This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van de Donk, N. W. C. J. Articles by Lokhorst, H. M. Search for Related Content PubMed PubMed Citation Articles by van de Donk, N. W. C. J. Articles by Lokhorst, H. M.

Protein Geranylgeranylation Is Critical for the Regulation of Survival and Proliferation of Lymphoma Tumor Cells

¹ Departments of Immunology,
² Haematology, and
³ Pathology, University Medical Center Utrecht, Utrecht, the Netherlands

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: Prenylation is essential for membrane localization and participation of proteins in various signaling pathways. The following study was conducted to examine the importance of protein farnesylation and geranylgeranylation for the regulation of lymphoma cell survival and proliferation.

Experimental Design: Lymphoma cells were treated with the ß-hydroxy-ß-methylglutaryl-CoA reductase inhibitor lovastatin, which inhibits protein farnesylation and geranylgeranylation by the depletion of intracellular pools of farnesylpyrophosphate and geranylgeranylpyrophosphate. In addition, farnesyl transferase and geranylgeranyl transferase activities were specifically inhibited by FTI-277 and GGTI-298, respectively.

Results: Only inhibition of geranylgeranylation by lovastatin led to reduction of cell viability in lymphoma cell lines and purified tumor cells from lymphoma patients in a time- and dose-dependent way. Reduction in the number of viable cells was mediated by both induction of apoptosis and inhibition of proliferation. In addition, GGTI-298 was more effective in induction of apoptosis and inhibition of proliferation than FTI-277. Apoptosis induced by inhibition of protein geranylgeranylation was associated with a reduction of Mcl-1 protein levels, collapse of the mitochondrial transmembrane potential, and caspase-3 activation. Inhibition of proliferation resulted from the induction of G₁ arrest. Furthermore, lovastatin at low concentrations sensitized lymphoma cells to dexamethasone, including cells resistant to this drug.

Conclusion: These results indicate that protein geranylgeranylation is critical for the regulation of lymphoma tumor cell survival and proliferation and that pharmacological agents such as lovastatin or geranylgeranyl transferase inhibitors, alone or in combination with other drugs, may be useful in the treatment of lymphoma.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The rate-limiting step of the mevalonate pathway is the conversion of HMG-CoA⁴ to mevalonate and is catalyzed by the enzyme HMG-CoA reductase (1) . Mevalonate is an intermediate in the synthesis of cholesterol, which is essential for membrane integrity; of dolichol, which is required for glycoprotein synthesis; of polyisoprenoid side chains of heme A and ubiquinone, which are involved in oxidative respiration; and of isopentyladenine, which is present in some tRNAs. Furthermore, mevalonate is also a precursor of the isoprenoids FPP and GGPP (1) . These isoprenoids are used for the post-translational modification of proteins, including Ras (farnesylation; Refs. 2 , 3 ) and the Rho family members Rac-1, RhoA, and Cdc42 (geranylgeranylation; Ref. 4 ). FTase and GGTase transfer farnesyl and geranylgeranyl moieties from FPP and GGPP, respectively, to the thiol group of conserved cysteine residues at or near the COOH terminus of target proteins. Prenylation is essential for membrane association (2) and participation of proteins in various signaling pathways regulating growth and survival (3 , 5, 6, 7) . Lovastatin is a potent competitive inhibitor of HMG-CoA reductase and thereby prevents the conversion of HMG-CoA to mevalonate and the synthesis of the other products of the mevalonate pathway. We and others have previously shown that lovastatin induces apoptosis and inhibits proliferation in various cancer cell lines (8, 9, 10, 11, 12) and in purified tumor cells derived from patients with multiple myeloma (12) or acute myeloid leukemia (11) .

NHL and B-CLL are characterized by initial sensitivity to cytotoxic drugs in the majority of the patients. However, multidrug-resistant disease will ultimately develop in many of these patients. Recent studies have shown that defects in apoptotic pathways contribute significantly to resistance of cancer cells to chemotherapeutic agents (13) . Antiapoptotic signaling pathways, such as PI-3K/Akt (14, 15, 16, 17, 18, 19, 20) or nuclear factor-B (21) , and expression of antiapoptotic Bcl-2 family members, including Bcl-2 (22, 23, 24, 25, 26, 27) , Mcl-1 (17 , 28, 29, 30) , and Bcl-XL (31) , are involved in the regulation of lymphoma tumor cell survival and protection against cytotoxic drugs. Therefore, interference with such pathways may induce apoptosis in lymphoma tumor cells or restore chemosensitivity in chemoresistant tumor cells.

This report shows that inhibition of protein geranylgeranylation by depletion of GGPP by lovastatin or by inhibition of GGTase I activity induces apoptosis and inhibits proliferation in lymphoma cell lines and tumor cells from patients. In addition, we found that lovastatin enhanced the sensitivity of lymphoma cells to dexamethasone. Altogether our results indicate that interference with the geranylgeranylation of proteins involved in survival and proliferation may be a new treatment strategy in lymphoma.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents.
Lovastatin and simvastatin were obtained from Merck & Co., Inc. (Rahway, NJ) and were chemically activated by alkaline hydrolysis before use as described previously (32) . Pravastatin Sodium was purchased from Bristol-Meyers Squibb (New Brunswick, NJ) and dissolved in PBS (20 mM). Atorvastatin Calcium was obtained from Pfizer GmbH (Freiburg, Germany) and dissolved in ethanol containing 3% DMSO (Riedel-de Haen, Seelze, Germany; final concentration of atorvastatin, 10 mM). Mevalonate and FOH were purchased from Sigma (St. Louis, MO), and GGOH was obtained from ICN Biomedicals, BV (Zoetermeer, the Netherlands). FOH and GGOH are metabolized in cells to FPP and GGPP, respectively (33) . FTI-277 and GGTI-298 were obtained from Calbiochem (Schwallbach, Germany).

Cell Lines.
The follicular lymphoma cell line DoHH2 was obtained from the German Collection of Microorganisms and Cell Cultures, and the follicular lymphoma cell line SU-DHL-6 (34) was kindly provided by Dr. J. Jansen (University Medical Center Nijmegen, Nijmegen, the Netherlands). The Burkitt’s lymphoma cell lines Raji, Ramos, and Daudi were purchased from the American Tissue Culture Collection. 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 µM ß-mercaptoethanol (growth medium).

Patients.
After receiving informed consent, we obtained mononuclear cells by Ficoll-Paque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) density centrifugation of peripheral blood from 13 patients suffering from leukemic NHL, B-CLL or hairy cell leukemia; of bone marrow from one patient with follicular lymphoma; and of pleural fluid from one patient with mantle cell lymphoma. The clinical characteristics of the patients are shown in Table 1 .

Tumor cells from patient 7 were purified from an axillary lymph node. The organ was cut into small pieces with a scalpel blade and incubated with 1 mg/ml collagenase IV (Sigma) and 0.1 mg/ml DNase I (Sigma) for 1 h at 37°C in 5% CO₂. The cell suspension was subsequently filtered through an open-filter chamber (NPBI; Emmer-Copascuum, the Netherlands), and mononuclear cells were obtained by Ficoll-Paque density centrifugation.

The percentages and monoclonality of the malignant cells were established by flow cytometry (FACSCalibur; BDIS) by analysis of the light chain distribution within the surface-bound immunoglobulins on CD19-positive cells or the CD4/CD8 ratio within CD3-positive T cells in case of patient 11. Percentages of clonal cells present in the samples after density centrifugation or MACS selection (patients 3 and 9) ranged from 75 to 99.4% of the nucleated cells (see Table 3 ). For experiments, tumor cells were resuspended in growth medium (see above).

Cell Proliferation.
Cells (3 x 10⁴) were seeded in 96-well flat-bottomed plates (Nunc) in 100 µl of growth medium with lovastatin (for concentrations, see figure legends) alone or in the presence of mevalonate (100 µM), FOH (10 µM), or GGOH (10 µM). Inhibition of FTase and GGTase I was accomplished by treating cells with FTI-277 and GGTI-298, respectively (for concentrations, see figure legends). After 32 and 80 h, [³H]thymidine (1 µCi/well; Amersham, Little Chalfont, United Kingdom) was added for the remaining 16 h of the assay. [³H]Thymidine incorporation was analyzed by liquid scintillation counting as described previously (12) .

Cell Cycle Analysis.
Lymphoma cells (1 x 10⁶ in 1.5 ml) were incubated with different concentrations of lovastatin (for concentrations, see figure legends) in the presence or absence of mevalonate (100 µM), FOH (10 µM), or GGOH (10 µM) in a 48-well plate (Nunc) for 2 days. Cells were then pulsed with 10 µM BrdUrd for 30 min at 37°C, after which the cells were washed twice with ice-cold PBS containing 1% BSA. Cells were subsequently fixed with 70% ethanol for 30 min at 4°C. After the ethanol was washed away, the cells were treated with 2 M HCl containing 0.5% Triton X-100 for 30 min at room temperature, followed by neutralization with 1 M sodium tetraborate (pH 8.5). After washing, cells were resuspended in PBS containing 0.5% Tween 20 and 1% BSA and incubated with anti-BrdUrd-FITC (BDIS) for 30 min at room temperature. After being washed with PBS containing 0.5% Tween 20 and 1% BSA, cells were stained with PI in PBS (5 µg/ml) and analyzed by flow cytometry.

Apoptosis Detection by Annexin V Staining.
Lymphoma cells (1.5 x 10⁵ in 0.5 ml) were incubated with different concentrations of lovastatin (for concentrations, see figure legends) alone or in the presence of mevalonate (100 µM), FOH (10 µM), or GGOH (10 µM) in a 48-well plate (Nunc). Inhibition of FTase and GGTase I was accomplished by FTI-277 and GGTI-298, respectively (for concentrations, see figure legends). After 2 or 4 days, cells were harvested, washed in ice-cold PBS, and directly stained with Annexin V-FITC (Nexins Research, Kattendijke, the Netherlands) and PI. After 10 min of incubation at room temperature in the dark, cells were analyzed by flow cytometry as described previously (12) . Apoptotic cells were defined as early apoptotic cells (Annexin V positive and PI negative) and late apoptotic cells (Annexin V and PI positive).

Caspase-3 Activity.
The caspase-3 activity assay (Roche) was used to determine caspase-3 activity. Briefly, cells were washed in ice-cold PBS and then resuspended in lysis buffer (1x DTT) and incubated for 1 min on ice. Supernatants were obtained after centrifugation at 14,000 rpm for 1 min at room temperature. Supernatants were added to anti-caspase-3-coated wells and incubated at 37°C for 1 h. After three washing steps, substrate solution (Ac-DEVD-AFC) was added, and the wells were incubated for 2 h at 37°C. Fluorescence was measured with a 400 nm excitation filter and a 505 nm emission filter.

Measurement of Mitochondrial Transmembrane Potential.
Changes in mitochondrial transmembrane potential (_m) were evaluated by staining with 40 nM DiOC₆[3] (Molecular Probes, Leiden, the Netherlands). Cells were incubated with DiOC₆[3] in PBS for 15 min at 37°C, washed, and resuspended in PBS. The cells were then analyzed on a flow cytometer (FACSCalibur).

Western Blotting.
Cells (1 x 10⁶ cells in 1.5 ml) were incubated for 2 days with lovastatin (for concentrations, see figure legends) in the presence or absence of mevalonate (100 µM), FOH (10 µM), or GGOH (10 µM). Inhibition of FTase and GGTase I was accomplished by FTI-277 and GGTI-298, respectively (for concentrations, see figure legends). After harvesting, whole-cell lysates were made by washing cells twice in ice-cold PBS and then resuspending them in lysis buffer [10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, and a cocktail of protease inhibitors] at 4°C for 20 min. Insoluble material was removed by centrifugation at 14,000 rpm for 6 min at 4°C. Protein concentrations were determined by the BCA assay (Pierce, Rockford, IL). Samples containing equal amounts of protein were mixed with 2x Laemmli sample buffer [0.125 M Tris (pH 6.9), 4% SDS, 20% glycerol, and 10% ß-mercaptoethanol] and boiled for 5 min. Proteins were subsequently fractionated by 10% SDS-PAGE at room temperature and electrically transferred from the gel to a polyvinylidene difluoride membrane (Bio-Rad). After blocking in 0.1% Tween 20, 5% skimmed powder milk, 2% BSA in 10 mM Tris–150 mM NaCl, the membranes were incubated with anti-Bcl-2 (Dako, Glostrup, Denmark), anti-Mcl-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-Bcl-XL (Santa Cruz Biotechnology), anti-Bax (Dako), anti-Rap1a (Santa Cruz Biotechnology), or anti-HDJ-2 (DnaJ; Neomarkers; Lab Vision, Fremont, CA). Antibody binding was visualized with enhanced chemiluminescence (Amersham) detection with hyperfilm ECL (Amersham) after incubation with a horseradish peroxidase-conjugated secondary antibody (Dako). Finally, the membranes were washed extensively in PBS and reprobed with anti--actin (Sigma) as a control for equal loading of protein. Relative amounts of protein were determined by densitometry and expressed as a percentage of the solvent control.

Statistics.
Data analysis was performed with the SPSS statistical software package (SPSS Inc., Chicago, IL). A two-sided Student’s t test or a Welch’s t test in case of unequal variances was used to determine differences between groups. Differences were considered statistically significant when P < 0.05. Data are plotted as means ± SE.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Lovastatin Reduces Cell Viability of Lymphoma Cell Lines.
The lymphoma cell lines (DoHH2, Raji, Daudi, Ramos, and SU-DHL-6) were incubated with different concentrations of lovastatin for 2 or 4 days. Lovastatin reduced viability in a dose- and time-dependent way. Representative examples from the DoHH2 and Raji cell lines are shown in Fig. 1, A and B . DoHH2 was the cell line most sensitive to lovastatin, whereas SU-DHL-6 was most resistant to lovastatin. The concentrations of lovastatin that reduced viability with 50% (MTT50) and 75% (MTT25) ranged from 4.7 to 45.7 and 9.7 to 118 µM at day 2, respectively. At day 4, MTT50 varied between 4.4 and 29.3 and MTT25 between 7.9 and 46.6 µM (Table 2) .

View larger version (23K): [in this window] [in a new window]   Fig. 1. Lovastatin reduces cell viability in cell lines and in purified tumor cells from lymphoma patients in a time- and dose-dependent way. Cell lines DoHH2 (A) and Raji (B), purified tumor cells from patient 1 (C), and purified tumor cells from patient 7 (D) were treated for 2 or 4 days with solvent control or with different concentrations of lovastatin (1–150 µM). The percentage of viable cells relative to the solvent-control-treated cells, was measured by MTT assay. Experiments were performed three times in triplicate in case of cell lines and one time in triplicate in case of purified tumor cells from patients. Data are presented as mean ± SE (bars). In some cases the SE is smaller than the symbol. FL, follicular lymphoma; MCL, mantle cell lymphoma.

Lovastatin Inhibits Protein Prenylation.
The effect of lovastatin on prenylation in DoHH2 and Raji cell lines was determined by analysis of the migratory behavior during electrophoresis of DnaJ, a protein prenylated exclusively by FTase (35 , 36) , and of Rap1a, a protein prenylated exclusively by GGTase I (37 ; Fig. 2A ). Inhibition of prenylation of these proteins can be monitored by immunoblotting because the nonprenylated forms of these proteins display reduced mobility in SDS-PAGE relative to their prenylated versions. In solvent-control-treated cells, DnaJ and Rap1a were in the processed, prenylated forms. Lovastatin inhibited the farnesylation of DnaJ and geranylgeranylation of Rap1a. Addition of mevalonate restored the processing of DnaJ and Rap1a. Treatment with lovastatin in the presence of GGOH (10 µM), which is metabolized to GGPP in the cells (33) , restored geranylgeranylation of Rap1a but had no effect on the inhibition of farnesylation of DnaJ. In contrast, FOH (10 µM), which is metabolized to FPP (33) , restored DnaJ farnesylation but not Rap1a geranylgeranylation.

View larger version (43K): [in this window] [in a new window]   Fig. 2. Lovastatin inhibits protein farnesylation and geranylgeranylation and induces apoptosis by depletion of intracellular pools of GGPP. A, DoHH2 and Raji cells were treated for 2 days with solvent control or lovastatin (30 µM) alone or in the presence of mevalonate (meva; 100 µM), GGOH (10 µM), or FOH (10 µM). After protein isolation, processing of DnaJ and Rap1a was determined by Western blot analysis. The faster migrating band represents mature and processed protein, the slower band represents nonprenylated, unprocessed protein. The data shown are representative of at least three independent experiments. B, DoHH2 cells were treated for 4 days with solvent control or lovastatin (30 µM) alone or in combination with mevalonate (meva; 100 µM), GGOH (10 µM), or FOH (10 µM). The percentage of apoptotic cells was examined by the Annexin V assay. The percentages of viable plasma cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-) and late apoptotic cells (Annexin V+/PI+) in each dot plot are indicated in the corresponding quadrants. Results are representative of three experiments performed in triplicate. C, DoHH2 and Raji cells were treated for 4 days with solvent control or lovastatin (5, 10, 40, 100, or 150 µM) in combination with mevalonate (meva; 100 µM), GGOH (10 µM), or FOH (10 µM), after which apoptosis was determined by Annexin V assay. Shown is the sum of the percentages of early and late apoptotic cells. Experiments were performed three times in triplicate. Data are presented as mean ± SE (bars). In some cases the SE is smaller than the symbol.

Apoptosis Induction by Lovastatin Is Associated with Mcl-1 Protein Reduction, Collapse of the Mitochondrial Transmembrane Potential, and Caspase-3 Activation.
Expression of Bcl-2 family proteins was determined in DoHH2 and Raji cells treated with lovastatin (30 µM) alone or in combination with mevalonate (100 µM), FOH (10 µM), or GGOH (10 µM) by Western blot analysis. Lovastatin treatment resulted in a significant reduction of Mcl-1 expression levels (Fig. 3A) . Only in DoHH2 cells did lovastatin reduce Bcl-XL and increase Bax protein expression. Although Bcl-2 protein levels remained unaltered, there was an increase of the proapoptotic 23-kDa Bcl-2 form, which could be visualized after long exposure of the film compared with Bcl-2. Importantly, mevalonate and GGOH prevented reduction of Mcl-1 and Bcl-XL expression and increases in Bax and the proapoptotic Bcl-2 form. In contrast, FOH had no effect.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Depletion of GGPP by lovastatin is associated with Mcl-1 protein reduction, collapse of the mitochondrial transmembrane potential, and caspase-3 activation. A, lymphoma cell lines DoHH2 and Raji were treated for 2 days with solvent control or lovastatin (30 µM) alone or in combination with mevalonate (meva; 100 µM), FOH (10 µM), or GGOH (10 µM). After protein isolation, Mcl-1, Bcl-XL, Bcl-2, and Bax levels were determined by Western blot analysis. In addition, the proapoptotic 23-kDa Bcl-2 form was detected after long exposure of the film. The data shown are representative of at least three independent experiments. B, DoHH2 cells were treated for 4 days with solvent control or lovastatin (30 µM) alone or in combination with mevalonate (meva; 10 µM), FOH (10 µM), or GGOH (10 µM). Collapse of the mitochondrial transmembrane potential was determined by staining the cells with DiOC6[3]. Results are representative of three experiments performed in triplicate. The fraction of cells with low (DiOC6[3]low) and the fraction of cells with intact (DiOC6[3]high) mitochondrial transmembrane potential are indicated in gates M1 and M2, respectively. C, DoHH2 cells were treated for 4 days with solvent control or different concentrations of lovastatin (10, 30, 50, 100, or 150 µM) alone or in combination with mevalonate (meva; 100 µM), FOH (10 µM), or GGOH (10 µM). Collapse of the mitochondrial transmembrane potential was determined by staining the cells with DiOC6[3]. Shown is the percentage of cells with low mitochondrial transmembrane potential. Experiments were performed three times in triplicate. Data are presented as mean ± SE (bars). In some cases the SE is smaller than the symbol. D, DoHH2 cells were exposed to solvent control or lovastatin (30 µM) alone or in combination with mevalonate (meva; 100 µM), FOH (10 µM), or GGOH (10 µM) for 2 days, at which time the cells were harvested and caspase-3 activation was assessed as described in "Materials and Methods." Experiments were performed three times in duplicate. Data are presented as mean ± SE (bars).

Treatment of DoHH2 and Raji cells with lovastatin in the presence of mevalonate or GGOH prevented collapse of the mitochondrial transmembrane potential and activation of caspase-3. However, incubation of cells with lovastatin in combination with FOH had no effect compared with lovastatin alone (Fig. 3, B–D) .

Synergism between Lovastatin and Dexamethasone in the Induction of Lymphoma Cell Death.
Cell lines were treated with dexamethasone (0, 5, 10, 50, or 100 µM) or doxorubicin (0, 10, 25, 50, 100, 200, or 500 nM) alone or in combination with lovastatin (0–30 µM). The combination of lovastatin with doxorubicin produced a significant additive effect (data not shown). Furthermore, low concentrations of lovastatin potently sensitized both dexamethasone-sensitive (DoHH2, SU-DHL-6, Ramos) and dexamethasone-resistant (Raji, Daudi) lymphoma cells to dexamethasone in a synergistic fashion (Fig. 4) .

View larger version (26K): [in this window] [in a new window]   Fig. 4. Lovastatin synergizes with dexamethasone in reducing cell viability. DoHH2 (A), Raji (B), Daudi (C), SU-DHL-6 (D), and Ramos cells (E) were incubated with different concentrations of dexamethasone (0–100 µM) in combination with lovastatin (lova; 2, 5, 10, 20, or 30 µM) or solvent control for 4 days. Cell viability was determined by MTT assay and compared with cells treated without dexamethasone and lovastatin. Results are representative of at least three experiments performed in triplicate and are presented as the mean ± SE (bars). In some cases the SE is smaller than the symbol. *, significantly higher reduction in cell viability than the sum of the effects observed in the corresponding lovastatin- and dexamethasone-treated groups (P < 0.05).

View larger version (58K): [in this window] [in a new window]   Fig. 5. Depletion of GGPP by lovastatin inhibits proliferation by the induction of a G1 block. A, DoHH2 and Raji cells were treated for 4 days with solvent control or different concentrations of lovastatin (1, 5, 10, 30, 50, 100, or 150 µM) alone or in combination with mevalonate (meva; 100 µM), FOH (10 µM), or GGOH (10 µM). Proliferation was determined by [3H]thymidine incorporation during the last 16 h of culture. Data are from three experiments performed in triplicate and are presented as mean ± SE (bars). In some cases the SE is smaller than the symbol. B, DoHH2 cells were treated with solvent control or lovastatin (lova; 10 or 30 µM) alone or in combination with mevalonate (meva; 100 µM), FOH (10 µM), or GGOH (10 µM) for 2 days, at which time the cells were harvested and cell cycle distribution was determined by BrdUrd (BrdU) assay. The percentage of apoptotic cells (quadrant 1) and the percentages of cells in G1 phase (quadrant 2), G2-M phase (quadrant 3), and S-phase (quadrant 4) of the cell cycle are indicated in each dot plot. Results are representative of three experiments performed in triplicate.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Treatment of purified tumor cells from lymphoma patients with lovastatin reduces cell viability, down-regulates Mcl-1 protein expression, and inhibits proliferation by reduction of intracellular pools of GGPP. A, tumor cells derived from patient 15 were treated for 4 days with solvent control or different concentrations of lovastatin (5, 10, 30, 50, 100, or 150 µM) alone or in the presence of mevalonate (meva; 100 µM), FOH (10 µM), or GGOH (10 µM). The percentage of viable cells relative to the solvent-control-treated cells was measured by MTT assay. Experiments were performed once in triplicate. Data are presented as mean ± SE (bars). In some cases the SE is smaller than the symbol. B, tumor cells derived from patient 15 were treated for 4 days with solvent control or lovastatin (30 µM) alone or in the presence of mevalonate (meva; 100 µM), FOH (10 µM), or GGOH (10 µM). After protein isolation, Mcl-1, Bcl-XL, Bcl-2, and Bax were determined by Western blot analysis. C, tumor cells derived from patient 7 were treated for 4 days with solvent control or different concentrations of lovastatin (5, 10, 30, 50, 100, or 150 µM) alone or in the presence of mevalonate (meva; 100 µM), FOH (10 µM), or GGOH (10 µM). Proliferation was determined by [3H]thymidine incorporation during the last 16 h of culture. Experiments were performed once in triplicate. Data are presented as mean ± SE (bars). In some cases the SE is smaller than the symbol.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Inhibition of GGTase I by GGTI-298 inhibits protein geranylgeranylation, resulting in Mcl-1 down-regulation and induction of apoptosis. DoHH2 and Raji cells were treated for 2 or 4 days with solvent control, the farnesyl transferase inhibitor FTI-277 (20 µM) or with the geranylgeranyl transferase I inhibitor GGTI-298 (20 µM). A, after protein isolation at day 2, processing of DnaJ and Rap1a was determined by Western blot analysis. The faster-migrating band represents prenylated protein; the slower band represents nonprenylated, unprocessed protein. The data shown are representative of at least three independent experiments. B, percentage of apoptotic cells determined by Annexin V assay. Shown is the sum of the percentages of early and late apoptotic cells. Experiments were performed three times in triplicate. Data are presented as mean ± SE (bars). C, after protein isolation at day 2, Mcl-1, Bcl-XL, Bcl-2, and Bax were determined by Western blot analysis. In addition, the proapoptotic 23-kDa Bcl-2 form was detected after long exposure of the film. The data shown are representative of at least three independent experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Inhibition of GGTase I by GGTI-298 inhibits proliferation. DoHH2 (A) and Raji (B) cells were treated for 2 or 4 days with solvent control, with different concentrations of FTI-277 (2.5, 5, 10, 15, 20, or 30 µM), or with different concentrations of GGTI-298 (2.5, 5, 10, 15, 20, or 30 µM). Proliferation was determined by [3H]thymidine incorporation during the last 16 h of culture. Data are from three experiments performed in triplicate and are presented as mean ± SE (bars). In some cases the SE is smaller than the symbol.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mevalonate is the precursor of various molecules, including the isoprenoids FPP and GGPP (1) . We found that geranylgeranylated protein(s) are important regulators of survival and proliferation in lymphoma tumor cells. Although lovastatin depletes intracellular pools of both GGPP and FPP, resulting in inhibition of geranylgeranylation of Rap1a and farnesylation of DnaJ, only addition of GGOH, which is converted to GGPP in cells (33) , abrogated the inhibition of geranylgeranylation by lovastatin and restored both cell viability and proliferation. In contrast, although addition of FOH, which is converted to FPP (33) , completely restored farnesylation of DnaJ, it had no effect on viability or proliferation. However, in some cases GGOH was less effective than mevalonate in rescuing lymphoma tumor cells from apoptosis and growth arrest. This suggests that depletion of other metabolites downstream of mevalonate may also partly contribute to the effects of lovastatin. The importance of geranylgeranylation for the regulation of growth and survival of lymphoma cells was further confirmed by specific inhibition of FTase by FTI-277 and GGTase I by GGTI-298. GGTI-298 inhibited protein geranylgeranylation and resulted in the induction of apoptosis and inhibition of proliferation, whereas inhibition of farnesylation by FTI-277 had no or only a small effect.

The Bcl-2 family consists of proapoptotic proteins, such as Bax, Bad, and Bak, and antiapoptotic family members, including Mcl-1, Bcl-XL, and Bcl-2. The balance of anti- and proapoptotic Bcl-2 family proteins determines the survival or death of cells (38 , 39) . The antiapoptotic Bcl-2 family members inhibit apoptosis by forming inactivating heterodimers with proapoptotic Bcl-2 family proteins and by preventing the collapse of the mitochondrial transmembrane potential and cytochrome c release from mitochondria into the cytosol (38 , 39) . In the cytosol, cytochrome c and Apaf-1 can activate procaspase-9, which subsequently activates caspase-3 (38 , 39) . Mcl-1 is expressed in various types of human leukemias and lymphomas, including CLL (28) , follicular lymphoma (31 , 42) , and anaplastic large cell lymphoma (43) . There is increasing evidence that Mcl-1 plays a prominent role in lymphomagenesis (44) and in the survival (17 , 30 , 45) and chemoresistance (28 , 29) of lymphoma tumor cells. We showed that apoptosis induced by inhibition of geranylgeranylation was associated with a reduction in Mcl-1 protein expression in lymphoma cell lines and patient cells, which in turn resulted in collapse of the mitochondrial transmembrane potential and caspase-3 activation.

Lovastatin treatment also led to an increase in the 23-kDa proapoptotic Bcl-2 form. Bcl-2 is a known substrate of caspase-3 (40 , 41) ; it is therefore likely that lovastatin-mediated stimulation of caspase-3 activity led to the increase in the proapoptotic Bcl-2 form. The proapoptotic 23-kDa Bcl-2 form is able to localize to mitochondria and stimulate the release of cytochrome c into the cytosol (41) . Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop. Only in DoHH2 cells was expression of the antiapoptotic protein Bcl-XL decreased and the proapoptotic protein Bax increased. Although the alterations in expression levels of these proteins were relatively small compared with the change in Mcl-1 expression, they may contribute partly to the induction of apoptosis induced by inhibition of geranylgeranylation in this cell line.

The identities of the geranylgeranylated target protein(s) of lovastatin and GGTI-298 that mediate protection against cell death and regulate the proliferation of lymphoma tumor cells remain unknown. Candidates, however, include RhoA, Rac-1, R-Ras, and Cdc42, which are involved in important cellular functions, including the regulation of apoptosis (4 , 46, 47, 48, 49, 50) . Several pathways, including PI-3K, JAK/STAT3, and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase, can stimulate Mcl-1 transcription (51) . Because RhoA (52 , 53) , Rac-1 (54) , R-Ras (49 , 55) , and Cdc42 (56) have been shown to activate the PI-3K pathway, inhibition of these proteins by lovastatin or GGTI-298 may be involved in Mcl-1 down-regulation and induction of apoptosis. Furthermore, RhoA, Rac-1, R-Ras, and Cdc42 have also been implicated as regulators of proliferation (4 , 47 , 48 , 50 , 57) . We showed that inhibition of geranylgeranylation led to a reduction of proliferation attributable to arrest in G₁ phase of the cell cycle. This indicates that geranylgeranylated proteins are critical for G₁-S transition in lymphoma tumor cells. Also in lung adenocarcinoma (58) and in mouse fibroblasts (37) , inhibition of geranylgeranylation arrested cells in the G₁ phase of their cycle. Rac-1 (59 , 60) , Cdc42 (60, 61, 62) , and RhoA (59 , 60) play an essential role in G₁-S-phase transition, and inhibition of processing of these proteins by lovastatin or GGTI-298 may, therefore, be responsible for the G₁ block. Ongoing studies are directed at identifying the geranylgeranylated target proteins.

Lovastatin augmented the sensitivity of both drug-sensitive and drug-resistant lymphoma cells to dexamethasone in a synergistic way. Moreover, additive cytotoxicity was observed between lovastatin and doxorubicin. Our findings are consistent with the ability of lovastatin to sensitize tumor cells to cytotoxic agents in other models (10 , 12 , 63, 64, 65) . Because Mcl-1 is an important regulator of response to cytotoxic agents (28 , 29 , 66) , the synergism between lovastatin and dexamethasone may be attributable, at least in part, to the lovastatin-mediated Mcl-1 down-regulation. Importantly, cell death and chemosensitization induced by lovastatin were observed at concentrations that could be achieved in vivo without significant tissue toxicity (67) .

In summary, we have shown that geranylgeranylation of proteins is critical for the survival and proliferation of lymphoma tumor cells. Inhibition of geranylgeranylation, either by depletion of intracellular pools of GGPP through the inhibition of HMG-CoA reductase by lovastatin or by the specific inhibition of GGTase I activity by GGTI-298, resulted in the induction of apoptosis and reduction of proliferation. Apoptosis induced by inhibition of geranylgeranylation was probably attributable to a reduction of the antiapoptotic Bcl-2 family member Mcl-1, which in turn resulted in collapse of the mitochondrial transmembrane potential and activation of caspase-3. The Mcl-1 down-regulation may also explain the chemosensitizing activity of lovastatin. Furthermore, inhibition of proliferation was associated with a G₁ block. These data suggest that inhibition of protein geranylgeranylation either alone or in combination with chemotherapy warrants further investigation as a new therapeutic strategy in lymphoma and CLL.

	FOOTNOTES

 
Grant support: This study was financially supported by a grant from the Dutch Cancer Society (KWF).

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: Dr. H. M. Lokhorst, Department of Hematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. Phone: 31 30 2506511; Fax: 31 30 2511893; E-mail: h.lokhorst{at}azu.nl

⁴ The abbreviations used are: HMG-CoA, ß-hydroxy-ß-methylglutaryl-CoA; FPP, farnesylpyrophosphate; GGPP, geranylgeranylpyrophosphate; FTase, farnesyl transferase; GGTase I, geranylgeranyl transferase I; NHL, non-Hodgkin’s lymphoma; B-CLL, B-cell chronic lymphocytic leukemia; PI-3K, phosphatidylinositol 3'-kinase; FOH, farnesol; GGOH, geranylgeraniol; BDIS, Becton Dickinson; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; BrdUrd, bromodeoxyuridine; PI, propidium iodide; DiOC₆[3], 3,3'-dihexyloxacarbocyanine iodide.

Received 6/27/03; revised 8/ 4/03; accepted 8/12/03.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Goldstein J. L., Brown M. S. Regulation of the mevalonate pathway. Nature (Lond.), 343: 425-430, 1990.[CrossRef][Medline]
2. Magee T., Marshall C. New insights into the interaction of Ras with the plasma membrane. Cell, 98: 9-12, 1999.[CrossRef][Medline]
3. Reuter C. W., Morgan M. A., Bergmann L. Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies?. Blood, 96: 1655-1669, 2000.[Abstract/Free Full Text]
4. Bishop A. L., Hall A. Rho GTPases and their effector proteins. Biochem. J, 348 (Pt. 2): 241-255, 2000.
5. Kato K., Cox A. D., Hisaka M. M., Graham S. M., Buss J. E., Der C. J. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc. Natl. Acad. Sci. USA, 89: 6403-6407, 1992.[Abstract/Free Full Text]
6. Gelb M. H. Protein prenylation, et cetera: signal transduction in two dimensions. Science (Wash. DC), 275: 1750-1751, 1997.[Free Full Text]
7. Rebollo A., Martinez A. Ras proteins: recent advances and new functions. Blood, 94: 2971-2980, 1999.[Free Full Text]
8. Dimitroulakos J., Yeger H. HMG-CoA reductase mediates the biological effects of retinoic acid on human neuroblastoma cells: lovastatin specifically targets P-glycoprotein-expressing cells. Nat. Med., 2: 326-333, 1996.[CrossRef][Medline]
9. Ghosh P. M., Ghosh-Choudhury N., Moyer M. L., Mott G. E., Thomas C. A., Foster B. A., Greenberg N. M., Kreisberg J. I. Role of RhoA activation in the growth and morphology of a murine prostate tumor cell line. Oncogene, 18: 4120-4130, 1999.[CrossRef][Medline]
10. Agarwal B., Bhendwal S., Halmos B., Moss S. F., Ramey W. G., Holt P. R. Lovastatin augments apoptosis induced by chemotherapeutic agents in colon cancer cells. Clin. Cancer Res., 5: 2223-2229, 1999.[Abstract/Free Full Text]
11. Dimitroulakos J., Nohynek D., Backway K. L., Hedley D. W., Yeger H., Freedman M. H., Minden M. D., Penn L. Z. Increased sensitivity of acute myeloid leukemias to lovastatin-induced apoptosis: a potential therapeutic approach. Blood, 93: 1308-1318, 1999.[Abstract/Free Full Text]
12. van de Donk N. W., Kamphuis M. M., Lokhorst H. M., Bloem A. C. The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells. Leukemia (Balt.), 16: 1362-1371, 2002.
13. O’Gorman D. M., Cotter T. G. Molecular signals in anti-apoptotic survival pathways. Leukemia (Balt.), 15: 21-34, 2001.
14. Borlado L. R., Redondo C., Alvarez B., Jimenez C., Criado L. M., Flores J., Marcos M. A., Martinez A., Balomenos D., Carrera A. C. Increased phosphoinositide 3-kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo. FASEB J., 14: 895-903, 2000.[Abstract/Free Full Text]
15. Slupianek A., Nieborowska-Skorska M., Hoser G., Morrione A., Majewski M., Xue L., Morris S. W., Wasik M. A., Skorski T. Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis. Cancer Res., 61: 2194-2199, 2001.[Abstract/Free Full Text]
16. Wickremasinghe R. G., Ganeshaguru K., Jones D. T., Lindsay C., Spanswick V. J., Hartley J. A., Wadhwa M., Thorpe R., Hoffbrand A. V., Prentice H. G., Mehta A. B. Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells. Br. J. Haematol., 114: 608-615, 2001.[CrossRef][Medline]
17. Bernal A., Pastore R. D., Asgary Z., Keller S. A., Cesarman E., Liou H. C., Schattner E. J. Survival of leukemic B cells promoted by engagement of the antigen receptor. Blood, 98: 3050-3057, 2001.[Abstract/Free Full Text]
18. Brennan P., Mehl A. M., Jones M., Rowe M. Phosphatidylinositol 3-kinase is essential for the proliferation of lymphoblastoid cells. Oncogene, 21: 1263-1271, 2002.[CrossRef][Medline]
19. Ringshausen I., Schneller F., Bogner C., Hipp S., Duyster J., Peschel C., Decker T. Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase C. Blood, 100: 3741-3748, 2002.[Abstract/Free Full Text]
20. Jones D. T., Ganeshaguru K., Anderson R. J., Jackson T. R., Bruckdorfer K. R., Low S. Y., Palmqvist L., Prentice H. G., Hoffbrand A. V., Mehta A. B., Wickremasinghe R. G. Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis. Blood, 101: 3174-3180, 2003.[Abstract/Free Full Text]
21. Furman R. R., Asgary Z., Mascarenhas J. O., Liou H. C., Schattner E. J. Modulation of NF-B activity and apoptosis in chronic lymphocytic leukemia B cells. J. Immunol., 164: 2200-2206, 2000.[Abstract/Free Full Text]
22. Cotter F. E., Johnson P., Hall P., Pocock C., al Mahdi N., Cowell J. K., Morgan G. Antisense oligonucleotides suppress B-cell lymphoma growth in a SCID-hu mouse model. Oncogene, 9: 3049-3055, 1994.[Medline]
23. McConkey D. J., Chandra J., Wright S., Plunkett W., McDonnell T. J., Reed J. C., Keating M. Apoptosis sensitivity in chronic lymphocytic leukemia is determined by endogenous endonuclease content and relative expression of BCL-2 and BAX. J. Immunol., 156: 2624-2630, 1996.[Abstract]
24. Thomas A., El Rouby S., Reed J. C., Krajewski S., Silber R., Potmesil M., Newcomb E. W. Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance. Oncogene, 12: 1055-1062, 1996.[Medline]
25. Pepper C., Hoy T., Bentley D. P. Bcl-2/Bax ratios in chronic lymphocytic leukaemia and their correlation with in vitro apoptosis and clinical resistance. Br. J. Cancer, 76: 935-938, 1997.[Medline]
26. Pepper C., Thomas A., Hoy T., Cotter F., Bentley P. Antisense-mediated suppression of Bcl-2 highlights its pivotal role in failed apoptosis in B-cell chronic lymphocytic leukaemia. Br. J. Haematol., 107: 611-615, 1999.[CrossRef][Medline]
27. Guinness M. E., Kenney J. L., Reiss M., Lacy J. Bcl-2 antisense oligodeoxynucleotide therapy of Epstein-Barr virus-associated lymphoproliferative disease in severe combined immunodeficient mice. Cancer Res., 60: 5354-5358, 2000.[Abstract/Free Full Text]
28. Kitada S., Andersen J., Akar S., Zapata J. M., Takayama S., Krajewski S., Wang H. G., Zhang X., Bullrich F., Croce C. M., Rai K., Hines J., Reed J. C. Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in vivo chemoresponses. Blood, 91: 3379-3389, 1998.[Abstract/Free Full Text]
29. Vrana J. A., Bieszczad C. K., Cleaveland E. S., Ma Y., Park J. P., Mohandas T. K., Craig R. W. An MCL1-overexpressing Burkitt lymphoma subline exhibits enhanced survival on exposure to serum deprivation, topoisomerase inhibitors, or staurosporine but remains sensitive to 1-ß-D-arabinofuranosylcytosine. Cancer Res., 62: 892-900, 2002.[Abstract/Free Full Text]
30. Pedersen I. M., Kitada S., Leoni L. M., Zapata J. M., Karras J. G., Tsukada N., Kipps T. J., Choi Y. S., Bennett F., Reed J. C. Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1. Blood, 100: 1795-1801, 2002.[Abstract/Free Full Text]
31. Ghia P., Boussiotis V. A., Schultze J. L., Cardoso A. A., Dorfman D. M., Gribben J. G., Freedman A. S., Nadler L. M. Unbalanced expression of bcl-2 family proteins in follicular lymphoma: contribution of CD40 signaling in promoting survival. Blood, 91: 244-251, 1998.[Abstract/Free Full Text]
32. Keyomarsi K., Sandoval L., Band V., Pardee A. B. Synchronization of tumor and normal cells from G₁ to multiple cell cycles by lovastatin. Cancer Res., 51: 3602-3609, 1991.[Abstract/Free Full Text]
33. Crick D. C., Andres D. A., Waechter C. J. Novel salvage pathway utilizing farnesol and geranylgeraniol for protein isoprenylation. Biochem. Biophys. Res. Commun., 237: 483-487, 1997.[CrossRef][Medline]
34. Siminovitch K. A., Jensen J. P., Epstein A. L., Korsmeyer S. J. Immunoglobulin gene rearrangements and expression in diffuse histiocytic lymphomas reveal cellular lineage, molecular defects, and sites of chromosomal translocation. Blood, 67: 391-397, 1986.[Abstract/Free Full Text]
35. Davis A. R., Alevy Y. G., Chellaiah A., Quinn M. T., Mohanakumar T. Characterization of HDJ-2, a human 40 kD heat shock protein. Int. J. Biochem. Cell Biol., 30: 1203-1221, 1998.[CrossRef][Medline]
36. Omer C. A., Chen Z., Diehl R. E., Conner M. W., Chen H. Y., Trumbauer M. E., Gopal-Truter S., Seeburger G., Bhimnathwala H., Abrams M. T., Davide J. P., Ellis M. S., Gibbs J. B., Greenberg I., Koblan K. S., Kral A. M., Liu D., Lobell R. B., Miller P. J., Mosser S. D., O’Neill T. J., Rands E., Schaber M. D., Senderak E. T., Oliff A., Kohl N. E. Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor. Cancer Res., 60: 2680-2688, 2000.[Abstract/Free Full Text]
37. Vogt A., Qian Y., McGuire T. F., Hamilton A. D., Sebti S. M. Protein geranylgeranylation, not farnesylation, is required for the G₁ to S phase transition in mouse fibroblasts. Oncogene, 13: 1991-1999, 1996.[Medline]
38. Gross A., McDonnell J. M., Korsmeyer S. J. BCL-2 family members and the mitochondria in apoptosis. Genes Dev., 13: 1899-1911, 1999.[Free Full Text]
39. Kroemer G., Reed J. C. Mitochondrial control of cell death. Nat. Med., 6: 513-519, 2000.[CrossRef][Medline]
40. Cheng E. H., Kirsch D. G., Clem R. J., Ravi R., Kastan M. B., Bedi A., Ueno K., Hardwick J. M. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science (Wash. DC), 278: 1966-1968, 1997.[Abstract/Free Full Text]
41. Kirsch D. G., Doseff A., Chau B. N., Lim D. S., Souza-Pinto N. C., Hansford R., Kastan M. B., Lazebnik Y. A., Hardwick J. M. Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c. J. Biol. Chem., 274: 21155-21161, 1999.[Abstract/Free Full Text]
42. Krajewski S., Bodrug S., Gascoyne R., Berean K., Krajewska M., Reed J. C. Immunohistochemical analysis of Mcl-1 and Bcl-2 proteins in normal and neoplastic lymph nodes. Am. J. Pathol., 145: 515-525, 1994.[Abstract]
43. Rassidakis G. Z., Lai R., McDonnell T. J., Cabanillas F., Sarris A. H., Medeiros L. J. Overexpression of Mcl-1 in anaplastic large cell lymphoma cell lines and tumors. Am. J. Pathol., 160: 2309-2310, 2002.[Free Full Text]
44. Zhou P., Levy N. B., Xie H., Qian L., Lee C. Y., Gascoyne R. D., Craig R. W. MCL1 transgenic mice exhibit a high incidence of B-cell lymphoma manifested as a spectrum of histologic subtypes. Blood, 97: 3902-3909, 2001.[Abstract/Free Full Text]
45. Zhou P., Qian L., Bieszczad C. K., Noelle R., Binder M., Levy N. B., Craig R. W. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood, 92: 3226-3239, 1998.[Abstract/Free Full Text]
46. Suzuki J., Kaziro Y., Koide H. An activated mutant of R-Ras inhibits cell death caused by cytokine deprivation in BaF3 cells in the presence of IGF-I. Oncogene, 15: 1689-1697, 1997.[CrossRef][Medline]
47. Van Aelst L., D’Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev., 11: 2295-2322, 1997.[Free Full Text]
48. Zohn I. M., Campbell S. L., Khosravi-Far R., Rossman K. L., Der C. J. Rho family proteins and Ras transformation: the RHOad less traveled gets congested. Oncogene, 17: 1415-1438, 1998.[CrossRef][Medline]
49. Osada M., Tolkacheva T., Li W., Chan T. O., Tsichlis P. N., Saez R., Kimmelman A. C., Chan A. M. Differential roles of Akt, Rac, and Ral in R-Ras-mediated cellular transformation, adhesion, and survival. Mol. Cell Biol., 19: 6333-6344, 1999.[Abstract/Free Full Text]
50. Bar-Sagi D., Hall A. Ras and Rho GTPases: a family reunion. Cell, 103: 227-238, 2000.[CrossRef][Medline]
51. Craig R. W. MCL1 provides a window on the role of the BCL2 family in cell proliferation, differentiation and tumorigenesis. Leukemia (Balt.), 16: 444-454, 2002.
52. Kumagai N., Morii N., Fujisawa K., Nemoto Y., Narumiya S. ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells. J. Biol. Chem., 268: 24535-24538, 1993.[Abstract/Free Full Text]
53. Tolias K. F., Cantley L. C., Carpenter C. L. Rho family GTPases bind to phosphoinositide kinases. J. Biol. Chem., 270: 17656-17659, 1995.[Abstract/Free Full Text]
54. Bokoch G. M., Vlahos C. J., Wang Y., Knaus U. G., Traynor-Kaplan A. E. Rac GTPase interacts specifically with phosphatidylinositol 3-kinase. Biochem. J., 315: 775-779, 1996.
55. Marte B. M., Rodriguez-Viciana P., Wennstrom S., Warne P. H., Downward J. R-Ras can activate the phosphoinositide 3-kinase but not the MAP kinase arm of the Ras effector pathways. Curr. Biol., 7: 63-70, 1997.[CrossRef][Medline]
56. Zheng Y., Bagrodia S., Cerione R. A. Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. J. Biol. Chem., 269: 18727-18730, 1994.[Abstract/Free Full Text]
57. Yu Y., Feig L. A. Involvement of R-Ras and Ral GTPases in estrogen-independent proliferation of breast cancer cells. Oncogene, 21: 7557-7568, 2002.[CrossRef][Medline]
58. Miquel K., Pradines A., Sun J., Qian Y., Hamilton A. D., Sebti S. M., Favre G. GGTI-298 induces G₀-G₁ block and apoptosis whereas FTI-277 causes G₂-M enrichment in A549 cells. Cancer Res., 57: 1846-1850, 1997.[Abstract/Free Full Text]
59. Khosravi-Far R., Solski P. A., Clark G. J., Kinch M. S., Der C. J. Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation. Mol. Cell Biol., 15: 6443-6453, 1995.[Abstract]
60. Olson M. F., Ashworth A., Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G₁. Science (Wash. DC), 269: 1270-1272, 1995.[Abstract/Free Full Text]
61. Qiu R. G., Abo A., McCormick F., Symons M. Cdc42 regulates anchorage-independent growth and is necessary for Ras transformation. Mol. Cell Biol., 17: 3449-3458, 1997.[Abstract]
62. Lin R., Bagrodia S., Cerione R., Manor D. A novel Cdc42Hs mutant induces cellular transformation. Curr. Biol., 7: 794-797, 1997.[CrossRef][Medline]
63. Soma M. R., Pagliarini P., Butti G., Paoletti R., Paoletti P., Fumagalli R. Simvastatin, an inhibitor of cholesterol biosynthesis, shows a synergistic effect with N,N'-bis(2-chloroethyl)-N-nitrosourea and ß-interferon on human glioma cells. Cancer Res., 52: 4348-4355, 1992.[Abstract/Free Full Text]
64. Soma M. R., Baetta R., De Renzis M. R., Mazzini G., Davegna C., Magrassi L., Butti G., Pezzotta S., Paoletti R., Fumagalli R. In vivo enhanced antitumor activity of carmustine [N,N'-bis(2-chloroethyl)-N-nitrosourea] by simvastatin. Cancer Res., 55: 597-602, 1995.[Abstract/Free Full Text]
65. Feleszko W., Mlynarczuk I., Balkowiec-Iskra E. Z., Czajka A., Switaj T., Stoklosa T., Giermasz A., Jakobisiak M. Lovastatin potentiates antitumor activity and attenuates cardiotoxicity of doxorubicin in three tumor models in mice. Clin. Cancer Res., 6: 2044-2052, 2000.[Abstract/Free Full Text]
66. Zhou P., Qian L., Kozopas K. M., Craig R. W. Mcl-1, a Bcl-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions. Blood, 89: 630-643, 1997.[Abstract/Free Full Text]
67. Thibault A., Samid D., Tompkins A. C., Figg W. D., Cooper M. R., Hohl R. J., Trepel J., Liang B., Patronas N., Venzon D. J., Reed E., Myers C. E. Phase I study of lovastatin, an inhibitor of the mevalonate pathway, in patients with cancer. Clin. Cancer Res., 2: 483-491, 1996.[Abstract]