mtor Is Activated in the Majority of Malignant Melanomas

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1 ORIGINAL ARTICLE mtor Is Activated in the Majority of Malignant Melanomas Magdalena Karbowniczek 1, Cynthia S. Spittle 2, Tasha Morrison 1, Hong Wu 3 and Elizabeth P. Henske 1 The objective of this study was to determine whether activation of the kinase mammalian target of rapamycin (mtor) is associated with human melanoma. We found moderate or strong hyperphosphorylation of ribosomal protein S6 in 78/17 melanomas (73%). In contrast, only 3/67 benign nevi (4%) were moderately positive, and none were strongly positive. These data indicate that mtor activation is very strongly associated with malignant, compared to benign, melanocytic lesions. Next, we tested six melanoma-derived cell lines for evidence of mtor dysregulation. Five of the six lines showed persistent phosphorylation of S6 after 18 hours of serum deprivation, and four had S6 phosphorylation after 3 minutes of amino-acid withdrawal, indicating inappropriate mtor activation. The proliferation of three melanoma-derived lines was blocked by the mtor inhibitor rapamycin, indicating that mtor activation is a growth-promoting factor in melanoma-derived cells. mtor is directly activated by the small guanosine triphosphatase Ras homolog enriched in brain (Rheb), in a farnesylation-dependent manner. Therefore, to investigate the mechanism of mtor activation, we used the farnesyl transferase inhibitor FTI-277, which partially blocked the growth of three of the six melanoma cell lines. Together, these data implicate activation of mtor in the pathogenesis of melanoma, and suggest that Rheb and mtor may be targets for melanoma therapy. Journal of Investigative Dermatology (28) 128, 98987; doi:1.138/sj.jid.57174; published online 4 October 27 1 Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA; 2 Population Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA and 3 Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA Correspondence: Dr Elizabeth P. Henske, Department of Medical Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111, USA. Elizabeth.Henske@fccc.edu Abbreviations: FTI, farnesyl transferase inhibitor; mtor, mammalian target of rapamycin; Rheb, Ras homolog enriched in brain; TMA, tissue microarray; TSC, tuberous sclerosis complex; TSC1/2, tuberous sclerosis complex 1/2 Received 31 January 27; revised 26 June 27; accepted 13 July 27; published online 4 October 27 INTRODUCTION In the United States in the past 4 years, the incidence of melanoma has increased 15-fold, more than any other malignancy (Chudnovsky et al., 25). Currently, 62,19 melanomas are diagnosed each year in the United States, with 7,9 deaths (Cancer Facts Figures ACS, 26). There have been many advances in melanoma pathogenesis (Rodolfo et al., 24; Chudnovsky et al., 25; Thompson et al., 25), including the identification of activating B-Raf mutations in the majority of tumors (Davies et al., 22; Gray-Schopfer et al., 25). However, no current treatment is known to enhance the survival of patients with advanced disease (Tsao et al., 24; Chudnovsky et al., 25; Thompson et al., 25). Therefore, to develop optimal targeted therapeutic agents, continuing to identify the cellular pathways that promote the growth and progression of melanoma remains a priority. The mammalian target of rapamycin (mtor) is a kinase that integrates growth factor stimulation and nutrient availability with protein synthesis and cell growth (Thomas and Hall, 1997; Hara et al., 1998; Avruch et al., 21; Sabatini, 26). Activation of mtor leads to phosphorylation and activation of p7 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1, thereby promoting increased protein translation and cell growth. Rapamycin has been previously found to inhibit the growth of three melanoma-derived cell lines and to inhibit the phosphorylation of ribosomal protein S6 in a melanoma-derived cell line (Molhoek et al., 25). mtor is activated by the small guanosine triphosphatase Ras homolog enriched in brain (Rheb) (Castro et al., 23; Garami et al., 23; Inoki et al., 23; Saucedo et al., 23; Stocker et al., 23; Tee et al., 23; Zhang et al., 23), which is the target of the guanosine triphosphatase-activating domain of the tuberous sclerosis complex 2 (TSC2) protein. In addition to Rheb s activation of mtor, Rheb also inhibits the activity of B-Raf and C-Raf kinase (Karbowniczek et al., 24, 26). Because Rheb activity is regulated by the TSC2 protein, Rheb and mtor are activated in tumor cells from patients with TSC. TSC is a tumor-suppressor gene disorder characterized by seizures, mental retardation, and benign tumors in multiple organs. The abnormal smooth muscle cells of renal angiomyolipomas and pulmonary lymphangioleiomyomatosis, two of the characteristic lesions of TSC, have been known for many years to express melanocytic antigens, including HMB-45, melan-a, CD63, and PNL2 (Chan et al., 1993; Makhlouf et al., 22; Tuna et al., 23; Zhe and Schuger, 24). This expression of melanoma-associated antigens in 98 Journal of Investigative Dermatology (28), Volume 128 & 27 The Society for Investigative Dermatology

2 TSC tumors, together with the role of TSC2 and Rheb in regulating the activity of B-Raf kinase (Karbowniczek et al., 24, 26), led us to ask whether the mtor pathway is activated in human melanoma. In this study, we investigated the presence of hyperphosphorylation of ribosomal protein S6, a marker of mtor activation, in human melanocytic lesions and melanoma-derived cell lines, and the impact of the mtor inhibitor rapamycin and the farnesyl transferase inhibitor (FTI)-277 on the growth of melanoma-derived cell lines. RESULTS Hyperphosphorylation of ribosomal protein S6 is associated with melanoma To determine whether malignant melanomas exhibit activation of mtor signaling in vivo, phospho-ribosomal protein S6 immunostaining was performed on tissue microarrays (TMAs) containing a total of 17 melanomas and 67 benign nevi. The phosphorylation of ribosomal protein S6 is widely used as an indicator of mtor activity in human tumor specimens (El-Hashemite et al., 23; Karbowniczek et al., 23). Of the benign nevi, 43 (64%) were negative for phospho-s6 staining, 21 (31%) were weakly (1 þ ) positive, and only 3 (4%) were moderately (2 þ ) positive (Figure 1a and Table 1). None of the benign lesions showed strong (3 þ ) phospho-s6 positivity. In striking contrast to the nevi, 78 (73%) of the melanomas were moderately or strongly positive: 1/13 in situ melanomas, 18/27 invasive melanomas, and 5/67 metastatic melanomas (Figure 1e and Table 1). Identical staining intensities were observed for the 38 tumors present in duplicate on the arrays. Staining for total S6 ribosomal protein showed similar amounts in benign nevi and melanomas (data not shown). These findings suggest that the mtor signaling pathway is activated in the majority of melanomas in vivo. We obtained full sections and compared anti-phospho- S6 immunostaining between the full sections and the TMA. The first tumor had 2 þ positivity in approximately 34% of the melanoma cells on the TMA; in the full section, there was 2 to 3 þ positivity in 56% of the cells. The second tumor had 2 þ positivity in 51% of the cells in the TMA; 1 to 2 þ positivity was present in 5% of the cells on the full section. The third tumor had 3 þ positivity in 5% of the cells on the TMA; 2 þ positivity was present in 3% of the cells on the full section. Thus, there was a 52% variation in the proportion of positive cells between the TMA and the full sections. To determine whether mtor activation is specific to tumors carrying B-Raf or N-Ras mutations, we tested a series of melanomas on the array for mutations within exon 15 of B- Raf or N-Ras, using pyrosequencing on DNA prepared from the archival paraffin-embedded sections. Sequencing was successful for both genes in 35 tumors (including 23 with moderate or strong hyperphosphorylation of ribosomal protein S6; Table 2). Two additional tumors with a B-Raf mutation (V6E) are included in Table 2 even though the N-Ras sequencing was not successful. B-Raf or Ras mutations were present in 25 of the 37 tumors (67.6%). Nineteen mutations were found in B-Raf (15 V6E, 1 V6K, 1 L597R, 1 V6R, and 1 K61E). Six mutations were found in N-Ras (15.8%), all of which were Q61K. No tumors had mutations in both genes. Of the moderate and strong phospho-s6 tumors, 1 had B-Raf mutations and 4 had N-Ras mutations (Table 2), suggesting that mtor activation is independent of B-Raf or N-Ras mutational status. The mtor pathway is activated in melanoma-derived cell lines We next asked whether inappropriate activation of the mtor pathway is present in melanoma-derived cell lines. We tested six cell lines, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, and M-14, and included mousederived IMCD3 cells as a control. Five of the melanomaderived cell lines (SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, and M-14) showed only a minor increase in phospho-s6 after serum stimulation (Figure 2, lane 2 vs 3), suggesting dysregulation of the mtor pathway. Rapamycin, which is a specific inhibitor of the mtor/raptor complex (Brown et al., 1994; Sabatini et al., 1994; Schmelzle and Hall, 2; Kim and Sabatini, 24), inhibited the phosphorylation of S6 in all cell lines, indicating that regulation of S6 in these cells is mtor complex 1-dependent. Rapamycin inhibits the proliferation of melanoma-derived cell lines To determine whether inappropriate activation of mtor promotes the growth of melanoma-derived cell lines, we tested the effect of mtor inhibition on proliferation. In these experiments, we focused on three cell lines with previously identified melanoma-associated mutations: SK-MEL-28 and Malme (both of which carry the V6E B-Raf mutation) and SK-MEL-2 (which has a Q61K N-Ras mutation). The cells were treated with 2 nm rapamycin or DMSO vehicle control for 3 days, and the incorporation of 3 H-thymidine was measured daily. Rapamycin strongly inhibited the growth of all three melanoma-derived cell lines. Both of the V6E B-Raf mutant lines showed a statistically significant difference in thymidine incorporation after 24 hours of rapamycin, whereas the N-Ras mutant line (SK-MEL-2) reached significance after 48 hours (Figure 3). FTI-277 inhibits the proliferation of melanoma-derived cells We treated six cell lines with 1 mm of the farnesylationtransferase inhibitor FTI-277 for 3 days. FTI-277 significantly inhibited the growth of three of the cell lines at the 3-day time point: SK-MEL-2 by 7%, UACC-62 by 5%, and UACC-257 by 4% (Figure 4a). The small guanosine triphosphatase Rheb directly activates mtor, and Rheb is farnesylated (Clark et al., 1997; Castro et al., 23; Li et al., 24; Basso et al., 25; Gau et al., 25). Whether farnesylation of Rheb is required for mtor activation is not yet completely understood. Farnesylation inhibitors have been shown to inhibit the phosphorylation of S6 in MCF-7 cells (Basso et al., 25) and in Tsc1- and Tsc2-null mouse embryonic fibroblasts (Gau et al., 25), although a farnesyl-deficient form of Rheb can also partially activate mtor (Li et al., 24). Using lysates from cells treated in parallel with those used in the proliferation assays, we found that phospho-s6 was 981

3 a c b d Dermal nevus Lentigo maligna e g i f h j Melanoma in situ Invasive melanoma Metastatic melanoma Figure 1. Phosphorylation of ribosomal protein S6 in dermal nevi and malignant melanoma. (ad) Hematoxylin and eosin (left panels) and phospho-ribosomal protein S6 (right panels) staining of a representative dermal nevus (a and b) and lentigo maligna melanoma (c and d). Weak phospho-s6 (1 þ ) staining is present in the dermal nevus (b, arrow), which contrasts with strong (3 þ ) staining in the epidermis (b and d, open arrow). Strong (3 þ ) phospho-s6 staining is evident in the lentigo maligna melanoma (d, arrow). (ej) Hematoxylin and eosin and phospho-s6 staining of melanoma in situ (e and f), invasive melanoma (g and h), and metastatic melanoma (i and j). Strong (3 þ ) cytoplasmic phospho-s6 staining is evident in melanoma cells (f, h, and j arrows). Bar ¼ 2 mm (aj). Bar ¼ 5 mm (insets). Table 1. results in benign nevi and malignant melanoma Number of cases ps6 negative ( )% (n) ps6 positive (1)% (n) ps6 positive (2)% (n) ps6 positive (3)% (n) Dermal nevi (43) 31.3 (21) 4.5 (3) Melanoma in situ (1) 15.3 (2) 46 (6) 31 (4) Invasive melanoma (2) 26 (7) 22.2 (6) 44.4 (12) Metastatic melanoma (3) 21 (14) 25.3 (17) 49.2 (33) Table 2. Mutational analysis in malignant melanoma B-Raf N-Ras ps6 staining n % wild type % mutant n % wild type % Q61K Seven mutations were V6E and one was V6R. 2 K61E. 3 Seven mutations were V6E, one was V6K, and one was L597R. significantly inhibited by FTI-277 in only SK-MEL-5 (Figure 4B). Therefore, these data suggest that the effects of the FTI on growth of melanoma cells are primarily independent of Rheb farnesylation. DISCUSSION Novel therapeutic strategies are urgently needed for patients with advanced melanoma. Identifying the molecular pathways that promote the progression and metastasis of melanoma is a key step toward this goal. We report here a strong association between the phosphorylation of ribosomal protein S6, an indicator of mtor activation, and malignant melanoma. Seventy-three percent of melanomas stained moderately or strongly positive for phosphorylation of ribosomal protein S6, compared with only 4% of benign nevi. These data indicate that mtor activation occurs during the pathogenesis of the majority of melanomas. To our knowledge, hyperphosphorylation of ribosomal protein S6 is more strongly associated with malignant versus benign melanocytic lesions than any other single marker. For example, phosphorylation of Akt, which is strongly associated with melanoma, has been reported in 71% of primary melanomas 982 Journal of Investigative Dermatology (28), Volume 128

4 Full serum: 24-hour serum deprivation: 1 minutes, 2% FBS: 3 minutes, amino-acid withdrawal: 24 hours, 2 nm rapamycin: but also in 254% of benign nevi (Dhawan et al., 22; Slipicevic et al., 25). It is interesting to speculate that Akt activation may be directly responsible for mtor activation in some melanomas, since Akt phosphorylates and inactivates TSC2 (Potter et al., 22; Manning et al., 22) resulting in activation of Rheb and mtor. However, since Akt is also phosphorylated in 254% of benign nevi, which lack mtor activation in our study, other stimuli in addition to Akt must be required for mtor activation. To investigate further the role of mtor activation in melanoma pathogenesis, we examined six melanomaderived cell lines. Five of the lines showed evidence of inappropriate mtor activation after overnight serum deprivation, and four of them had persistent S6 phosphorylation after an additional 3 minutes of amino-acid deprivation. Three cell lines were treated with the mtor inhibitor rapamycin, which completely blocked their growth. This UACC-257 SK-MEL-28 SK-MEL-2 SK-MEL-5 UACC-62 M-14 IMCD3 Figure 2. mtor activation in melanoma-derived cell lines. Whole-cell lysates from six melanoma-derived cell lines and mouse IMCD3 cells (as a control for the antibody specificity) were immunoblotted with a phospho-specific antibody to Ser 235/236 of ribosomal protein S6, in conditions of steady-state growth in full serum, 24 hours serum deprivation, 24 hours serum deprivation followed by 1 minutes stimulation with 2% fetal bovine serum, 24 hours serum deprivation followed by 3 minutes amino-acid withdrawal, or 24 hours rapamycin in full serum. Of the six melanoma lines, only UACC-257 showed the expected increase of phospho- S6 after serum stimulation (compare second and third lanes). In all cell lines, phospho-s6 was strongly inhibited by rapamycin (fifth lane). The mouse IMCD3 cells show an increase of phospho-s6 after serum stimulation and a decrease after amino-acid withdrawal and rapamycin treatment. growth inhibition is consistent with previous work by Molhoek et al. (25), who showed that rapamycin inhibited the growth of three other melanoma patient-derived cell lines (VMN5A, VMN18, and VMN39). We next tested all six lines for growth inhibition by the farnesylation inhibitor FTI-277. Three of the lines were partially inhibited by FTI-277, but the phosphorylation of S6 was not inhibited, suggesting that the growth effects of FTI-277 are not mediated by the Rheb mtor pathway. The absence of a strong effect of FTI on the phosphorylation of S6 appears to be consistent with the literature, in which strong effects of FTI on mtor activity have been observed almost exclusively in cells with Rheb overexpression or TSC1/TSC2 deletion (Clark et al., 1997; Castro et al., 23; Li et al., 24; Basso et al., 25; Gau et al., 25) and rarely in cells with endogenous expression levels of TSC1, TSC2, and Rheb. We hypothesize that activation of Rheb and mtor is a common mechanism through which mutations and/or constitutive activation of upstream genes and proteins contribute to melanoma progression. If this hypothesis is correct, then therapy targeted at Rheb or mtor could benefit patients with a variety of upstream lesions. For example, mutations in phosphatase and tensin homolog deleted on chromosome ten, which occur in approximately 1% of melanomas (Gray- Schopfer et al., 25), would be predicted to activate Akt, which is known to inactive TSC2 (Inoki et al., 22; Manning et al., 22; Potter et al., 22), resulting in activation of Rheb and mtor. Similarly, Ras-induced activation of phosphatidylinositol 3 -kinase/akt (Okano et al., 2) and ribosomal S6 kinase would be predicted to inactivate TSC2 (Roux et al., 24), resulting in activation of Rheb and mtor. Interestingly, B-Raf activation should also theoretically result in mtor activation, because p42 mitogen-activated protein kinase, which is a downstream effector of B-Raf, directly phosphorylates and inactivates TSC2 (Ma et al., 25). However, we found that mtor activation was uncommon in benign nevi, despite the fact that the majority of benign nevi carry activating B-Raf mutations. It is possible that B-Raf activation, although not sufficient to activate mtor, synergizes with subsequent cellular events during melanoma progression, resulting in the high level of mtor activation in malignant lesions. Further studies will be needed to test this model. In conclusion, we found that activation of mtor is strongly associated with malignant melanocytic lesions in vivo, that the majority of melanoma-derived cell lines display inappropriate mtor activation in serum-deprivation conditions, and that inhibition of mtor blocks the proliferation of melanoma-derived cell lines. These data suggest that mtor inhibition may have clinical benefit in patients with advanced melanoma, either alone or in combination with another targeted agent. Since mtor activation is strongly associated with malignant versus benign melanocyte lesions, mtor inhibition could also prove beneficial in the adjuvant setting for patients with high-risk, mtor-positive tumors. Taken together, our data strongly support further investigation of mtor and Rheb as targets for therapy of advanced melanoma

5 (c.p.m. x1 3 ) Day Day 1 Day 2 Day 3 nm rapamycin 2 nm rapamycin SK-MEL-2 (c.p.m. x1 3 ) Day Day 1 Day 2 Day 3 nm rapamycin 2 nm rapamycin SK-MEL-28 (c.p.m. x1 3 ) Day Day 1 Day 2 Day 3 nm rapamycin 2 nm rapamycin Malme Figure 3. Rapamycin inhibits the proliferation of melanoma-derived cell lines. Treatment of the SK-MEL-2, SK-MEL-28, and Malme melanoma cell lines with 2 nm rapamycin (dashed lines) inhibited growth, compared with DMSO vehicle control (solid lines). Po.5 (Student s t-test). MATERIALS AND METHODS Tissue microarrays and immunohistochemistry Tissue microarrays were generated by the Fox Chase Cancer Center Tumor Bank Facility, with the approval of the Institutional Review Board. A total of 67 benign nevi, 13 melanomas in situ, 27invasive melanomas, and 67 metastatic melanomas were included, with 38 lesions present in duplicate as an internal control. Four-micrometer sections were deparaffinized in xylene and rehydrated in a gradient series of ethanol. For antigen retrieval, sections were boiled in citric buffer (1 mm sodium citrate-trisodium salt dehydrate; Sigma, St Louis, MO), ph 6., for 1 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 15 minutes at room temperature. The sections were then incubated in a humidified chamber with a rabbit polyclonal anti-phospho-s6 antibody (Ser235/ 236; Cell Signaling Technology, Beverly, MA) overnight at 41C or with goat IgG, as a negative control (Santa Cruz Technology, Santa Cruz, CA). The slides were then washed, incubated with an enhanced, human-absorbed, biotinylated affinity-purified goat secondary antibody from the Histostain-Plus Kit (Zymed, San Francisco, CA) for 1 minutes at room temperature, then washed and incubated with enhanced horseradish peroxidase-conjugated streptavidin (Zymed) for 1 minutes at room temperature. After washing, the slides were developed using AEC Chromogen Solution (Zymed) and lightly counterstained with hematoxylin (Biomeda, Foster City, CA). Staining intensity was scored semiquantitatively as negative (no staining), 1 þ (weakly positive), 2 þ (moderately positive), or 3 þ (strongly positive). Cells and immunoblots Melanoma cell lines were maintained in RPMI (Invitrogen, Carlsbad, CA). IMCD3 cell line, used a control cell line, was maintained in DMEM (Invitrogen). Where indicated, cells were serum-deprived for 24 hours and/or treated for 24 hours with 2 nm rapamycin (Biomol Research Laboratories, Plymouth Meeting, PA) or with 1 mm FTI-277 (Calbiochem, La Jolla, CA). Cells were rinsed once in ice-cold 1 phosphate-buffered saline and then lysed in PTY buffer (5 mm HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), ph 7.5, 5 mm NaCl, 5 mm EDTA, 5 mm NaF, 1 mm Na 4 P 2 O 7, and 1% Triton X-1) supplemented with phosphatase inhibitor cocktail I and II (Sigma). Cell lysates were resolved by SDS-PAGE and transferred onto Immobilon P membranes (Millipore, Bedford, MA). The following antibodies were used: anti-phospho-s235/236 S6 ribosomal protein and anti-s6 ribosomal protein (Cell Signaling Technology), and anti-b-actin (Sigma). Mutational analysis DNA was isolated from formalin-fixed, paraffin-embedded tissue sections using the PicoPure TM DNA Extraction Kit (Arcturus, 984 Journal of Investigative Dermatology (28), Volume 128

6 4,5 4, 3,5 3, 2,5 2, 1,5 1, 5 Day Day 1 Day 2 Day 3 μm FTI 1 μm FTI SK-MEL-2 Full serum: 24-hour serum deprivation: 24 hours, 1 μm FTI: 24 hours, 2 nm rapamycin: SK-MEL-5 9, 8, 7, 6, 5, 4, 3, 2, 1, Day Day 1 Day 2 Day 3 μm FTI 1 μm FTI SK-MEL-5 SK-MEL-28 UACC-62 1,2 1, 8, 6, 4, 2, 9, 8, 7, 6, 5, 4, 3, 2, 1, 12, 1, 8, 6, 4, 2, 12, 1, 8, 6, 4, 2, Day Day 1 Day 2 Day 3 Day Day 1 Day 2 Day 3 Day Day 1 Day 2 Day 3 Day Day 1 Day 2 Day 3 μm FTI 1 μm FTI μm FTI 1 μm FTI μm FTI 1 μm FTI μm FTI 1 μm FTI SK-MEL-28 UACC-62 UACC-257 M-14 UACC-257 M-14 Figure 4. FTI-277 inhibits the proliferation of melanoma-derived cell lines. (a) Treatment of the SK-MEL-2, UACC-62, and UACC-257 cell lines with 1 mm FTI-277 (dashed lines) inhibited growth by 7, 5, and 4%, respectively, compared with DMSO vehicle control (solid lines). Po.5 (Student s t-test). (b) Cells treated and harvested in parallel with those in panel (a) were lysed and analyzed by immunoblot. FTI-277 (third lane in each panel) partially inhibited the phosphorylation of ribosomal protein S6 in SK-MEL-5 but not in the other lines. In all cell lines, the phosphorylation of S6 was strongly inhibited by rapamycin (fourth lane). Mountain View, CA) according to the manufacturer s instructions. A 228 bp region of B-Raf exon 15 was amplified by seminested PCR. The second round of amplification was performed with the reverse primer 5 -biotinylated to facilitate single-strand DNA template isolation for the pyrosequencing reaction. Successful and specific amplification was verified by agarose gel electrophoresis. Preparation of the single-stranded DNA template for pyrosequencing was performed using streptavidin-coated Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ). The pyrosequencing primer for B-Raf was designed to screen codons 662 within exon 15 (B-Raf PYROSEQ: 5 -TGATTTTGGTCTAGCTACAG). The pyrosequencing primers for N-Ras were reported previously (Sivertsson et al., 22). The sequencing-by-synthesis reaction of the complementary strand was performed on a PSQ 96MA instrument (Pyrosequencing AB, Uppsala, Sweden). For selected tumors, the B-Raf PCR amplicon generated for pyrosequencing was also directly sequenced on the 985

7 ABI Prism s 31 system (B-Raf PCR forward: 5 -ATGCTTGCTCT GATAGGAA, B-Raf PCR reverse: 5 -GCATCTCAGGGCCAAA). Proliferation assays Melanoma cell lines were maintained in RPMI (Invitrogen, Foster City, CA). When indicated, cells were serum-deprived for 24 hours, amino acid deprived for 3 minutes, or treated for 24 hours with 2 nm rapamycin (Biomol Research Laboratories) or 1 mm FTI-277 (Calbiochem). For thymidine incorporation assays, melanoma cells were plated in triplicate in 24-well plates. Twenty-four hours after plating, 2 nm rapamycin or 1 mm FTI or DMSO vehicle control was added. After either 72 or 96 hours of growth at 371C ina 5% CO 2 humidified atmosphere, cells were labeled for 6 hours with 1 mci of 3 H-thymidine directly added to the media. Six hours later, cells were washed with phosphate-buffered saline, fixed in 5% trichloroacetic acid for 3 minutes at 41C, and lysed in.5 ml of.5 N NaOH/.5% SDS. 3 H-thymidine incorporation was measured by scintillation counting. 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