Download Geranylgeranylated Proteins are Involved in the Regulation of

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
Evert H.J. Nijhuis,2 Marloes M.J. Kamphuis,1 and
Andries C. Bloem1
Departments of 1Immunology, 2Pulmonary Diseases, and 3Hematology,
University Medical Center Utrecht, Utrecht, the Netherlands
ABSTRACT
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.
Received 6/29/04; revised 10/5/04; accepted 10/14/04.
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.
Requests for reprints: Andries C. Bloem, Department of Immunology,
University Medical Center Utrecht, Heidelberglaan 100, 3584 CX
Utrecht, the Netherlands. Phone: 31-30-2506511; Fax: 31-30-2517107;
E-mail: [email protected].
D2005 American Association for Cancer Research.
INTRODUCTION
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-nB (9 – 11), and phosphatidylinositol 3V-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-nB (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 posttranslational 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
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430 Geranylgeranylated Proteins Mediate Myeloma Cell Growth
this pathway is the conversion of 3-hydroxy-3-methylglutarylCoA (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 HMGCoA 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
Reagents
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 TatRac-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).
Cell Lines
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 Ag/mL streptomycin, and 10 Amol/L h-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.
Cell Proliferation
Myeloma cells (3 104) were seeded in 96-well flatbottomed plates (Nunc, Roskilde, Denmark) in 100 AL 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 Amol/L), farnesol (10 Amol/L), or geranylgeraniol (10 Amol/L) were used. These concentrations have
proven to be optimal in rescuing myeloma cells from lovastatininduced 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 TatRac-1 proteins (for concentrations, see figure legends). After 32
and 80 hours, [3H]thymidine (Amersham, Little Chalfont, United
Kingdom; 1 ACi/well) was added for the remaining 16 hours of
the assay. [3H]thymidine incorporation was analyzed by liquid
scintillation counting as described previously (40).
Cell Viability
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, dominantnegative 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).
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Clinical Cancer Research 431
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 Ag/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 f95% 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 SDSPAGE and immunoblotting using anti-hemagglutinin and antiRac-1 antibodies. Protein solution was aliquoted and frozen
at 80jC.
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 Ag/mL FITC-labeled Tat-Rac-1
Q61L, FITC-labeled Tat-Rac-1 N17, or FITC-labeled BSA for
15 minutes at 37jC. After incubation, cells were harvested,
washed thrice in ice-cold PBS, and treated with TO-PRO-3
(0.2 Amol/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 37jC. 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 icecold 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 4jC for
5 minutes. After centrifugation (16,000 g for 15 minutes
at 4jC), the supernatant was added to a spin column
containing an immobilized glutathione disc and 20 Ag GSTPak1-PBD and incubated at 4jC 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 AL) containing 5% hmercaptoethanol was added to the resin. The samples were
boiled at 100jC for 5 minutes. After a centrifugation step at
7,200 g for 2 minutes, the samples were electrophoresed
on a gel.
Western Blotting
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.
RESULTS
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 Amol/L), farnesol (10
Amol/L), or geranylgeraniol (10 Amol/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 doseand time-dependent way as determined by detection of
[3H]thymidine incorporation (Fig. 1A). Inhibition of proliferation by 30 Amol/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.
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432 Geranylgeranylated Proteins Mediate Myeloma Cell Growth
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; 10 Amol/L), or geranylgeraniol (GGOH; 10 Amol/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; 10 Amol/L), or geranylgeraniol (G; 10 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. 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
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]thymimidine incorporation. Reduction of spontaneous proliferation
induced by 30 Amol/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
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Clinical Cancer Research 433
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 timedependent way (Fig. 3A). FTI-277 had no effect or inhibited
proliferation only to a small extent when compared with GGTI298. 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 dosedependent 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 ADPribosylating Asp41 (39). Treatment of myeloma cell lines with
C3 exoenzyme (50 Ag/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
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.
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434 Geranylgeranylated Proteins Mediate Myeloma Cell Growth
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
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.
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-6induced proliferation coincided with a significant increase in Rac1-GTP levels in purified myeloma tumor cells (Fig. 5C and D).
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Clinical Cancer Research 435
Table 1
Effect of C. difficile toxin B on viability, proliferation, and apoptosis of myeloma cell lines
L363
No. viable cells (% control)
Proliferation (% control)
% Apoptotic cells
RPMI-8226
XG-1
U266
Day 2
Day 4
Day 2
Day 4
Day 2
Day 4
Day 2
Day 4
53.1
61.5
31.9/36.8
39.6
65.2
42.8/41.6
93.1
93.8
30.2/33.4
79.8
83.7
48.1/50.5
42.9
39.8
30.2/38.1
13.7
22.7
41.5/48.8
85.8
39.6
10.9/12.7
59.6
36.3
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,
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 FITClabeled 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 TatRac-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).
DISCUSSION
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
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.
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436 Geranylgeranylated Proteins Mediate Myeloma Cell Growth
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
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 seruminduced 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-6responsive purified myeloma cells from patients. However,
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Clinical Cancer Research 437
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-Rac1 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-
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, dominantnegative 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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Research.
438 Geranylgeranylated Proteins Mediate Myeloma Cell Growth
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/mitogenactivated 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, Rac1 may be activated by phosphorylated Vav. In myeloma cells, IL6 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-nB
(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.
ACKNOWLEDGMENTS
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.
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Geranylgeranylated Proteins are Involved in the Regulation
of Myeloma Cell Growth
Niels W.C.J. van de Donk, Henk M. Lokhorst, Evert H.J. Nijhuis, et al.
Clin Cancer Res 2005;11:429-439.
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