27 in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium or they remained untreated and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and were analyzed by Western blotting. (A) Analysis of the expression and phosphorylation of MEK1/2 was performed with anti–phospho-MEK1/2 (P-MEK1/2) and anti-MEK1 (MEK1) antibodies. (B) Analysis of the expression of cyclin D1 and p27 by Western blotting. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 6.
 
Effects of 17-AAG on the expression and activation levels of MEK1/2 and on the expression levels of cyclin D1 and p27 in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium or they remained untreated and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and were analyzed by Western blotting. (A) Analysis of the expression and phosphorylation of MEK1/2 was performed with anti–phospho-MEK1/2 (P-MEK1/2) and anti-MEK1 (MEK1) antibodies. (B) Analysis of the expression of cyclin D1 and p27 by Western blotting. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 7.
 
Effects of 17-AAG on the expression and activation levels of Akt and c-Kit. Effects of the inhibition of Akt kinase activity on uveal melanoma cell lines. WTB-Raf cells (92.1 [AC], Mel270 [A, left]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A, left]) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium, or they remained untreated and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting. (A) Western blotting analysis of Akt expression and phosphorylation in untreated cells over a 48-hour culture period and effects of 17-AAG–treated cells over a 72-hour culture period. (B) Melanoma cells were treated with the two Akt inhibitors, triciribine (10 μM) and Akt inhibitor II (10 μM), and cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. Effects of the MEK1/2 inhibitor UO126 (10 μM) on cell proliferation were measured in parallel. The percentage of growth inhibition was calculated with respect to control cells. (C) Analysis of the expression of c-Kit was performed with an anti–c-Kit (c-Kit) antibody in 17-AAG–treated and untreated cells. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 7.
 
Effects of 17-AAG on the expression and activation levels of Akt and c-Kit. Effects of the inhibition of Akt kinase activity on uveal melanoma cell lines. WTB-Raf cells (92.1 [AC], Mel270 [A, left]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A, left]) were cultured for 3 days in complete growth medium. Then cells were treated with 300 nM 17-AAG in 5% FCS culture medium, or they remained untreated and were lysed at various times after serum stimulation. Equal amounts of protein extracts were resolved on SDS-PAGE and analyzed by Western blotting. (A) Western blotting analysis of Akt expression and phosphorylation in untreated cells over a 48-hour culture period and effects of 17-AAG–treated cells over a 72-hour culture period. (B) Melanoma cells were treated with the two Akt inhibitors, triciribine (10 μM) and Akt inhibitor II (10 μM), and cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. Effects of the MEK1/2 inhibitor UO126 (10 μM) on cell proliferation were measured in parallel. The percentage of growth inhibition was calculated with respect to control cells. (C) Analysis of the expression of c-Kit was performed with an anti–c-Kit (c-Kit) antibody in 17-AAG–treated and untreated cells. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 8.
 
Effect of cdc37 depletion on ERK1/2 activation, proliferation, and cell cycle in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured in complete medium. Twenty-four hours later, cells were treated with lipofectamine (Ct siRNA) or with lipofectamine and cdc37-specific siRNA (Cdc37 siRNA), or they remained untreated (siRNA), as described in Experimental Procedures. (A) Expression of cdc37, Raf-1, B-Raf, phospho-ERK1/2 (P-(ERK1/2), and total ERK1/2 was analyzed by Western blotting 3 days after cell transfection. (B) Effects of 17-AAG, alone or in combination with cdc37 depletion on cell proliferation, was measured by the MTT colorimetric assay 3 days after cell transfection. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 8.
 
Effect of cdc37 depletion on ERK1/2 activation, proliferation, and cell cycle in uveal melanoma cell lines. WTB-Raf cells (92.1) and V600EB-Raf cells (OCM-1) were cultured in complete medium. Twenty-four hours later, cells were treated with lipofectamine (Ct siRNA) or with lipofectamine and cdc37-specific siRNA (Cdc37 siRNA), or they remained untreated (siRNA), as described in Experimental Procedures. (A) Expression of cdc37, Raf-1, B-Raf, phospho-ERK1/2 (P-(ERK1/2), and total ERK1/2 was analyzed by Western blotting 3 days after cell transfection. (B) Effects of 17-AAG, alone or in combination with cdc37 depletion on cell proliferation, was measured by the MTT colorimetric assay 3 days after cell transfection. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in three independent experiments.
Figure 9.
 
Effects of 17-DMAG on cell proliferation, expression levels of B-Raf and ERK1/2, and ERK1/2 activation. WTB-Raf cells (92.1 [AC], Mel270 [A]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A]) were cultured for 3 days in complete growth medium, and then cells were treated with 17-DMAG at the indicated concentrations in 5% FCS culture medium. (A) Cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. (B) Equal amounts of protein extract were separated on SDS-PAGE and analyzed by Western blotting with anti–Raf-1, anti–B-Raf, anti–phospho-ERK1/2 (P-ERK1 and P-ERK2), and anti–ERK1/2 (ERK1 and ERK2) antibodies. (C) Comparison of the effects of 17-AAG and 17-DMAG on B-Raf expression levels in WTB-Raf cells and V600EB-Raf cells. Expression levels of B-Raf were quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in five (A) and three (B, C) independent experiments.
Figure 9.
 
Effects of 17-DMAG on cell proliferation, expression levels of B-Raf and ERK1/2, and ERK1/2 activation. WTB-Raf cells (92.1 [AC], Mel270 [A]) and V600EB-Raf cells (OCM-1 [AC], TP31 [A]) were cultured for 3 days in complete growth medium, and then cells were treated with 17-DMAG at the indicated concentrations in 5% FCS culture medium. (A) Cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. (B) Equal amounts of protein extract were separated on SDS-PAGE and analyzed by Western blotting with anti–Raf-1, anti–B-Raf, anti–phospho-ERK1/2 (P-ERK1 and P-ERK2), and anti–ERK1/2 (ERK1 and ERK2) antibodies. (C) Comparison of the effects of 17-AAG and 17-DMAG on B-Raf expression levels in WTB-Raf cells and V600EB-Raf cells. Expression levels of B-Raf were quantified. Results in both sets of WTB-Raf cells (92.1 and Mel270) were similar, as were the results for both sets of V600EB-Raf cells (OCM-1 and TP31); data not shown. Similar results were obtained in five (A) and three (B, C) independent experiments.
Figure 10.
 
17-DMAG sensitizes uveal melanoma cell lines to imatinib mesylate. WTB-Raf (92.1 and Mel270) and V600EB-Raf cells (OCM-1 and TP31) were cultured for 3 days in complete growth medium. Cells were treated with 17-DMAG alone at the respective concentrations corresponding to the IC50 or in combination with 1 μM imatinib mesylate, and cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. Similar results were obtained in three independent experiments.
Figure 10.
 
17-DMAG sensitizes uveal melanoma cell lines to imatinib mesylate. WTB-Raf (92.1 and Mel270) and V600EB-Raf cells (OCM-1 and TP31) were cultured for 3 days in complete growth medium. Cells were treated with 17-DMAG alone at the respective concentrations corresponding to the IC50 or in combination with 1 μM imatinib mesylate, and cell proliferation was measured with the MTT colorimetric assay on day 3 of treatment. The percentage of growth inhibition was calculated with respect to control cells. Similar results were obtained in three independent experiments.
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