Purpose: Prenylation is essential for membrane localization and participation of proteins in various signaling pathways. This study examined the role of farnesylated and geranylgeranylated proteins in the regulation of myeloma cell proliferation.
Experimental Design: Antiproliferative and apoptotic effects of various modulators of farnesylated and geranylgeranylated proteins were investigated in myeloma cells.
Results: Depletion of geranylgeranylpyrophosphate inhibited myeloma cell proliferation through accumulation of cells in G1 phase of the cell cycle and loss of cells in S phase. In contrast, depletion of farnesylpyrophosphate had no or only minor effects. Furthermore, inhibition of geranylgeranyl transferase I activity was more effective in reducing myeloma cell growth when compared with inhibition of farnesyl transferase activity. This indicates that protein geranylgeranylation is important for myeloma cell proliferation and cell cycle progression through G1. Geranylgeranylated target proteins involved in the control of proliferation include GTPases, such as Rac-1, Cdc42, and RhoA. Inhibition of Rho, Rac, and Cdc42 GTPases by toxin B reduced proliferation, without affecting cell viability, whereas specific inhibition of Rho GTPases by C3 exoenzyme was without effect. This suggests a role for Rac and/or Cdc42 GTPases in myeloma cell growth. Rac-1 activity was found in all myeloma cell lines and was suppressed by the depletion of intracellular pools of geranylgeranylpyrophosphate, whereas interleukin-6 rapidly induced Rac-1 activation. Furthermore, dominant-negative Tat-Rac-1 reduced myeloma cell proliferation, whereas constitutively active Tat-Rac-1 enhanced proliferation.
Conclusion: These results indicate that protein geranylgeranylation is essential for myeloma cell proliferation and suggest that Rac-1 is a regulator of myeloma cell growth.
Multiple myeloma is characterized by the accumulation of slowly proliferating monoclonal plasma cells in the bone marrow. Via the production of growth factors, such as 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 resistance of tumor cells for apoptosis-inducing signals (7). Multiple signaling pathways are involved in the regulation of growth and survival of myeloma tumor cells. Activation of the Janus-activated kinase-signal transducers and activators of transcription (8), nuclear factor-κB (9–11), and phosphatidylinositol 3′-kinase (PI-3K; refs. 4, 12, 13) pathways has been implicated in the protection against apoptosis, whereas activation of the PI-3K (4, 12, 13), nuclear factor-κB (10, 11), and mitogen-activated protein kinase pathways (14) induces proliferation in myeloma cell lines.
GTPases of the Ras and Rho families cycle between an inactive GDP-bound form and a GTP-bound form with affinity for various effector proteins that control signal transduction cascades regulating multiple cellular processes, including migration, cytoskeletal reorganization, stimulation of cell proliferation, and survival. Activating Ras mutations are frequently detected in myeloma (15–17) and contribute to reduced apoptosis (18, 19), increased cell proliferation (18, 19), and an adverse clinical outcome (15, 17). Rac-1, Cdc42, and RhoA have been implicated in the regulation of cell cycle progression through G1 phase of the cell cycle. Constitutively activated mutants of Rac-1, Cdc42, and RhoA caused G1 progression and stimulation of DNA synthesis in fibroblasts (20–22). Furthermore, expression of active forms of Rac-1, Cdc42, and RhoA can transform fibroblasts (23, 24), and activation of Rac-1, RhoA, and Cdc42 is required for full Ras transforming activity (25–27). The role of Rac, Cdc42, and RhoA in multiple myeloma is currently unknown.
Participation of Ras and Rho family proteins in signaling pathways depends on their proper subcellular localization to the plasma membrane, which is facilitated by a series of post-translational modifications of the carboxyl terminus (28–30). This includes the addition of a farnesyl lipid side chain by 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 acid. The protein will be farnesylated when X is methionine, serine, cysteine, or glutamine and geranylgeranylated when X is leucine or isoleucine. Target proteins include Ras, which is predominantly farnesylated, and Rho family proteins, such as Rac-1, Cdc42, and RhoA, which are geranylgeranylated. The farnesyl and geranylgeranyl lipids used for protein prenylation are derived from farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), respectively. FPP and GGPP are produced in the mevalonate pathway. The rate-limiting step of this pathway is the conversion of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, which is catalyzed by the enzyme HMG-CoA reductase (31). Mevalonate is an intermediate in the synthesis of, among others, cholesterol, dolichol, and the isoprenoid molecules FPP and GGPP. Inhibitors of HMG-CoA reductase, such as lovastatin, are widely used to treat patients with hypercholesterolemia (32).
In this report, we show that inhibition of protein geranylgeranylation either by depletion of GGPP by lovastatin or by specific inhibition of geranylgeranyl transferase I activity inhibits proliferation of myeloma cells. Furthermore, our data suggest that the geranylgeranylated GTP-binding protein Rac-1 is involved in the control of myeloma cell growth.
MATERIALS AND METHODS
Lovastatin and simvastatin were obtained from Merck & Co., Inc. (Rahway, NJ) and chemically activated by alkaline hydrolysis before use as described previously (33). Mevalonate and farnesol were purchased from Sigma (St. Louis, MO), and geranylgeraniol was obtained from ICN Biomedicals BV (Zoetermeer, the Netherlands). FTI-277 (Calbiochem, Schwallbach, Germany) and GGTI-298 (Calbiochem) are CAAX peptidomimetics, which are highly selective inhibitors of farnesyl transferase and geranylgeranyl transferase I, respectively. Tat-Rac-1 Q61L (constitutively active) and Tat-Rac-1 N17 (dominant-negative) vectors (34) were a kind gift of Dr. S. Dowdy (Howard Hughes Medical Institute, Department of Pathology, Washington University School of Medicine, St.Louis, MO). The Clostridium botulinum C3 exoenzyme was purchased from List Biological Laboratories, Inc. (Campbell, CA), and Clostridium difficile toxin B was obtained from Sigma. Mouse monoclonal antibodies directed against Rac-1 were purchased from Pierce (Rockford, IL).
Plasma cell lines RPMI-8226 and U266 were obtained from the American Tissue Culture Collection (Manassas, VA), and L363 was from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The IL-6-dependent plasma cell line XG-1 was a kind gift of Dr. B. Klein (Institute for Molecular Genetics, Montpellier, France; ref. 35). Cell lines were cultured in RPMI 1640 (Life Technologies, Breda, the Netherlands) supplemented with 10% FCS (Integro, Zaandam, the Netherlands), 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10 μmol/L β-mercaptoethanol (growth medium). The IL-6-dependent cell line XG-1 was cultured in the continuous presence of exogenous IL-6 (1.25 ng/mL recombinant human IL-6, Roche, Almere, the Netherlands).
Isolation of Myeloma Tumor Cells
Myeloma plasma cells were obtained from bone marrow aspirates taken from the posterior iliac crest in seven patients and from peripheral blood in one patient with plasma cell leukemia (patient 4) after obtaining informed consent. There were four males and four females. Median age was 59 years, with a range of 50 to 76 years. Two patients had stage I disease, one had stage II, and five had stage III. Two patients had chemosensitive disease and six patients were refractory to conventional chemotherapy. The plasma cell percentage in the patient samples varied from 15% to 96% of the mononuclear cells as determined by coexpression of CD38 (anti-CD38-FITC, Immunotech, Marseilles,France) and CD138 (anti-CD138-PE, Immunotech) by flow cytometric analysis (FACSCalibur, Becton Dickinson Immunocytometry Systems, Erembodegem, Belgium). Except for patient 3, who had 96% myeloma cells in her bone marrow, tumor cells were purified ex vivo by magnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) based on CD138 expression as described previously (36). Samples obtained in this way contained >95% myeloma plasma cells as determined by analysis of CD38/CD138 coexpression. For experiments, myeloma cells were resuspended in growth medium (see above). Approval was obtained from the University Medical Center Utrecht Institutional Review Board for these studies (01/051-E). This study was done according to the Helsinki agreement.
Myeloma cells (3 × 104) were seeded in 96-well flat-bottomed plates (Nunc, Roskilde, Denmark) in 100 μL growth medium with different concentrations of lovastatin (for concentrations, see figure legends) alone or in the presence of mevalonate, farnesol, or geranylgeraniol. Fixed concentrations of mevalonate (100 μmol/L), farnesol (10 μmol/L), or geranylgeraniol (10 μmol/L) were used. These concentrations have proven to be optimal in rescuing myeloma cells from lovastatin-induced inhibition of proliferation. Specific inhibition of farnesyl transferase and geranylgeranyl transferase I was accomplished with FTI-277 and GGTI-298, respectively (for concentrations, see figure legends; ref. 37). C. difficile toxin B specifically inhibits Rho, Rac, and Cdc42 GTPases (38, 39), whereas C. botulinum C3 exoenzyme selectively inactivates Rho GTPases (39). Their effect on myeloma cell proliferation was studied (for concentrations, see figure legends). The effect of Rac-1 on myeloma cell proliferation was investigated by treating cells with dominant-negative Tat-Rac-1 or constitutively active Tat-Rac-1 proteins (for concentrations, see figure legends). After 32 and 80 hours, [3H]thymidine (Amersham, Little Chalfont, United Kingdom; 1 μCi/well) was added for the remaining 16 hours of the assay. [3H]thymidine incorporation was analyzed by liquid scintillation counting as described previously (40).
Viability of cells was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (40).
Apoptosis Detection by Annexin V Staining
Myeloma cells (1.5 × 105 in 0.5 mL) were incubated with C. difficile toxin B, C. botulinum C3 exoenzyme, dominant-negative Tat-Rac-1, or constitutively active Tat-Rac-1 (for concentrations, see figure legends). After 2 or 4 days, cells were harvested and apoptosis was determined by using the Annexin V assay as described previously (40).
Cell Cycle Analysis
Cell cycle analysis of myeloma cells was done by detection of DNA content and incorporated bromodeoxyuridine after propidium iodide and anti-bromodeoxyuridine-FITC staining, respectively, as described previously (41).
Treatment of Cells with C. botulinum C3 Exoenzyme and C. difficile Toxin B
Cells were washed in PBS and resuspended in buffer (114 mmol/L KCl, 15 mmol/L NaCl, 5.5 mmol/L MgCl2, 10 mmol/L Tris) in the presence of solvent control or 50 μg/mL C.botulinum C3 exoenzyme for 1 hour at room temperature as described previously (42, 43). Cells were washed and resuspended in medium. C. difficile toxin B (50 ng/mL) was directly given to the cells. Toxin B and C3 exoenzyme were used at concentrations that inhibited proliferation of various cell lines as shown previously (43, 44).
Tat-Rac-1 Protein Isolation
Polyhistidine-tagged Tat-Rac-1 Q61L (constitutively active) and Tat-Rac-1 N17 (dominant-negative) constructs [gift from Dr. S. Dowdy (34)] were expressed in BL21 bacteria (Novagen, Madison, WI). Expression and isolation of Tat-Rac-1 Q61L and 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 under nonreducing conditions. Lipopolysaccharide was removed from the protein elution by a method described previously (46). Purity of the proteins was ∼95% as determined by SDS-PAGE and subsequent Coomassie blue staining. Protein expression of Tat-Rac-1 Q61L and Tat-Rac-1 N17 was confirmed by SDS-PAGE and immunoblotting using anti-hemagglutinin and anti-Rac-1 antibodies. Protein solution was aliquoted and frozen at −80°C.
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 w/w) in PBS with 100 mmol/L NaHCO3 (pH 9.0) for 1 hour at room temperature. The nonconjugated FITC was removed by dialysis with PBS. Myeloma cell lines (0.5 × 106 cells/mL) were incubated with 12 μg/mL FITC-labeled Tat-Rac-1 Q61L, FITC-labeled Tat-Rac-1 N17, or FITC-labeled BSA for 15 minutes at 37°C. After incubation, cells were harvested, washed thrice in ice-cold PBS, and treated with TO-PRO-3 (0.2 μmol/L, Molecular Probes, Leiden, the Netherlands). Green fluorescence of viable (TO-PRO-3-negative) cells was determined by flow cytometric analysis (Becton Dickinson Immunocytometry Systems). Uptake of FITC-labeled proteins was confirmed in cytocentrifuged myeloma cells by confocal laser scanning microscopy.
Detection of Rac-1 Activity
Rac-1 activity was determined by using the EZ-Detect Rac-1 activation kit (Pierce). Active Rac-1 binds specifically to the p21-binding domain (PBD) of p21-activated protein kinase 1 (Pak1). A glutathione S-transferase (GST; recombinant Schistosoma japonicum GST) fusion protein containing PBD of human Pak1 (GST-Pak1-PBD) was used to specifically pull-down active Rac-1. Cells were treated for 2 days with lovastatin alone or in the presence of mevalonate, farnesol, or geranylgeraniol (for concentrations, see figure legends). Before stimulation with IL-6, myeloma cell lines were cultured overnight in serum-free RPMI 1640. After purification of myeloma cells from patients, cells were washed and resuspended in serum-free medium and incubated for 1 hour at 37°C. Cell lines and purified tumor cells from myeloma patients were stimulated with 10 ng/mL IL-6. Equal numbers of cells (10 × 106-15 × 106) were washed in ice-cold PBS and then resuspended in 1 mL lysis buffer [25 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L MgCl2, 1% NP40, 1 mmol/L DTT, 5% glycerol] at 4°C for 5 minutes. After centrifugation (16,000 × g for 15 minutes at 4°C), the supernatant was added to a spin column containing an immobilized glutathione disc and 20 μg GST-Pak1-PBD and incubated at 4°C for 60 minutes with gentle rocking. The columns were centrifuged at 7,200 × g for 2 minutes. The resin was washed four times with lysis buffer, and 2× SDS sample buffer (50 μL) containing 5% β-mercaptoethanol was added to the resin. The samples were boiled at 100°C for 5 minutes. After a centrifugation step at 7,200 × g for 2 minutes, the samples were electrophoresed on a gel.
Western blotting procedure was done as described previously (37). In short, cell lysates containing equal amounts of protein were fractionated in 10% SDS-PAGE and then electrically transferred from the gel to a polyvinylidene difluoride membrane. After blocking, the membranes were incubated with anti-Rac-1. Antibody binding was visualized with enhanced chemiluminescence (Amersham) detection with Hyperfilm enhanced chemiluminescence after incubation with a horseradish peroxidase–conjugated secondary antibody.
Depletion of GGPP Inhibits Proliferation by Inducing G1 Arrest in Myeloma Cell Lines
To investigate the effect of depletion of FPP and GGPP on myeloma cell proliferation, myeloma cell lines were incubated for 2 or 4 days with different concentrations of lovastatin alone or in the presence of mevalonate (100 μmol/L), farnesol (10 μmol/L), or geranylgeraniol (10 μmol/L). Geranylgeraniol and farnesol are metabolized to GGPP and FPP in the cells, respectively (47). In a previous study, we have shown that lovastatin under these conditions, and in these cell lines, effectively prevented prenylation of target proteins (37). Addition of mevalonate to lovastatin-treated myeloma cells restored both farnesylation and geranylgeranylation, whereas addition of geranylgeraniol and farnesol resulted in the specific rescue of geranylgeranylation and farnesylation, respectively (37). Lovastatin inhibited proliferation of RPMI-8226 and L363 cells and the IL-6-dependent U266 and XG-1 cell lines in a dose- and time-dependent way as determined by detection of [3H]thymidine incorporation (Fig. 1A). Inhibition of proliferation by 30 μmol/L lovastatin varied from 64.1% to 98.2% at day 2, whereas proliferation was reduced by >95% in all four cell lines tested at day 4. Addition of mevalonate to lovastatin-treated cell lines restored cell proliferation. This indicates that lovastatin inhibits proliferation through the decrease of mevalonate production because the specific inhibition of HMG-CoA reductase and not through nonspecific cell toxicity. Geranylgeraniol restored proliferation in lovastatin-treated myeloma cells. In contrast, farnesol had no effect or only partial protective effects (Fig. 1A). Cell cycle analysis showed that lovastatin treatment caused accumulation of cells in G1 phase of the cell cycle and a loss of cells in S phase in a dose-dependent way. Lovastatin also induced apoptosis as determined by the presence of a sub-G1 population, except for U266 cells at day 2 (<5% apoptosis), which is in agreement with previous data (37). A representative example is shown for U266 cells in Fig. 1B. Addition of mevalonate or geranylgeraniol, but not farnesol, prevented the G1-S-phase cell cycle arrest as shown for U266 cells in Fig. 1C. Addition of mevalonate, geranylgeraniol, or farnesol to myeloma cells in the absence of lovastatin had no effect on cell cycle distribution.
Depletion of GGPP Inhibits Proliferation of Purified Tumor Cells Derived from Myeloma Patients
Similar to myeloma cell lines, lovastatin inhibited both spontaneous and IL-6-induced proliferation of purified myeloma tumor cells (Fig. 2A) as determined by detection of [3H]thymidine incorporation. Reduction of spontaneous proliferation induced by 30 μmol/L lovastatin varied between 61.9% to 99.8% and 73.0% to 99.9% at days 2 and 4, respectively (n = 7). Addition of exogenous IL-6 enhanced myeloma cell proliferation but did not affect lovastatin-induced inhibition of proliferation (n = 4) as shown in Fig. 2A at day 4 for two myeloma patients' samples. However, proliferation was restored by addition of mevalonate or geranylgeraniol to lovastatin-treated myeloma tumor cells, whereas farnesol had no effect (n = 4) as shown in Fig. 2B for two representative myeloma patients' samples. These data suggest that depletion of intracellular pools of GGPP results in inhibition of myeloma cell proliferation.
Inhibition of Geranylgeranyl Transferase I Activity Reduces Proliferation of Myeloma Cells
We have shown previously that in myeloma cells GGTI-298 specifically inhibited geranylgeranylation, whereas farnesylation was specifically inhibited by FTI-277 (37). To confirm that geranylgeranylation is critical for the regulation of myeloma cell proliferation, myeloma cell lines L363, RPMI-8226, XG-1, and U266 were incubated with FTI-277 and GGTI-298 for 2 or 4 days. GGTI-298 inhibited proliferation in a dose- and time-dependent way (Fig. 3A). FTI-277 had no effect or inhibited proliferation only to a small extent when compared with GGTI-298. Cell cycle analysis showed that GGTI-298 treatment caused myeloma cells to arrest at the G1 phase of the cell cycle and reduced the number of cells in S phase (Fig. 3B).
Rho Family Members Are Involved in the Regulation of Myeloma Cell Proliferation
The data presented thus far support the involvement of geranylgeranylated proteins in myeloma cell growth. Potential geranylgeranylated target proteins are the Rho family members RhoA, Rac-1, and Cdc42, which are involved in a host of cellular processes, including cell proliferation (20–24). To discriminate between these proteins, we studied the effect of toxin B and C3 exoenzyme. C. difficile toxin B specifically glycosylates and inactivates Rho, Rac, and Cdc42 but not other small molecular weight GTPases, such as Ras, Rab, or Arf (38, 39). Treatment of myeloma cell lines with toxin B for 2 or 4 days reduced the number of viable cells in a dose-dependent way (data not shown) and time-dependent way (Table 1; Fig. 4A). This was caused by a dose-dependent (data not shown) and time-dependent inhibition of myeloma cell proliferation, because the percentage of apoptotic cells was not affected by toxin B (Table 1; Fig. 4A). C. botulinum C3 exoenzyme selectively inactivates Rho GTPases by ADP-ribosylating Asp41 (39). Treatment of myeloma cell lines with C3 exoenzyme (50 μg/mL) for 2 or 4 days had no effect on apoptosis or proliferation as shown for the XG-1 cell line in Fig. 4B. In addition, when C3 exoenzyme was introduced in myeloma cells using reversible permeabilization with streptolysin-O, no effect on apoptosis or proliferation was observed (data not shown). Under these conditions, RhoA protein levels were reduced (data not shown), indicating that C3 exoenzyme had successfully entered the cells. A similar reduction of RhoA protein levels in C3 exoenzyme-treated cells has been described previously (42). These data suggest that in myeloma cells Rac and/or Cdc42 small GTPases rather than Rho proteins might be involved in the regulation of myeloma cell proliferation. Because Rac-1 is required for proliferation, but not survival, of BCR/ABL-expressing myeloid precursor cells (48) and plays a role in the regulation of invasion and metastasis of lymphoma tumor cells (49), we explored the involvement of Rac-1 in myeloma proliferation.
|.||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|
|.||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.
Lovastatin Reduces Rac-1 Activity in Myeloma Cell Lines
In myeloma cell lines, we analyzed whether lovastatin, through inhibition of protein geranylgeranylation, reduced Rac-1 activity. The activation of Rac-1 was measured by specifically coprecipitating the GTP-bound form of Rac-1 using a recombinant fusion protein of GST and amino acid residues 59 to 145 of Pak1, including the Rac binding domain (GST-Pak1-PBD). Rac-1 was activated in all four myeloma cell lines tested as shown for XG-1 cells in Fig. 5A. Lovastatin reduced Rac-1 activity to near background levels. Rac-1 activity was restored by addition of mevalonate or geranylgeraniol to lovastatin-treated cells, whereas farnesol had no effect (Fig. 5A).
Activation of Rac-1 by IL-6 in Myeloma Cell Lines and Tumor Cells Derived from Patients
IL-6 is an important growth and survival factor for myeloma tumor cells. We observed that IL-6 rapidly stimulated Rac-1 activity in IL-6-deprived, serum-starved myeloma cell lines. This activation was already observed after 1 minute and was maintained for at least 15 minutes as shown in Fig. 5B. Total Rac-1 levels were not affected significantly by IL-6 (Fig. 5B). Myeloma tumor cells derived from patients were used to confirm that Rac-1 activity was induced by IL-6. Similar to cell lines, IL-6-induced proliferation coincided with a significant increase in Rac-1-GTP levels in purified myeloma tumor cells (Fig. 5C and D).
Dominant-Negative Tat-Rac-1 Reduces Proliferation, whereas Constitutively Active Tat-Rac-1 Enhances Proliferation of Myeloma Cells
Previous studies have shown that Tat fusion proteins rapidly enter cells following their addition to cell culture medium (45, 50); therefore, Tat fusion proteins offer a novel method for transduction of proteins into cells. In this study, Tat-hemagglutinin was fused with N17 dominant-negative or Q61L constitutively active Rac-1. Uptake of Tat-Rac-1 proteins was studied in XG-1 and U266 cells. Myeloma cell lines were incubated with FITC-labeled Tat-Rac-1 Q61L, FITC-labeled Tat-Rac-1 N17, or FITC-labeled BSA for 15 minutes. Flow cytometric analysis showed that >80% of the myeloma cells had taken up the FITC-labeled Tat-Rac-1 Q61L and Tat-Rac-1 N17 proteins. In contrast, only 10% of the cells were positive for FITC-labeled BSA not linked to the Tat peptide, demonstrating that the Tat-linked fusion proteins had entered the myeloma cells (shown in Fig. 6A and B for U266 cells). Localization of Tat-Rac-1 mutant proteins was analyzed by confocal laser scanning microscopy. Both Tat-Rac-1 Q61L and Tat-Rac-1 N17 showed intracellular fluorescence in both nuclear and cytoplasmic compartments. FITC-labeled BSA was not detected in cells (data not shown). The effect of dominant-negative Tat-Rac-1 N17 or constitutively active Tat-Rac-1 Q61L transduced into cells on myeloma cell proliferation was analyzed in U266 and XG-1 cells. Myeloma cells were incubated with the Tat-Rac-1 proteins for 2 or 4 days after which proliferation was determined by detection of [3H]thymidine incorporation. Transduction of myeloma cells with dominant-negative Tat-Rac-1 N17 decreased proliferation when compared with solvent control-treated cells. In contrast, cells transduced with constitutively active Tat-Rac-1 Q61L showed increased proliferation (Fig. 6C). However, both dominant-negative and constitutively active Tat-Rac-1 protein had no effect on survival of myeloma cells as determined by the Annexin V/propidium iodide assay (Fig. 6D).
In this study, we investigated the importance of protein prenylation for the regulation of myeloma cell growth. In a previous study, we showed that inhibition of HMG-CoA reductase in myeloma cells by statins effectively inhibited farnesylation and geranylgeranylation of target proteins by depletion of the isoprenoids FPP and GGPP, respectively (37). Depletion of GGPP or specific inhibition of geranylgeranyl transferase I activity in myeloma cells resulted in the induction of apoptosis via down-regulation of the antiapoptotic protein Mcl-1 (37). These results implied that geranylgeranylated proteins are involved in the regulation of myeloma cell survival. In addition, it has recently been shown that geranylgeranylation is necessary for cell adhesion–mediated drug resistance in multiple myeloma (51). In this article, we show for the first time that inhibition of protein geranylgeranylation reduces myeloma cell growth through the accumulation of myeloma cells in the G1 phase of the cell cycle and loss of cells in S phase. This is in agreement with studies on lung adenocarcinoma cell lines (52) and mouse fibroblasts (53). Addition of geranylgeraniol, which is metabolized to GGPP in cells (47), to lovastatin-treated myeloma cells restored protein geranylgeranylation (37), G1-S-phase cell cycle progression, and proliferation. Farnesol, which is metabolized to FPP in cells (47), completely restored protein farnesylation (37) but had no or only minor effects on proliferation in lovastatin-treated myeloma cells. Furthermore, specific inhibition of geranylgeranyl transferase I activity was more effective in reducing myeloma cell proliferation when compared with inhibition of farnesyl transferase activity. This indicates that farnesylated proteins are not involved in the regulation of myeloma cell proliferation or are unable to support proliferation in the absence of geranylgeranylated proteins. These data support a role for geranylgeranylated proteins in the progression of myeloma cells from G1-S phase of the cell cycle.
Geranylgeranylated target proteins that regulate proliferation include Rac-1, RhoA, and Cdc42 (20–24). C. difficile toxin B specifically glycosylates and inactivates Rho, Cdc42 and Rac GTPases (38, 39). Toxin B did not induce apoptosis but inhibited the proliferation of myeloma cells. C. botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho GTPases (39), had no effect on myeloma cell growth or survival. This suggests that reduced cell growth by inhibition of geranylgeranylation is likely due, at least in part, to inhibition of Rac and/or Cdc42 function. Furthermore, these data indicate that the regulation of proliferation by these geranylgeranylated proteins is independent of effects on survival. Because Rac-1 is required for proliferation, but not for survival, of BCR/ABL-expressing myeloid precursor cells (48) and plays a role in the regulation of invasion and metastasis of lymphoma tumor cells (49), we investigated the potential role of Rac-1 in myeloma cell proliferation. Rac-1 is a regulator of diverse cellular processes, including the control of cytoskeleton organization, membrane trafficking, cellular adhesion, and gene expression (23, 24). In addition, Rac-1 plays an essential role in cell cycle progression through G1 (20, 21). Studies in fibroblasts with activated Rac-1 mutants showed that activation of Rac-1 alone was sufficient to initiate cell cycle progression (20, 21), whereas dominant-negative versions of Rac-1 blocked serum-induced DNA synthesis (20).
Depletion of GGPP by lovastatin reduced Rac-1 activity in myeloma cell lines, whereas the myeloma growth factor IL-6 induced activation of Rac-1 in both myeloma cell lines and IL-6-responsive purified myeloma cells from patients. However, activation of Rac-1 by IL-6 could not be detected in myeloma cells derived from patients, which did not respond to IL-6 (data not shown). The role of Rac-1 in myeloma was investigated by using Tat-Rac-1 mutant proteins. We showed that myeloma cells can be transduced with a Tat fusion of the N17 dominant-negative Rac-1 and a Tat fusion of the Q61L constitutively active Rac-1. Tat-Rac-1 mutant proteins transduced into myeloma cells did not affect cell survival. However, we showed that dominant-negative Tat-Rac-1 reduced proliferation, whereas constitutively active Tat-Rac-1 stimulated proliferation of myeloma cell lines. These opposite actions of the Tat-Rac-1 mutant proteins make a nonspecific effect of the Tat peptide unlikely. Comparable results were obtained with antisense oligodeoxynucleotides complementary to a sequence shared by Rac-1 and Rac-2 genes (54, 55). Sequence-specific reduction of Rac protein levels by oligodeoxynucleotides reduced proliferation of myeloma cells, whereas cell viability was not affected (data not shown). The positive and negative effects of the Tat-Rac-1 mutant proteins on proliferation were not profound; the negative effect was smaller than the inhibitory effect of lovastatin or GGTI-298. This can be explained by the absence of a carboxyl-terminal geranylgeranyl lipid modification of the Tat-Rac-1 mutant proteins. This probably interferes with their functional effectiveness. However, it cannot be excluded that in addition to Rac-1 other geranylgeranylated proteins play a role in the regulation of myeloma cell proliferation. Candidates include Cdc42 (20–22), R-Ras (56), and Rap1 (57).
The data presented in this article suggest for the first time that Rac-1 is involved in the regulation of myeloma cell proliferation and that IL-6 induces myeloma cell proliferation, at least in part, through Rac-1-dependent pathways. In myeloma, it is at present unclear by which pathway(s) Rac-1 is activated and in turn which downstream effectors are activated by Rac-1. Several studies have indicated that the Ras/Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (14, 58, 59) and PI-3K/Akt (4, 12, 59) pathways are involved in IL-6-induced proliferation. Rac-1 may be a downstream component of Ras and/or PI-3K signaling pathways in myeloma cells, because both Ras (25, 26, 60) and PI-3K (60–62) have been shown to activate Rac-1. Alternatively, Rac-1 may be activated by phosphorylated Vav. In myeloma cells, IL-6 induces tyrosine phosphorylation of Vav (63), which is a GDP/GTP exchange factor for members of the Rho family of GTPases, including Rac-1 (64, 65). Many targets of Rac-1 have been identified, including PI-3K (66, 67) and nuclear factor-κB (68, 69), which are both implicated in the regulation of myeloma cell growth (4, 10–13). Future work will focus on signaling pathways that lead to the activation of Rac-1 and on the identification of Rac-1 downstream effectors involved in the regulation of proliferation of myeloma cells.
In conclusion, our findings indicate that protein geranylgeranylation is essential for myeloma cell proliferation through the induction of G1-S progression. We have identified the geranylgeranylated GTP-binding protein Rac-1 as a regulator of myeloma tumor cell proliferation without affecting cell viability. These results together with the role of geranylgeranylated proteins in mediating myeloma cell survival and cell adhesion–mediated drug resistance suggest that inhibition of protein geranylgeranylation may be a new treatment strategy in multiple myeloma.
Grant support: Dutch Cancer Society.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. S. Bhakdi (Institute of Medical Microbiology and Hygiene, Johannes Gutenberg University, Mainz, Germany) for the kind gift of streptolysin-O and Dr. P. Coffer (Department of Pulmonary Diseases, University Medical Center Utrecht, Utrecht, the Netherlands) for helpful discussions.