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Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, 28034 Madrid; and Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá de Henares, Spain
1Correspondence: Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, Ctra. de Colmenar, km 9, 28034 Madrid, Spain. E-mail: miguel.a.lasuncion{at}hrs.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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demethylase.
Through HPLC analyses with on-line radioactivity detection, we found
that SKF 104976 efficiently blocked the [14C]-acetate
incorporation into cholesterol, resulting in an accumulation of
lanosterol and dihydrolanosterol, without affecting the synthesis of
mevalonic acid. The inhibitor also produced a rapid and intense
inhibition of cell proliferation (IC50 = 0.1 µM), as
assessed by both [3H]-thymidine incorporation into DNA
and cell counting. Flow cytometry and morphological examination showed
that treatment with SKF 104976 for 48 h or longer resulted in the
accumulation of cells specifically at G2 phase, whereas both the G1
traversal and the transition through S were unaffected. The G2 arrest
was accompanied by an increase in the hyperphosphorylated form of
p34cdc2 and a reduction of its activity, as determined by
assaying the H1 histone phosphorylating activity of p34cdc2
immunoprecipitates. The persistent deficiency of cholesterol induced
apoptosis. However, supplementing the medium with cholesterol, either
in the form of LDL or free cholesterol dissolved in ethanol, completely
abolished these effects, whereas mevalonate was ineffective. Caffeine,
which abrogates the G2 checkpoint by preventing p34cdc2
phosphorylation, reduced the accumulation in G2 when added to cultures
containing cells on transit to G2, but was ineffective in cells
arrested at G2 by sustained cholesterol starvation. Cells arrested in
G2, however, were still viable and responded to cholesterol provision
by activating p34cdc2 and resuming the cell cycle. We
conclude that in both lymphoblastoid and promyelocytic cells,
cholesterol availability governs the G2 traversal, probably by
affecting p34cdc2 activity.—Martínez-Botas, J.,
Suárez, Y., Ferruelo, A. J., Gómez-Coronado, D.,
Lasunción, M. A. Cholesterol starvation decreases
P34cdc2 kinase activity and arrests the cell cycle at G2.
Key Words: SKF 104976 • cholesterol synthesis • cell proliferation • low density lipoprotein • caffeine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and several other enzymes
participating in cholesterol synthesis—HMG-CoA synthase (2)
, farnesyl
diphosphate synthase (3
4)
, squalene synthase (5)
—as well as the LDL
receptor are down-regulated at the transcriptional level by the
intracellular cholesterol levels (6
, 7)
through a mechanism that
involves the transcription factors SREBP (SRE binding proteins) and the
participation of SCAP, a protein that senses the changes of cholesterol
levels in the endoplasmic reticulum (8)
.
As originally pointed out by Chen et al. (9)
and Brown and
Goldstein (10)
, cholesterol synthesis is tightly related to cell
proliferation. Phytohemagglutinin-stimulated mouse lymphocytes undergo
a sixfold increase in cholesterol synthesis just before the cells enter
the S phase of the cell cycle (11)
. In synchronized BHK-21 cells, it
was also observed that cholesterol synthesis reached a maximum during
the G1 phase, concurrent with the activation of HMG-CoA reductase (12)
.
In general, thus, proliferating cells display both a high rate of
cholesterol synthesis and an elevated LDL receptor activity (13)
, which
warrant the provision of this lipid. On the other hand, inhibition of
HMG-CoA reductase with vastatins, which depletes the cells of both
cholesterol and other mevalonate derivatives, results in a dramatic
reduction of DNA synthesis, inducing the arrest of the cell cycle at G1
(14
15
16)
. This block of the cell cycle is specific because
supplementing the medium with mevalonic acid can readily reverse it
(14
, 17
). Recent studies indicate that in addition to cholesterol,
several other mevalonate derivatives are necessary for DNA synthesis,
such as the phosphorylated forms of isopentane (18)
, farnesol (19
, 20
),
geranylgeraniol (21
, 22
), and dolichol (23
, 24
).
Aside from vastatins, other cholesterol synthesis inhibitors also block
cell proliferation. Thus, ketoconazole, which is an inhibitor of
P450 enzymes and lanosterol conversion into
cholesterol, has been shown to produce an arrest of a minor portion of
cells in G2 (25)
. Fluoromevalonate [an inhibitor of
pyrophosphatemevalonate decarboxylase (26
, 27
)], azole derivatives,
and oxysterols (14
, 28
) also reduce DNA synthesis but their action on
the cell cycle has not been elucidated.
Eukaryotic cell cycle is governed by cyclin-dependent kinases (cdk)
coupled to specific cyclins. The protein–kinase complex
p34cdc2-cyclin B controls the traversal of both
the G2 and the M phases (29
, 30
). Expression levels of cyclin B change
along the cell cycle, increasing during S and G2 phases and abruptly
decreasing at the metaphase/anaphase transition (31)
. Moreover, the
activity of p34cdc2 is regulated by
phosphorylation/dephosphorylation reactions mediated by different
kinases (wee1, mik1, myt1) and phosphatase cdc25, respectively (32)
.
Finally, entry into mitosis is caused by degradation of cyclin B and
p34cdc2 inactivation (33)
.
It has been reported recently that lovastatin increases the levels of
cyclin-dependent kinase inhibitors p21 and p27, which results in a
decrease of cdk2 kinase activity (34
35
36)
. This finding agrees with the
G1 block this drug produces (15
, 16
). However, the potential role of
cholesterol in the cell cycle could not be established from these
studies since vastatins compromise the provision of both cholesterol
and nonsterol mevalonate derivatives.
Here we document the effect of cholesterol starvation and readdition on the cell cycle progression and the activity of p34cdc2 in both MOLT-4 and HL-60 cells. The results indicate that cholesterol is essential for the transition from G2 to mitosis and that cholesterol availability affects p34cdc2 activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Cell culture
MOLT-4 and HL-60 cells were maintained in RPMI 1640 supplemented
with 10% fetal calf serum, 100 U penicillin/ml, 100 µg
streptomycin/ml, and 10 µg gentamicin/ml at 37°C in a humidified
atmosphere of 5% CO2. Before each experiment,
the cells were washed three times with RPMI 1640 to remove fetal calf
serum and grown in RPMI 1640 supplemented with 10%
lipoprotein-deficient serum (LPDS) (final cholesterol concentration in
medium <0.2 µg/ml). The cell viability was greater than 90–95% in
all experiments, as determined by the trypan blue exclusion test.
Metabolic labeling and high-performance liquid chromatography
(HPLC) analysis of isoprene lipids
MOLT-4 (15 x 106 cells) were
preincubated in 2 ml RPMI 1640, 10% LPDS with or without SKF 104976
for 2 h and supplemented with 40 µCi of
[2-14C]-acetate (53 mCi/mmol) (Amersham) for a
further 8 h. The cells were agitated on a rocking platform at
37°C in a 5% CO2 atmosphere. At the end of the
incubation, the cells were washed with ice-cold phosphate-buffered
saline (PBS) and resuspended in 0.5 ml of 10% KOH. The samples were
sequentially treated with chloroform:methanol (2:1) and distilled water
to obtain the lipidic and aqueous-soluble fractions (38)
. The lipid
extract was further subfractionated into the saponifiable and the
nonsaponifiable fractions, as reported (39)
. The nonsaponifiable lipids
were analyzed by reverse phase HPLC with a Nova
Pack-C18 column (150 x 3.9 mm, 4 µM,
Waters Chromatography Division, Millipore Corp.). Lipids were eluted
with methanol-acetonitrile-water (65:25:10) during the first 9 min and
then with methanol-acetonitrile (10:90) at a flow rate of 1.2 ml/min.
The column effluent was monitored simultaneously by UV (Beckman 168
variable wavelength detector; Beckman Instruments, Palo Alto, Calif.)
and on-line radioactivity detection (LB-506 C-1, Berthold, Bad
Widbad, Germany). The aqueous-soluble fractions were analyzed by
HPLC by using an ion exclusion column Aminex HPX-87H (300 mm x
7.8 mm, Bio-Rad Lab, Richmond, Calif.) with a cation-H refill cartridge
(30 mm x 4.6 mm). The organic acids were eluted with 5 mM
sulfuric acid at a flow rate of 0.6 ml/min at 42°C and radioactivity
was monitored on-line, as before.
Measurement of cell proliferation
Incubations were carried out in sterile 96-well plates
(Multiscreen-HV), each well containing 36 x
103 cells in a final volume of 225 µl. The
culture medium (RPMI 1640 with 10% LPDS) was supplemented with either
the cholesterol synthesis inhibitor SKF 104976 dissolved in dimethyl
sulfoxide (final concentration in the wells, 0.05%), LDL, mevalonate,
or free cholesterol dissolved in ethanol (final concentration, 0.05%),
as indicated. DNA synthesis was assessed by measuring the incorporation
of [3H-methyl]-thymidine into DNA as reported
elsewhere (40)
. Cell number and viability were determined by trypan
blue dye exclusion using a hemocytometer.
Cell cycle analysis
Cells sampled for flow cytometry were harvested by
centrifugation and washed three times with PBS. Cells were then fixed
in 70% cold ethanol for 30 min at -20°C, washed three times with
PBS, and incubated for 1 h at 37°C in PBS containing 100 µg/ml
ribonuclease A and 50 µg/ml propidium iodide. Cells were analyzed
with a FACScan flow cytometer (Becton-Dickinson, San Jose, Calif.)
using a 488 nm argon ion laser. Data were acquired using selective
gating excluding doublet cells and analyzed with Cell FIT software
(Becton-Dickinson). The fraction of cells in each cell cycle
compartment was calculated according to the SOBR analysis model.
To ascertain whether cells in S phase were actively synthesizing DNA, 2–4 x 106 cells were incubated with 100 µM bromodeoxyuridine (BrdU) for 1 h in the CO2 incubator at 37°C, fixed in 70% cold ethanol at -20°C, and washed three times with PBS. After a 20 min incubation at room temperature with 2 N HCl, cells were washed twice with PBS and incubated for 15 min at room temperature in PBS, 0.5% Tween 20, 1% normal goat serum (Vector Laboratories, Inc., Burlingame, Calif.). Subsequently, cells were centrifuged, resuspended in 0.5% Tween 20, 1% normal goat serum containing 20 µl of Anti-BrdU FITC (Becton Dickinson) for 1 h at room temperature, stained with propidium iodide, and analyzed by flow cytometry.
Analysis of apoptosis
For morphological analysis, cells were stained with Hoechst
33258 and examined with a fluorescence microscope (Olympus IX70,
Hamburg, Germany) as reported (40)
. Apoptosis was also assessed by a
TUNEL assay with the Apoptosis Detection System kit (Promega
Corporation, Madison, Wis.), following the manufacturer's
instructions, and analyzed by flow cytometry.
Gel electrophoresis and immunoblots
Cells extracts were obtained by using lysis buffer containing 50
mM Tris-HCl pH 8, 120 mM NaCl, 0.5% Nonidet P40, 100 mM NaF, 40
µg/ml PMSF, 40 µg/ml aprotinin, and 40 µg/ml leupeptin. Equal
amounts of protein (41)
were run in each lane of sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (12%
acrylamide) and transferred to nitrocellulose paper. The
p34cdc2 protein was detected by using an
anti-cdc2 polyclonal antibody (Ab-1, Calbiochem, Cambridge, Mass.) and
then probed with horseradish peroxidase-conjugated anti-rabbit
immunoglobulin G. Immunoreactive bands were detected after addition of
ECL reagent (ECL kit; Amersham), followed by exposure to photographic
film.
Assessment of p34cdc2 kinase activity
p34cdc2 kinase activity was assayed as
described by Poon et al. (34)
. Briefly, equal protein amounts
of cell extracts were resuspended in immunoprecipitation buffer (50 mM
Tris-HCl pH 8, 120 mM NaCl, 30 mM NaF, 30 mM pNPP, 1% Nonidet P40, 1%
Triton X-100, 1 mM benzamide, 0.1 mM PMSF) containing 1 µg of
anti-cdc2 polyclonal antibody Ab-1. The samples were precipitated with
25 µl of protein A-Sepharose. The immunoprecipitates were resuspended
in 25 µl of kinase buffer (80 mM Na-ß-glycerophosphate, 20 mM EGTA,
15 mM Mg(OAc)2, 1 mM DTT, 32 µM ATP, 6.4
µCi[
-32P] ATP, and 5 µg histone H1) and
incubated for 30 min at 30°C. The reaction was terminated by the
addition of 12.5 µl of SDS sample buffer. The samples were subjected
to 12% SDS-PAGE gel electrophoresis, transference to nitrocellulose
membrane, and autoradiography. Phosphorylation was quantified by
scanning the film and image analysis (Diversity One Software Package,
PDI, New York).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, in the control condition most of the radioactivity appearing in
nonsaponifiable lipids corresponded to cholesterol. By contrast, in
cells treated with SKF 104976, the predominant
14C-labeled sterols were lanosterol and
dihydrolanosterol, with practically undetectable radioactivity in
cholesterol. Since it was reported that secondary to the inhibition of
lanosterol 14- demethylase, SKF 104976 also reduced HMG-CoA
reductase (42)
, we measured the incorporation of
[14C]-acetate into organic acids (Fig. 2
) and found that [14C]-mevalonate levels in
cells treated with SKF 104976 were similar to those in the control
(49,460±4938 and 52,352±3045 dpm/well, respectively; n=5,
not significant).
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Effect of SKF 104976 on MOLT-4 and HL-60 cell proliferation
To study cell proliferation, the incorporation of
[3H]-thymidine into DNA was first determined.
SKF 104976 inhibited the proliferation of MOLT-4 and HL-60 cell lines
in a dose-dependent manner when incubated in a cholesterol-free medium
with an IC50 of ~0.1 µM (Fig. 3
). Time course studies revealed that DNA synthesis was significantly
reduced at 24 h of treatment and practically undetectable at
48 h or longer (Fig. 4
).
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To determine whether this effect on cell proliferation was specific to
the inhibition of lanosterol 14- demethylase simultaneous with the
addition of the inhibitor, the cultures were supplemented with LDL,
free cholesterol, and mevalonate in different combinations. As shown in
Fig. 5
for HL-60 cells, both LDL and free cholesterol restored cell
proliferation in a dose-dependent manner, LDL being more efficient than
free cholesterol. Similar results were observed with MOLT-4 cells (data
not shown). In contrast, neither mevalonate, all-trans
farnesol, nor all-trans geranylgeraniol prevented the
inhibitory effect of SKF 104976 or affected the reversal due to LDL or
free cholesterol (data not shown). Viable cells were counted at
different incubation times (Fig. 6
). At 24 h, cell counts in cultures containing SKF 104976 did not
differ from the controls. Whereas the controls displayed an ascending
pattern, in the presence of the drug the number of viable cells
declined to practically zero at 72 h (Fig. 6)
. LDL or free
cholesterol supplementation abolished the effect of SKF 104976 on cell
growth, whereas mevalonate was ineffective. On the other hand,
mevalonate did not produce any benefit over cholesterol supplementation
(Fig. 6)
. Altogether, these results demonstrate that the inhibition of
cell growth by SKF 104976 was attributable to the deficiency in
cholesterol but not in mevalonic acid and/or its nonsterol derivatives.
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Effects of SKF 104976 on cell cycle and rescue by LDL or free
cholesterol
The effect of SKF 104976 on cell cycle distribution and its
prevention by LDL, free cholesterol, and mevalonate was then studied
with flow cytometry after propidium iodide DNA staining. As observed in
Fig. 7
, SKF 104976 treatment resulted in a progressive increase in the
proportion of cells in G2/M phase and a decrease in G1. The
simultaneous addition of either LDL or free cholesterol prevented the
accumulation of cells in G2 and allowed the cell cycle to progress
normally despite the continuous presence of the inhibitor (Fig. 7)
.
Mevalonate supplementation did not alter the effect of SKF 104976 on
cell cycle nor its reversion by LDL or free cholesterol (Fig. 7)
.
Similar results were found with MOLT-4 cells (data not shown). In the
absence of any exogenous source of cholesterol, prolonging incubation
with the inhibitor for 72 h or longer resulted in generalized cell
death, as indicated by the prominent Sub G1 (Fig. 7)
. To confirm that
cell death corresponded to apoptosis, a TUNEL assay coupled to flow
cytometry was performed. As observed in Fig. 8
, cells treated with the drug for 72 h had much more fluorescence
than controls, which demonstrates apoptosis-induced DNA fragmentation,
whereas supplementing the medium with LDL totally prevented this
effect. In keeping with this, many condensed and fragmented nuclei were
visualized by fluorescence microscopy in cultures exposed to SKF 104976
after staining with Hoechst 33258 (data not shown). On the other hand,
this technique also revealed that the intact, not fragmented nuclei
remaining after 48 h of treatment with SKF 104976 were larger than
the average in the control condition and showed the complete absence of
mitotic figures (separating chromosomes) (data not shown). The latter
results, in combination with those derived from flow cytometry,
indicated that SKF 104976 arrested the cell cycle at G2 rather than at
M.
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To determine whether the G2 arrest could be released by
cholesterol, HL-60 cells were preincubated with SKF 104976 for 48 h and then the medium was supplemented with placebo, LDL, or free
cholesterol. As shown in Fig. 9
, LDL produced a rapid increase of the number of cells in G1 and
complete restoration of the cell cycle distribution 24 h after its
addition. Free cholesterol also allowed the traversal of G2/M phase but
less efficiently than LDL, since the cell cycle required almost 48 h to be entirely reestablished (Fig. 9)
. In addition, the number of
cells increased after supplementing the medium with cholesterol, which
confirmed release of the cell cycle arrest (data not shown). In
contrast, supplementing the medium with mevalonate either alone or in
combination with LDL or free cholesterol exerted no effect on the
release of the G2 block (data not shown). Altogether, these results
demonstrate that cholesterol enables the cells to surpass the G2 block
induced by SKF 104976, allowing the cell cycle to be resumed.
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Changes in p34cdc2 activity associated to the G2 arrest
induced by SKF 104976
In eukaryotic cells, the G2 to M transition is known to be
controlled by the protein kinase complex
p34cdc2-cyclin B. To determine whether the SKF
104976-induced G2 arrest was associated with changes in this kinase
activity, we analyzed p34cdc2 protein levels by
Western blotting and H1 kinase activity in
p34cdc2 immunoprecipitates. As depicted in
Fig. 10
, p34cdc2 protein levels increased during
treatment in parallel to the increase in the proportion of cells in G2
phase (Fig. 8)
, which is in accordance with previous results by others
using other G2-arrest inductors (43)
. Notably, the relative proportion
of the slow-migrating band, which corresponds to the
hyperphosphorylated, inactive form of the enzyme (44
45)
, increased
with time. Consistent with this, p34cdc2 kinase
activity showed a biphasic pattern, with a transient increase at
36 h of treatment and a marked decrease thereafter (Fig. 10)
. At
60 h of treatment, despite the fact that 72% of live cells were
in G2 phase in this experiment, the p34cdc2
kinase activity was even lower than in the control (Fig. 10)
. Thus,
apparently, the G2 arrest induced by SKF 104976 was associated with
diminished p34cdc2 kinase activity.
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We next studied whether cholesterol provision could restore
p34cdc2 activity in previously arrested cells. We
found that p34cdc2 activity increased upon
supplementing the medium with either cholesterol source (Fig. 11
). This recovery was faster with LDL (Fig. 11A
) than with
free cholesterol (Fig. 11B
), which is in keeping with their
effects in releasing the G2 arrest as determined by flow cytometry
(Fig. 10)
.
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Caffeine release of the SKF 104976-induced G2 arrest
To further analyze the level at which cholesterol availability was
acting in the regulation of the cell cycle, we took advantage of the
well-known effect of caffeine in promoting the unscheduled entry into
mitosis by preventing the phosphorylation-mediated
p34cdc2 inactivation (46
, 47
). HL-60 cells were
incubated in the presence of SKF 104976 for 36 h, a time when the
S phase was very active as determined by BrdUr incorporation (data not
shown), and 60 h, when most of the cells were arrested at G2 (Fig. 9)
; then the medium was supplemented with 5 mM caffeine or placebo. As
shown in Fig. 12
, caffeine reduced the proportion of cells in G2 and raised the
proportion in G1. This effect of caffeine was preceded by a rapid and
transient stimulation of p34cdc2 activity
(Fig. 13
), which is in accordance with previous results by others in other
systems (43
, 48
, 49
). The effect of caffeine on cell cycle progression,
however, depended on the cell cycle distribution at the time of
application. For example, in a culture treated with SKF 104976 for
60 h (when the cell cycle distribution was 20.3% in G1, 17.0% in
S, and 62.7% in G2), the addition of caffeine did not produce any
significant change after 12 h (21.0% in G1, 18.1% in S, and
60.9% in G2 for 12 h). Thus, the different effects probably
relied on the proportion of cells in S phase in transit to G2, caffeine
being ineffective on cells previously arrested at G2 by cholesterol
deprivation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To explore the role of cholesterol on cell cycle, MOLT-4 and HL-60 cell
lines were cultured in a cholesterol-deficient medium and
simultaneously exposed to SKF 104976, which is a specific inhibitor of
lanosterol 14- demethylase (42)
. We demonstrated that SKF 104976
efficiently blocks the incorporation of
[14C]-acetate into cholesterol, resulting in
the accumulation of both lanosterol and dihydrolanosterol, without
affecting significantly the synthesis of mevalonic acid.
The strong inhibitory effect of SKF 104976 on cell growth, which is
described here for the first time, is in keeping with previous
observations indicating that cells deficient in lanosterol 14-
demethylase require cholesterol for normal cell growth (50)
. The effect
of SKF 104976 was fully restored by either LDL or free cholesterol,
whereas mevalonate was ineffective. These results demonstrate that SKF
104976-treated cells were not deprived of the nonsterol mevalonate
derivatives essential for cell proliferation (17
18
19
20
21
22
23
24)
, but just of
cholesterol.
Flow cytometry and morphological examination clearly showed that SKF
104976 treatment arrested the cells specifically at the G2 phase,
whereas both G1 traversal and the transition through S were unaffected.
A similar block in G2/M was observed in lanosterol 14-
demethylase-deficient cells when incubated in a cholesterol-poor medium
(51)
. It has been reported that other cholesterol synthesis inhibitors
also disturb cell cycle progression. Ketoconazole, which is an
inhibitor of cytochrome P450-dependent enzymes,
including lanosterol 14- demethylase, blocks the cell cycle
primarily at G1, although a small accumulation of cells in G2 is also
observed when used at high concentration (25)
. Both vastatins and
dehydroepiandrosterone, which deplete endogenous mevalonate pools, have
been shown to arrest the cell cycle predominantly at G1, but they also
delayed the cells in G2/M transitorily (15
, 52
, 53
). These observations
and our results suggest that the accumulation of cells in G2/M is due
to the cholesterol deficiency these drugs produce. On other hand, the
block in G1 is probably the result of either reduced protein
prenylation (53
, 54
) or depletion of certain nonsterol isoprene
derivatives (i.e., dolichol, isopentenyladenine) (18
, 23
, 24
).
One of the main findings of this study is the requirement of cholesterol for G2 traversal. In fact, cells arrested at G2 entered into mitosis and the cell cycle resumed on provision of cholesterol, either in the form of LDL or free cholesterol dissolved in ethanol, despite the continuous presence of the inhibitor. This reinforces the concept that cholesterol is essential for G2 traversal. Given that SKF 104976-treated cells are temporarily viable and the cell cycle can easily be resumed by cholesterol addition, this model offers a valuable tool for studying the mechanisms governing the traversal of G2 phase.
The G2 arrest that occurs in response to DNA damage or other insults is
imposed by transient inactivation of p34cdc2
kinase, the universal determinant of entry into mitosis (29
30
31
32)
. The
observation that p34cdc2 in cells arrested at G2
by prolonged cholesterol starvation is predominantly in its
hyperphosphorylated form and inactive (Fig. 10)
, and that cholesterol
provision to these cells increases p34cdc2
activity (Fig. 11)
and releases the block (Fig. 9)
, suggests that the
cell cycle arrest was mediated by the reduced
p34cdc2 activity. We found that caffeine
activated p34cdc2 and partially reduced the
accumulation of cells in G2 provided it was added when the culture
contained a substantial number of cells in S phase, on transit to G2.
By contrast, cells arrested at G2, not responding to caffeine, still
responded to cholesterol by p34cdc2 activation
and proliferation. Therefore, the results suggest that cholesterol acts
downstream of caffeine on the regulation of the cell cycle. This
hypothesis and the extrapolation of present results to other cell types
deserve further verification.
The finding that cholesterol is required specifically for the traversal
of the G2 phase extends previous studies that demonstrated the
necessity of this sterol for cell growth (9
, 10
). Whether
p34cdc2 activity responds to other sterols as it
does to cholesterol was not directly addressed here. However, the fact
that cells treated with SKF 104976 accumulated lanosterol and
dihydrolanosterol suggests that the regulatory response herein
described is stringent for cholesterol. Previous studies in
Mycoplasma capricolum (55
, 56
) and Saccharomyces
cerevisiae (57
, 58
) have led to the notion that sterols serve a
dual role in cells: one structural, ensuring the integrity of
membranes; another of regulatory nature (59)
. The structural role is
accomplished by the amount of the cellular sterol. Although the
preferred sterol is the one synthesized by each organism (i.e.,
cholesterol in mammals, ergosterol in yeast, sitosterol and others in
plants), this role has a relatively broad specificity; in fact, other
exogenously supplied sterols can be used for membrane formation
provided there is some compensatory change in membrane fatty acids
(60
61
62)
. In contrast, the regulatory role is restricted to the sterol
that is characteristic for the organism (55
, 56
). These two actions
explain the synergistic effects of different sterols on cell growth as
observed in several species (57
, 58
). It was reported that extremely
low concentrations of ergosterol (< 1 nM) in the budding yeast
activated a pp60v-src-related protein kinase involved in the cell cycle
control division (59)
. This finding allowed the suggestion that this
kinase activity might be part of the signal transduction process that
senses the availability of sterol needed for cell growth and commits
the cell to a new mitotic division (59)
.
Similar to that reported for ergosterol in yeast, we observed that
cholesterol provision in cholesterol-deprived mammalian cells leads to
the rapid stimulation of p34cdc2, a cell
cycle-related protein kinase. How the cell senses cholesterol
availability and how this signal is transduced are still unknown.
Cholesterol is an important regulator of the expression of several
lipid metabolism-related genes, which contain the so-called sterol
regulatory elements (SRE) in their promoter regions (8)
. When the
intracellular cholesterol levels decrease, the endoplasmic
reticulum-bound SCAP activates the proteolytic processing of SRE
binding proteins to form transcription factors (SREBP-1a, -1c, -2),
which migrate to the nucleus and activate gene transcription by binding
to the SRE (8)
. Whether an analogous mechanism is involved in the
modulation of p34cdc2 activity by cholesterol
deprivation remains to be established.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication April 9, 1998.
Revision received February 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 J. W. Allen, D. C. Wolf, M. H. George, S. D. Hester, G. Sun, S.-F. Thai, D. A. Delker, T. Moore, C. Jones, G. Nelson, et al. Toxicity Profiles in Mice Treated with Hepatotumorigenic and Non-Hepatotumorigenic Triazole Conazole Fungicides: Propiconazole, Triadimefon, and Myclobutanil Toxicol Pathol, December 1, 2006; 34(7): 853 - 862. [Abstract] [Full Text] [PDF]
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 C. Fernandez, M. Martin, D. Gomez-Coronado, and M. A. Lasuncion Effects of distal cholesterol biosynthesis inhibitors on cell proliferation and cell cycle progression J. Lipid Res., May 1, 2005; 46(5): 920 - 929. [Abstract] [Full Text] [PDF]
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 Y. Hase, S. Fujioka, S. Yoshida, G. Sun, M. Umeda, and A. Tanaka Ectopic endoreduplication caused by sterol alteration results in serrated petals in Arabidopsis J. Exp. Bot., April 1, 2005; 56(414): 1263 - 1268. [Abstract] [Full Text] [PDF]
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 Y. Suarez, C. Fernandez, D. Gomez-Coronado, A. J. Ferruelo, A. Davalos, J. Martinez-Botas, and M. A. Lasuncion Synergistic upregulation of low-density lipoprotein receptor activity by tamoxifen and lovastatin Cardiovasc Res, November 1, 2004; 64(2): 346 - 355. [Abstract] [Full Text] [PDF]
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 M. T. Montero, J. Matilla, E. Gomez-Mampaso, and M. A. Lasuncion Geranylgeraniol Regulates Negatively Caspase-1 Autoprocessing: Implication in the Th1 Response against Mycobacterium tuberculosis J. Immunol., October 15, 2004; 173(8): 4936 - 4944. [Abstract] [Full Text] [PDF]
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M. E. Higgins and Y. A. Ioannou Apoptosis-induced release of mature sterol regulatory element-binding proteins activates sterol-responsive genes J. Lipid Res., December 1, 2001; 42(12): 1939 - 1946. [Abstract] [Full Text] [PDF]
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) and HL-60
(
) cells were incubated in RPMI 1640, 10% LPDS in the presence of
increasing concentrations of SKF 104976 for 72 h and then received
an 18 h pulse of [3H]-thymidine. Cells were
harvested and radioactivity incorporated into DNA was counted. Results
are expressed as % of the control (cells incubated in the absence of
the inhibitor): MOLT-4, 170378 ± 6034 dpm/well; HL-60,
152918 ± 885 dpm/well. Data correspond to the means ±
SE of four replicates from a representative experiment.
) or
presence of 1.5 µM SKF 104976 (
, 
) or presence of 1.5
µM SKF 104976 (
), 30 µg/ml LDL plus 1 mM
mevalonate (•), or 30 µg/ml free cholesterol plus 1 mM mevalonate
(
