From the Laboratory of Cell Regulation, School of Pharmaceutical Sciences, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8131 and the § Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan
Received for publication, July 12, 2000, and in revised form, September 20, 2000
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ABSTRACT |
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Constitutive activation of the ERK pathway is
associated with the neoplastic phenotype of a relatively large number
of human tumor cells. Blockade of the ERK pathway by treatment with
PD98059, a specific inhibitor of mitogen-activated protein (MAP)
kinase/ERK kinase (MEK), completely suppressed the growth of tumor
cells in which the pathway is constitutively activated (RPMI-SE
and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G1 cell cycle
arrest, followed by a modest apoptotic response, in these tumor cells.
Selective up-regulation of p27Kip1 was observed after
PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in
RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP
kinase phosphatase-3 also induced up-regulation of p27Kip1.
The up-regulation of p27Kip1 correlated with increased
association of p27Kip1 with cyclin
E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease
in the phosphorylation state of retinoblastoma protein, which would
culminate in the marked G1 cell cycle arrest observed in
these tumor cells. These results suggest that the complete growth
suppression that follows specific blockade of the ERK pathway in tumor
cells in which the pathway is constitutively activated is mediated by
up-regulation of p27Kip1.
The 41-/43-kDa mitogen-activated protein
(MAP)1 kinase pathway, also
called the extracellular signal-regulated kinase (ERK) pathway, is
activated in a variety of cell types by diverse extracellular stimuli
and is among the most thoroughly studied of signaling pathways that
connect different membrane receptors to the nucleus (1, 2). Activation
of the ERK pathway involves the activation of Ras at the plasma
membrane, and the sequential activation of a series of protein kinases.
Initially, Ras interacts with and activates Raf-1, which in turn
activates MAP kinase/ERK kinase (MEK)-1 and -2 by serine
phosphorylation. MEK-1/2 then catalyze the phosphorylation of 41- and
43-kDa MAP kinases (ERK2 and ERK1, respectively) on tyrosine and
threonine residues, and these activated MAP kinases can phosphorylate
cytoplasmic and nuclear targets. The ERK pathway participates in a wide
range of cellular programs including proliferation, differentiation,
and movement (1, 2).
Aberrant activation of signal transducing proteins has been linked with
cancer. For example, constitutively active mutants of Ras (3) and Raf-1
(4) have been observed in several human tumors, and constitutively
active mutants of MEK-1 have been shown to transform mammalian cells
(5, 6). We recently examined whether constitutive activation of the ERK
pathway is associated with the neoplastic phenotype of human tumor
cells. Constitutive activation of ERKs and MEK was observed in a
relatively large number of tumors; tumor cells derived from pancreas,
colon, lung, ovary, and kidney tissues showed especially high
frequencies (30-50%) and a high degree of kinase activation (7, 8).
Activation of the ERKs is also associated with prostate cancer
progression (9). The precise cause of constitutive activation of the
ERK pathway in many of these tumor cells remains unclear. However, such
high frequencies of ERK/MEK activation in human tumors indicate that
specific inhibitors might be developed against these protein kinases
for cancer therapy, especially for treatment of tumors showing
constitutive activation of the ERK pathway.
In the present study, we have examined the effect of blockade of the
ERK pathway on the proliferation of human tumor cells. We utilized
small molecule inhibitors of this pathway, PD98059 (10) and U0126 (11),
which specifically inhibit MEK activity. Our results demonstrate that
these MEK inhibitors induce a remarkable G1 cell cycle
arrest, followed by a modest apoptotic response, in tumor cells in
which the ERK pathway is constitutively activated. Up-regulation of the
CDK inhibitor p27Kip1 was observed in these
G1-arrested tumor cells.
Cell Culture--
Human cell lines A-172 (glioblastoma), TGW
(neuroblastoma), GOTO (neuroblastoma), HT1080 (fibrosarcoma), RPMI-SE
(renal cell carcinoma), Colo320 (colon adenocarcinoma), and TIG-3
(diploid fibroblasts) (7) were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum.
Antibodies and Reagents--
The polyclonal anti-ERK antibody
has been described previously (12, 13). Antibodies against
p16INK4a (SC-1661), p19INK4d (SC-1063),
p21Cip1 (SC-6246), p27Kip1 (SC-528),
p57Kip2 (SC-1040), cyclin A (SC-239), cyclin D1 (SC-6281),
cyclin E (SC-247), and pRb (SC-102) were obtained from Santa Cruz
Biotechnology. Anti-cyclin B1 antibody (CC-03) was from Calbiochem.
2-(2-Amino-3-methoxyphenyl) chromone, which is identical to the
published compound PD98059 (10), was synthesized as described
previously (14). U0126 (11) was purchased from Promega. Other chemicals
and reagents were of the purest grade available.
Cell Growth Analysis--
For monolayer growth, cells were
plated at a density of 1 × 104 cells per 35-mm dish
and incubated for 24 h at 37 °C. Cells were then mock-treated
or treated with 50 µM PD98059 or 20 µM
U0126 for up to 5 days. Cells were harvested by trypsinization, and viable cells which excluded trypan blue were counted using a
hemocytometer. For anchorage-independent growth, 1 × 104 cells were suspended in 3 ml of 0.33% Difco agar in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
in the presence or absence of 50 µM PD98059, overlaid on
a 5-ml layer of 0.5% agar in the respective medium in a 60-mm dish,
and cultured for 14 days.
Immunoblotting--
Cells were scraped off plates in IB cell
lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 2 mM EDTA, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, 2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin) and lysed by sonication for 60 s. Lysates were cleared
by centrifugation at 15,000 × g for 30 min, and
protein concentrations were determined using the BCA protein assay
reagent (Pierce). Cell lysates (50 µg of protein) were separated by
SDS-PAGE, electrophoretically transferred to an Immobilon-P membrane
(Millipore Corp), and probed with the appropriate primary antibody and
horseradish peroxidase-conjugated secondary antibody. Immunoreactive
bands were visualized with the enhanced chemiluminescence system
(Amersham Pharmacia Biotech) (7, 15).
ERK Assay--
ERK activity was measured in an immune complex
kinase assay as described previously (7, 12, 13). Briefly, cell lysates prepared as described above (10 µg of protein) were
immunoprecipitated by incubating for 3 h at 4 °C with
polyclonal anti-ERK antibody preadsorbed to protein-A Sepharose
(Amersham Pharmacia Biotech). After washing twice with kinase buffer A
(50 mM Tris-HCl, pH 8.0, 25 mM
MgCl2, 1 mM dithiothreitol, 0.5 mM
EGTA, and 10% glycerol), each immunoprecipitate was incubated for 30 min at 30 °C with 20 µM ATP, 1 µCi of
[ Flow Cytometry--
Cells were fixed in 70% ethanol, treated
with 100 µg/ml RNase A (DNase-free; Sigma), and stained with 20 µg/ml propidium iodide (16). At least 1 × 104 cells
from each sample were analyzed for DNA content using a Coulter EPICS XL
flow cytometer (Coulter Electronics). Percentages of cells in
G1, S, and G2/M phases were determined using
Multicycle AV software (Phoenix Flow Systems).
Nuclear Staining--
Cells grown on glass coverslips were
treated with 50 µM PD98059 for 96 h. After fixing
with 3.7% paraformaldehyde, apoptotic cells with condensed or
fragmented nuclei were visualized by DAPI staining (0.4 mg/ml, 30 min).
DNA Fragmentation Analysis--
Cells were lysed in TET lysis
buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, and
0.5% Triton X-100) on ice for 10 min. After centrifugation,
supernatants were treated with 100 µg/ml of RNase A for 60 min. DNA
was extracted and resolved by electrophoresis on 2% agarose gels as
described (17). Pulsed-field gel electrophoresis was performed on 1%
agarose gels as described (18) using a CHEF system (Bio-Rad).
Immunoprecipitation and CDK2/Cyclin E-associated Kinase
Assay--
Cells were lysed in IP cell lysis buffer (50 mM
Hepes, pH 7.3, 150 mM NaCl, 10% glycerol, 0.1% Tween 20, 1 mM NaF, 0.1 mM sodium orthovanadate, 50 mM glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) on ice for 60 min. Cell lysates (400 µg of protein) were incubated with 2 µg of
anti-CDK2 antibody (SC-163, Santa Cruz Biotechnology) or 3 µg of
anti-cyclin E antibody (14761A, PharMingen) for 3 h at 4 °C.
Immune complexes were collected on protein A-Sepharose beads, washed
twice with kinase buffer B (50 mM Hepes, pH 7.3, 10 mM MgCl2 and 1 mM dithiothreitol),
and resuspended in 30 µl of kinase buffer B supplemented with 2 µg
of histone H1 (Roche Molecular Biochemicals), 25 µM ATP,
and 10 µCi of [ Plasmid and Transient Transfection--
The expression vector
for MKP-3 (pMT-SM-Myc-MKP-3) (19) was kindly provided by Dr. Steve
Arkinstall (Serono Pharmaceutical Research Institute), and the plasmid
expressing a kinase-negative form of MEK1
(pcDNA1neo-Myc- MEK1(AA); phosphorylation sites Ser-218 and
Ser-222 replaced with alanine) was kindly provided by Dr. Emmanuel Van
Obberghen (INSERM Unit 145, France). Transfection of these plasmids (1 µg) into RPMI-SE cells (1.5 × 104 cells/well of
24-well plate) was carried out using LipofectAMINE 2000 Reagent (Life
Technologies, Inc.) according to the manufacturer's protocol. Thirty
hours after transfection, cells were immunostained as described (20)
using polyclonal anti-c-Myc antibody (SC-789, Santa Cruz
Biotechnology), monoclonal anti-p27Kip1 antibody (K25020,
Transduction Laboratories), or monoclonal anti-phospho-ERKs antibody
(M8159, Sigma) as the primary antibody, and AlexaTM 488 goat anti-rabbit IgG conjugate (A-11034, Molecular Probes) or
AlexaTM 546 goat anti-mouse conjugate (A-11030, Molecular
Probes) as the secondary antibody. For BrdUrd incorporation
assay, 20 µM BrdUrd was added to the culture 30 h
after transfection, and then cells were incubated for 24 h. To
detect BrdUrd-positive cells, cells were fixed, treated with 2.5 N HCl to denature DNA, and then immunostained with
fluorescein isothiocyanate-conjugated monoclonal anti-BrdUrd antibody
(BU5.1, Progen Biotechnik).
MEK Inhibitor Completely Suppresses the Growth of Human Tumor Cells
in Which the ERK Pathway Is Constitutively Activated--
We have
recently proposed that human tumor cells can be classified into four
groups with regard to the activation of the ERK pathway (7). Tumor
cells in which constitutive activation of the ERK pathway is detected
are classified as type III or type II; type III tumor cells are those
in which the degree of activation of the ERK pathway is especially
high. Tumor cells in which constitutive activation of the ERK pathway
is not detected are classified as type I or type 0. Type I tumor cells
are those in which the ERK pathway is markedly activated when
serum-starved cells are growth-stimulated with 10% serum; this
response is identical to that observed in normal diploid fibroblasts.
Type 0 tumor cells are abnormal with respect to activation of the ERK
pathway, i.e. significant activation of the pathway is
undetectable even when serum-starved cells are growth-stimulated with
10% serum.
We first examined the inhibitory effect of two MEK inhibitors, PD98059
and U0126, on the activation of ERKs in exponentially growing tumor
cells. Activation of ERKs was determined by two different assay
procedures as follows: by performing a direct in vitro
kinase assay of the immunoprecipitates using myelin basic protein as
the substrate, and by measuring the appearance of the phosphorylated/activated forms of ERKs, which show reduced mobility in
SDS-PAGE. As described previously (7), type III (RPMI-SE and HT1080)
and type II (A-172) tumor cells exhibited a significantly high degree
of ERK activation under exponentially growing conditions. Treatment of
these tumor cells with U0126 or PD98059 suppressed the activation of
ERKs in a dose-dependent manner; U0126 inhibited ERK
activation more efficiently than PD98059, with virtually complete suppression being observed at 20 or 50 µM, respectively.
Although only a limited degree of ERK activation was detected in
exponentially growing type I (TGW) and type 0 (Colo320 and GOTO) tumor
cells, treatment with PD98059 or U0126 also reduced the degree of ERK activation in these cells. Fig.
1C shows the
PD98059/U0126-mediated inhibition of ERK activation in RPMI-SE and
Colo320 cells.
Treatment with PD98059 or U0126 inhibited the growth of all of the
tumor cells examined (Fig. 1A). These results seemed
reasonable, because the ERK pathway is the major cytoplasmic kinase
pathway and is activated commonly by numerous mitogenic stimuli that
interact with a diversity of structurally distinct receptors. In
addition, activation of the ERK pathway has been shown to be necessary
for fibroblast proliferation (5, 6, 21). However, susceptibility to
these MEK inhibitors showed distinct differences among cell types. The
growth of tumor cells in which the ERK pathway was constitutively
activated was totally abolished (type III tumor cells) or almost
completely inhibited (type II tumor cells) by 50 µM
PD98059 or 20 µM U0126. PD98059/U0126 inhibited the
growth of TGW type I tumor cells and TIG-3 diploid fibroblasts (data not shown) to a similar extent; the inhibition was considerable but not complete.
Although proliferation of type 0 tumor cells appeared not to depend on
the activation of the ERK pathway, PD98059/U0126 slightly inhibited the
growth of these cells. Basal ERK activity (Fig. 1C) may be
partially responsible for the proliferation of type 0 tumor cells;
inhibition of the basal ERK activity by PD98059/U0126 treatment might
have resulted in the slight growth inhibition observed in these cells.
Consistent with its antiproliferative effect on monolayer growth,
PD98059 completely inhibited the anchorage-independent growth of
RPMI-SE type III tumor cells but did not significantly affect Colo320
type 0 tumor cells (Fig. 1B).
MEK Inhibitor Induces Marked G1 Cell Cycle Arrest in
Tumor Cells in Which the ERK Pathway Is Constitutively
Activated--
To investigate the mechanism underlying the
antiproliferative effect of MEK inhibitors, cells were treated with 50 µM PD98059 for 0-96 h, stained with propidium iodide,
and subjected to flow cytometric analysis of cell cycle distribution.
Consistent with the marked inhibitory effect on cell proliferation,
PD98059 induced remarkable G1 cell cycle arrest in type III
tumor cells (Fig. 2). The onset of
G1 phase-arrest was apparent as early as 12 h after
PD98059 treatment of RPMI-SE and HT1080 cells, and almost complete
G1 cell cycle arrest was observed by 24 h, at which
time the proportion of cells in G1 phase had increased from
57.5 to 93.2% or 45.1 to 77.4%, and the proportion of cells in S
phase had declined from 27.8 to 2.4% or 34.8 to 2.9% in RPMI-SE or
HT1080 cells, respectively. Furthermore, PD98059 treatment induced in these tumor cells a significant increase in the proportion of dead
cells with fractional DNA content, which is a characteristic feature of
apoptosis (22). The proportion of such dead cells 96 h after
PD98059 treatment was 10.7% in RPMI-SE cells and 26.1% in HT1080
cells; the proportions in mock-treated control cells were 2.3 and
3.5%, respectively. PD98059 also induced prominent G1 cell
cycle arrest in A-172 type II tumor cells. However, the accumulation of
PD98059-treated A-172 cells in G1 phase was slower than
that observed in type III tumor cells; the proportion of A-172 cells in
G1 phase had increased from 38.4 to 87.4% by 96 h, at
which time the proportion of cells in S phase had decreased from 40.3 to 7.6%.
In contrast, MEK inhibition affected the cell cycle distribution of TGW
type I tumor cells and TIG-3 diploid fibroblasts (data not shown) to
only a small extent. PD98059 induced a slight increase (at most 5-7%)
in the proportion of these cells in G1 phase and a slight
decrease in the proportion of cells in S phase. This modest effect of
PD98059 treatment on the cell cycle distribution of these cells
appeared to result in the considerable growth suppression described
above (Fig. 1A). PD98059 did not significantly affect the
cell cycle distribution of Colo320 or GOTO type 0 tumor cells.
MEK Inhibitor Induces a Modest Apoptotic Response in Tumor Cells in
Which the ERK Pathway Is Constitutively Activated--
To characterize
the cell death caused by PD98059, we examined the nuclear morphology of
dying RPMI-SE and HT1080 cells with a fluorescent DNA-binding agent,
DAPI. After treatment with 50 µM PD98059 for 120 h,
~10% of the RPMI-SE cell population and ~15% of the HT1080 cell
population clearly exhibited condensed and fragmented nuclei,
indicative of apoptotic cell death (Fig. 3). No such nuclear morphology was
detected in mock-treated control cells. PD98059 treatment also induced
internucleosomal DNA fragmentation in HT1080 cells; this was most
evident after 120 h. PD98059 induced degradation of RPMI-SE DNA
not into internucleosomal fragments but into fragments of high
molecular weight. Pulsed-field gel electrophoresis revealed the
presence of 10-40-kbp DNA fragments in PD98059-treated RPMI-SE cells
after 120 h. It has recently been proposed, however, that the
appearance of large DNA fragments (20-300 kbp) occurs prior to the
appearance of internucleosomal DNA fragmentation and that such large
DNA fragments serve as precursors for the smaller DNA fragments (18,
23). Thus, apoptosis appeared to be the major mechanism of
PD98059-induced cell death in RPMI-SE and HT1080 tumor cells.
MEK Inhibitor Induces Selective Increase in the CDK Inhibitor
p27Kip1 and Inhibition of CDK2 Kinase Activity--
To
investigate the molecular mechanism of the G1 cell cycle
arrest observed in PD98059-treated RPMI-SE and HT1080 cells, we examined whether changes in G1-associated regulatory
proteins had occurred in these cells, focusing on the retinoblastoma
protein (Rb) (24). As shown in Fig. 4,
PD98059 treatment decreased the phosphorylation state of Rb; the
hypophosphorylated (pRb) species began to increase by 6 h, and
virtually complete loss of the hyperphosphorylated (ppRb) species was
observed 24-48 h after PD98059 treatment of RPMI-SE and HT1080 cells.
In addition, PD98059 treatment markedly reduced the levels of cyclin A
and B1. This is consistent with PD98059-induced inhibition of entry
into S and G2/M phases, where peak expression of these
cyclins is known to occur. Although the ERK pathway has been shown to
regulate positively the expression of cyclin D1 (25), PD98059
inhibition of the pathway did not significantly affect the level of
either cyclin D1 or cyclin E in these tumor cells.
We next explored possible reasons for Rb hypophosphorylation, focusing
on the CDK inhibitors p21Cip1, p27Kip1,
p57Kip2, p16INK4a, and p19INK4d
(26). Exponentially growing RPMI-SE and HT1080 cells expressed only
limited levels of these CDK inhibitors. PD98059 treatment induced a
marked increase in p27Kip1 in these tumor cells, which
began at 12-24 h and reached a maximum by 48 h. Immunostaining
clearly revealed the up-regulation of p27Kip1 in the nuclei
of PD98059-treated RPMI-SE cells (Fig. 6). In contrast, PD98059 did not
induce any significant increase in p21Cip1,
p57Kip2, p16INK4a, or p19INK4d in
either of the tumor cell lines (data not shown).
Up-regulation of p27Kip1 has been shown to favor its
association with G1-specific cyclin-CDK complexes such as
cyclin E-CDK2, resulting in kinase inhibition and contributing to Rb
hypophosphorylation (26). To investigate the functional significance of
p27Kip1 up-regulation in PD98059-treated RPMI-SE cells,
cell extracts were immunoprecipitated with antibodies against CDK2 or
cyclin E, and CDK2 kinase activity and cyclin E-associated kinase
activity were measured using histone H1 as a substrate. PD98059
treatment of the cells inhibited both CDK2 kinase activity and cyclin
E-associated kinase activity, with complete inhibition being apparent
by 48 h (Fig. 5). Immunoblot
analysis of the immunoprecipitated proteins revealed an increase in
p27Kip1 in both the CDK2 complexes and the cyclin E
complexes after PD98059 treatment, indicating increased binding of
p27Kip1 to cyclin E/CDK2 in response to PD98059. PD98059
treatment did not significantly affect the amount of either CDK2 or
cyclin E in the immunoprecipitates.
In Colo320 and GOTO type 0 tumor cells, PD98059 treatment did not
induce a significant change in the phosphorylation state of Rb or in
the levels of G1-associated regulatory proteins such as
p27Kip1, cyclin A, cyclin B1, cyclin D1, or cyclin E; this
was also the case in TGW type I tumor cells (Fig. 4 and data not
shown). These results were in consistent with the slight inhibitory
effect of PD98059 on the growth of these tumor cells (Fig. 1).
Overexpression of a Kinase-negative form of MEK1 or Wild-type MKP-3
Induces Up-regulation of p27Kip1 in RPMI-SE Cells--
To
confirm further that specific blockade of the ERK pathway induced
up-regulation of p27Kip1, RPMI-SE cells were transiently
transfected with an expression vector encoding either Myc-tagged
MEK1(AA) (a kinase-negative form of MEK1) or Myc-tagged MKP-3. MKP-3 is
a member of the dual-specificity MAP kinase phosphatase family and is
unique in that it is localized predominantly in the cytoplasm and
specifically inactivates ERKs, in contrast to c-Jun
NH2-terminal kinase or p38 MAP kinase (19). Double
immunofluorescence using anti-Myc and anti-phospho (activated)-ERKs antibodies revealed that overexpressed MEK1(AA) and MKP-3 were localized in the cytoplasm and completely prevented the activation of
ERKs. Importantly, up-regulation of p27Kip1 was clearly
observed in the nuclei of MEK1(AA)-/MKP-3-overexpressing RPMI-SE cells
in the absence of PD98059 treatment (Fig.
6). Furthermore, overexpression of
MEK1(AA) or MKP-3 (data not shown) appeared to prevent DNA synthesis in
the cells, as determined by the incorporation of BrdUrd into the
nucleus. In contrast, RPMI-SE cells that did not express the
kinase-negative form of MEK1 or exogenous MKP-3 exhibited ERK
activation throughout the cells as well as BrdUrd incorporation into
the nucleus; up-regulation of p27Kip1 was observed in these
cells only after treatment with PD98059 for more than 24 h.
In the present study, we examined the effect of a specific
blockade of the ERK pathway on the growth of human tumor cells in
vitro, using the specific MEK inhibitors PD98059 and U0126 (10,
11). Both inhibitors efficiently suppressed ERK activation in all of
the tumor cells examined (Fig. 1C). This essentially complete inhibition of the ERK pathway suppressed the growth of all
tumor cells examined. However, the susceptibility of cells to the
blockade of the ERK pathway showed distinct differences among cell
types and appeared to depend on the activation state of ERKs (7). The
growth of tumor cells with constitutively high levels of ERK activation
(type III tumor cells) was totally suppressed by PD98059/U0126, whereas
the growth of tumor cells with barely detectable levels of ERK
activation (type 0 tumor cells) was only slightly suppressed by the MEK
inhibitors (Fig. 1A). The different effects of PD98059 on
the anchorage-independent growth of type III and type 0 tumor cells are
clearly shown in Fig. 1B. These results reinforce our
previous observation that the requirement for the ERK pathway in
proliferation differs markedly among human tumor cells; type III tumor
cells depend absolutely on the activation of the ERK pathway for
proliferation, whereas proliferation of type 0 tumor cells appears not
to depend on the ERK pathway (7).
Consistent with its prominent antiproliferative effect, PD98059 induced
striking G1 cell cycle arrest in tumor cells in which the
ERK pathway is constitutively activated (Fig. 2). These results are
consistent with the idea that activation of the ERK pathway is
essential for cells to pass the G1 restriction point (21). It has recently been reported, however, that the ERK pathway functions not only in the G1/S transition but also in the transition
from G2 to M phase in mammalian fibroblasts (27). In this
respect, populations of HT1080 cells treated with 50 µM
PD98059 for more than 24 h remained at G2/M;
essentially complete depletion of the PD98059-treated cells in S phase
was confirmed by pulsing the cells with BrdUrd and then double-staining
with a fluorescein isothiocyanate-conjugated anti-BrdUrd antibody and
propidium iodide for flow cytometric analysis (data not shown). Cells
remaining at G2/M phase were not observed in
PD98059-treated RPMI-SE or A-172 cell populations. Requirements for the
ERK pathway in G2/M transition may differ significantly
among tumor cells.
Orderly progression through the cell cycle is cooperatively regulated
by several classes of CDKs, whose activities are in turn constrained by
CDK inhibitors (26). We demonstrated that PD98059 induced selective
up-regulation of p27Kip1 in RPMI-SE and HT1080 cells.
PD98059 did not induce a significant increase in p27Kip1 in
tumor cells in which constitutive activation of the ERK pathway is
undetectable (Fig. 4). Furthermore, overexpression in RPMI-SE cells of
either a kinase-negative form of MEK1 or wild-type MKP-3 clearly
induced up-regulation of p27Kip1 (Fig. 6). These results
suggest that specific blockade of the ERK pathway induces marked
up-regulation of p27Kip1 only in tumor cells in which the
pathway is constitutively activated.
The pivotal role of p27Kip1 in controlling CDK function and
thus cell cycle progression is well established (26).
p27Kip1 mediates cell cycle arrest in response to various
anti-mitogenic signals such as transforming growth factor- Regulation of p27Kip1 is mediated by transcriptional,
post-transcriptional, and post-translational mechanisms. We did not
observe any significant increase in p27Kip1 mRNA levels
in RPMI-SE cells treated with PD98059 for up to 48 h (data not
shown),2 by which time
p27Kip1 reaches a maximum level (Fig. 4). Given the recent
demonstration that cyclin E-CDK2 directly phosphorylates
p27Kip1 and promotes its destruction by the ubiquitin
pathway (26, 28), it seems likely that the accumulation of
p27Kip1 induced by inhibiting the ERK pathway is a result
of stabilization of p27Kip1 at the protein level.
PD98059 treatment induced a significant increase in the proportion of
cells with condensed and fragmented nuclei in tumor cells in which the
ERK pathway is markedly activated (Fig. 3). PD98059 induced the
degradation of chromosomal DNA into internucleosomal DNA fragments in
HT1080 cells or into 10-40-kbp DNA fragments in RPMI-SE cells. This
fragmentation was not observed if the cells were preincubated with 50 µM benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a general caspase inhibitor (data not
shown).3 Furthermore, PD98059
treatment induced a significant increase in the caspase-3-like protease
activity in these tumor cells, which was determined using
N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide as a
substrate.3 These results suggest that specific blockade of
the ERK pathway by PD98059 induces apoptosis in HT1080 and RPMI-SE
cells. In this respect, recent reports have demonstrated that
overexpression of p27Kip1 leads to apoptosis in various
cancer cells (29, 30). Thus, the accumulation of p27Kip1
observed in PD98059-treated HT1080/RPMI-SE cells could play a role, at
least in part, in the induction of apoptosis in these tumor cells.
Moreover, blockade of the ERK pathway would cause serious metabolic
disorders in HT1080 and RPMI-SE cells, which could culminate in the
induction of apoptosis; these type III tumor cells appear to depend
absolutely on the ERK pathway for growth, and probably for survival
(31). The exact mechanism(s) responsible for the induction of
apoptosis and of up-regulation of p27Kip1 following
specific blockade of the ERK pathway in tumor cells in which the
pathway is constitutively activated remains to be determined.
Induction of apoptosis is considered a possible therapy for human
cancers (32). Here, we have shown that specific blockade of the ERK
pathway induces apoptosis in tumor cells in which the pathway is
constitutively activated. PD98059 and U0126 inhibited the proliferation
of human diploid fibroblasts to a considerable degree but not
completely. Importantly, PD98059/U0126 caused no increase in the
proportion of diploid fibroblasts with fractional DNA content even
after 10 days. Moreover, growth-inhibited diploid fibroblasts
reinitiated proliferation soon after removal of the inhibitors (data
not shown). Taken together, our results strongly suggest that the ERK
pathway is a potential therapeutic target in a group of tumor cells in
which the pathway is constitutively activated. This possibility is
further supported by a recent report showing that a new MEK inhibitor,
PD184352, inhibited the growth of human colon tumor xenografts in
vivo (33).
In conclusion, we have demonstrated in this report that specific
blockade of the ERK pathway completely suppresses the growth of tumor
cells in which the pathway is constitutively activated, and we
presented evidence suggesting that this prominent growth inhibition is
mediated by the up-regulation of p27Kip1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (Amersham Pharmacia Biotech), and 7.5 µg
of myelin basic protein (Sigma) in 30 µl of kinase buffer A. Radioactivity incorporated into myelin basic protein was determined by
liquid scintillation spectrometry.
-32P]ATP. Reaction mixtures were
incubated for 30 min at 30 °C, and the extent of histone H1
phosphorylation was determined by SDS-PAGE and autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Inhibitory effect of PD98059/U0126 on the
proliferation of human tumor cells. A, type III (HT1080
and RPMI-SE), type II (A-172), type I (TGW), or type 0 (Colo320 and
GOTO) tumor cells were cultured for the indicated times in the absence
(open symbols) or in the presence of 50 µM
PD98059 (closed symbols) or 20 µM U0126
(closed symbols, dashed line). Viable cell numbers are
expressed as a percentage of initial cell numbers. Each value
represents the mean of duplicate measurements performed on separate
dishes (cell number variations were within 10% at each data point).
B, RPMI-SE or Colo320 cells were suspended in 0.33% Difco
agar in the absence ( ) or presence (+) of 50 µM PD98059
(PD) and overlaid on a 0.5% agar layer. Colonies were
photographed after 14 days. C, RPMI-SE cells or Colo320
cells were treated with 50 µM PD98059 (PD) or
20 µM U0126 for the indicated times. Upper,
ERK activity was determined by performing a direct in vitro
kinase assay of the immunoprecipitates using myelin basic protein as
the substrate. Radioactivity incorporated into myelin basic protein is
shown. Each value represents the mean ± S.D. of duplicate
determinations. Lower, cell lysates (10 µg of protein)
were resolved by SDS-PAGE, followed by immunoblot analysis using
anti-ERK antibody. pp43/pp41, activated (phosphorylated)
forms of ERK1/2. Data shown are representative of 3-4 separate
experiments.
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Fig. 2.
Flow cytometric analysis of tumor cells
treated with PD98059. Exponentially growing cells were incubated
without (Control) or with 50 µM PD98059 for
the indicated times. Cells were fixed, stained with propidium iodide,
and analyzed by flow cytometry. Experiments were repeated 3-4 times
with similar results.
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Fig. 3.
Induction of apoptotic responses by
PD98059 in HT1080 and RPMI-SE cells. HT1080 or RPMI-SE cells were
treated with 50 µM PD98059 for the indicated times.
Left, DNA extracted from the cells was resolved by
electrophoresis on 2% agarose gels (HT1080 cells) or by pulsed-field
gel electrophoresis on 1% agarose gels (RPMI-SE cells).
Right, cells were fixed with paraformaldehyde and then
stained with DAPI. Data shown are representative of three separate
experiments.
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Fig. 4.
Immunoblot analysis of
G1-associated regulatory proteins in tumor cells treated
with PD98059. RPMI-SE, HT1080, Colo320, or GOTO cells were
mock-treated (C) or treated with 50 µM PD98059
for the indicated times. Cell lysates (50 µg of protein) were
resolved by SDS-PAGE, and proteins were detected by immunoblotting with
the indicated antibodies. Square brackets indicate
hyperphosphorylated species of Rb (ppRb), and arrowheads
indicate hypophosphorylated species of Rb (pRb). Data shown are
representative of three separate experiments.
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Fig. 5.
Inactivation of cyclin E-CDK2 complexes by
binding of p27Kip1 in PD98059-treated RPMI-SE cells.
RPMI-SE cells were mock-treated (C) or treated with 50 µM PD98059 (PD) for the indicated times. After
cell lysates (400 µg of protein) were immunoprecipitated
(IP) with an antibody against CDK2 or cyclin E, CDK2 and
cyclin E-associated kinase activities were measured using histone H1
(H1) as a substrate. The amounts of CDK2, cyclin E, or
p27Kip1 in the CDK2/cyclin E immunoprecipitates were
analyzed by immunoblotting (IB). Data shown are
representative of two separate experiments.
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Fig. 6.
Up-regulation of p27Kip1 induced
by overexpression of MEK1(AA) or MKP-3 in RPMI-SE cells. RPMI-SE
cells were mock-transfected or transfected with an expression vector
encoding Myc-tagged MEK1(AA) or Myc-tagged MKP-3. Mock-transfected
cells were then treated with 50 µM PD98059 for 48 h.
For BrdUrd incorporation assays, the transfected cells were incubated
with 20 µM BrdUrd for 24 h. Cells expressing either
MEK1(AA) or exogenous MKP-3 were detected by immunofluorescence using
the anti-Myc antibody. Localization of the phosphorylated (activated)
ERKs was revealed by immunofluorescence using anti-phospho-ERKs
antibody (pERKs), and localization of p27Kip1
was revealed with anti-p27Kip1 antibody. BrdUrd-positive
cells were detected with fluorescein isothiocyanate-conjugated
monoclonal anti-BrdUrd antibody (BrdU). The nuclei of all
cells were visualized with DAPI. Arrowheads indicate cells
expressing either MEK1(AA) or exogenous MKP-3. Experiments were
repeated 3-5 times with similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, serum
withdrawal, and cell-cell contact. p27Kip1 associates with
and inhibits the catalytic activities of G1-specific cyclin-CDK complexes such as cyclin E-CDK2, whose activity is essential
for entry of cells into S phase. One major target of cyclin E-CDK2 is
Rb, which upon hyperphosphorylation dissociates from bound
transcription factors such as E2F, enabling them to activate genes
required for DNA replication. We demonstrated that the up-regulation of
p27Kip1 observed in PD98059-treated RPMI-SE cells
correlated with an increase in p27Kip1 associated with
cyclin E-CDK2 complexes, a concomitant inhibition of cyclin E-CDK2
kinase activity, and a consequent decrease in the phosphorylation of
Rb, which would culminate in the G1 cell cycle arrest of
these cells. Taken together, our results suggest that the complete
growth suppression that follows specific blockade of the ERK pathway in
tumor cells in which the pathway is constitutively activated is
mediated by up-regulation of p27Kip1.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Steve Arkinstall and Emmanuel Van Obberghen for supplying plasmids, Dr. Kei-ichi Ozaki for the analysis of p27Kip1 mRNA, and Dr. Patrick Hughes for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and a grant from the Naito Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the Japan Society for the Promotion
of Science for Japanese Junior Scientists.
¶ To whom all correspondence should be addressed: Laboratory of Cell Regulation, School of Pharmaceutical Sciences, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8131, Japan. Tel.: 81-95-849-2693; Fax: 81-95-849-2671; E-mail: kohnom@net.nagasaki-u.ac.jp.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M006132200
2 K. Ozaki, R. Hoshino, and M. Kohno, unpublished observations.
3 R. Hoshino and M. Kohno, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; CDK, cyclin-dependent kinase; Rb, retinoblastoma protein; PAGE, polyacrylamide gel electrophoresis; MKP, MAP kinase phosphatase; DAPI, 4',6'-diamidino-2-phenylindole; BrdUrd, bromodeoxyuridine; kbp, kilobase pair.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Robinson, M. J., and Cobb, M. H. (1997) Curr. Opin. Cell Biol. 9, 180-186[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Widmann, C.,
Gibson, S.,
Jarpe, B.,
and Johnson, G. L.
(1999)
Physiol. Rev.
79,
143-180 |
3. | Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-827[CrossRef][Medline] [Order article via Infotrieve] |
4. | Nakatsu, Y., Nomoto, S., Oh-uchida, M., Shimizu, K., and Sekiguchi, M. (1985) Cold Spring Harbor Symp. Quant. Biol. 51, 1001-1008 |
5. | Mansour, S. J., Mattern, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukasawa, K., Vande Woude, G. F., and Ahn, N. G. (1994) Science 265, 966-970[Medline] [Order article via Infotrieve] |
6. | Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841-852[Medline] [Order article via Infotrieve] |
7. | Hoshino, R., Chatani, Y., Yamori, T., Tsuruo, T., Oka, H., Yoshida, O., Shimada, Y., Ari-i, S., Wada, H., Fujimoto, J., and Kohno, M. (1999) Oncogene 18, 813-822[CrossRef][Medline] [Order article via Infotrieve] |
8. | Oka, H., Chatani, Y., Hoshino, R., Ogawa, O., Kakehi, Y., Terachi, T., Okada, Y., Kawaichi, M., Kohno, M., and Yoshida, O. (1995) Cancer Res. 55, 4182-4187[Abstract] |
9. |
Gioeli, D.,
Mandell, J. W.,
Petroni, G. R.,
Frierson, H. F., Jr.,
and Weber, M. J.
(1999)
Cancer Res.
59,
279-284 |
10. | Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689[Abstract] |
11. |
Favata, M. F.,
Horiuchi, K. Y.,
Manos, E. J.,
Daulerio, A. J.,
Stradley, D. A.,
Feeser, W. S.,
Van Dyk, D. E.,
Pitts, W. J.,
Earl, R. A.,
Hobbs, F.,
Copeland, R. A.,
Magolda, R. L.,
Scherle, P. A.,
and Trzaskos, J. M.
(1998)
J. Biol. Chem.
273,
18623-18632 |
12. |
Chatani, Y.,
Tanaka, E.,
Tobe, K.,
Hattori, A.,
Sato, M.,
Tamemoto, H.,
Nishizawa, N.,
Nomoto, H.,
Takeya, T.,
Kadowaki, T.,
Kasuga, M.,
and Kohno, M.
(1992)
J. Biol. Chem.
267,
9911-9916 |
13. |
Chatani, Y.,
Tanimura, S.,
Miyoshi, N.,
Hattori, A.,
Sato, M.,
and Kohno, M.
(1995)
J. Biol. Chem.
270,
30686-30692 |
14. | Tanimura, S., Chatani, Y., Hoshino, R., Sato, M., Watanabe, S., Kataoka, T., Nakamura, T., and Kohno, M. (1998) Oncogene 17, 57-65[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Iwasaki, S.,
Iguchi, M.,
Watanabe, K.,
Hoshino, R.,
Tsujimoto, M.,
and Kohno, M.
(1999)
J. Biol. Chem.
274,
26503-26510 |
16. | Darzynkiewicz, Z., Gong, J., and Traganos, F. (1994) Methods Cell Biol. 41, 421-435[Medline] [Order article via Infotrieve] |
17. |
Sellins, K. S.,
and Cohen, J. J.
(1987)
J. Immunol.
139,
3199-3206 |
18. |
Samejima, K.,
Tone, S.,
Kottke, T. J.,
Enari, M.,
Sakahira, H.,
Cooke, C. A.,
Durrieu, F.,
Martins, L. M.,
Nagata, S.,
Kaufmann, S. H.,
and Earnshaw, W. C.
(1998)
J. Cell Biol.
143,
225-239 |
19. |
Muda, M.,
Boschert, U.,
Dickinson, R.,
Martinou, J. C.,
Martinou, I.,
Camps, M.,
Schlegel, W.,
and Arkinstall, S.
(1996)
J. Biol. Chem.
271,
4319-4326 |
20. |
Brunet, A.,
Roux, D.,
Lenormand, P.,
Dowd, S.,
Keyse, S.,
and Pouyssegur, J.
(1999)
EMBO J.
18,
664-674 |
21. |
Pages, G.,
Lenormand, P.,
L'Allemain, G.,
Chambard, J. C.,
Meloche, S.,
and Pouyssegur, J.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8319-8323 |
22. | Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. (1997) Cytometry 27, 1-20[CrossRef][Medline] [Order article via Infotrieve] |
23. | Oberhammer, F., Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Wakeling, A. E., Walker, P. R., and Sikorska, M. (1993) EMBO J. 12, 3679-3684[Abstract] |
24. | Taya, Y. (1997) Trends Biochem. Sci. 22, 14-17[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Lavoie, J. N.,
L'Allemain, G.,
Brunet, A.,
Muller, R.,
and Pouyssegur, J.
(1996)
J. Biol. Chem.
271,
20608-20616 |
26. |
Sherr, C. J.,
and Roberts, J. M.
(1999)
Genes Dev.
13,
1501-1512 |
27. |
Wright, J. H.,
Munar, E.,
Jameson, D. R.,
Andreassen, P. R.,
Margolis, R. L.,
Seger, R.,
and Krebs, E. G.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11335-11340 |
28. | Sheaff, R., Groudine, M., Gordon, M., Roberts, J., and Clurman, B. (1997) Genes Dev. 11, 1464-1478[Abstract] |
29. | Katayose, Y., Kim, M., Rakkar, A. N. S., Li, Z., Cowan, K. H., and Seth, P. (1997) Cancer Res. 57, 5441-5445[Abstract] |
30. | Wang, X., Gorospe, M., Huang, Y., and Holbrook, N. J. (1997) Oncogene 15, 2991-2997[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Bonni, A.,
Brunet, A.,
West, A. E.,
Datta, S. R.,
Takasu, M. A.,
and Greenberg, M. E.
(1999)
Science
286,
1358-1362 |
32. |
Evan, G.,
and Littlewood, T.
(1998)
Science
281,
1317-1322 |
33. | Sebolt-Leopold, J. S., Dudley, D. T., Herrera, R., Van Becelaere, K., Wiland, A., Gowan, R. C., Tecle, H., Barrett, S. D., Bridges, A., Przybranowski, S., Leopold, W. R., and Saltiel, A. R. (1999) Nat. Med. 5, 810-816[CrossRef][Medline] [Order article via Infotrieve] |