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Geranylgeranylated Proteins Are Involved in the Regulation of Myeloma Cell Growth

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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 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. 430 Geranylgeranylated Proteins Mediate Myeloma Cell Growth 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
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