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Vol. 303, Issue 1, 74-81, October 2002
Department of Pharmacology (R.R.M, R.E.M.), Wayne State University, Detroit, Michigan; Programs in Molecular Biology and Genetics (R.R.M.) and Proteases (J.J.R.), Barbara Ann Karmanos Cancer Institute, Detroit, Michigan; Department of Medicinal Chemistry and Molecular Pharmacology (R.A.G.), College of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana; and Institute of Environmental Health Sciences (J.J.R.), Wayne State University, Detroit, Michigan
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Statins, which inhibit 3-hydroxy-3-methylglutaryl-CoA
reductase and thus the synthesis of cholesterol, are remarkably
effective in the treatment of cardiovascular disease. In addition to
their favorable effect on lipid profile, these drugs may also prevent the proliferation of vascular smooth muscle that is characteristic of
atherosclerosis. We hypothesize that statins prevent the
post-translational prenylation, and thus inhibit the function, of
critical small GTPases in vascular smooth muscle cells. We have
therefore assayed the effect of lovastatin on both the growth of A10
vascular smooth muscle cells and the status of their Ras and RhoB
proteins. We find that 
1 µM lovastatin potently inhibits the
proliferation of A10 cultures, and higher concentrations (
3 µM)
induce apoptosis. We have also tested the effect of 3-allylfarnesol
(3-alFOH), an inhibitor of farnesyl transferase (FTI). The data show
that although 10 µM 3-alFOH is required for a cytostatic effect,
the action of 3 µM 3-alFOH can be greatly potentiated by even
nanomolar levels of lovastatin. We also find that lovastatin and
3-alFOH exhibit synergism to cause the up-regulation and relocalization
of RhoB from the membrane to cytosolic compartments. This
relocalization of RhoB, which is presumed to reflect an inhibition of
its prenylation, correlates with the proapoptotic activities of
combined 3-alFOH and lovastatin treatment. These data suggest that RhoB
may be a valuable pharmacological target in cardiovascular disease, and that combinations of statins and certain FTIs may be of value in
treatment of disorders that are characterized by excess cell proliferation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Hyperplasia
of vascular smooth muscle is responsible for the intimal thickening
that occurs after arterial wall damage. It may also contribute to the
atherosclerotic process (Raines and Ross, 1993
). Strategies that
prevent inappropriate proliferation of vascular smooth muscle cells may
therefore be of significant value in the treatment of cardiovascular
disease. The drugs termed statins, which inhibit hepatic
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and thus
reduce the production of cholesterol, are remarkably effective in the
management of hypercholesteremia, with proven efficacy to improve lipid
profile, to reduce risk of myocardial infarction and stroke, and to
decrease mortality. It is becoming clear, however, that the beneficial
effects of statins may also be due to effects other than the inhibition
of hepatic HMG-CoA reductase. For example, there is poor correlation between improved clinical endpoints and effects on baseline low-density lipoprotein levels in several major clinical trials (for review, see Kolovou, 2001). An additional therapeutic mechanism for the action of statins could be the inhibition of vascular smooth muscle proliferation (Negre-Aminou et al., 1997; Raiteri et al., 1997). Indeed, lovastatin is known to be effective in prevention of the restenosis of coronary arteries after angioplasty (Sahni et al., 1991).
Inhibition of HMG-CoA reductase causes a reduction in supply of
mevalonate metabolites that are critical not only for the synthesis of
cholesterol but also for the post-translational modification of certain
proteins with prenyl groups (Khwaja et al., 2000). Considerable effort
has been invested in the development of selective inhibitors of protein
prenylation after the recognition that Ras farnesylation is essential
for its ability to stimulate cell proliferation even after its
oncogenic activation (Gibbs et al., 1994). Several inhibitors of
protein farnesylation are now in advanced clinical trial for the
therapy of various malignancies (Gibbs, 2000). Current evidence
suggests that other members of the Ras superfamily of proteins,
particularly RhoB, may be more relevant clinical targets for these
farnesyl transferase inhibitors (FTIs) (Lebowitz et al., 1995; Aznar
and Lacal, 2001).
There is some evidence that blockade of the function of Rho proteins,
through inhibition of their prenylation, may also contribute directly
and indirectly to the effects of statins on vascular smooth muscle
cells. For example, statins induce an increase in endothelial
nitric-oxide synthase activity that is dependent on the inhibition of
Rho (Laufs et al., 2000). Statins have been reported to directly
affect the prenylation of Rho proteins in vascular smooth muscle cells,
although at high drug concentrations (Guijarro et al., 1998). We
hypothesize that if the effect of statins on vascular smooth muscle
cell proliferation is mediated through blockade of Rho protein
prenylation, statins should exhibit synergy with selective inhibitors
of prenylation (Fig. 1). Furthermore, if
such a mechanism is to be relevant to the clinical use of statins, the
effects should occur at drug concentrations that can be
pharmacologically achieved. We therefore tested the effects of single
and combined treatments of A10 vascular smooth muscle cells with
lovastatin and an inhibitor of farnesyltransferase. The results
demonstrate a correlation between their synergistic effects to suppress
proliferation, to induce apoptosis, and to block the prenylation of
RhoB.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Drugs
Lovastatin was purchased from Sigma-Aldrich (St. Louis, MO). The
3-allylfarnesol (3-alFOH) was synthesized as described previously (Gibbs et al., 1999). Both drugs were maintained as 100 mM stocks in
dimethyl sulfoxide (DMSO) in aliquots under nitrogen at 
80°C. AMC
and acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin were purchased from
BD PharMingen (San Diego, CA).
Cells
The A10 rat vascular smooth muscle cell line was obtained from the American Type Culture Collection (Rockville, MD). The cells were maintained in a humidified incubator with 5% CO2 and grown on 100-mm culture dishes in Dulbecco's modified Eagle's medium (DMEM) with high glucose (Invitrogen, Carlsbad, CA), that was supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). The cells were split 1:10 when approaching confluence. Aliquots in DMEM/fetal bovine serum/DMSO (4:5:1) were prepared for storage in liquid nitrogen, and all experiments were performed on cells grown between three and 10 passages from receipt.
Cell Proliferation Assay
A10 cells were plated at 100,000 cells/60-mm culture dish. Cells attached and spread within 2 h of plating. The media were then removed and fresh growth media with drugs were added, as noted in the figure legends. The medium was replaced every 2 days (with fresh drug addition as required) until the number of viable cells on the dish was counted with a hemocytometer after trypsinization. Ability to exclude trypan blue was used as a measurement of viability. Data are presented as mean ± S.E.M. of the number of independent experiments shown in each Figure. Differences from control were assessed by two-way analysis of variance with Bonferroni post tests when multiple comparisons were required (GraphPad Prism 3.0a; GraphPad Software, San Diego, CA).
Cell Cycle Analysis
A10 cells were plated at 250,000 cells/100-mm culture dish and
allowed to attach overnight. The media were then removed and fresh
growth media with drugs were added. The medium was replaced every 2 days until the time of analysis. The medium was carefully removed and
combined with a 1-ml phosphate-buffered saline (PBS) rinse to
constitute the "detached sample". The adherent cells were
trypsinized and suspended in growth medium. Samples of the adherent and
detached suspensions were counted and assessed for viability by
exclusion of trypan blue. The remaining cell suspensions were washed
1× with PBS and then suspended in PBS before being fixed and processed
for flow cytometric analyses of DNA content as described previously
(Reiners et al., 1999). Percentages of apoptotic cells and cells in the
G1, S, and G2/M stages of
the cell cycle were determined with a DNA histogram-fitting program (MODFIT; Verity Software, Topsham, ME). Attempts were made to collect a
minimum of 104 events/sample for subsequent analyses.
Subcellular Fractionation
A10 cells were plated at approximately 50% confluence and grown
for 24 h. The media were then replaced with fresh media plus drugs
as required (two plates per condition). Approximately 18 h later,
the cultures were rinsed twice with hypotonic lysis buffer (HLB) (10 mM
HEPES, pH 7.4, with NaOH; 5 mM MgCl2; 1 mM
EDTA·Na; 20 µg/ml aprotinin; 20 µg/ml leupeptin; and 100 µM
phenylmethylsulfonyl fluoride) and then scraped with 0.5 ml/plate of
HLB into microcentrifuge tubes on ice. The cells were allowed to swell
for 10 min and then disrupted by sonication for 5 s and six
passages through a 26-gauge syringe needle, combining material from
duplicate plates. KCl was added to 150 mM final concentration and the
nuclei removed by a 10-min centrifugation at 1,000g. The
postnuclear supernatant was centrifuged at 10,000g for 15 min to prepare an organelle fraction. The postorganellar supernatant
was centrifuged at 100,000g for 45 min to prepare a
microsomal fraction. All pellet fractions were resuspended in 40 µl
of HLB. The supernatant from the 100,000g spin was used as
the soluble/cytosolic fraction, mixed with an equal volume of 300 mM
NaCl, 2% Nonidet-40, 1% sodium deoxycholate, 0.2% SDS, and 100 mM
Tris pH 7.4, and precipitated by addition of 10% trichloracetic acid
on ice for 1 h. The precipitate was washed three times with cold
acetone and allowed to dry in a fume hood. The precipitate was
dissolved in 40 µl of 1 M NaHCO3, pH 8.8, at
37°C. All fractions were then mixed with 40 µl of 2× sample loading buffer (Mattingly et al., 2001a) and separated by
SDS-polyacrylamide gel electrophoresis (PAGE).
Transfection
Calcium phosphate precipitation-mediated transfection of COS-7
and human embryonic kidney (HEK)293 cells was performed as described
previously (Mattingly et al., 1994), using expression vectors pKH3RhoA
(Beqaj et al., 2002) and pCMV3HARhoB (Lebowitz et al., 1997a) that
express RhoA and RhoB with hemagglutinin (HA) tags at their N termini.
A10 cells were transfected using the FuGENE 6 reagent according to the
manufacturer's protocol (Roche Applied Science, Indianapolis, IN).
Cells were grown for 48 h and then whole cell lysates were
prepared in boiling Laemmli sample buffer as described previously
(Mattingly et al., 2001a).
Western Blotting
Primary antibodies used were polyclonal and monoclonal anti-RhoB
(Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100, monoclonal anti-Ras at 1:250 and monoclonal anti-ERK mitogen-activated protein kinase at 1:5000 (Transduction Laboratories, Lexington, KY), polyclonal anti-cathepsin B (Moin et al., 1992) at 1:4000, and 12CA5 monoclonal anti-hemagglutinin (Mattingly, 1999) at 1:5000. Secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnology) were diluted to 1:25,000, and detection was performed with Dura enhanced chemiluminescence reagents (Pierce Chemical, Rockford, IL). Data were
collected using an LAS 1000 plus imaging system (Fujifilm, Tokyo,
Japan) and exposure to film. For sequential probing of the membranes
with different primary antibodies, the blots were stripped as described
previously (Mattingly et al., 2001b).
To verify the selectivity and sensitivity of the commercial anti-RhoB
antibodies they were tested against HA-tagged RhoB and RhoA proteins
that were overexpressed in COS-7 and HEK293 cells (Fig.
2). The results show that the polyclonal
anti-RhoB antibody shows greater sensitivity for the detection of RhoB
than does the monoclonal antibody, with detection of both the
overexpressed and endogenous proteins present in whole cell extracts of
COS-7 cells (Fig. 2A). The polyclonal antibody to RhoB can selectively recognize RhoB and not RhoA (Fig. 2B). The ability of the antibody to
recognize both endogenous and overexpressed RhoB in lysates of A10
cells was then confirmed (Fig. 2C).
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Caspase-3 Assay
At the time of harvest the medium was removed and saved.
Cultures were washed twice with PBS, and the washings were combined with the medium. After centrifugation, the pelleted detached cells were
washed once with PBS and suspended in lysis buffer (10 mM Tris pH 7.5, 130 mM NaCl, 1% Triton X-100, 10 mM NaF, 10 mM
NaPi, and 10 mM NaPPI).
Adherent cells were covered with lysis buffer, dislodged with a cell
scraper, and transferred to small tubes. Adherent and detached cell
populations were stored at 80°C until the day of assay. On the day
of assay, cell lysates were sonicated for 1 s and centrifuged for
5 min at 10,000g at 4°C. Caspase-3 activity in the
supernatant fluids was assayed by monitoring the release of AMC from
the caspase-3 substrate
acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin. The assay has been
described in detail previously (Reiners and Clift, 1999). Fluorescence
was monitored on a SpectraMax Gemini plate reader (Molecular Devices,
Sunnyvale, CA). Caspase-3-specific activities are reported as nanomoles
of AMC released per minute per milligram of protein. The bicinchoninic
acid assay was used for the determination of protein content. Bovine
serum albumin was used as the standard.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Lovastatin Potently Inhibits Proliferation of A10 Rat Vascular
Smooth Muscle Cells.
To characterize the effects of prenylation
inhibitors on the growth of vascular smooth muscle cells, the A10 cell
line was selected because it provides a uniform population of cells
that exhibit significant phenotypic resemblance to neointimal cells (Rao et al., 1997). Because statins are clinically used in chronic treatment regimens, we investigated the effect of lovastatin on the
growth of A10 cells over an 8-day period (Fig.
3A). In agreement with an earlier study
that used human aortic smooth muscle cells from the internal mammary
artery (Negre-Aminou et al., 1997), concentrations of lovastatin as low
as 1 µM were cytostatic to A10 cultures (p < 0.05),
whereas 3 µM lovastatin was cytotoxic and reduced cell numbers to
values below that plated.
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Suppression of A10 Proliferation by a Farnesyl Transferase
Inhibitor.
Previous studies have indicated that 3-alFOH acts as a
cell-permeable precursor of a farnesyl pyrophosphate (FPP)-competitive inhibitor of farnesyl transferase (Gibbs et al., 1999). Treatment of
A10 vascular smooth muscle cells with 3-alFOH required a concentration of 10 µM to produce a significant (p < 0.05)
suppression of cell proliferation (Fig.
6A). Treatment with 30 µM 3-alFOH
reduced cell numbers to a value below that plated (Fig. 6A). Flow
cytometric analyses of DNA contents suggested that the cytotoxic
effects of this higher concentration reflected the induction of
apoptosis (data not shown).
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10 µM, its effects on the subcellular localization of Ras were determined (Fig. 6B). Treatment of A10 cells with either 30 µM 3-alFOH for 24 h or 10 µM 3-alFOH for 48 h was able to induce the appearance of Ras in the soluble fraction of A10 cells. Cytosolic Ras presumably reflects the precursor form of the protein, before prenylation (Gibbs et al.,
1999). Thus, the data in Fig. 6B support 3-alFOH as an inhibitor of
farnesylation in this system.
3-alFOH Potentiation of Cytostatic/Cytotoxic Activities of Lovastatin. Combinations of low concentrations of lovastatin and 3-alFOH were very effective at inhibiting A10 proliferation (Fig. 6A). For example, 0.3 µM lovastatin plus 3 µM 3-alFOH (neither of which produced a measurable effect on cell number at 8 days as single agents) reduced cell numbers by one-third (p < 0.05). Similarly, the combination of 1 µM lovastatin plus 3 µM 3-alFOH reduced cell numbers to a value below that plated. This latter result was the consequence of at least two activities. First, flow cytometric analyses of DNA contents demonstrated that cotreatment of cultures with lovastatin and 3 µM 3-alFOH potentiated the loss of S-phase cells (Figs. 4A and 5, C and D). Cultures cotreated with 1 µM lovastatin + 1 µM 3-alFOH had S-phase contents comparable with that obtained with 3 µM lovastatin (Fig. 4). Second, the combination treatments resulted in the detachment of cells (Fig. 5B). Flow cytometric analyses of the 96-h detached populations from 1 µM lovastatin + 3 or 10 µM 3-alFOH treatment groups demonstrated a predominant population having subdiploid DNA content, suggestive of an apoptotic population (Fig. 4B). Cotreatment with the higher concentration of 3-alFOH (10 µM) resulted in a higher percentage of cells having subdiploid DNA contents.
The subdiploid DNA contents of apoptotic cells reflect the actions of an endonuclease that is activated by caspase-3. Detached cells generally constituted ~15% of the cellularity of nontreated and DMSO-treated A10 cultures. Of this population ~40% were trypan blue-permeable. In DMSO-treated cultures the caspase-3-specific activities of the detached cell population were ~3- to 4-fold higher than the activity measured in the adherent population (Fig. 7). Exposure to 3 or 10 µM 3-alFOH neither induced the detachment of A10 cells (Fig. 5) nor the activation caspase-3 (Fig. 7). In contrast, concentration-dependent increases in caspase-3 activities were seen in both attached and detached A10 cells after exposure to 1 and 3 µM lovastatin (Fig. 7). The effects of lovastatin were markedly potentiated by cotreatment with concentrations of 3-alFOH (3 or 10 µM) that had no detectable effect by themselves. Specifically, 3-alFOH synergized with lovastatin to increase caspase-3-specific activities in both adherent and detached A10 cells (Fig. 7)
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Lovastatin and 3-alFOH Induce an Up-Regulation of RhoB into
Cytosolic Compartment of A10 Vascular Smooth Muscle Cells.
To
investigate whether the effects of lovastatin and 3-alFOH on cell
proliferation and apoptosis correlate with alterations in protein
prenylation in A10 vascular smooth muscle cells, subcellular fractionation was performed, and the fractions were immunoblotted for
RhoB and Ras proteins (Fig. 8). Properly
processed Ras and RhoB would be expected to be in membrane fractions,
whereas unprenylated precursors would be soluble in the cytosol
(Guijarro et al., 1998; Gibbs et al., 1999). The unprenylated
precursors also typically migrate more slowly on SDS-PAGE gels (Gibbs
et al., 1999). Lovastatin induced the relocalization of almost all the
RhoB and a very small fraction of the Ras from the microsomal fraction
to the soluble/cytosolic portion of A10 cells after an overnight
treatment. Inhibition of farnesylation by 3-alFOH alone was without any
apparent effect on either Ras or RhoB. This lack of effect may be a
consequence of RhoB being alternatively geranylgeranylated after FTI
treatment, and so it would remain attached to the membrane. The lack of
effect on Ras is probably due to both alternative geranylgeranylation, and to the relative stability of the prenylated Ras protein pool that
existed before drug addition. Note that this overnight incubation time
with 10 µM 3-alFOH is much shorter than the 48-h treatment that did
produce an effect on Ras (Fig. 6B). Combinations of lovastatin and
3-alFOH showed a greater ability to cause the relocalization of Ras
than did lovastatin alone, although the effect was still less than
50%. However, combination treatment caused a marked increase in the
amount of RhoB, with the increased protein being in the soluble
fraction.
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). Mature cathepsin B was found in the organelle fraction, as
expected. However, cathepsin B cross-reacting material was also found
in the soluble fraction and may represent lysosome breakage that occurred during the fractionation protocol.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Inhibition of Farnesyl Transferase.
Prenylation of a cysteine
residue in a subset of mammalian proteins, including almost all members
of the Ras superfamily of small GTPases, is now known to be an
essential step in the functional maturation of these proteins (Khwaja
et al., 2000). The discovery that farnesylation remains necessary for
the growth-promotion functions of Ras proteins, even after their
prevalent oncogenic activation in human cancer progression (Gibbs et
al., 1994), has provided the stimulus for the development of selective
inhibitors of farnesyl transferase, the enzyme responsible for this
post-translational modification (Gibbs, 2000). As these agents have
moved through preclinical and now clinical trials, significant
questions have arisen as to whether Ras proteins are the most relevant
target for the cytostatic action of FTIs. Prendergast and colleagues have argued that the lack of correlation between the cytostatic action
of FTIs and their effects on the prenylation of Ras proteins suggests
that another farnesylated protein in cells, in particular RhoB, may be
the most relevant target for FTIs (Lebowitz et al., 1995). In support
of this hypothesis are the facts that RhoB has a short half-life in
cells (as opposed to the highly stable Ras proteins) and thus is more
vulnerable to drugs that inhibit its synthesis (Lebowitz et al., 1995),
and that fibroblasts from mice that have had their RhoB gene deleted
are resistant to the effects of FTIs (Liu et al., 2001). A
prenylation-independent form of RhoB can also confer resistance to FTI
treatment (Lebowitz et al., 1997b). Transformation of RhoB-deficient
fibroblasts with oncogenic H-Ras generates a model that is resistant to
both FTIs and DNA-damaging agents (Liu et al., 2001). A connection
between RhoB and apoptosis has also been made in other systems,
including central nervous system neurons (Trapp et al., 2001), NIH-3T3
fibroblasts (Fritz and Kaina, 2000), and rat thoracic aorta smooth
muscle cells (Guijarro et al., 1998).
Synergism between FTIs and Lovastatin.
RhoB is distinct within
Ras-like small GTPase superfamily in that it can be found in two forms
in cells, one modified with the farnesyl prenyl group, and one modified
with geranylgeranyl (Adamson et al., 1992). The shift to predominantly
the geranylgeranylated form that occurs in cells treated with FTIs
(Lebowitz et al., 1997a) has been proposed to be a critical event as
geranylgeranylated-RhoB may be proapoptotic (Du and Prendergast, 1999;
but also see Chen et al., 2000). The failure of 3-alFOH, as a single
agent, to cause relocalization of RhoB to the soluble fraction in the
current study would be consistent with a shift of RhoB to the
geranylgeranylated form under this treatment condition. Exposure to
3-alFOH did result in cytotoxicity and apoptosis at concentrations
sufficient to inhibit farnesyl transferase. However, the apoptotic
effects of 3-alFOH could be markedly potentiated by combined treatment
with lovastatin. Note that 3-alFOH is unusual among FTIs, in that its active form is an FPP-competitive, rather than a peptide-competitive, inhibitor of protein farnesyltransferase (Gibbs, 2000). It would be
expected that an FPP-competitive FTI such as 3-alFOH would exhibit a
more synergistic effect with lovastatin, which blocks endogenous FPP
synthesis, than other peptide-competitive FTIs (Yonemoto et al., 1998).
; Gampel et al., 1999), but
any link between this function and cell death is unclear. RhoB is also
known to be a product of an immediate-early gene that is induced by DNA
damage (Fritz et al., 1995) and that may play a role in stress
responses through modulation of signal transduction pathways (Fritz and
Kaina, 2001). Inhibition of Ras prenylation may also be significant
because Ras is known to play an important antiapoptotic role in
vascular smooth muscle cells (Miyamoto and Fox, 2000).
Inhibition of Vascular Smooth Muscle Proliferation by Statins.
The beneficial effect of statins on cardiovascular morbidity and
mortality may reflect activities in addition to their reversal of
hypercholesteremia via inhibition of hepatic HMG-CoA reductase (Kolovou, 2001). An additional mechanism of action might be inhibition of the proliferation of neointimal vascular smooth muscle cells either
directly (Negre-Aminou et al., 1997) or indirectly via their ability to
cause sensitization to proapoptotic signals (Knapp et al., 2000). Such
effects of statins in smooth muscle systems may be associated with
their ability to inhibit the prenylation of critical cellular proteins,
such as RhoB (Guijarro et al., 1998) or RhoA (Laufs et al., 1999). Our
investigation of the effects of lovastatin on the proliferation of the
A10 neointimal-like cell line demonstrates that lovastatin can potently
inhibit both the growth of these cells and the prenylation of RhoB,
with pronounced effects occurring at 1 to 3 µM lovastatin, a
concentration much closer to what can be achieved therapeutically
(Duggan et al., 1989) than the 100 µM used previously (Guijarro et
al., 1998). Exposure of A10 cells to 1 to 3 µM lovastatin resulted in
the loss of S-phase cells, and an accumulation of
G1 and/or G2/M phase cells.
Such accumulations require the activation of both
G1 and G2/M checkpoints.
The cytostatic effect of simvastatin in human aortic smooth muscle
cells has been ascribed to a blockade of 
-dependent down-regulation
of the cyclin-dependent kinase inhibitor p27kip1 (Laufs et al., 1999),
a potent inducer of the G1 checkpoint. Inhibition
of the prenylation of CENP-E may be responsible for the accumulation of
G2/M cells observed in the current study. CENP-E
is a kinetochore motor that is required for completion of mitosis. It
is only functional when farnesylated (Ashar et al., 2000).
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Acknowledgments |
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We thank Drs. George Prendergast and Bonnie Sloane for the generous gifts of pCMV3HARhoB and polyclonal anti-cathepsin B, respectively, Irina Laer and Bhadrani Chelladurai for technical assistance, Dr. Todd Zahn and Raj Amin for the synthesis of 3-alFOH, and Drs. T. Kocarek and R. Yamazaki for helpful discussion.
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Footnotes |
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Accepted for publication June 4, 2002.
Received for publication March 11, 2002.
This work was supported by an Atorvastatin Research Award Award from Pfizer, Inc., Grant DAMD17-00-1-0544 from the Department of the Army, and a Faculty Development Award from the PhRMA Foundation (to R.R.M.), and by National Institutes of Health Grants CA-78819 (to R.A.G.) and ES-09392 (to J.J.R.). This work was aided by the Cell Imaging and Cytometry Facility Core, which is supported by Center Grants from the National Institute of Environmental Health Sciences (P30 ES06639) and National Cancer Institute (P30 CA22453). R.E.M. was supported in part by a Training Program in Cancer Biology (T32-CA09531-15). The content of the information does not necessarily reflect the position or policy of the U.S. Government, and no official endorsement should be inferred.
DOI: 10.1124/jpet.102.036061
Address correspondence to: Raymond R. Mattingly, Department of Pharmacology, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201. E-mail: r.mattingly{at}wayne.edu
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Abbreviations |
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. HMG, 3-hydroxy-3-methylglutaryl; FTI, inhibitor of farnesyl transferase; 3-alFOH, 3-allylfarnesol; DMSO, dimethyl sulfoxide; AMC, 7-amino-4-methyl-coumarin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HLB, hypotonic lysis buffer; PAGE, polyacrylamide gel electrophoresis; HEK, human embryonic kidney; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; FPP, farnesyl pyrophosphate.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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B signaling.
J Biol Chem
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