Farnesyltransferase Inhibitor SCH66336 Induces Rapid Phosphorylation of Eukaryotic Translation Elongation Factor 2 in Head and Neck Squamous Cell Carcinoma Cells

Research Article

Hening Ren,1 Shyh-Kuan Tai,1,3 Fadlo Khuri,4 Zuming Chu,1 and Li Mao1,2

1Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center; 2Cancer Biology Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas; 3Department of Otolaryngology, National Yang Ming University, Taipei Veteran General Hospital, Taipei, Taiwan; and 4Department of Oncology/Hematology, Winship Cancer Institute, Emory University, Atlanta, Georgia

Abstract and protein kinases, nuclear lamins, and centromere protein F (4). Farnesyltransferase inhibitors (FTIs) are a class of therapeutic The ras protein plays a critical role in transducing growth signals agents designed to target tumors with mutations of the ras from cell surface receptors to cytosol and nucleus. Activation oncogene. However, the biological effect of FTIs is often mutations of ras are frequently detected in various types of independent of ras mutation status, which suggests the human cancers (5, 6) and its constitutive activation helps trans- existence of additional mechanisms. In this study, we form normal cells in both in vitro and in vivo models thereby investigated the molecular effects of SCH66336, an FTI, in leading to tumor formation (7, 8). The discovery that prenyla- head and neck squamous cell carcinoma cells using proteomic tion is a necessary step in the functional maturation of ras (9) approaches. We showed that SCH66336 induced phosphoryla- prompted the development of farnesyltransferase inhibitors tion (inactivation) of eukaryotic translation elongation factor (FTIs) as targeted therapeutic agents in cancers with a ras muta- 2 (eEF2), an important molecule for protein synthesis, as early tion (10–12). as 3 hours after SCH66336 administration. Protein synthesis In a clinical study of patients with head and neck squamous cell carcinoma (HNSCC), we observed antitumor activity of SCH66336, was subsequently reduced in the cells. Paradoxically, activa- a potent nonpeptide tricyclic inhibitor of farnesyltransferase (13). tion of eEF2 kinase (eEF2K), the only known kinase that This FTI has also been shown to have antitumor activity in vitro regulates eEF2, was observed only at 12 hours after SCH66336 and in vivo for other tumors with or without a ras mutation treatment. Consistent with this observation, the inhibition of (14, 15). However, the mechanism of this activity is poorly phosphorylated-MEK and phosphorylated-p70S6K, the two understood. The effect of SCH66336 on cell growth inhibition is key signaling molecules responsible for activation of eEF2K, often observed within a few hours after administration, although also occurred at least 12 hours after SCH66336 administra- the cellular half-life of ras is f24 hours (16). In light of this tion. Our data suggest that inhibition of protein synthesis discrepancy and the possibility that FTIs may inhibit the activity of through inactivation of eEF2 is a novel mechanism of farnesylated proteins other than ras, FTIs may work through more SCH66336-mediated growth inhibition and that this effect is than one pathway for their antitumor activity. Using proteomic independent of ras-MEK/p70S6K-eEF2K signaling cascades. approaches, we explored how SCH66336 affects the growth of (Cancer Res 2005; 65(13): 5841-7) HNSCC cells and whether the effects were dependent on ras signaling. We found that SCH6636 may induce growth inhibition of Introduction HNSCC cells by delaying their entry into and accumulation in the Protein prenylation is a posttranslational modification in which G1 phase of the cell cycle. Evidence emerged that SCH66336 a farnesyl or geranylgeranyl isoprenoid is linked to a specific induces rapid inactivation of eukaryotic translation elongation cystine residue of proteins through a thioether bond (1). The factor 2 (eEF2) through its phosphorylation and subsequent housekeeping enzymes farnesyltransferase and glycerol-3-phos- reduction of protein synthesis. Furthermore, the inactivation of phare cytidylyltransferase I and II catalyze the addition of a prenyl eEF2 was independent of ras-MEK-eEF2 kinase (eEF2K) and ras- group to a conserved cystine residue in proteins that contain the PI3K/p70S6K-eEF2K signaling cascades. motif CaaX, CC, or CxC at or near the COOH terminal of their nascent proteins (2). Materials and Methods Comprising up to 0.5% of all proteins in mammalian tissues (3), Cell lines and culture conditions. Eight human HNSCC cell lines prenylated proteins have diverse functions in cell growth, (UMSCC14B, UMSCC17B, UMSCC21A, UMSCC22A, UMSCC38, MDA1186, differentiation, cytoskeleton structure, and vesicle trafficking MDA886, and TR146) were used in this study. The cells were grown in (2, 4). Examples of such proteins are the ras family of small monolayer culture in a 1:1 mixture of DMEM and Ham’s F12 medium GTP-binding proteins, Rho family proteins, certain phosphatases supplemented with heat inactivated 5% fetal bovine serum and antibiotics

at 37jC in a humidified atmosphere consisting of 95% air and 5% CO2.For synchronized culture, cells were grown exponentially to 40% confluence Requests for reprints: Li Mao, Molecular Biology Laboratory, Department of and starved in serum-free DMEM/Ham’s F12 medium for 24 hours before Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson serum-containing medium was added back. Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363; Fax: 713-796-8655; E-mail: [email protected]. Cell cycle analysis. UMSCC38 cells were grown to 30% confluence and I2005 American Association for Cancer Research. grown for 18 hours in medium with 5% serum and with serum-free medium www.aacrjournals.org 5841 Cancer Res 2005; 65: (13). July 1, 2005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research for 24 hours. Serum-containing medium was then added back, and cells were harvested at different times, fixed in 70% cold ethanol, and stored at 4jC until cell cycle analysis. The FTI SCH66336 dissolved in DMSO was added to the cell culture medium, and cells were harvested at different times. The cells were then stained with 50 Amol/L/mL propidium iodide in PBS buffer containing 50 Ag/mL RNase A. DNA content was measured using an EPICS 752 flow cytometer (Coulter Corp., Hialeah, FL). Data analysis was done using the Multi series (Phoenix Flow Systems, San Diego, CA) and Summit software (Cytomation, Fort Collins, CO). Protein extraction and Western blot analysis. Cells were washed in cold PBS and incubated for 15 minutes on ice in a buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1% SDS, and 1% Triton X-100 supplemented with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). The cell lysates were spun in a centrifuge at 12,000 Â g for 5 minutes. The protein concentration of the supernatant was determined using a detergent-compatible protein assay kit (Bio-Rad, Hercules, CA). Proteins (10 Ag) were separated through a 10% polyacrylamide gel in a Mini-Protean II apparatus (Bio-Rad) and transferred to a nitrocellulose membrane (BA83; Schleicher & Shuell BioScience, Keene, Figure 1. Change of G1 phase distribution after SCH66336 treatment. NH). Membranes were blocked with 2% casein in PBS and probed with UMSCC38 cell growth was synchronized by serum starvation. The cells then cultured with 5% serum and treated with 8 Amol/L SCH66336 when most of antibodies. Specific antibody binding was detected using an enhanced them were rolled out from the G1 phase. The percentage of cells in the G1 phase chemiluminescence kit (Pierce, Rockford, IL) according to the manufac- was determined using flow cytometry. Treatment with SCH66336 (FTI) turer’s protocol. slowed entry of cells into G1 and subsequent prolonged accumulation in the G1 For Western blotting, antibodies were obtained from Cell Signaling phase compared with cells treated with DMSO (Control). Technology (Beverly, MA) against phospho-(serine/threonine) protein kinase A (PKA) substrate, phospho-(serine) protein kinase C (PKC) substrate, laser desorption/ionization time-of-flight mass spectrometry (MALDI- phospho-eEF2 (Thr56), eEF2, phospho-eEF2K (Ser366), eEF2K, mitogen- TOF), AXIMA-CFR (Kratos Analytical, Manchester, United Kingdom). activated protein kinase kinase (MEK), phosphor-MEK1/2 (Ser217/221), and Protein identification based on peptide fingerprints was achieved using phospho-p70S6K/p-85S6K (Thr389). Monoclonal anti-actin antibody (AC-15) online search engine: Mascot.5 was obtained from Sigma Chemical (St. Louis, MO). Metabolic labeling. Cells were cultured in 60-mm plastic dishes to Two-dimensional gel electrophoresis. Cells grown in monolayer were 30% confluence, synchronized by serum starvation for 24 hours, and washed in cold PBS thrice, and proteins were extracted by the addition of grown in serum-containing medium for another 24 hours. The two-dimensional gel electrophoresis sample buffer containing 8 mol/L urea, synchronized cells were then treated with SCH66336 in full culture 4% CHAPS, and 25 mmol/L DTT. An aliquot of cell lysates containing an medium for 1 hour. The medium was changed to cystine- and equivalent of 5 Â 105 cells was applied to a 17-cm immobilized pH gradient methionine-free DMEM with 5% serum for 30 minutes and then 100 strip (pH 5 to 8, Bio-Rad) for 12 hours and focused under 48,000 V hours at ACi [35S] trans-label mixture (Amersham Biosciences) was added. Cells 18jC in an IPGphor isoelectric focusing unit (Amershan Biosciences, were harvested 4 hours later, and proteins were extracted in the two- Piscataway, NJ). After focusing, the immobilized pH gradient strips were dimensional gel electrophoresis sample buffer. The extracted cellular treated sequentially with 2% DTT followed by 2.5% iodoacetamide in SDS- proteins were analyzed for total proteins and newly synthesized proteins PAGE equilibration buffer [6 mol/L urea, 0.375 mol/L Tris (pH 8.8), 2% SDS, as described above. and 20% glycerol] for 15 minutes each. Focused proteins were then separated in a 10% SDS-polyacrylamide gel. For two-dimensional gel electrophoresis Western blotting, the separated proteins were transferred to Results nitrocellulose membranes, blocked, and probed with antibodies as The replication cycle of the HNSCC cell line UMSCC38 was f30 described above. For analysis of newly synthesized (radiolabeled) proteins, hours when cultured in DMEM and Ham’s F12 with 5% serum. We the gels were fixed and stained with a Silver Stain Plus kit (Bio-Rad) treated these cells with 8 Amol/L SCH66336 when most of them according to the manufacturer’s protocol and dried on filter paper followed had completed one replication after synchronization. We observed by exposure to autoradiography. To quantitate the level of protein expression by two-dimensional gel electrophoresis, the autoradiography a slowed cell accumulation at the G1 phase of the cell cycle after 6 or gel image was scanned using Amersham-Pharmacia ImageScanner. The hours following SCH66336 treatment but a prolonged G1 phase integrated absorbance of all recognized protein spots was obtained by (Fig. 1). We did not observe an emergence of a sub-G1 population analyzing the gel image with ImageMaster 2D image analysis software in the drug-treated cells but an early accumulation of G2-M phase (Amersham Biosciences). (data not shown). These results suggest a delayed G1 entry and G1 Peptide mapping for protein identification. After two-dimensional arrest by SCH66336 in UMSCC38 cells. gel electrophoresis separation of cellular proteins, the gels were stained To identify which protein expression were affected by using colloidal Coomassie brilliant blue (Bio-Rad) in 17% ammonium SCH66336 treatment, we did two-dimensional gel electrophoresis sulfate and 15% methanol, as previously described (17). Protein spots were analysis to compare expression levels in UMSCC38 cells before excised, destained in 50% methanol, and dehydrated in acetonitrile. The and after treatment. The expression levels of the most of the dried gel slots were rehydrated and digested in 25 AL of 25 mmol/L proteins remained similar. The exceptions were a protein of ammonium carbonate containing 2 Ag/mL sequencing grade modified f trypsin (Roche Applied Science) at 37jC overnight. The digest products 100 kDa with an isoelectric point (pI) of 7.1 and another protein of f100 kDa with a pI of 7.4, which increased and were purified using C18 microbed chromatography (ZipTip, Millipore, Billerica, MA) according to the manufacturer’s protocol. The purified decreased, respectively, after SCH66336 treatment (Fig. 2A and B). peptides were eluted in 50% acetonitrile and 0.1% trifluoroacetic acid In a separate attempt to identify proteins whose phosphorylation saturated with a-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, St. Louis, MO), and 1.5 AL of the peptide mix were spotted on a sample plate for analysis. Peptide fragments were determined by using a matrix-assisted 5 http://www.matrixscience.com/references/725.html

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. FTI Induces eEF2 Phosphorylation was affected by FTI treatment, we stained two-dimensional PKC substrate motif coincided with the 100-kDa (pI = 7.1) protein protein blots with several phosphorylation-specific antibodies, spot seeing increased after FTI treatment by chromogenic including antibodies specific to phosphorylated substrates of PKA staining. Using mass spectrometer-based peptide fingerprinting, and PKC. We found that the level of protein phosphorylation was we identified both 100-kDa proteins as eEF2 (Fig. 3). To confirm generally reduced after SCH66336 treatment, but the phosphor- the proteins were indeed eEF2, we did two-dimensional gel ylation of a few protein spots, including a 100-kDa protein with a electrophoresis and Western blotting using antibodies specific to pI of 7.1 was increased (Fig. 2C and D). The 100-kDa (pI = 7.1) eEF2 and phospho-eEF2 (Thr56). The protein spot with a pI of 7.1 protein spot recognized by anti-phospho-PKA and anti-phospho- reacted with anti-phospho-eEF2 antibody, which increased after

Figure 2. Changes in protein levels and modifications after SCH66336 treatment. UMSCC38 cells at 60% confluence were treated with DMSO (Control)or8Amol/L SCH66336 for 24 hours. Total protein (5 Â 105 cell equivalent) was subjected to two-dimensional gel electrophoresis analysis, and separated proteins were transferred to nitrocellulose membranes. Total proteins on the membranes were visualized by staining with dye (A and B); phosphorylated proteins were detected by using antibodies specific to phosphorylated substrates of PKA and PKC (C and D). Total eEF2 and P-eEF2 were detected by using specific antibodies (E). The thinner solid arrows (right) indicate unphosphorylated eEF2 whereas the thicker solid arrows (left) indicate P-eEF2. F, peptides after trypsin digestion measured by MALDI-TOF. Matched peptides are listed based on the sizes and their locations within the protein.

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SCH66336 treatment, whereas both protein spots of pI 7.1 and 7.4 Ser221 by ras-activated Raf-1 activity (23). The levels of P-eEF2K reacted with anti-eEF2 antibody (Fig. 2E). These data show that (Ser336) and P-MEK1/2 (Ser217/221) were decreased at 12 and eEF2 was predominantly unphosphorylated in exponentially 24 hours after SCH66336 administration, respectively, whereas the growing UMSCC38 cells and that SCH66336 induced its P-eEF2 level sharply increased as early as 3 hours after SCH66336 phosphorylation. administration, and this increased level was maintained for up to To determine whether the treatment effect is universal in 30 hours (Fig. 5). We found it interesting that the total eEF2 level HNSCC, we examined the effect on seven other HNSCC cell lines. did not changed over time but that the levels of MEK and eEF2K Increased P-eEF2 was observed in all but the UMSCC17B line as were reduced at 24 and 30 hours after the treatment. The P-MEK the SCH66336 concentration increased from 2 to 8 Amol/L level was transiently reduced at 3 hours but rebounded by 6 hours (Fig. 4A). Because UMSCC38 is among the most sensitive ones to with corresponding change in P-eEF2K level (Fig. 5). SCH66336 induced eEF2 phosphorylation, it was selected for In contrast to the dramatic changes in the FTI treated cells, the further analysis to determine potential mechanism of the levels of P-eEF2 (Thr56) and P-eEF2K (Ser336) level in vehicle phosphorylation. Using UMSCC38 cells as a model, we then (DMSO)–treated cells did not change significantly over time, analyzed how soon eEF2 phosphorylation occurs after SCH66336 whereas the transient depression of P-MEK 1/2(Ser217/221) were treatment. Increased P-eEF2 level was observed as early as 3 hours seen. Furthermore, in the serum-starved cells, the level of P-MEK 217/221 after administration of 5 to 8 Amol/L SCH66336 (Fig. 3B). These 1/2 (Ser ) reduced dramatically, with corresponding decrease 336 56 data indicate SCH66336 can induce rapid phosphorylation of Thr56 in P-eEF2K (Ser ) level and increase in P-eEF2 (Thr ) level in eEF2 in majority of HNSCC cell lines. (Fig. 5). These results suggest SCH66336 induced eEF2 phosphor- Because phosphorylation at Thr56 inactivates eEF2 (18), we next ylation is independent of ras-MEK-eEF2K pathway. examined whether the increased P-eEF2 (Thr56) expression after Phosphorylation of eEF2 could also occur through the ras-PI3K/ 389 SCH66336 treatment inhibits protein synthesis. By adding 35S- p70S6K-eEF2K pathway (12, 22). Phosphorylation of Thr in labeled methionine and cystine into the culture medium of p70S6K is critical for its kinase activity in vivo (24, 25). Therefore, we analyzed P-p70S6K (Thr389) status in UMSCC38 cells after UMSCC38 cells in the G1 phase 1 hour after SCH66336 treatment, we found that the amount of newly synthesized proteins in the treatment with SCH66336. The level of P-p70S6K was transiently following 4 hours was substantially reduced compared with that in reduced at 3 hours but rebounded by 6 hours before declining at cells not treated with SCH66336 but that the reduction of total 12 hours and 30 hours. Similar changes were seen in cells treated proteins was insubstantial (Fig. 4). with vehicle (DMSO). In contrast, the changes of P-p70S6K level Because phosphorylation of eEF2 at Thr56 is mainly caused by were greatly reduced in serum-starved cells (Fig. 5). These results the activity of eEF2K (19, 20) which itself is negatively regulated by suggest the induction of P-eEF2 is also independent of the ras- phosphorylation through the ras-MEK signaling pathway (21, 22), PI3K/p70S6K-eEF2K pathway. one may expect that the inhibition of ras activity would result in the activation of eEF2K by reducing eEF2K phosphorylation, Discussion thereby increasing the P-eEF2 level. To determine whether the In synchronized UMSCC38 cells, SCH66336 treatment induced effect of SCH66336 on eEF2 is through the ras-MEK-eEF2K a delay of the G1-phase entry starting at about 6 hours and pathway, we analyzed the levels of MEK and eEF2K proteins and subsequent G1 arrest. A previous study showed that SCH66336 their phosphorylated forms after SCH66336 treatment. Activation induced G1 arrest in cells transformed by H-ras or cells with an 217 of MEK1 and MEK2 occurs through phosphorylation at Ser and activated H-ras but induced G2-M phase accumulation in cells

Figure 3. Induction of eEF2 phosphorylation in HNSCC cell lines after SCH66336 treatment. HNSCC cells with 40% confluence were treated with 1 to 8 Amol/L SCH66336 for 24 hours. Total eEF2 and P-eEF2 were measured by Western blot using specific antibodies. eEF2 and P-eEF2 levels at different time points with different SCH66336 concentrations in UMSCC38 cells. h-Actin was used as a loading control. SF, serum free.

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Figure 4. Reduction of protein synthesis in UMSCC38 cells after SCH66336 treatment. Synchronized cells were grown to 30% confluence before treatment with DMSO (control) or 8 Amol/L SCH66336 for 1 hour. Medium was then changed to cystine- and methionine-free DMEM with 100 ACi 35S and cultured for 4 hours. Total protein was extracted and separated by two-dimensional gel electrophoresis. Proteins on the two-dimensional gel electrophoresis gels were visualized by either silver staining (A and B; representing total protein) or autoradiography (C and D; representing newly synthesized proteins). The total protein quantity on each gel was quantified and compared (E).

without activated H-ras (26). However, mutations of H-ras are rare further investigation, but it is unlikely to be the mechanism for in HNSCC, and no mutation in H-ras and K-ras genes was eEF2 phosphorylation because P-eEF2K level was not reduced identified in any of the HNSCC cell lines analyzed,6 suggesting before 12 hours and the high P-eEF2 level did not fluctuate that the G1 effect we observed in the current study was between 3 and 30 hours. Because eEF2K is the only known kinase independent of H-ras mutation status. Chun et al. (27) recently for eEF2, our data suggest the presence of a novel mechanism reported a G2-M arrest in a HNSCC cell line (SqCC/Y1) after mediated by SCH66336 to inactivate eEF2 and thereby inhibit SCH66336 treatment. These data suggest the presence of two protein synthesis. In supporting of our hypothesis, we found that distinct mechanisms of this FTI in cell growth inhibition in HNSCC. serum starvation leads to an increase in P-eEF2 (Thr56) In this study, we showed that SCH66336 induced a rapid level, accompanied by substantial decrease in the level of P-MEK inactivation of eEF2 and inhibition of protein synthesis in HNSCC (Ser217/221), P-p70S6K (Thr389), and P-eEF2K (Ser336). cells. eEF2, also known as polypeptidyl-tRNA translocase, is a key Two possibilities may explain our observations. First, SCH66336 enzyme in protein biosynthesis. It catalyzes the translocation of might affect farnesyl-dependent proteins other than ras and result peptidyl tRNA from the A site to the P site on the ribosome, and in increased eEF2 phosphorylation through an unidentified kinase. the activity of eEF2 is regulated through phosphorylation by eEF2K, The substantial changes in phosphorylation of proteins other than a unique Ca2+/calmodulin-dependent kinase (18–20). The principle eEF2 after SCH66336 treatment observed suing two-dimensional site of phosphorylation by eEF2 is Thr56 (28). The phosphorylation gel electrophoresis and Western blotting indicate the involvement inactivates eEF2 activity by preventing it from binding to ribosome of other signaling molecules responsible for the cellular response (18), resulting in reduced protein synthesis. The eEF2K activity is to SCH66336. Identification and characterization of these mole- regulated by growth factors through either the MEK/extracellular cules may help reveal the precise mechanism of the signaling signal-regulated kinase or PI3K/p70S6K signaling pathways (21–25, cascade affecting eEF2 after SCH66336 treatment. The combined 29–31). Phosphorylation of eEF2K at Ser366 inactivates the kinase, two-dimensional gel electrophoresis and Western blot approach leading to dephosphorylation of eEF2 (Thr56) and increased protein allowed us to observe proteins at very low and otherwise unde- synthesis (21, 25). ectable quantities, presumably because of the use of high-affinity The increased P-eEF2 level observed in this study cannot be antibodies. The antibodies we used are specific to the phosphor- simply explained by the decreased P-eEF2K level in SCH66336- ylated substrates of PKA and PKC; thus, the unknown protein treated cells, because this decrease was observed 12 hours after kinase might belong to PKA or PKC signaling cascades. treatment, whereas the increased P-eEF2 level was detected at as The other possibility is that SCH66336 inhibits the activity of a early as 3 hours and the high level was maintained thereafter. The protein phosphatase and reduces the rate of eEF2 depho‘sphory- reduced P-eEF2K level after 12 hours may be explained by lation. Previous studies have shown that P-eEF2 may be reduced by SCH66336-mediated inhibition of ras signaling. Consistent with growth stimuli (32, 33), but inhibition of serine-threonine protein this view, P-MEK and P-p70S6K levels were also reduced 12 hours phosphatase 2A (PP2A), a complicated protein complex, may or later after SCH66336 treatment. The transient reduction of P- attenuate the reduction of P-eEF2 level (34). If SCH66336 inhibits MEK and P-p70S6K levels at 3 hours is interesting and warrants PP2A activity, then the P-eEF2 level may be elevated despite the lack of activation of eEF2K. However, inhibition of PP2A has been shown to increase cell proliferation and tumorigenicity (35), which 6 L. Mao, unpublished data. is inconsistent with the phenotypic functions of SCH66336 in www.aacrjournals.org 5845 Cancer Res 2005; 65: (13). July 1, 2005

Figure 5. Effect of SCH66336 on eEF2 signaling in UMSCC38 cells. UMSCC38 cells at 40% confluence were treated with 8 Amol/L SCH66336 or DMSO. Cells under serum starvation were used as positive control of eEF2 signaling. Total protein was extracted at different time points and subjected to Western blot analysis using specific antibodies. h-Actin served as a loading control. cancer cells (12, 13). Furthermore, PP2A function has been found underlying mechanism of the FTI’s antitumor activity. A better to be impaired in some human cancers (36), which supports its understanding how eEF2 function is controlled and which role in antiproliferation and antitransformation. Nevertheless, the proteins are affected by eEF2 may allow us to develop novel functional status of the PP2A complex in HNSCC may help strategies to target protein synthesis for treating or preventing elucidate the involvement of this complex in SCH66336-induced HNSCC and other human cancers. cellular responses. The reduced protein synthesis after SCH66336 treatment is Acknowledgments consistent with the increased level of P-eEF2, which affects only Received 8/31/2004; revised 4/1/2005; accepted 4/13/2005. the synthesis of new proteins. Although we have not determined Grant support: Department of Defense grant DAMD17-01-1-01689-1 and National the identity of the proteins whose synthesis was affected by Cancer Institute grants PO1 CA106451, PO1 CA91844, and U01 CA 86390. The costs of publication of this article were defrayed in part by the payment of page SCH66336 treatment, we can predict that their reduced level have charges. This article must therefore be hereby marked advertisement in accordance effected the cellular functions, which may be part of the with 18 U.S.C. Section 1734 solely to indicate this fact.

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