Geranylgeranylated Proteins Are Involved in the Regulation of Myeloma Cell Growth

Vol. 11, 429–439, January 15, 2005 Clinical Cancer Research 429

Geranylgeranylated Proteins are Involved in the Regulation of Myeloma Cell Growth

Niels W.C.J. van de Donk,1 Henk M. Lokhorst,3 INTRODUCTION 2 1 Evert H.J. Nijhuis, Marloes M.J. Kamphuis, and Multiple myeloma is characterized by the accumulation Andries C. Bloem1 of slowly proliferating monoclonal plasma cells in the bone Departments of 1Immunology, 2Pulmonary Diseases, and 3Hematology, marrow. Via the production of growth factors, such as University Medical Center Utrecht, Utrecht, the Netherlands interleukin-6 (IL-6) and insulin-like growth factor-I (1–4), and cellular interactions (5, 6), the local bone marrow microenvironment sustains tumor growth and increases the ABSTRACT resistance of tumor cells for apoptosis-inducing signals (7). Purpose: Prenylation is essential for membrane locali- Multiple signaling pathways are involved in the regulation of zation and participation of proteins in various signaling growth and survival of myeloma tumor cells. Activation of the pathways. This study examined the role of farnesylated and Janus-activated kinase-signal transducers and activators of geranylgeranylated proteins in the regulation of myeloma transcription (8), nuclear factor-nB (9–11), and phosphatidy- cell proliferation. linositol 3V-kinase (PI-3K; refs. 4, 12, 13) pathways has been Experimental Design: Antiproliferative and apoptotic implicated in the protection against apoptosis, whereas effects of various modulators of farnesylated and geranyl- activation of the PI-3K (4, 12, 13), nuclear factor-nB (10, 11), geranylated proteins were investigated in myeloma cells. and mitogen-activated protein kinase pathways (14) induces Results: Depletion of geranylgeranylpyrophosphate proliferation in myeloma cell lines. inhibited myeloma cell proliferation through accumulation GTPases of the Ras and Rho families cycle between an of cells in G phase of the cell cycle and loss of cells in S phase. 1 inactive GDP-bound form and a GTP-bound form with affinity In contrast, depletion of farnesylpyrophosphate had no or for various effector proteins that control signal transduction only minor effects. Furthermore, inhibition of geranylger- cascades regulating multiple cellular processes, including anyl transferase I activity was more effective in reducing migration, cytoskeletal reorganization, stimulation of cell myeloma cell growth when compared with inhibition of proliferation, and survival. Activating Ras mutations are farnesyl transferase activity. This indicates that protein frequently detected in myeloma (15–17) and contribute to geranylgeranylation is important for myeloma cell prolifer- reduced apoptosis (18, 19), increased cell proliferation (18, 19), ation and cell cycle progression through G . Geranylgerany- 1 and an adverse clinical outcome (15, 17). Rac-1, Cdc42, and lated target proteins involved in the control of proliferation RhoA have been implicated in the regulation of cell cycle include GTPases, such as Rac-1, Cdc42, and RhoA. progression through G phase of the cell cycle. Constitutively Inhibition of Rho, Rac, and Cdc42 GTPases by toxin B 1 activated mutants of Rac-1, Cdc42, and RhoA caused G reduced proliferation, without affecting cell viability, where- 1 progression and stimulation of DNA synthesis in fibroblasts as specific inhibition of Rho GTPases by C3 exoenzyme was (20–22). Furthermore, expression of active forms of Rac-1, without effect. This suggests a role for Rac and/or Cdc42 Cdc42, and RhoA can transform fibroblasts (23, 24), and GTPases in myeloma cell growth. Rac-1 activity was found in activation of Rac-1, RhoA, and Cdc42 is required for full Ras all myeloma cell lines and was suppressed by the depletion of transforming activity (25–27). The role of Rac, Cdc42, and intracellular pools of geranylgeranylpyrophosphate, whereas RhoA in multiple myeloma is currently unknown. interleukin-6 rapidly induced Rac-1 activation. Furthermore, dominant-negative Tat-Rac-1 reduced myeloma cell prolif- Participation of Ras and Rho family proteins in signaling eration, whereas constitutively active Tat-Rac-1 enhanced pathways depends on their proper subcellular localization to the proliferation. plasma membrane, which is facilitated by a series of post- Conclusion: These results indicate that protein gera- translational modifications of the carboxyl terminus (28–30). nylgeranylation is essential for myeloma cell proliferation This includes the addition of a farnesyl lipid side chain by and suggest that Rac-1 is a regulator of myeloma cell growth. farnesyl transferase (farnesylation) or a geranylgeranyl lipid side chain by geranylgeranyl transferase I (geranylgeranylation) to a conserved cysteine residue at the carboxyl terminus of proteins ending in CAAX, where C is cysteine and A is an aliphatic amino Received 6/29/04; revised 10/5/04; accepted 10/14/04. acid. The protein will be farnesylated when X is methionine, Grant support: Dutch Cancer Society. serine, cysteine, or glutamine and geranylgeranylated when X The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked is leucine or isoleucine. Target proteins include Ras, which is advertisement in accordance with 18 U.S.C. Section 1734 solely to predominantly farnesylated, and Rho family proteins, such as indicate this fact. Rac-1, Cdc42, and RhoA, which are geranylgeranylated. The Requests for reprints: Andries C. Bloem, Department of Immunology, farnesyl and geranylgeranyl lipids used for protein prenylation University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. Phone: 31-30-2506511; Fax: 31-30-2517107; are derived from farnesylpyrophosphate (FPP) and geranylger- E-mail: [email protected]. anylpyrophosphate (GGPP), respectively. FPP and GGPP are D2005 American Association for Cancer Research. produced in the mevalonate pathway. The rate-limiting step of

this pathway is the conversion of 3-hydroxy-3-methylglutaryl- The plasma cell percentage in the patient samples varied from 15% CoA (HMG-CoA) to mevalonate, which is catalyzed by the to 96% of the mononuclear cells as determined by coexpression of enzyme HMG-CoA reductase (31). Mevalonate is an intermedi- CD38 (anti-CD38-FITC, Immunotech, Marseilles, France) and ate in the synthesis of, among others, cholesterol, dolichol, and CD138 (anti-CD138-PE, Immunotech) by flow cytometric the isoprenoid molecules FPP and GGPP. Inhibitors of HMG- analysis (FACSCalibur, Becton Dickinson Immunocytometry CoA reductase, such as lovastatin, are widely used to treat Systems, Erembodegem, Belgium). Except for patient 3, who had patients with hypercholesterolemia (32). 96% myeloma cells in her bone marrow, tumor cells were purified In this report, we show that inhibition of protein ex vivo by magnetic cell sorting (Miltenyi Biotec, Bergisch geranylgeranylation either by depletion of GGPP by lovastatin Gladbach, Germany) based on CD138 expression as described or by specific inhibition of geranylgeranyl transferase I activity previously (36). Samples obtained in this way contained >95% inhibits proliferation of myeloma cells. Furthermore, our data myeloma plasma cells as determined by analysis of CD38/CD138 suggest that the geranylgeranylated GTP-binding protein Rac-1 coexpression. For experiments, myeloma cells were resuspended is involved in the control of myeloma cell growth. in growth medium (see above). Approval was obtained from the University Medical Center Utrecht Institutional Review Board for MATERIALS AND METHODS these studies (01/051-E). This study was done according to the Reagents Helsinki agreement. Lovastatin and simvastatin were obtained from Merck Cell Proliferation & Co., Inc. (Rahway, NJ) and chemically activated by alkaline Myeloma cells (3 Â 104) were seeded in 96-well flat- hydrolysis before use as described previously (33). Mevalonate bottomed plates (Nunc, Roskilde, Denmark) in 100 AL growth and farnesol were purchased from Sigma (St. Louis, MO), and medium with different concentrations of lovastatin (for concen- geranylgeraniol was obtained from ICN Biomedicals BV trations, see figure legends) alone or in the presence of (Zoetermeer, the Netherlands). FTI-277 (Calbiochem, mevalonate, farnesol, or geranylgeraniol. Fixed concentrations Schwallbach, Germany) and GGTI-298 (Calbiochem) are of mevalonate (100 Amol/L), farnesol (10 Amol/L), or geranyl- CAAX peptidomimetics, which are highly selective inhibitors geraniol (10 Amol/L) were used. These concentrations have of farnesyl transferase and geranylgeranyl transferase I, proven to be optimal in rescuing myeloma cells from lovastatin- respectively. Tat-Rac-1 Q61L (constitutively active) and Tat- induced inhibition of proliferation. Specific inhibition of farnesyl Rac-1 N17 (dominant-negative) vectors (34) were a kind gift of transferase and geranylgeranyl transferase I was accomplished Dr. S. Dowdy (Howard Hughes Medical Institute, Department of with FTI-277 and GGTI-298, respectively (for concentrations, Pathology, Washington University School of Medicine, St. Louis, see figure legends; ref. 37). C. difficile toxin B specifically MO). The Clostridium botulinum C3 exoenzyme was purchased inhibits Rho, Rac, and Cdc42 GTPases (38, 39), whereas C. from List Biological Laboratories, Inc. (Campbell, CA), and botulinum C3 exoenzyme selectively inactivates Rho GTPases Clostridium difficile toxin B was obtained from Sigma. Mouse (39). Their effect on myeloma cell proliferation was studied (for monoclonal antibodies directed against Rac-1 were purchased concentrations, see figure legends). The effect of Rac-1 from Pierce (Rockford, IL). on myeloma cell proliferation was investigated by treating cells Cell Lines with dominant-negative Tat-Rac-1 or constitutively active Tat- Rac-1 proteins (for concentrations, see figure legends). After 32 Plasma cell lines RPMI-8226 and U266 were obtained from 3 the American Tissue Culture Collection (Manassas, VA), and and 80 hours, [ H]thymidine (Amersham, Little Chalfont, United Kingdom; 1 ACi/well) was added for the remaining 16 hours of L363 was from the German Collection of Microorganisms and 3 Cell Cultures (Braunschweig, Germany). The IL-6-dependent the assay. [ H]thymidine incorporation was analyzed by liquid plasma cell line XG-1 was a kind gift of Dr. B. Klein (Institute scintillation counting as described previously (40). for Molecular Genetics, Montpellier, France; ref. 35). Cell lines Cell Viability were cultured in RPMI 1640 (Life Technologies, Breda, the Viability of cells was examined by the 3-(4,5-dimethylth- Netherlands) supplemented with 10% FCS (Integro, Zaandam, iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay the Netherlands), 100 IU/mL penicillin, 100 Ag/mL streptomy- as described previously (40). cin, and 10 Amol/L h-mercaptoethanol (growth medium). The IL-6-dependent cell line XG-1 was cultured in the continuous Apoptosis Detection by Annexin V Staining presence of exogenous IL-6 (1.25 ng/mL recombinant human Myeloma cells (1.5 Â 105 in 0.5 mL) were incubated with IL-6, Roche, Almere, the Netherlands). C. difficile toxin B, C. botulinum C3 exoenzyme, dominant- negative Tat-Rac-1, or constitutively active Tat-Rac-1 Isolation of Myeloma Tumor Cells (for concentrations, see figure legends). After 2 or 4 days, cells Myeloma plasma cells were obtained from bone marrow were harvested and apoptosis was determined by using the aspirates taken from the posterior iliac crest in seven patients and Annexin V assay as described previously (40). from peripheral blood in one patient with plasma cell leukemia (patient 4) after obtaining informed consent. There were four Cell Cycle Analysis males and four females. Median age was 59 years, with a range of Cell cycle analysis of myeloma cells was done by detection 50 to 76 years. Two patients had stage I disease, one had stage II, of DNA content and incorporated bromodeoxyuridine after and five had stage III. Two patients had chemosensitive disease propidium iodide and anti-bromodeoxyuridine-FITC staining, and six patients were refractory to conventional chemotherapy. respectively, as described previously (41).

Treatment of Cells with C. botulinum C3 Exoenzyme and C. hour at 37jC. Cell lines and purified tumor cells from difficile Toxin B myeloma patients were stimulated with 10 ng/mL IL-6. Equal Cells were washed in PBS and resuspended in buffer numbers of cells (10 Â 106-15 Â 106) were washed in ice-

(114 mmol/L KCl, 15 mmol/L NaCl, 5.5 mmol/L MgCl2, cold PBS and then resuspended in 1 mL lysis buffer 10 mmol/L Tris) in the presence of solvent control or 50 Ag/mL [25 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/ C. botulinum C3 exoenzyme for 1 hour at room temperature as L MgCl2, 1% NP40, 1 mmol/L DTT, 5% glycerol] at 4jC for described previously (42, 43). Cells were washed and resus- 5 minutes. After centrifugation (16,000 Â g for 15 minutes pended in medium. C. difficile toxin B (50 ng/mL) was directly at 4jC), the supernatant was added to a spin column given to the cells. Toxin B and C3 exoenzyme were used at containing an immobilized glutathione disc and 20 Ag GST- concentrations that inhibited proliferation of various cell lines as Pak1-PBD and incubated at 4jC for 60 minutes with gentle shown previously (43, 44). rocking. The columns were centrifuged at 7,200 Â g for 2 minutes. The resin was washed four times with lysis buffer, Tat-Rac-1 Protein Isolation and 2Â SDS sample buffer (50 AL) containing 5% h- Polyhistidine-tagged Tat-Rac-1 Q61L (constitutively active) mercaptoethanol was added to the resin. The samples were and Tat-Rac-1 N17 (dominant-negative) constructs [gift from Dr. boiled at 100jC for 5 minutes. After a centrifugation step at S. Dowdy (34)] were expressed in BL21 bacteria (Novagen, 7,200 Â g for 2 minutes, the samples were electrophoresed Madison, WI). Expression and isolation of Tat-Rac-1 Q61L and on a gel. Tat-Rac-1 N17 were done exactly as has been described by Hall et al. (45), except that the isolation of the proteins was done Western Blotting under nonreducing conditions. Lipopolysaccharide was removed Western blotting procedure was done as described previ- from the protein elution by a method described previously (46). ously (37). In short, cell lysates containing equal amounts of Purity of the proteins was f95% as determined by SDS-PAGE protein were fractionated in 10% SDS-PAGE and then and subsequent Coomassie blue staining. Protein expression of electrically transferred from the gel to a polyvinylidene Tat-Rac-1 Q61L and Tat-Rac-1 N17 was confirmed by SDS- difluoride membrane. After blocking, the membranes were PAGE and immunoblotting using anti-hemagglutinin and anti- incubated with anti-Rac-1. Antibody binding was visualized Rac-1 antibodies. Protein solution was aliquoted and frozen with enhanced chemiluminescence (Amersham) detection with at À80jC. Hyperfilm enhanced chemiluminescence after incubation with a horseradish peroxidase–conjugated secondary antibody. Analysis of Tat-Rac-1 Uptake Tat-Rac-1 Q61L, Tat-Rac-1 N17, and a control protein [bovine serum albumin (BSA)] were labeled with FITC (1:3 RESULTS w/w) in PBS with 100 mmol/L NaHCO3 (pH 9.0) for 1 hour at room temperature. The nonconjugated FITC was removed Depletion of GGPP Inhibits Proliferation by Inducing G1 by dialysis with PBS. Myeloma cell lines (0.5 Â 106 cells/ Arrest in Myeloma Cell Lines mL) were incubated with 12 Ag/mL FITC-labeled Tat-Rac-1 To investigate the effect of depletion of FPP and GGPP on Q61L, FITC-labeled Tat-Rac-1 N17, or FITC-labeled BSA for myeloma cell proliferation, myeloma cell lines were incubated 15 minutes at 37jC. After incubation, cells were harvested, for 2 or 4 days with different concentrations of lovastatin alone washed thrice in ice-cold PBS, and treated with TO-PRO-3 or in the presence of mevalonate (100 Amol/L), farnesol (10 (0.2 Amol/L, Molecular Probes, Leiden, the Netherlands). Amol/L), or geranylgeraniol (10 Amol/L). Geranylgeraniol and Green fluorescence of viable (TO-PRO-3-negative) cells was farnesol are metabolized to GGPP and FPP in the cells, determined by flow cytometric analysis (Becton Dickinson respectively (47). In a previous study, we have shown that Immunocytometry Systems). Uptake of FITC-labeled proteins lovastatin under these conditions, and in these cell lines, was confirmed in cytocentrifuged myeloma cells by confocal effectively prevented prenylation of target proteins (37). laser scanning microscopy. Addition of mevalonate to lovastatin-treated myeloma cells restored both farnesylation and geranylgeranylation, whereas Detection of Rac-1 Activity addition of geranylgeraniol and farnesol resulted in the specific Rac-1 activity was determined by using the EZ-Detect rescue of geranylgeranylation and farnesylation, respectively Rac-1 activation kit (Pierce). Active Rac-1 binds specifically (37). Lovastatin inhibited proliferation of RPMI-8226 and L363 to the p21-binding domain (PBD) of p21-activated protein cells and the IL-6-dependent U266 and XG-1 cell lines in a dose- kinase 1 (Pak1). A glutathione S-transferase (GST; recombi- and time-dependent way as determined by detection of nant Schistosoma japonicum GST) fusion protein containing [3H]thymidine incorporation (Fig. 1A). Inhibition of prolifera- PBD of human Pak1 (GST-Pak1-PBD) was used to specifi- tion by 30 Amol/L lovastatin varied from 64.1% to 98.2% at day cally pull-down active Rac-1. Cells were treated for 2 days 2, whereas proliferation was reduced by >95% in all four cell with lovastatin alone or in the presence of mevalonate, lines tested at day 4. Addition of mevalonate to lovastatin-treated farnesol, or geranylgeraniol (for concentrations, see figure cell lines restored cell proliferation. This indicates that lovastatin legends). Before stimulation with IL-6, myeloma cell lines inhibits proliferation through the decrease of mevalonate were cultured overnight in serum-free RPMI 1640. After production because the specific inhibition of HMG-CoA purification of myeloma cells from patients, cells were washed reductase and not through nonspecific cell toxicity. Geranylger- and resuspended in serum-free medium and incubated for 1 aniol restored proliferation in lovastatin-treated myeloma cells.

Fig. 1 Lovastatin reduces proliferation of myeloma cell lines through the depletion of GGPP. A, L363, RPMI-8226, XG-1, and U266 cells were treated for 2 or 4 days with solvent control or different concentrations of lovastatin (lova; 1, 5, 10, 30, 50, 100, and 150 Amol/L) alone or in combination with mevalonate (meva; 100 Amol/L), farnesol (FOH;10Amol/L), or geranylgeraniol (GGOH;10Amol/L). Proliferation was determined by [3H]thymidine incorporation during the last 16 hours of culture. Points, mean of three experiments in triplicate; bars, SE. In some cases, the SE was smaller than the symbol. B and C, U266 cells were treated with solvent control (0 Amol/L) or lovastatin (L; 5, 10, 30, 100, or 150 Amol/L) alone or in combination with mevalonate (M; 100 Amol/L), farnesol (F;10Amol/L), or geranylgeraniol (G;10Amol/L) for 2 days, at which time the cells were harvested and cell cycle distribution was determined by bromodeoxyuridine assay. Percentages of cells in G1,G2-M, and S phases of the cell cycle. Columns, mean of triplicate determinations from a single experiment (representative of three independent experiments); bars, SE.

In contrast, farnesol had no effect or only partial protective Depletion of GGPP Inhibits Proliferation of Purified Tumor effects (Fig. 1A). Cell cycle analysis showed that lovastatin Cells Derived from Myeloma Patients treatment caused accumulation of cells in G1 phase of the cell Similar to myeloma cell lines, lovastatin inhibited both cycle and a loss of cells in S phase in a dose-dependent way. spontaneous and IL-6-induced proliferation of purified myeloma Lovastatin also induced apoptosis as determined by the presence tumor cells (Fig. 2A) as determined by detection of [3H]thymi- of a sub-G1 population, except for U266 cells at day 2 (<5% midine incorporation. Reduction of spontaneous proliferation apoptosis), which is in agreement with previous data (37). A induced by 30 Amol/L lovastatin varied between 61.9% to 99.8% representative example is shown for U266 cells in Fig. 1B. and 73.0% to 99.9% at days 2 and 4, respectively (n = 7). Addition of mevalonate or geranylgeraniol, but not farnesol, Addition of exogenous IL-6 enhanced myeloma cell proliferation prevented the G1-S-phase cell cycle arrest as shown for U266 but did not affect lovastatin-induced inhibition of proliferation cells in Fig. 1C. Addition of mevalonate, geranylgeraniol, or (n = 4) as shown in Fig. 2A at day 4 for two myeloma patients’ farnesol to myeloma cells in the absence of lovastatin had no samples. However, proliferation was restored by addition of effect on cell cycle distribution. mevalonate or geranylgeraniol to lovastatin-treated myeloma

tumor cells, whereas farnesol had no effect (n = 4) as shown in geranylgeranylated target proteins are the Rho family members Fig. 2B for two representative myeloma patients’ samples. These RhoA, Rac-1, and Cdc42, which are involved in a host of data suggest that depletion of intracellular pools of GGPP results cellular processes, including cell proliferation (20–24). To in inhibition of myeloma cell proliferation. discriminate between these proteins, we studied the effect of toxin B and C3 exoenzyme. C. difficile toxin B specifically Inhibition of Geranylgeranyl Transferase I Activity Reduces glycosylates and inactivates Rho, Rac, and Cdc42 but not Proliferation of Myeloma Cells other small molecular weight GTPases, such as Ras, Rab, or We have shown previously that in myeloma cells GGTI-298 Arf (38, 39). Treatment of myeloma cell lines with toxin B specifically inhibited geranylgeranylation, whereas farnesylation for 2 or 4 days reduced the number of viable cells in a dose- was specifically inhibited by FTI-277 (37). To confirm that dependent way (data not shown) and time-dependent way geranylgeranylation is critical for the regulation of myeloma cell (Table 1; Fig. 4A). This was caused by a dose-dependent (data proliferation, myeloma cell lines L363, RPMI-8226, XG-1, and not shown) and time-dependent inhibition of myeloma cell U266 were incubated with FTI-277 and GGTI-298 for 2 or proliferation, because the percentage of apoptotic cells was not 4 days. GGTI-298 inhibited proliferation in a dose- and time- affected by toxin B (Table 1; Fig. 4A). C. botulinum C3 dependent way (Fig. 3A). FTI-277 had no effect or inhibited exoenzyme selectively inactivates Rho GTPases by ADP- proliferation only to a small extent when compared with GGTI- ribosylating Asp41 (39). Treatment of myeloma cell lines with 298. Cell cycle analysis showed that GGTI-298 treatment caused C3 exoenzyme (50 Ag/mL) for 2 or 4 days had no effect on myeloma cells to arrest at the G1 phase of the cell cycle and reduced the number of cells in S phase (Fig. 3B). apoptosis or proliferation as shown for the XG-1 cell line in Fig. 4B. In addition, when C3 exoenzyme was introduced in Rho Family Members Are Involved in the Regulation of myeloma cells using reversible permeabilization with strepto- Myeloma Cell Proliferation lysin-O, no effect on apoptosis or proliferation was observed The data presented thus far support the involvement of (data not shown). Under these conditions, RhoA protein levels geranylgeranylated proteins in myeloma cell growth. Potential were reduced (data not shown), indicating that C3 exoenzyme

Fig. 2 Lovastatin reduces proliferation of purified myeloma cells from patients through depletion of GGPP. Plasma cells from myeloma patients were purified from bone marrow mononuclear cells by magnetic cell sorting based on CD138 expression. Plasma cell percentage was >95% after purification. A, myeloma tumor cells were treated for 4 days with lovastatin (30 Amol/L) or solvent control in the presence or absence of IL-6 (10 ng/mL). Proliferation was determined by [3H]thymidine incorporation during the last 16 hours of culture and expressed as a percentage of the solvent control-treated cells. Columns, mean of experiments in triplicate; bars, SE. B, myeloma cells were treated for 4 days with solvent control or lovastatin (30 Amol/L) alone or in combination with mevalonate (100 Amol/L), farnesol (10 Amol/L), or geranylgeraniol (10 Amol/L). Proliferation was determined by [3H]thymidine incorporation during the last 16 hours of culture. Columns, mean of experiments in triplicate; bars, SE.

Fig. 3 Inhibition of geranylgeranyl transferase I activity inhibits proliferation of myeloma cell lines. A, L363, RPMI-8226, XG-1, and U266 cells were treated for 2 or 4 days with solvent control or different concentrations of FTI-277 (2.5, 5, 10, 15, 20, and 30 Amol/L) or GGTI-298 (2.5, 5, 10, 15, 20, and 30 Amol/L) for 2 or 4 days. Proliferation was determined by [3H]thymidine incorporation during the last 16 hours of culture. Columns, mean of three experiments in triplicate; bars, SE. In some cases, the SE was smaller than the symbol. B, U266 cells were treated with solvent control, FTI-277 (20 Amol/L), or GGTI-298 (20 Amol/L) for 2 days, at which time the cells were harvested and cell cycle distribution was determined by bromodeoxyuridine assay. Percentages of cells in G1,G2-M, and S phases of the cell cycle are shown. Columns, mean of triplicate determinations from a single experiment (representative of three independent experiments); bars, SE.

had successfully entered the cells. A similar reduction of was activated in all four myeloma cell lines tested as shown for RhoA protein levels in C3 exoenzyme-treated cells has been XG-1 cells in Fig. 5A. Lovastatin reduced Rac-1 activity to near described previously (42). These data suggest that in myeloma background levels. Rac-1 activity was restored by addition of cells Rac and/or Cdc42 small GTPases rather than Rho mevalonate or geranylgeraniol to lovastatin-treated cells, whereas proteins might be involved in the regulation of myeloma cell farnesol had no effect (Fig. 5A). proliferation. Because Rac-1 is required for proliferation, but not survival, of BCR/ABL-expressing myeloid precursor cells Activation of Rac-1 by IL-6 in Myeloma Cell Lines and (48) and plays a role in the regulation of invasion and Tumor Cells Derived from Patients metastasis of lymphoma tumor cells (49), we explored the IL-6 is an important growth and survival factor for myeloma involvement of Rac-1 in myeloma proliferation. tumor cells. We observed that IL-6 rapidly stimulated Rac-1 activity in IL-6-deprived, serum-starved myeloma cell lines. This Lovastatin Reduces Rac-1 Activity in Myeloma Cell Lines activation was already observed after 1 minute and was In myeloma cell lines, we analyzed whether lovastatin, maintained for at least 15 minutes as shown in Fig. 5B. Total through inhibition of protein geranylgeranylation, reduced Rac-1 Rac-1 levels were not affected significantly by IL-6 (Fig. 5B). activity. The activation of Rac-1 was measured by specifically Myeloma tumor cells derived from patients were used to confirm coprecipitating the GTP-bound form of Rac-1 using a recombi- that Rac-1 activity was induced by IL-6. Similar to cell lines, IL-6- nant fusion protein of GST and amino acid residues 59 to 145 of induced proliferation coincided with a significant increase in Rac- Pak1, including the Rac binding domain (GST-Pak1-PBD). Rac-1 1-GTP levels in purified myeloma tumor cells (Fig. 5C and D).

Table 1 Effect of C. difficile toxin B on viability, proliferation, and apoptosis of myeloma cell lines L363 RPMI-8226 XG-1 U266 Day 2 Day 4 Day 2 Day 4 Day 2 Day 4 Day 2 Day 4 No. viable cells (% control) 53.1 39.6 93.1 79.8 42.9 13.7 85.8 59.6 Proliferation (% control) 61.5 65.2 93.8 83.7 39.8 22.7 39.6 36.3 % Apoptotic cells 31.9/36.8 42.8/41.6 30.2/33.4 48.1/50.5 30.2/38.1 41.5/48.8 10.9/12.7 11.3/16.7 NOTE. Myeloma cell lines were treated for 2 or 4 days with solvent control or toxin B (50 ng/mL). The number of viable cells was determined by MTT assay, the percentage of apoptotic cells was evaluated by Annexin V/propidium iodide assay, and proliferation was determined by [3H]thymidine incorporation. The number of viable cells and proliferation are expressed as a percentage of the solvent control-treated cells. The percentage of early (Annexin V–positive and propidium iodide–negative) and late (Annexin V–positive and propidium iodide–positive) apoptotic cells is shown for solvent control (left) and toxin B-treated cells (right). Data were from three experiments done in triplicate.

Dominant-Negative Tat-Rac-1 Reduces Proliferation, fluorescence in both nuclear and cytoplasmic compartments. whereas Constitutively Active Tat-Rac-1 Enhances FITC-labeled BSA was not detected in cells (data not shown). Proliferation of Myeloma Cells The effect of dominant-negative Tat-Rac-1 N17 or constitutively Previous studies have shown that Tat fusion proteins rapidly active Tat-Rac-1 Q61L transduced into cells on myeloma cell enter cells following their addition to cell culture medium (45, proliferation was analyzed in U266 and XG-1 cells. Myeloma 50); therefore, Tat fusion proteins offer a novel method for cells were incubated with the Tat-Rac-1 proteins for 2 or 4 days transduction of proteins into cells. In this study, Tat-hemagglu- after which proliferation was determined by detection of tinin was fused with N17 dominant-negative or Q61L constitu- [3H]thymidine incorporation. Transduction of myeloma cells tively active Rac-1. Uptake of Tat-Rac-1 proteins was studied in with dominant-negative Tat-Rac-1 N17 decreased proliferation XG-1 and U266 cells. Myeloma cell lines were incubated with when compared with solvent control-treated cells. In contrast, FITC-labeled Tat-Rac-1 Q61L, FITC-labeled Tat-Rac-1 N17, or cells transduced with constitutively active Tat-Rac-1 Q61L FITC-labeled BSA for 15 minutes. Flow cytometric analysis showed increased proliferation (Fig. 6C). However, both showed that >80% of the myeloma cells had taken up the FITC- dominant-negative and constitutively active Tat-Rac-1 protein labeled Tat-Rac-1 Q61L and Tat-Rac-1 N17 proteins. In contrast, had no effect on survival of myeloma cells as determined by the only 10% of the cells were positive for FITC-labeled BSA not Annexin V/propidium iodide assay (Fig. 6D). linked to the Tat peptide, demonstrating that the Tat-linked fusion proteins had entered the myeloma cells (shown in Fig. 6A and B DISCUSSION for U266 cells). Localization of Tat-Rac-1 mutant proteins In this study, we investigated the importance of protein was analyzed by confocal laser scanning microscopy. Both Tat- prenylation for the regulation of myeloma cell growth. In a Rac-1 Q61L and Tat-Rac-1 N17 showed intracellular previous study, we showed that inhibition of HMG-CoA reductase

Fig. 4 Toxin B inhibits proliferation but has no effect on the survival of myeloma cell lines. A, XG-1 cells were treated for 2 or 4 days with solvent control or C. difficile toxin B (50 ng/mL). B, XG-1 cells were treated for 2 or 4 days with solvent control or C. botulinum C3 exoenzyme (50 Ag/mL). Percentage of viable cells relative to the solvent control was measured by using MTT assay. Proliferation was determined by [3H]thymidine incorporation during the last 16 hours of culture and expressed as a percentage of the solvent control-treated cells. Percentage of apoptotic cells was determined by Annexin V assay. Percentage of early (Annexin V–positive and propidium iodide–negative) and late (Annexin V–positive and propidium iodide–positive) apoptotic cells. Columns, mean of three experiments in triplicate; bars, SE.

Fig. 5 Lovastatin inhibits Rac-1 activity, and IL-6 induces activation of Rac-1 in myeloma cell lines and purified tumor cells from patients. A, XG-1 cells were treated for 2 days with solvent control or lovastatin (5 Amol/L) alone or in combination with mevalonate (100 Amol/L), farnesol (10 Amol/L), or geranylgeraniol (10 Amol/L). Rac-1 activation state was examined using GST-Pak1-PBD pull-down assays. Representative of three experiments. B, XG-1 cells were stimulated with IL-6 for 1, 5, and 15 minutes. Before stimulation with IL-6, XG-1 cells were cultured overnight in serum-free growth medium without IL-6. Rac-1 activation state was examined using GST-Pak1-PBD pull-down assays. Representative of three experiments. C, plasma cells from myeloma patient 6 were purified from bone marrow mononuclear cells by magnetic cell sorting based on CD138 expression. Plasma cell percentage was >95% after purification. Purified myeloma tumor cells were incubated for 1 hour at 37jC in serum-free RPMI 1640 and then incubated with 10 ng/mL IL-6 or solvent control for 15 minutes. Rac-1 activation state was examined using GST-Pak1-PBD pull-down assays. D, purified myeloma cells from patient 6 were incubated for 2 days in growth medium in the presence or absence of IL-6. Proliferation was determined by [3H]thymidine incorporation during the last 16 hours of culture. Columns, mean of experiments in triplicate; bars, SE.

in myeloma cells by statins effectively inhibited farnesylation and progression of myeloma cells from G1-S phase of the cell cycle. geranylgeranylation of target proteins by depletion of the Geranylgeranylated target proteins that regulate proliferation isoprenoids FPP and GGPP, respectively (37). Depletion of include Rac-1, RhoA, and Cdc42 (20–24). C. difficile toxin B GGPP or specific inhibition of geranylgeranyl transferase I specifically glycosylates and inactivates Rho, Cdc42 and Rac activity in myeloma cells resulted in the induction of apoptosis via GTPases (38, 39). Toxin B did not induce apoptosis but inhibited down-regulation of the antiapoptotic protein Mcl-1 (37). These the proliferation of myeloma cells. C. botulinum C3 exoenzyme, results implied that geranylgeranylated proteins are involved in which ADP-ribosylates and inactivates Rho GTPases (39), had no the regulation of myeloma cell survival. In addition, it has recently effect on myeloma cell growth or survival. This suggests that been shown that geranylgeranylation is necessary for cell reduced cell growth by inhibition of geranylgeranylation is likely due, at least in part, to inhibition of Rac and/or Cdc42 function. adhesion–mediated drug resistance in multiple myeloma (51). Furthermore, these data indicate that the regulation of prolifera- In this article, we show for the first time that inhibition of protein tion by these geranylgeranylated proteins is independent of effects geranylgeranylation reduces myeloma cell growth through the on survival. Because Rac-1 is required for proliferation, but not for accumulation of myeloma cells in the G1 phase of the cell cycle survival, of BCR/ABL-expressing myeloid precursor cells (48) and loss of cells in S phase. This is in agreement with studies on and plays a role in the regulation of invasion and metastasis of lung adenocarcinoma cell lines (52) and mouse fibroblasts (53). lymphoma tumor cells (49), we investigated the potential role of Addition of geranylgeraniol, which is metabolized to GGPP in Rac-1 in myeloma cell proliferation. Rac-1 is a regulator of cells (47), to lovastatin-treated myeloma cells restored protein diverse cellular processes, including the control of cytoskeleton geranylgeranylation (37), G1-S-phase cell cycle progression, and organization, membrane trafficking, cellular adhesion, and gene proliferation. Farnesol, which is metabolized to FPP in cells (47), expression (23, 24). In addition, Rac-1 plays an essential role in completely restored protein farnesylation (37) but had no or only cell cycle progression through G1 (20, 21). Studies in fibroblasts minor effects on proliferation in lovastatin-treated myeloma cells. with activated Rac-1 mutants showed that activation of Rac-1 Furthermore, specific inhibition of geranylgeranyl transferase I alone was sufficient to initiate cell cycle progression (20, 21), activity was more effective in reducing myeloma cell proliferation whereas dominant-negative versions of Rac-1 blocked serum- when compared with inhibition of farnesyl transferase activity. induced DNA synthesis (20). This indicates that farnesylated proteins are not involved in the Depletion of GGPP by lovastatin reduced Rac-1 activity in regulation of myeloma cell proliferation or are unable to support myeloma cell lines, whereas the myeloma growth factor IL-6 proliferation in the absence of geranylgeranylated proteins. These induced activation of Rac-1 in both myeloma cell lines and IL-6- data support a role for geranylgeranylated proteins in the responsive purified myeloma cells from patients. However,

activation of Rac-1 by IL-6 could not be detected in myeloma cells actions of the Tat-Rac-1 mutant proteins make a nonspecific effect derived from patients, which did not respond to IL-6 (data not of the Tat peptide unlikely. Comparable results were obtained with shown). The role of Rac-1 in myeloma was investigated by using antisense oligodeoxynucleotides complementary to a sequence Tat-Rac-1 mutant proteins. We showed that myeloma cells can be shared by Rac-1 and Rac-2 genes (54, 55). Sequence-specific transduced with a Tat fusion of the N17 dominant-negative Rac-1 reduction of Rac protein levels by oligodeoxynucleotides reduced and a Tat fusion of the Q61L constitutively active Rac-1. Tat-Rac- proliferation of myeloma cells, whereas cell viability was not 1 mutant proteins transduced into myeloma cells did not affect cell affected (data not shown). The positive and negative effects of the survival. However, we showed that dominant-negative Tat-Rac-1 Tat-Rac-1 mutant proteins on proliferation were not profound; the reduced proliferation, whereas constitutively active Tat-Rac-1 negative effect was smaller than the inhibitory effect of lovastatin stimulated proliferation of myeloma cell lines. These opposite or GGTI-298. This can be explained by the absence of a carboxyl-

Fig. 6 Dominant-negative Tat-Rac-1 inhibits proliferation and constitutively active Tat-Rac-1 induces proliferation of myeloma cells. A and B, constitutively active Tat-Rac-1 Q61L, dominant-negative Tat-Rac-1 N17, and BSA were labeled with FITC as described in MATERIALS AND METHODS. U266 cells were incubated with 12 Ag/mL FITC-labeled Tat-Rac-1 N17 (A; bold line) or FITC-labeled Tat-Rac-1 Q61L (B; bold line) for 15 minutes at 37jC. As a control for protein bound to the cellular exterior, FITC-labeled BSA was incubated with U266 cells under identical conditions (A and B; thin line). Uptake was studied by flow cytometry. Representative of three independent experiments. C, U266 and XG-1 cells were treated for 2 or 4 days with solvent control, dominant-negative Tat-Rac-1 N17 (18.7 Ag/mL), or constitutively active Tat-Rac-1 Q61L (18.7 Ag/mL). Proliferation was determined by [3H]thymidine incorporation during the last 16 hours of culture and expressed as a percentage of the solvent control-treated cells. Columns, mean of three experiments in triplicate; bars, SE. D, U266 and XG-1 cells were treated for 2 or 4 days with solvent control, dominant- negative Tat-Rac-1 N17 (18.7 Ag/mL), or constitutively active Tat-Rac-1 Q61L (18.7 Ag/mL). Percentage of apoptotic cells was determined by Annexin V assay. Percentage of early (Annexin V–positive and propidium iodide–negative) and late (Annexin V–positive and propidium iodide–positive) apoptotic cells. Columns, mean of experiments done thrice in triplicate; bars, SE.

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