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INTRODUCTION |
Differentiation-inducing factors
(DIFs)1 were identified in
Dictyostelium discoideum as the morphogens required for
stalk cell differentiation of Dictyostelium (1). In the DIF
family, DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) was the
first to be identified, and DIF-3, the monochlorinated analogue of
DIF-1, is a natural metabolite of DIF-1 in Dictyostelium
(2). However, the actions of DIFs are not limited to
Dictyostelium. They also have strong effects on mammalian
cells. DIF-1 and/or DIF-3 strongly inhibit proliferation and induce
differentiation in several leukemia cells, such as the murine
erythroleukemia cell line B8, human leukemia cell line K562, and human
myeloid leukemia cell line HL-60 (3, 4). DIF-3 has been reported to
have the most potent antiproliferative effect on mammalian leukemia
cells among DIF analogues examined to date (5). Recently, we found that
DIF-1 strongly inhibits proliferation and induces differentiation in
human vascular smooth muscle cells, indicating that cells sensitive to
DIFs are not limited to transformed cells (6).
However, the target molecule (receptor) of DIFs is unknown, and it is
not clear even in Dictyostelium how DIFs induce an
antiproliferative effect and cell differentiation. DIFs are small
hydrophobic molecules and are therefore expected to be able to cross
cell membranes without requiring channels or carriers. Also, the
rapidity with which DIFs induce prestalk cell-specific gene expression
suggests that they directly regulate gene expression. Therefore, the
target molecule(s) for DIFs may be located in cytoplasm or nucleus (7). Although the precise mechanisms underlying their antiproliferative and
differentiation-inducing effects are not yet known, we found that DIF-1
induces cell cycle arrest at G0/G1 phase by
suppressing the expression of cyclin D1 (6). Cyclin D1 is synthesized
early in G1 phase and plays a key role in the initiation
and progression of this phase. When cells enter the S phase, cyclin D1
is rapidly degraded by ubiquitin-proteasome-dependent
proteolysis (8).
Therefore, in the present study, we investigated the mechanism
underlying the DIF-induced inhibition of cyclin D1 expression. We
particularly paid attention to the possible involvement of glycogen
synthase kinase-3
(GSK-3
), because this serine/threonine protein
kinase has been shown to regulate not only cyclin D1 gene transcription
by phosphorylating
-catenin but also cyclin D1 proteolysis by
directly phosphorylating cyclin D1 itself (9-12). Here we show for the
first time that DIF-3, which is more effective than DIF-1 in inhibiting
cell proliferation, induces the rapid degradation of cyclin D1 and the
activation of GSK-3
.
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MATERIALS AND METHODS |
Chemicals--
DIF-1
(1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) was
purchased from Affiniti Research Products. DIF-3 (1-(3-chloro-2, 6-dihydroxy-4-methoxyphenyl)-1-hexanone) was synthesized by Toyama Chemical Co. (Tokyo, Japan). U0126 was purchased from Cell Signaling Technology. N-acetyl-Leu-Leu-norleucinal and
wortmannin were purchased from Sigma.
Cell Culture and Transfection--
HeLa cells and bovine aortic
endothelial cells (BAECs) were grown in Dulbecco's modified Eagle's
medium (Sigma) supplemented with 10% fetal bovine serum, 100 units/ml
penicillin G, and 0.1 µg/ml streptomycin. The cells were plated on
plastic tissue culture dishes or coverslips. Human umbilical vein
endothelial cells were plated on 0.1% gelatin-coated dishes and
maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented
with 20% fetal bovine serum, 5 ng/ml bovine fibroblast growth factor,
100 units/ml penicillin G, and 0.1 µg/ml streptomycin. Wild-type
human cyclin D1 cDNA was provided by Dr. K. Tamai (Medical and
Biological Laboratories Co., Nagano, Japan) and subcloned into
pcDNA3 (Invitrogen). Transfection was carried out using TransIT-LT1
(Mirus), and transfected cells were maintained in growth medium for
16 h before stimulation.
Cell Proliferation Assay--
The cells were plated on 24-well
plates (0.5 × 104 cells/well) and treated with or
without various amounts of DIF-1 or DIF-3 for given periods. Cells were
harvested by the trypsin/EDTA treatment and enumerated.
Flow Cytometry--
Cells harvested by the trypsin/EDTA
treatment were suspended in hypotonic fluorochrome solution containing
50 µg/ml of propidium iodide, 0.1% sodium citrate, and 0.1% Triton
X-100 (13). Cells (5 × 103) for each sample were
analyzed for fluorescence by a Becton-Dickinson FACScalibur (Franklin
Lakes, NJ).
mRNA Expression Analysis--
Total cellular RNA was
extracted with Isogen (Nippon Gene). Using 1 µg of the RNA, the
expression of cyclin D1 mRNA was analyzed by reverse
transcription-polymerase chain reaction (RT-PCR) using Ready-To-Go
RT-PCR Beads (Amersham Biosciences) (6).
Purification of Nucleic and Cytoplasmic Proteins--
Nucleic
and cytoplasmic proteins were purified from cells cultured in 100-mm
plates using NE-PERTM nuclear and cytoplasmic extraction
reagents (Pierce). Five µg of each sample was subjected to Western
blot analysis.
Immunoblotting--
Samples were separated by 12% SDS-PAGE and
transferred to a polyvinylidene difluoride membrane using a semidry
transfer system (1 h, 15 V). After blocking with 5% skim milk or 5%
bovine serum albumin for 1 h, the membrane was probed with a first
antibody. For the polyclonal anti-cyclin D1 antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), the polyclonal anti-cyclin D2
antibody (Santa Cruz Biotechnology), the polyclonal
anti-phospho-GSK-3
(Ser9) antibody (Cell Signaling
Technology), the polyclonal anti-phospho-Akt (Ser473)
antibody (Cell Signaling Technology), and the polyclonal
anti-phospho-p90RSK (Ser380) antibody (Cell
Signaling Technology), the incubation was carried out overnight at
4 °C. For the monoclonal anti-phosphotyrosine antibody (PY-20; Santa
Cruz Biotechnology), the monoclonal anti-GSK-3
antibody (BD
Transduction Laboratories), and the monoclonal anti-cyclin D3 antibody
(Santa Cruz Biotechnology), the incubation was carried out for 1 h
at room temperature. The membrane was washed three times and incubated
with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse
IgG (Bio-Rad) for 1 h. Immunoreactive proteins on the membrane
were visualized by treatment with a detection reagent (LumiGLO, Cell
Signaling Technology). An optical densitometric scan was performed
using Science Lab 99 Image Gauge Software (Fuji Photo Film).
Immunoprecipitation--
HeLa cells (1 × 106
cells) were incubated with or without DIF-3 for the periods indicated.
Cells were lysed on ice for 1 h in 1 ml of the lysis buffer (50 mM NaCl, 5 mM NaF, 2 mM
Na3VO4, 10 mM Tris/HCl, pH 7.4, 1 mM EDTA, 1% (v/v) Triton X-100, and 2 mM
phenylmethylsulfonyl fluoride), and insoluble cell debris was removed
by centrifugation at 5,000 rpm for 3 min. The cell lysate was
precleared with protein A-Sepharose CL-4B (Amersham Biosciences) and
then incubated with an anti-GSK-3
antibody (1 µg) and protein
A-Sepharose CL-4B at 4 °C for 3 h. After incubation, proteins
bound to the antibody/protein A-Sepharose complex were precipitated by
centrifugation at 15,000 rpm for 5 min and washed three times with the
lysis buffer.
GSK-3
Activity Assay--
For the in vitro kinase
assay, immunoprecipitated samples were washed twice with lysis buffer
and twice with a kinase assay buffer (20 mM Tris/HCl, pH
7.4, 5 mM MgCl2, and 1 mM
dithiothreitol). Kinase activity was measured by mixing
immunoprecipitated GSK-3
with 50 µl of kinase assay buffer
containing 20 mM Tris/HCl (pH 7.4), 5 mM
MgCl2, 1 mM dithiothreitol, 250 µM ATP, 5 µCi of [
-32P]ATP (Amersham
Biosciences), and 10 µM GSK-3
substrate peptide (Upstate Biotechnology, Inc., Lake Placid, NY). The samples were incubated at 30 °C for 30 min, and the reaction was terminated by
adding 10 µl of 50% trichloroacetic acid. Samples were then centrifuged at 15,000 rpm for 5 min, and 40 µl of supernatant was
spotted on P81 phosphocellulose filter paper (Whatman). The filters
were washed five times with 0.75% phosphoric acid and twice with
acetone and analyzed by scintillation counting.
Fluorescence Microscopy--
Cells plated on
CELL-TAKR (Collaborative Biomedical Products)-treated
coverslips were incubated with or without DIF-3 (30 µM) for given periods and washed with phosphate-buffered saline. The cells were fixed and permeabilized in ice-cold methanol/acetone (1:1)
for 15 min at
20 °C and then washed twice with phosphate-buffered saline. After blocking with 2% bovine serum albumin in
phosphate-buffered saline for 30 min, the coverslips were incubated
with polyclonal or monoclonal anti-GSK-3
antibody (1:200) overnight
at 4 °C. The coverslips were washed twice with phosphate-buffered
saline and incubated with anti-mouse IgG or anti-rabbit IgG + IgA + IgM-biotin (Nichirei) for 1 h at room temperature followed by
streptavidin-fluorescein isothiocyanate conjugate (1:50 dilution;
Invitrogen) for 1 h at room temperature. The cells were
examined under a fluorescence microscope (Olympus).
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RESULTS |
DIFs Inhibited HeLa Cell Proliferation--
DIFs exhibit powerful
antiproliferative effect in leukemia cells (3-5). In the present
study, we first examined whether DIFs also inhibit the proliferation of
HeLa cells. As shown in Fig. 1,
A and B, DIF-1 and DIF-3 both strongly inhibited
HeLa cell proliferation in a dose-dependent fashion,
suggesting that DIFs are also effective in solid tumors. These
antiproliferative effects were unlikely to be caused by cytotoxicity,
because the number of dead cells indicated by the trypan blue exclusion
test was not increased by the treatment with DIFs (data not shown).
Consistent with the result obtained in leukemia cells (5), DIF-3 was
more effective than DIF-1 in HeLa cells. Therefore, we used DIF-3 in the subsequent experiments. We next examined the cell cycle
distribution using flow cytometry. Although the cell populations in S
and G2/M phases decreased after the treatment with DIF-3,
the population in G0/G1 phase significantly
increased (Fig. 1C), indicating that DIF-3 induced
G0/G1 arrest in HeLa cells. This result was
consistent with our previous study as to the effect of DIF-1 on
vascular smooth muscle cell cycle (6).

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Fig. 1.
Antiproliferative effect of DIF-1 and DIF-3
on HeLa cells. HeLa cells seeded on a 24-well plate (0.5 × 104 cells/well) were incubated with various concentrations
of DIF-1 or DIF-3. Cells were harvested by the trypsin/EDTA treatment
at the times indicated and enumerated. A, the time course of
the increase in cell numbers. B, the increase in cell
numbers at 72 h are shown as percentages of the control cell
number. Values are means ± S.E. for three independent
experiments. *, p < 0.005; **, p < 0.001 compared with the control (Student's t test).
C, flow cytometry. HeLa cells were incubated with DIF-3 (30 µM) for the indicated periods and then harvested by the
trypsin/EDTA treatment. After the trypsin/EDTA treatment, cells were
stained with propidium iodide (PI), and fluorescence of
nuclei was measured. The percentages of cell number in the cell cycle
phases are also shown. The results are means ± S.E. of six
independent experiments.
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DIF-3 Suppressed the Expression of Cyclins D1, D2, and D3 in HeLa
Cells--
We reported that DIF-1 induced
G0/G1 arrest, suppressing the expression of
cyclins D1, D2, and D3 in human vascular smooth muscle cells (6). In
HeLa cells, DIF-3 also reduced both the mRNA and protein levels of
cyclin D1 (Fig. 2, A and
B). RT-PCR analyses showed that the cyclin D1 mRNA level
was not significantly affected after a 1-h incubation with
DIF-3 (Fig. 2A). Although it began to slowly
decrease from 3 h, a considerable amount of cyclin D1 mRNA was
still expressed until 6 h. Despite this slow decrease in the level
of mRNA, DIF-3 rapidly reduced the protein level of cyclin D1 (Fig.
2B). Cyclin D1 protein markedly decreased after 1 h of
incubation with DIF-3 and nearly completely disappeared by 3 h.
This rapid decrease in the amount of protein was not explained by
suppression of mRNA expression. The protein levels of cyclins D2
and D3 also significantly decreased after 1 h of treatment with
DIF-3 and almost disappeared after 6 h of treatment (Fig. 2C).

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Fig. 2.
The effect of DIF-3 on D-type cyclin
expression. HeLa cells were incubated with or without DIF-3 (30 µM) for the periods indicated. A, RT-PCR
analysis for cyclin D1. Total RNA (1 µg) was subjected to RT-PCR for
cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). PCR cycle numbers were 24 for cyclin D1 and 20 for
glyceraldehyde-3-phosphate dehydrogenase. The result demonstrated is
representative of three other experiments. B, Western blot
analysis for cyclin D1. Samples were separated by 12% SDS-PAGE, and
immunoblot analysis was performed using an anti-cyclin D1 antibody.
Demonstrated are the results representative of three other experiments.
C, Western blot analysis for cyclins D2 and D3. Samples were
separated by 12% SDS-PAGE and immunoblotted using an anti-cyclin D2
antibody and an anti-cyclin D3 antibody. Demonstrated are the results
representative of three other experiments.
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DIF-3-induced G0/G1 Arrest Was Rescued by
the Overexpression of Cyclin D1--
To clarify whether the
overexpression of cyclin D1 is able to rescue cells from cell cycle
arrest induced by DIF-3, wild-type human cyclin D1 cDNA was
transfected to BAECs, since transfection efficiency was the highest in
BAECs among mammalian cell species we examined including HeLa cells. As
shown in Fig. 3A, the
expression levels of cyclin D1 in untransfected cells and in cells
transfected with empty pcDNA3 were reduced after 24 h of
incubation with DIF-3, demonstrating that DIF-3 showed the same effect
in BAECs as in HeLa cells. However, the expression level of cyclin D1
in cells transfected with pcDNA3/cyclin D1 was not significantly
changed by DIF-3 treatment. We then examined the cell cycle
distribution using untransfected and transfected cells. Although DIF-3
induced G0/G1 arrest in untransfected and
pcDNA3-transfected cells, there was no significant difference in
pcDNA3/cyclin D1-transfected cells between the absence and presence
of DIF-3 treatment (Fig. 3B). Therefore, DIF-3 was likely to
induce cell cycle arrest by reducing the expression level of cyclin
D1.

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Fig. 3.
Overexpression of cyclin D1 disabled DIF-3
from inhibiting cell cycle. A, BAECs untransfected or
transfected with the plasmids indicated were incubated with or without
DIF-3 (30 µM) for 24 h. Protein samples were
subjected to immunoblot analysis using an anti-cyclin D1 antibody. The
pcDNA3/cyclin D1-transfected cells expressed an ~15-fold
excessive amount of cyclin D1 compared with untransfected cells.
Demonstrated are the results representative of three other experiments.
B, cell cycle distribution. Untransfected and transfected
BAECs were incubated with or without DIF-3 (30 µM) for
12 h. Cells were then stained with propidium iodide
(PI), and the nuclear fluorescent levels were measured with
a flow cytometer. The percentages of cell number in the cell cycle
phases are shown as means ± S.E. of three independent experiments
performed in duplicate.
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DIF-3 Induced Cyclin D1 Proteolysis--
To determine the
mechanism for the decrease in cyclin D1 protein, we used
N-acetyl-Leu-Leu-norleucinal, which inhibits
ubiquitin-proteasome-dependent degradation of cyclins (14). As
shown in Fig. 4A,
N-acetyl-Leu-Leu-norleucinal prevented the DIF-3-induced
loss of cyclin D1. We then performed a chase experiment to
determine whether the turnover rate of cyclin D1 is changed by the
treatment with DIF-3. As shown in Fig. 4B, DIF-3
significantly decreased the half-life of cyclin D1 in the presence of
cycloheximide. These results indicated that DIF-3 rapidly reduced the
amount of cyclin D1 protein by accelerating ubiquitin-proteasome-dependent proteolysis.

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Fig. 4.
DIF-3 induced proteolysis of cyclin D1.
A, the effect of a proteasome inhibitor. Cells pretreated
with or without N-acetyl-Leu-Leu-norleucinal (20 µM) for 3 h were incubated in the presence or
absence of DIF-3 (30 µM) for 3 h, and immunoblot
analysis was performed using an anti-cyclin D1 antibody. The result is
representative of three other experiments. B, the rate of
cyclin D1 degradation. HeLa cells were incubated with or without DIF-3
(30 µM) in the presence of cycloheximide (5 µM). Cells were lysed in SDS sample buffer at the times
indicated and subjected to immunoblot analysis for cyclin D1. The
expression levels of cyclin D1 were quantified by densitometry and
statistically analyzed. Values are means ± S.E. of three
independent experiments. *, p < 0.01 compared with the
value at time 0 (Student's t test).
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Lithium Inhibited DIF-3-induced Effects--
It has been reported
that cyclin D1 undergoes ubiquitin-proteasome-dependent
proteolysis upon phosphorylation by GSK-3
(12). To investigate
whether GSK-3
is involved in the DIF-3-induced degradation of cyclin
D1 and inhibition of cell proliferation, we examined the effect of
lithium chloride on these DIF-3-induced effects, because lithium ion is
a specific inhibitor for GSK-3
(15). As shown in Fig.
5A, lithium chloride (20 mM) completely inhibited the degradation of cyclin D1
induced by DIF-3. We next examined the effect of lithium chloride on
the inhibition of cell proliferation induced by DIF-3 (Fig. 5,
B and C). Since 20 mM lithium
chloride strongly inhibited cell proliferation on its own probably due
to cytotoxicity, we reduced the concentrations of lithium chloride to
2-5 mM. Although 5 mM lithium chloride inhibited cell proliferation by about 30%, it partially prevented the
effect of DIF-3 (Fig. 5B). Fig. 5C shows more
clearly that lithium chloride dose-dependently inhibited
the effect of DIF-3. Based on these results, we hypothesized that DIF-3
activates GSK-3
to induce proteolysis of cyclin D1 and
antiproliferative effect.

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Fig. 5.
Lithium chloride inhibited the effects of
DIF-3. A, the effect of lithium chloride on DIF-3-induced
cyclin D1 degradation. HeLa cells were pretreated with or without
lithium chloride (20 mM) for 3 h and then incubated in
the presence or absence of DIF-3 (30 µM) for 3 h.
Protein samples were separated by 12% SDS-PAGE and immunoblotted for
cyclin D1. The result demonstrated is representative of three other
experiments. B, the effect of lithium chloride on cell
proliferation. Cells were treated with or without lithium chloride (20 mM) for 3 h and then incubated in the presence or
absence of DIF-3 (30 µM) for 24 h. Cells were
enumerated after harvested by the trypsin/EDTA treatment. Values are
means ± S.E. for three independent experiments. C,
data presented in B are differently expressed. The numbers
of cells treated with DIF-3 are shown as percentages of the cell
numbers obtained in the absence of DIF-3. Values are means ± S.E.
for three independent experiments. *, p < 0.05; **,
p < 0.001 (Student's t test).
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DIF-3 Activated GSK-3
--
The activity of GSK-3
was
measured using an in vitro kinase assay. DIF-3 (30 µM) elevated GSK-3
activity by 1.9-fold after 30 min
incubation, and GSK-3
was still activated at 3 h (Fig. 6A). In the presence of
lithium chloride, however, DIF-3 was not able to activate GSK-3
(Fig. 6B). Since GSK-3
is activated by the
dephosphorylation of Ser9 (9-11), the level of GSK-3
Ser9 phosphorylation was examined using an
anti-phospho-GSK-3
(Ser9) antibody. As shown in Fig.
6C, DIF-3 dramatically reduced the phosphorylation level of
Ser9 on GSK-3
after the incubation with DIF-3 for 30 min, and the phosphorylation level of Ser9 was slowly
recovered, looking like a mirror image of the time course of GSK-3
activity (Fig. 6A). Further, we examined the tyrosine
phosphorylation level of GSK-3
, since the activity of this enzyme has been reported to be increased by phosphorylation of
Tyr216 (9-11). As shown in Fig. 6D, DIF-3
significantly elevated the tyrosine phosphorylation level of GSK-3
by 2.0-fold after the incubation with DIF-3 for 30 min. These results
strongly indicated that DIF-3 activates GSK-3
.

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Fig. 6.
DIF-3 activates
GSK-3 . HeLa cells were incubated with or
without DIF-3 (30 µM) for the periods indicated.
A, in vitro kinase assay. GSK-3 was
immunoprecipitated from cell lysates and measured for kinase activity
using a substrate peptide derived from the sequence of glycogen
synthase. The results are means ± S.E. of three independent
experiments performed in duplicate. *, p < 0.01 compared with the control at time 0 (Student's t test).
B, the effect of lithium chloride on GSK-3 activity.
GSK-3 immunoprecipitated from cell lysates was measured for kinase
activity using a substrate peptide derived from the sequence of
glycogen synthase in the presence or absence of 10 mM
lithium chloride. The results are means ± S.E. of three
independent experiments. C, phosphorylation of
Ser9 on GSK-3 . Cell lysates were subjected to immunoblot
analysis using an anti-phospho-GSK-3 (Ser9) antibody.
The levels of Ser9 phosphorylation on GSK-3 were
quantified by densitometry and shown as percentages of the levels in
the control cells. Values are means ± S.E. of three independent
experiments. *, p < 0.01 compared with the control
(Student's t test). D, tyrosine phosphorylation
of GSK-3 . Immunoprecipitated GSK-3 was subjected to immunoblot
analysis using an anti-phosphotyrosine antibody (PY-20). The membrane
was reprobed with an anti-GSK-3 antibody. The levels of
tyrosine-phosphorylated GSK-3 were quantified and shown as
percentages of the levels in the control cells. Values are means ± S.E. of three independent experiments. *, p < 0.01 compared with the control (Student's t test).
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DIF-3 Induced Nuclear Translocation of GSK-3
--
GSK-3
is a
cytosolic protein; however, it is translocated into the nucleus when
activated (9-11). GSK-3
thereby accumulated in the nucleus
phosphorylates cyclin D1 and excludes it from nucleus, resulting in its
degradation in the cytoplasm (12). To test whether DIF-3-activated
GSK-3
is able to target cyclin D1, we examined the subcellular
distribution of GSK-3
after stimulation with DIF-3.
Immunofluorescent staining for GSK-3
revealed that GSK-3
was most
present in the cytoplasm and that there was only a small amount in
nuclei in unstimulated cells; however, it was markedly translocated
into nuclei after stimulation with DIF-3 (Fig.
7A). Importantly, the time
course of GSK-3
translocation into nuclei was similar to that of
cyclin D1 degradation. As shown in Fig. 7B, a similar result
was obtained using another anti-GSK-3
antibody. The nuclear
translocation of GSK-3
was confirmed by immunoblotting of
subcellular protein fractions. As shown in Fig. 7C, the
treatment with DIF-3 markedly increased GSK-3
in the nuclear
fraction. Its reduction in the cytosolic fraction may not be very
clear, because we applied 5 µg of each protein sample, although the
amounts of cytosolic proteins extracted were ~5 times those of
nuclear proteins. These results clearly demonstrated that cytosolic
GSK-3
was translocated into the nucleus in response to DIF-3.

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Fig. 7.
DIF-3 induces GSK-3
nuclear accumulation. A and B,
immunofluorescence. Cells were plated on coverslips and incubated with
or without DIF-3 (30 µM) for the periods indicated.
Immunofluorescent staining with a monoclonal (A) or a
polyclonal (B) anti-GSK-3 antibody was performed as
described under "Materials and Methods." The results are
representative of three other experiments. C, Western blot
analysis. Nuclear and cytoplasmic proteins were purified from HeLa
cells incubated with or without DIF-3 (30 µM) for 2 h. Western blot analysis was carried out using a monoclonal
anti-GSK-3 antibody. The results are representative of three other
experiments.
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The Phosphatidylinositol 3-Kinase (PI3K)/Akt Pathway and the
Mitogen-activated Protein Kinase (MAPK) Cascade Were Not Involved in
DIF-3-induced Cyclin D1 Degradation--
GSK-3
has been reported to
be phosphorylated on Ser9 by several protein kinases
(e.g. Akt, which is activated by PI3K, and p90 ribosomal S6
kinase (p90RSK), which is activated by the MAPK cascade)
(16). We therefore examined the effect of DIF-3 on the phosphorylation
levels of Akt and p90RSK to determine whether DIF-3
inhibits these kinases. Although DIF-3 did not affect the
phosphorylation level of Akt, it significantly induced phosphorylation
on Ser380 of p90RSK (Fig.
8A). We also examined the
effects of a PI3K inhibitor wortmannin and a mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase inhibitor U0126 on
cyclin D1 degradation induced by DIF-3. However, as shown in Fig.
8B, neither prevented the effect of DIF-3 on cyclin D1.
Therefore, the PI3K/Akt pathway and the MAPK cascade did not seem to be
involved in DIF-3-induced cyclin D1 degradation.

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Fig. 8.
Involvement of the PI3K/Akt pathway and the
MAPK cascade in DIF-3 signaling. A, the effect of DIF-3
on the phosphorylation of Akt and p90RSK. Cells were
treated with or without 30 µM DIF-3 for the periods
indicated. Protein samples were subjected to immunoblot analysis using
anti-phospho-Akt (Ser473) or
anti-phospho-p90RSK (Ser380) antibody. The
results are representative of three other experiments. B,
the effects of a PI3K inhibitor and a mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase 1/2 inhibitor on
cyclin D1 degradation induced by DIF-3. Cells were treated with 300 nM of wortmannin (PI3K inhibitor) for 30 min or 10 µM of U0126 (mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase 1/2 inhibitor) for
2 h and then incubated with or without DIF-3 (30 µM)
for 3 h. Protein samples were subjected to immunoblot analysis
using an anti-cyclin D1 antibody. The results are representative of
three other experiments.
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The Effect of DIF-3 on Untransformed Mammalian Cells--
In human
umbilical vein endothelial cells as well, DIF-3 inhibited cell
proliferation (Fig. 9A),
induced G0/G1 arrest (Fig. 9B), and
induced the degradation of cyclin D1 and the activation of GSK-3
(Fig. 9C), indicating that these effects of DIF-3 are not
limited to transformed cells but common between transformed and
untransformed cells.

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Fig. 9.
The effect of DIF-3 on vascular endothelial
cells. A, cell number. Human umbilical vein endothelial
cells were enumerated after incubated with or without DIF-3 (30 µM) for 24 h. The increases in cell number are shown
as means ± S.E. of three independent experiments. B,
cell cycle distribution. Endothelial cells were incubated with DIF-3
(30 µM) for 24 h and analyzed for cell cycle by flow
cytometer. The percentages of cell number in the cell cycle phases are
shown as means ± S.E. of three independent experiments performed
in duplicate. C, the effect of DIF-3 on the levels of cyclin
D1 and phospho-GSK-3 . Endothelial cells were incubated with or
without DIF-3 (30 µM) for the periods indicated and
subjected to immunoblot analysis. The results are representative of
three other experiments.
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 |
DISCUSSION |
In the present study, we showed that DIF-3 accelerated the
degradation of cyclin D1 by activating GSK-3
mainly using HeLa cells. However, this effect of DIF-3 was also found in other cell species including transformed and normal (untransformed) cells. As an
example, we have demonstrated the results obtained in vascular endothelial cells. Therefore, DIF-3 may ubiquitously activate GSK-3
in the wide variety of cell types. Since HeLa cells express human
papilloma virus antigens E6 and E7, which inactivate retinoblastoma protein (8), it is not clear whether cyclin D1 is required for their
cell cycle progression. However, lithium chloride prevented both the
degradation of cyclin D1 and the inhibition of cell proliferation induced by DIF-3. Therefore, cyclin D1 may be required for
proliferation, and the disappearance of cyclin D1 may be responsible
for the anti-proliferative effect of DIF-3 in HeLa cells. Furthermore, overexpression of cyclin D1 prevented DIF-3-induced cell cycle arrest
in untransformed endothelial cells. Taken together, cyclin D1
degradation seemed to be a main mechanism for DIF-3 to induce cell
cycle arrest.
DIF-3, naturally generated from DIF-1 as its first metabolite, is much
weaker than DIF-1 in the ability to induce stalk cell differentiation
in Dictyostelium (2). However, in contrast, the
antiproliferative effect of DIF-3 was significantly stronger than that
of DIF-1 in HeLa cells, consistent with a previous report that DIF-3 is
more effective than DIF-1 at inhibiting proliferation in K562 human
leukemia cells (5). This species difference in the sensitivity to DIFs
may be caused by a difference in the nature of the target molecule
(such as the affinity for DIFs) between mammalian and
Dictyostelium cells.
We found that DIF-3 not only elevated the activity of GSK-3
but also
induced nuclear translocation of the kinase. GSK-3
was
initially considered to be a soluble protein expressed in cytoplasm;
however, GSK-3
would have no access to nuclear proteins such as
cyclin D1 if it resided in cytoplasm. Recent evidence has indicated
that GSK-3
in nucleus phosphorylates nuclear proteins, such as
cyclin D1 (12), nuclear factor of activated T-cells (17), heat shock
factor-1 (18), and cAMP-response element-binding protein (19). Indeed,
GSK-3
has been identified from the nuclei of cell cycle-arrested
NIH-3T3 cells (12), cardiomyocytes stimulated with endothelin-1 (20),
and heat shock- and staurosporine-treated SH-SY5Y human neuroblastoma
cells (21). Therefore, DIF-3 seems to make it possible for GSK-3
to
phosphorylate cyclin D1 by translocating GSK-3
into the nucleus.
Not only a rapid proteolysis but also a reduction in cyclin
D1 mRNA expression was induced by DIF-3, although this latter effect took much longer time. Cyclin D1 gene expression is activated by
-catenin, the degradation of which is also initiated by GSK-3
(22). Therefore, activation of GSK-3
was expected to lead to a
reduction in both protein and mRNA levels of cyclin D1 through independent pathways. Our results agreed well with this rationale. The
proteolysis and mRNA reduction both seemed to be explained by
DIF-3-induced GSK-3
activation.
The target molecule for DIFs is still unknown even in
Dictyostelium. Since DIF-3 activates GSK-3
, a target for
DIF-3 may be a protein closely related to the regulation of GSK-3
activity. The activity of GSK-3
is decreased by phosphorylation of
Ser9 and increased by phosphorylation of Tyr216
(9-11). We found that DIF-3 decreased the phosphorylation level of
Ser9 and increased the phosphorylation level of
Tyr216 on GSK-3
. Akt activated by PI3K and
p90RSK activated by the MAPK cascade are the candidate
molecules to modulate GSK-3
activity by Ser9
phosphorylation (16). Although DIF-3 did not affect the
Ser473 phosphorylation of Akt, it strongly induced
phosphorylation on Ser380 of p90RSK. However,
we were not able to elucidate the role of DIF-3-induced p90RSK activation, since this kinase did not seem to be
involved in cyclin D1 degradation induced by DIF-3 (Fig.
6B). DIF-3 also enhanced tyrosine phosphorylation on
GSK-3
. Recently, a novel nonreceptor tyrosine kinase, ZAK-1, has
been found to directly activate GSK-3 in Dictyostelium (23).
However, a ZAK-1 counterpart in mammals has not yet been discovered. A
target molecule for DIF-3 might be a novel protein kinase or
phosphatase controlling GSK-3
activity in mammalian cells. Recently,
it has been reported that not only phosphorylation but also
distribution regulates GSK-3
activity (21). Thus, DIF-3-induced
accumulation of GSK-3
in nuclei might have an important role to
regulate GSK-3
activity. In addition, if mammalian cells have a
target molecule for DIFs as Dictyostelium, one could
hypothesize that mammals also produce DIF-like substances to control
growth and differentiation.
The Wnt signaling pathway is essential for embryonic development, cell
proliferation, cell differentiation, microtubule dynamics, and cell
motility. Several mutations have been identified in the components of
the Wnt pathway in a variety of malignant tumors. For instance, most
human colon cancers have mutations in the APC gene
that result in the accumulation of
-catenin (24). Mutations in
-catenin, which amplify the accumulation of
-catenin itself, also
have been identified in colon cancers (25), malignant melanomas (26),
prostate cancers (27), and hepatocellular carcinomas (28).
-Catenin
accumulated to an abnormal level would produce an excessive amount of
cyclin D1 mRNA and promote tumor growth (29). Therefore, chemicals
that activate GSK-3
, such as DIFs, may be useful for the treatment
of several cancers.
Recently, it has been reported that proapoptotic stimuli,
such as heat shock and staurosporine, activate GSK-3
and induce its
accumulation in nucleus (21). However, cytotoxic or proapoptotic agents
damage not only cancer cells but also normal cells, which would cause
severe adverse drug events. Distinct from staurosporine and other
proapoptotic agents, DIF-3 did not induce the activation of caspase-3
(data not shown). Therefore, the antiproliferative effect of DIF-3 did
not seem to be caused by the induction of apoptosis. DIF-3 is a unique
compound that activates GSK-3
but does not induce apoptotic cell death.