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INTRODUCTION |
Interleukin-1
(IL-1),1 originally defined
as a monocyte-derived factor mitogenic for thymocytes, is now known to
affect many biological activities, including the ability to alter
immunologic, inflammatory, hematopoietic, and homeostatic responses in
the host system. In vitro, IL-1 has been shown to inhibit
the growth of certain tumor cells (1-3). A role for IL-1 in host
defense against tumors has been further suggested by its ability to
augment natural killer cell activity (4), monocyte-mediated tumor
cytotoxicity (5), and T- and B-cell responses (6) and to induce tumor regression in mice (7). These properties have made IL-1 an attractive
candidate for potential application for certain solid tumors (8-12),
although several problems including various adverse effects such as
fever and hypotension remain unresolved. Elucidation of the molecular
mechanisms that mediate the antiproliferative effect of IL-1 on tumor
cells would not only be of help in resolving these problems; it would
also provide valuable information on how cell proliferation is
regulated negatively by extracellular signals.
In cell culture, various types of tumor cells, such as melanoma (4,
13-19), breast carcinoma (20), myeloid leukemia (21, 22), ovarian
carcinoma (23), and lung adenocarcinoma (24) cells, have been shown to
be susceptible to the antiproliferative action of IL-1. A highly
susceptible human melanoma cell line, A375-C6, is commonly used to
study the mechanism of the IL-1-mediated growth arrest. The action of
IL-1 in A375-C6 cells has been documented to be mediated through
specific cell surface receptors (25). The binding of IL-1 to its
receptor results in activation of a variety of second-messenger
signaling pathways (reviewed in Ref. 13) and a unique primary gene
expression program characterized by the induction of a composite set of
immediate early genes such as gro-
,
gro-
, c-jun,
nur77/NGF1-B/NAK1,
IRG-9/TIS11, and Egr-1 (16, 17, 19).
IL-1 action in A375-C6 cells is also characterized by inhibition of
ornithine decarboxylase, a rate-limiting enzyme in polyamine synthesis,
leading to inhibition of DNA synthesis (18, 26). Although these events
are thought to mediate the antiproliferative effect of IL-1, further
investigation is required to elucidate the direct mechanisms
responsible for initiation and/or maintenance of the growth-arrested
state. Since cell growth and proliferation are ultimately regulated by
a highly conserved set of cell cycle-regulatory proteins, a fruitful
approach to the study of the antiproliferative mode of action of IL-1
is to examine the influence of IL-1 on the regulation of the cell
cycle-regulating machinery.
The eukaryotic cell cycle is regulated by the action of the
cyclin-dependent kinases (CDKs) and their activating
subunits, the cyclins (27, 28). In mammalian cells, Cdk6 and Cdk4 are associated with the D-type cyclins and regulate G1
progression. Cdk2 is associated with E- and A-type cyclins, and the
respective complexes are believed to control G1-S
transition and S phase progression, respectively. Cdc2 is associated
with B-type cyclins and regulates G2-M phase. The most well
studied substrates of the CDKs operating during G1 and S
phases are the retinoblastoma family of proteins (29, 30). This family
consists of pRb and the related proteins p107 and p130, collectively
termed the pocket proteins. Progression from G1 to S
requires inactivation of the retinoblastoma family proteins by
phosphorylation and the consequent release of a number of factors
including the E2F family of transcription factors. These transcription
factors then activate transcription of various genes that promote cell
cycle progression. Thus, the phosphorylation state of retinoblastoma
family proteins is a critical determinant in the execution of the
progression from G1 to S. Recently, Muthukkumar et
al. (15) documented that growth arrest by IL-1 was linked to
suppression of pRb phosphorylation and suggested that
hypophosphorylated pRb may possibly mediate the action of IL-1. Since
pRb is phosphorylated by the action of the CDKs, it is conceivable that
the IL-1-induced pRb hypophosphorylation is the result of negative
regulation of the CDKs. However, the effect of IL-1 on the activity or
the regulation of the CDKs has not been defined as yet. In mammalian
cells, regulation of CDKs is achieved by several mechanisms including
alteration of CDK levels; changes in the expression of the cyclins with
which CDKs interact; activation and inactivation of CDKs by
phosphorylation/dephosphorylation events; and the abundance and action
of two families of CDK inhibitors, the Cip/Kip family (p21cip1,
p27kip1, and p57kip2) and the Ink4 family
(p16ink4a, p15ink4b, p18ink4c, and
p19ink4d) (reviewed in Refs. 27, 31, and 32). Modulation at any of these levels of regulation could regulate pRb phosphorylation.
The current study was undertaken to investigate the molecular
mechanisms, which mediate the antiproliferative effect of IL-1, through
a detailed analysis of the IL-1 effects on the cell cycle-regulating molecules, such as CDKs, cyclins, CDK inhibitors, and pRb family proteins. By using A375S2 human melanoma cells (33), which are as
highly sensitive to the antiproliferative effect of IL-1 as A375-C6
cells, we have systematically analyzed the changes that occur in kinase
activities, expression levels, interactions, and phosphorylation status
of the cell cycle-regulating molecules and compared them to the timing
of onset and completion of the growth-arresting process. We conclude
from these experiments that IL-1 arrests A375S2 cells at G1
and G2 phases of the cell cycle by inhibiting kinase
activities of cyclin E-Cdk2 and cyclin B1-Cdc2 complexes, and the
inhibitory mechanism for each complex is different. Furthermore, data
presented here suggest that IL-1 exerts its growth-arresting effects
via p107 and p130 pathways rather than through pRb.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
A375S2 human melanoma cells (obtained from
Otsuka Pharmaceutical Co., Ltd., Cellular Technology Institute,
Tokushima, Japan) were cultured in minimum essential medium (Life
Technologies, Inc. Oriental Co., Tokyo, Japan) supplemented with 10%
heat-inactivated fetal calf serum (Life Technologies) in a humidified
incubator at 37 °C in 5% CO2. Human recombinant
IL-1 was purchased from R & D Systems (Minneapolis, MN). To
assure exponential cell growth, A375S2 cell cultures were set up
48 h prior to the addition of IL-1; 0.6 × 106
cells were plated in 100-mm plastic dishes and incubated for 24 h,
the culture medium was then replaced, and the incubation was continued
for another 24 h before the addition of IL-1 (1.0 ng/ml, unless
otherwise indicated). Cells were harvested at different times after the
addition of IL-1 and processed for cyclin-dependent kinase
assays, Western blotting, immunoprecipitation, or Northern blotting, as
described below. For the cell proliferation assay, cells at 1.2 × 105 per 60-mm dish were treated with IL-1 at different
concentrations. At various experimental intervals, cells were
trypsinized and counted using a cell counter (Sysmex model F-500,
Sysmex Co., Kobe, Japan). In a parallel experiment, the number of
viable cells was also determined by the Trypan blue dye exclusion test.
For cell cycle analysis, trypsinized cells were stained with propidium iodide by using the Cycle TEST PLUS kit (Nippon Becton Dickinson Co.,
Ltd., Tokyo, Japan) and analyzed for DNA content by using the Becton
Dickinson fluorescence-activated cell sorting system (FACSCalibur).
Cell cycle distribution was determined using ModFit LT Software (Verity
Software House, Popsham, ME). All experiments were repeated at least twice.
Antibodies--
Antibodies used and their sources were as
follows: Cdk2 (M2), Cdk4 (C-22), Cdk6 (C-21), cyclin A (BF683 and
H-432), cyclin B1 (H-433), cyclin D (HD11), cyclin E (HE111), p15
(K-18), p16 (C-20), p18 (N-20), p19 (M-167), p21 (C-19), p57 (C-20),
p107 (C-18), and phosphotyrosine (PY99) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); cyclin A (14591A), Cdc2 (14391A), cyclin E
(14591A), and p21 (15431E) from PharMingen (San Diego, CA); p27
(K25020) and p130 (R27020) from Transduction Laboratories (Lexington,
KY); cyclin D (06-137) from Upstate Biotechnology, Inc. (Lake Placid,
NY); p21 (Ab-1) and p53 (Ab-2) from Oncogene Research Products
(Cambridge, MA); pRb C-terminal (9032) from New England Biolabs Inc.
(Beverly, MA). The antibodies 15431E for p21, H-432 for cyclin A, HE111
for cyclin E, and HD11 for cyclin D were used only for
immunoprecipitation. The antibody Ab-1 for p21 was used for immunodepletion.
Immunoprecipitation and Kinase Assay--
Cell monolayers were
washed twice with ice-cold phosphate-buffered saline and then scraped
into ice-cold Nonidet P-40 lysis buffer (50 mM HEPES (pH
7.5), 250 mM NaCl, 0.1% Nonidet P-40, with 0.5 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 10 mM
-glycerophosphate, 1 mM NaF, 0.1 mM Na3VO4,
10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 µg/ml pepstatin A
added just before use). Cell lysates were sonicated and clarified by
centrifugation at 19,000 × g for 15 min at 4 °C,
aliquots were removed for analysis of protein concentration (measured
using the DC Protein Assay kit; Nippon Bio-Rad Laboratories, Osaka,
Japan), and samples were adjusted to equivalent protein concentrations.
After precleaning with protein G-Sepharose beads (Sigma-Aldrich Japan,
Tokyo, Japan), equal amounts of protein (150 µg) were incubated with
the appropriate antibody for 2 h at 4 °C. Antibody complexes
were recovered on protein G-Sepharose beads and washed four times with
Nonidet P-40 lysis buffer and twice with 50 mM HEPES buffer
(pH 7.5) containing 1 mM dithiothreitol. The beads were
then incubated at 30 °C for 20 min in 25 µl of reaction buffer (50 mM HEPES (pH 7.5), 10 mM MgCl2, 20 µM ATP, 1 mM dithiothreitol, 2 mM
glutathione, 10 mM
-glycerophosphate, 1 mM
NaF, 0.1 mM Na3VO4) in the presence
of either 2.5 µg of histone H1 (Roche Molecular Biochemicals) or 1 µg of GST-pRb substrate (Santa Cruz Biotechnology) and 5 µCi of
[
-32P]ATP (Amersham Pharmacia Biotech) per reaction.
The reactions were stopped by the addition of 2× SDS sample buffer,
and samples were analyzed by 12.5% SDS-PAGE followed by autoradiography.
Western Blot Analysis--
Lysis buffer and immunoprecipitation
were as described above. Whole cell lysates or immunoprecipitates were
resolved by 12.5% SDS-PAGE (7.5% in the case of pRb family proteins),
the resolved proteins were transferred to nitrocellulose membranes
(Nippon Bio-Rad Laboratories) using a semidry transfer apparatus
(Nippon Bio-Rad Laboratories), and the membranes were blocked by
incubating them overnight in TBST (Tris-buffered saline (pH 7.6)
containing 5% nonfat dry milk and 0.05% Tween 20). The blots were
then rinsed three times in TBST and incubated with primary antibody for
2 h. After a wash, the nitrocellulose was incubated with a
secondary antibody conjugated to horseradish peroxidase (Amersham
Pharmacia Biotech) for 30 min. Finally, the protein was visualized by
ECL-based autoradiography as recommended by the manufacturer (Amersham
Pharmacia Biotech).
Immunodepletion--
Whole cell extracts were prepared with
Nonidet P-40 lysis buffer and subjected to four rounds of
immunodepletion, using either normal rabbit serum or a rabbit
polyclonal antibody raised against p21 (15431E; PharMingen) immobilized
on protein G-Sepharose beads. Following depletion, supernatants were
subjected to Western blot analysis as described above to show the
presence of remaining proteins.
Northern Blot Analysis--
Total cellular RNA was isolated by
using RNAzol (Biotecx Laboratories, Inc., Houston, TX), and RNA samples
(10 µg) were fractionated by electrophoresis through 1.3% agarose
gels containing 6% formaldehyde, 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA and blotted
onto a synthetic nylon membrane (Biodyne A; Pall BioSupport Corp., East
Hills, NY) in 20× SSC (1× SSC: 150 mM NaCl, 15 mM sodium citrate) and baked at 80 °C for 2 h. The
blots were prehybridized and then hybridized to a
32P-labeled antisense RNA probe for human p21 at 65 °C.
The probe was synthesized in the presence of [
-32P]UTP
(Amersham Pharmacia Biotech) using a Riboprobe Gemini II system
(Promega KK, Tokyo, Japan). As a template for the antisense RNA probe,
the full-length cDNA of human p21 (kindly provided by Dr. A. Noda,
Kobe University, Kobe, Japan) was used. The membrane was washed twice
in 2× SSC plus 0.1% SDS for 5 min and then twice in 0.1× SSC plus
0.1% SDS for 30 min at 65 °C and exposed to x-ray film at
80 °C using an intensifying screen. After detection of p21
mRNA, the membranes were rehybridized with a
32P-labeled antisense RNA probe for human
glyceraldehyde-3-phosphate dehydogenase (Ambion Inc., Austin, TX) to
verify the equal loading of RNA.
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RESULTS |
Initial Characterization of the Antiproliferative Action of IL-1 on
A375S2 Cells--
To determine the optimal IL-1 concentration to
inhibit A375S2 cell proliferation completely, exponentially growing
cells were treated with various concentrations of IL-1 for 96 h,
and the total number of live cells was counted. As shown in Fig.
1A, more than 1 ng/ml of IL-1
inhibited the cell proliferation almost completely. A trypan blue dye
exclusion test indicated little toxicity associated with this dose of
IL-1 (>90% viability after 96 h). We therefore used 1 ng/ml IL-1
for subsequent experiments. Fig. 1B shows the time course of
the antiproliferative effect of IL-1 on A375S2 cells. A slight
inhibition of cellular proliferation was apparent as early as 24 h
after IL-1 treatment, and thereafter the proliferation was inhibited
almost completely, indicating that the action of IL-1 was exerted
rapidly. To further characterize the antiproliferative action of IL-1,
an analysis of the distribution of cells in the various phases of the
cell cycle was performed by flow cytometry as a function of time
following IL-1 treatment (Fig. 2). No
change in cell cycle distribution was apparent during the first 4 h following exposure to IL-1. A decrease in the percentage of cells in
S phase was noticeable between 6 and 8 h. There was a remarkable
decrease in the S-phase fraction after 24 h (to 3% or less)
accompanied by an increase in the percentage of cells in both
G1 and G2/M. By 48 h, the percentage of
cells in S phase had decreased to <1%. In microscopic analysis of
Wright-Giemsa-stained cell preparations, no mitotic cell was observed
after 48 h of IL-1 treatment (data not shown). These results
showed that IL-1 arrested A375S2 cells at G1 and
G2 phases of the cell cycle. It was also shown that the
process resulting in the blockage at two different cell cycle points
began to take effect within the first 6-8 h and was nearly completed
by 24 h following IL-1 treatment. We therefore focused on the
mechanisms inhibiting cell cycle progression during the early phase
(<6-8 h) of IL-1 treatment.

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Fig. 1.
Effect of IL-1 on the growth of A375S2 human
melanoma cells. A, exponentially growing A375S2 cells
(1.2 × 105 cells/60-mm dish) were left untreated or
treated with various concentrations (0.001-1 ng/ml) of IL-1 for
96 h. At the end of culture, the cells were harvested and cell
numbers were determined with a cell counter (Sysmex). The cell numbers
were corrected by subtracting the 0-h values, and percentage of growth
(% Growth) was calculated: (corrected cell number of
IL-1-treated cells/corrected cell number of untreated cells) × 100. B, exponentially growing cells (1.2 × 105 cells/60-mm dish) were left untreated or treated with
1.0 ng/ml IL-1 for the indicated time. At the end of culture, the cells
were harvested, and cell numbers were determined.
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Fig. 2.
Changes in cell cycle phase distribution
following IL-1 treatment. Exponentially growing A375S2 cells were
treated at time 0 with 1.0 ng/ml IL-1. At intervals thereafter, cells
were harvested and stained for DNA content, and the relative percentage
of total cells in G1, S, and G2 + M phases of
the cell cycle were determined. Data shown are representative of three
independent experiments and are the mean values of duplicates for each
time point.
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Effects of IL-1 on Cyclin-CDK Kinase Activities--
Since CDKs
complexed with their catalytic subunit cyclins regulate the cell cycle
progression in different phases, we first investigated whether the
IL-1-induced blockage at two different cell cycle points was associated
with changes in the kinase activities of various cyclin-CDK complexes.
Results of the in vitro kinase assays using appropriate
immunoprecipitates and substrates (GST-pRb or histone H1) are shown in
Fig. 3. Cdk6- and cyclin D-associated kinase activities both elevated from 2 to 4 h following IL-1
treatment, peaked at 6-8 h, and thereafter declined and fell below
control levels at 24-48 h. Cdk4 was expressed at very low levels in
all control and IL-1-treated A375S2 cells (see below), and no kinase activities could be detected in Cdk4 immunoprecipitates (data not
shown). The levels of cyclin A-associated kinase activity did not
change significantly during the first 8 h of IL-1 treatment, but
most of the activity had disappeared after 24 h of treatment. The
kinase activities associated with the other Cdks and cyclins we tested,
namely Cdk2, Cdc2, cyclin E, and cyclin B, all decreased gradually from
4 to 8 h and almost disappeared by 24 h after IL-1 treatment.
These results demonstrate that IL-1 treatment results in a temporary
increase in cyclin D-Cdk6 activity and consistent decreases of cyclin
E-Cdk2 and cyclin B1-Cdc2 activities. It should be noted that the
decreases of cyclin E-Cdk2 and cyclin B1-Cdc2 activities were both
already detectable after 4 h of IL-1 treatment, before any changes
in cell cycle distribution could be detected (Fig. 2). The kinase
activities of cyclin E-Cdk2 and cyclin B-Cdc2 are known to be critical
for G1-S and G2-M transition, respectively. Hence, it is likely that down-regulation of cyclin E-Cdk2 and cyclin
B1-Cdc2 activities from 4 h is a mechanism of braking the cell
cycle at their respective points during the early phase of IL-1
treatment.

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Fig. 3.
Effect of IL-1 on cyclin- and CDK-associated
kinase activities. Exponentially growing A375S2 cells were treated
with 1.0 ng/ml IL-1 at time 0. At intervals thereafter, whole cell
lysates were prepared and immunoprecipitated (I.P.) with
antibodies to either Cdk6 (C-21), cyclin D (HD11), Cdk2 (M2), cyclin E
(HE111), cyclin A (H-432), Cdc2 (14391A), or cyclin B1 (H-433), and
then the kinase activity of the immunoprecipitates was determined by
phosphorylation of GST-pRb or histone H1. Phosphorylated substrates
were separated by SDS-PAGE and detected by autoradiography.
Representative data from several independent experiments are
shown.
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Effects of IL-1 on the Protein Levels of Cyclins and CDKs--
We
next examined whether the early changes in cyclin-CDK kinase activities
are attributable to changes in the protein levels of cyclins and CDKs.
Results of Western analysis are shown in Fig.
4. IL-1 did not reduce the expression of
any of the cyclins or CDKs for at least 8 h, indicating that the
changes of cyclin D-CDK6, cyclin E-Cdk2, or cyclin B1-Cdc2 kinase
activities observed in the early phase of IL-1 treatment (Fig. 3) were
not due to changes in protein expression levels of the respective
components. At 24 h, the expression of Cdk6 and cyclin D was
decreased significantly. It was further decreased at 48 h. The
expression of cyclin A, cyclin B1, and Cdc2 was also decreased markedly
at 24 h, and these components had mostly disappeared at 48 h.
The decreased expression of these components after 24 h may
correspond to the decrease in the respective cyclin-CDK activities
during the late phase of IL-1 treatment (Fig. 3). In contrast, the
protein expression of Cdk2 and cyclin E remained unchanged up to
48 h despite the complete loss of cyclin E-Cdk2 kinase activity
after 24 h of IL-1 treatment (Fig. 3). For Cdk2, however, a slower
migrating form was observed to increase at 24 and 48 h. Since the
form represents the Thr-160-dephosphorylated inactive form of Cdk2
(34), the increased dephosphorylation of Cdk2 was likely to contribute
to the down-regulation of Cdk2-associated kinase activities after 24 h of IL-1 treatment. Cdk4 was expressed at very low levels in
these cells with no detectable change in the expression following IL-1
treatment.

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Fig. 4.
Western blot analysis of cyclin and CDK
expression. Exponentially growing A375S2 cells were treated at
time 0 with 1.0 ng/ml IL-1. At intervals thereafter, whole cell lysates
were prepared, subjected to SDS-PAGE, and immunoblotted with antibodies
to either Cdk6 (C-21), Cdk4 (C-22), cyclin D (06-137), Cdk2 (M2),
cyclin E (14591A), cyclin A (BF683), Cdc2 (14391A), or cyclin B1
(H-433). The asterisk beside the Cdk2
panel indicates the more slowly migrating form of Cdk2 that
is dephosphorylated on Thr-160. Representative data from several
independent experiments are shown.
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Further experiments examined the possibility that altered association
of cyclins with their CDK partners might contribute to the changes in
cyclin-CDK kinase activities during the early phase of IL-1 treatment.
In these experiments, cyclins were immunoprecipitated, and the
immunoprecipitates were then immunoblotted for the respective CDK
partners. As shown in Fig. 5, levels of
cyclin D-associated Cdk6 elevated from 2 to 4 h following IL-1
treatment, peaked at 6-8 h, and thereafter declined. The changes in
cyclin D-Cdk6 association were similar in timing and magnitude to the
changes in cyclin D-Cdk6 kinase activity (Fig. 3), suggesting that the
temporary increase of cyclin D-Cdk6 activity between 2 and 8 h
reflected the increased formation of cyclin D-Cdk6 complex. On the
other hand, no change was observed in levels of cyclin E-associated Cdk2 over the 48 h of treatment. Levels of cyclin A-associated Cdk2 and cyclin B1-associated Cdc2 were unaltered for the first 8 h following IL-1 treatment, but thereafter both declined to low levels
at 24 h, paralleling the temporal changes in total protein levels
of cyclin A, cyclin B1, and Cdc2 (Fig. 4).

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Fig. 5.
Effect of IL-1 on cyclin-CDK complex
formation. Exponentially growing A375S2 cells were treated at time
0 with 1.0 ng/ml IL-1. At intervals thereafter, whole cell lysates were
prepared, and the levels of cyclin D-bound Cdk6, cyclin E- or A-bound
Cdk2, and cyclin B1-bound Cdc2 were examined by immunoprecipitation
with anti-cyclin D (HD11), anti-cyclin E (HE111), anti-cyclin A
(H-432), and anti-cyclin B1 (H-433) antibodies and subsequent
immunoblotting with antibodies for Cdk6 (C-21), Cdk2 (M2), and Cdc2
(14391A). Representative data from three independent experiments are
shown.
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Effects of IL-1 on the Expression of CDK Inhibitors--
As
described above, the early decreases in cyclin E-Cdk2 and cyclin
B1-Cdc2 activities were not accompanied by decreases in the expression
of respective cyclins or CDKs or by decreases in their complex
formation (Figs. 3-5). Then, to investigate other possible causes of
the decrease in kinase activities, the expression of various CDK
inhibitors was examined by Western analysis. Data in Fig.
6 show that IL-1 treatment resulted in a
rapid induction of p21. The protein levels of p21 began to increase in
the first 2 h of treatment and continued to increase up to 48 h. On the other hand, the protein levels of p27, which was expressed at very low levels in untreated A375S2 cells, remained unchanged during
the early phase of IL-1 treatment, and an obvious increase was observed
only after 24 h of treatment. The expression of other CDK
inhibitors, p57, p15, p16, p18, and p19, was only detected at very low
levels in cells that were untreated or treated with IL-1 (Fig. 6).

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Fig. 6.
Western blot analysis of CDK-inhibitor
expression following IL-1 treatment. Exponentially growing A375S2
cells were treated at time 0 with 1.0 ng/ml IL-1. At intervals
thereafter, whole cell lysates were prepared, subjected to SDS-PAGE,
and immunoblotted with antibodies to either p21 (15431E), p27 (K25020),
p57 (C-20), p16 (C-20), p15 (K-18), p18 (N-20), or p19 (M-167).
Representative data from two independent experiments are shown.
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To determine whether the increase of p21 protein expression is
regulated at the transcriptional level, p21 mRNA was quantitated by
Northern blot analysis using a full-length antisense RNA probe for
human p21. As shown in Fig. 7, a small
increase in p21 mRNA levels was detectable within 30 min of IL-1
treatment, and the levels continued to increase up to 48 h,
paralleling the changes in p21 protein levels. These results indicated
that the IL-1-induced increase in p21 protein levels is regulated
transcriptionally at least in part. p21 expression is known to be under
the control of both p53-dependent and p53-independent
mechanisms. To examine whether p53 protein contributes to the
IL-1-mediated induction of p21, the protein levels of p53 were analyzed
after treatment of cells with IL-1. As shown in Fig. 7, p53 protein was
barely detectable in normal growing A375S2 cells, and no change was
observed in its expression levels following IL-1 treatment. On the
other hand, when we treated A375S2 cells with camptothecin, a
topoisomerase inhibitor that induces double-stranded DNA-breaks, we
observed the induction of both p53 and p21 proteins, indicating that
the function of p53, required for induction of p21 by DNA damage, is
retained in these cells (data not shown). Thus, the present results
suggested that IL-1 induces p21 independently of p53.

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Fig. 7.
IL-1 induces p21 mRNA expression
independently of p53. Exponentially growing A375S2 cells were
treated at time 0 with 1.0 ng/ml IL-1. A, total RNA was
prepared from the cells and examined by Northern blot analysis using an
antisense RNA probe for p21. The Northern blot was subsequently probed
with an antisense RNA probe for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) to verify the equal loading of RNA.
B, whole cell lysates were prepared, subjected to SDS-PAGE,
and immunoblotted with antibodies to p53 (Ab-2).
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Association of p21 with Cyclin-CDK Complexes--
To determine
whether p21 is responsible for the inhibition of cyclin E-Cdk2 and
cyclin B-Cdc2 complexes during the early phase of IL-1 treatment, we
next investigated whether there was any change in the association of
p21 with cyclin-CDK complexes after IL-1 treatment. Cell extracts were
immunoprecipitated with antibodies against various cyclins or CDKs and
Western blotted for p21 (Fig. 8). We
found that a certain amount of p21 was coimmunoprecipitated with
anti-Cdk6, -Cdk2, -cyclin D, -cyclin E, and -cyclin A antibodies even
from extracts of untreated growing A375S2 cells. The relative amount of
p21 associated with cyclin E was increased gradually from 4 to 48 h following treatment with IL-1. Cyclin A-associated p21 was increased
slightly from 4 to 8 h, although cyclin A-Cdk2 kinase activity was
not changed during the early phase of IL-1 treatment (Fig. 3).
Cdk2-associated p21 increased up to 8 h and thereafter decreased
gradually, consistent with the observed changes in the p21 association
with cyclins E and A, partners of Cdk2. For Cdc2 and cyclin B1, a p21
association was not detectable in untreated growing cells but became
detectable faintly at 2-4 h following IL-1 treatment and thereafter
tended to increase slightly up to 24 h. These findings indicate
that the level of p21 associated with cyclin E-Cdk2, cyclin A-Cdk2, and
cyclin B1-Cdc2 was increased during the early phase of IL-1
treatment.

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Fig. 8.
Association of p21 with cyclin-CDK
complexes. Exponentially growing A375S2 cells were treated with
1.0 ng/ml IL-1 at time 0. At intervals thereafter, whole cell lysates
were prepared and immunoprecipitated (I.P.) with antibodies
to either Cdk6 (C-21), cyclin D (HD11), Cdk2 (M2), cyclin E (HE111),
cyclin A (H-432), Cdc2 (14391A), or cyclin B1 (H-433), and the
immunoprecipitates were subjected to SDS-PAGE and immunoblotted with
antibodies for p21 (C-19). Representative data from three independent
experiments are shown.
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We next asked whether the increased level of p21 associated with these
cyclin-CDK complexes was sufficient to inhibit their kinase activities.
To answer this question, we determined what fraction of each cyclin-CDK
complex was associated with p21 by immunodepletion experiments. Cell
extracts from untreated cells or from cells treated with IL-1 for
8 h were immunodepleted by three successive rounds of
immunoprecipitation with either anti-p21 antiserum or normal rabbit
serum as a control. What remained in the depleted extracts was then
examined by immunoblotting with specific antibodies against cyclins and
CDKs (Fig. 9). Quantitation of p21 levels
indicated that anti-p21 antiserum successfully removed p21 from control
or IL-1-treated extracts. We found that nearly all of cyclin E was
depleted with anti-p21 antiserum not only from IL-1-treated extracts
but also from control extracts, indicating that all of the cyclin
E-Cdk2 complex was already bound by p21 before treatment with IL-1 in
A375S2 cells. This finding was surprising in light of the fact that
cyclin E-Cdk2 complex is active in growing cells and that the amount of
p21 bound to cyclin E-Cdk2 increased still further following IL-1
treatment (Fig. 8). A possible interpretation of these findings is that
p21 can associate with the cyclin E-Cdk2 complex in a noninhibitory
mode and that multiple molecules of p21 can associate with the complex.
Indeed, it has been proposed that the first molecule of p21 that
associates with a cyclin-CDK complex does not inhibit its activity and
that the binding of a second p21 molecule is required to inhibit its
kinase activity (35, 36). For Cdk2, a small proportion of the protein
was depleted from both control and IL-1-treated extracts, consistent with the fact that Cdk2 is expressed in excess over cyclin E and A in
the cell and that the binding affinity of p21 to free Cdk2 is much
lower than to cyclin-Cdk2 complex (35, 37-39). In contrast with the
case of cyclin E, no depletion of cyclin A, cyclin B1, or Cdc2 was
observed either before or after treatment with IL-1, suggesting that
only a minor portion of the cyclin A-Cdk2 or cyclin B-Cdc2 complexes
was associated with p21 even after IL-1 treatment. On the basis of
these results, we conclude that p21 binds preferentially to cyclin
E-Cdk2 complex and that the increased level of p21 after IL-1 treatment
is effectual for the inhibition of cyclin E-Cdk2 but has little or no
effect on cyclin A-Cdk2 and cyclin B1-Cdc2.

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Fig. 9.
p21 binds preferentially to almost all of the
cyclin E-Cdk2 complexes after and even before IL-1 treatment.
Whole cell lysates were prepared from A375S2 cells before (time 0) or
8 h after treatment with IL-1 (1.0 ng/ml). The cell lysates were
immunodepleted with normal rabbit serum (NRS) or anti-p21
antiserum (Ab-1). The supernatants after immunodepletion were then
subjected to SDS-PAGE and immunoblotted with antibodies against p21
(C-19), Cdk6 (C-21), cyclin D (HD11), Cdk2 (M2), cyclin E (HE111),
cyclin A (H-432), Cdc2 (14391A), and cyclin B1 (H-433) as indicated.
Representative data from several independent experiments are
shown.
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We also found that the relative amount of Cdk6- and cyclin D-associated
p21 increased 2-4 h following IL-1 treatment, peaked at 6-8 h, and
thereafter declined (Fig. 8). Immunodepletion experiments performed
with anti-p21 antiserum verified that a certain proportion of cyclin D,
possibly complexed with Cdk6, was indeed associated with p21 in cells
treated with IL-1 for 8 h. These findings were surprising since,
as seen in Fig. 3, the kinase activity of cyclin D-Cdk6 increased
during the early phase of IL-1 treatment. It is noteworthy that the
time kinetics of the increase in the association of p21 with cyclin
D-Cdk6 complexes (Fig. 8) coincided with the increase of kinase
activity (Fig. 3), resulting probably from the increase of complex
formation between cyclin D and Cdk6 (Fig. 5). These findings may be
explained by the recent suggestion that p21 acts not only as a CDK
inhibitor but also as an assembly factor for cyclin-CDK complex
formation (36, 40).
IL-1 Induces Tyrosine Phosphorylation of Cdc2--
Another
important mechanism for controlling CDK activity is phosphorylation of
conserved threonine and tyrosine residues of CDKs. The kinase activity
of both Cdk2 and Cdc2 is regulated negatively by phosphorylation of
Tyr-15 and Thr-14 (34, 41). To examine the possibility that such
phosphorylation might contribute to the inhibition of Cdk2 and Cdc2
during the early phase of IL-1 treatment, we analyzed the relative
content of phosphotyrosine in these two CDKs by immunoblotting with an
anti-phosphotyrosine antibody. As shown in Fig.
10, no tyrosine phosphorylation was detected in Cdk2 immunoprecipitated with anti-cyclin E antibodies from
either the control or IL-1-treated cell extracts. On the other hand,
phosphotyrosine was detected at low levels in cyclin B1-associated Cdc2
before treatment with IL-1, and the levels increased gradually from 4 to 8 h following IL-1 treatment (Fig. 10), coincident with the
observed decrease in cyclin B1-Cdc2 kinase activity (Fig. 3). These
findings suggest that Tyr-15 phosphorylation is a mechanism that
inhibits the activity of cyclin B1-Cdc2 but is not involved in the
inhibition of cyclin E-Cdk2 during the early phase of IL-1
treatment.

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Fig. 10.
Analysis of tyrosine phosphorylation of Cdk2
and Cdc2. Exponentially growing A375S2 cells were treated with 1.0 ng/ml IL-1 at time 0. At intervals thereafter, whole cell lysates were
prepared and cyclin E-bound Cdk2 and cyclin B1-bound Cdc2 were
coimmunoprecipitated with antibodies to cyclin E (HE111) and cyclin B1
(H-433), respectively. The immunoprecipitates were resolved by SDS-PAGE
and immunoblotted with antibodies against phosphotyrosine
(p-Tyr; PY99). Representative data from two independent
experiments are shown.
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Analysis of Phosphorylation Status of pRb Family Proteins--
As
shown above, the kinase activity of cyclin E-Cdk2 started to decrease
4 h following IL-1 treatment (Fig. 3). The best studied G1 cyclin-CDK substrate is the product of the
retinoblastoma tumor suppresser gene, pRb. It has become clear that pRb
is a negative regulator that acts in the G1 phase of the
cell cycle, and its activity appears to be modulated by
phosphorylation. Then we determined whether pRb underwent
dephosphorylation at times compatible with the decrease in cyclin
E-Cdk2 activities. The reduction in mobility of pRb on SDS-PAGE and
Western blot analysis is a widely accepted indicator of pRb
phosphorylation. In asynchronously growing A375S2 cells, pRb was found
in both hyper- and hypophosphorylated forms, which migrated as multiple
bands in the molecular mass range of 110-115 kDa (Fig.
11; detected by an antibody recognizing
a pRb C-terminal protein). Contrary to our expectation, no change in the proportions of hyper- and hypophosphorylated pRb was recognized in
the early phase (up to 8 h) of IL-1 treatment, and disappearance of the slow-migrating hyperphosphorylated forms of pRb was observed only 24 h after treatment (Fig. 11). Then, we next investigated whether IL-1 affected the phosphorylation state of the two known pRb-related proteins, p107 and p130. As for the p107 protein, a slight
downward mobility shift, which seemed to represent the dephosphorylation of the protein, was first seen at 4 h of IL-1 treatment, and a more marked shift was evident at 8 h (Fig. 11). The protein expression level of p107 was not affected during the first
8 h of treatment, but after 24 h, when the vast majority of
cells have exited the cell cycle, the p107 protein almost disappeared. On the other hand, p130 was present as both hyper- and
hypophosphorylated forms in asynchronously growing control cells. In
contrast with p107, the total protein level of p130 was increased by
treatment with IL-1. The increase was first seen at 6 h and peaked
at 24 h. Interestingly, independent of the increase in the total
protein levels, the ratio between the hyper- and hypophosphorylated
forms of p130 was constant during the first 8 h, and a decrease in
the hyperphosphorylated form with an accumulation of the
hypophosphorylated form was observed only at the 24- and 48-h time
points. Taken together, these results suggest that IL-1 exerts its
growth-inhibitory effects not through pRb but by regulating the
phosphorylation state of p107 and the abundance of p130.

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Fig. 11.
Western blot analysis of phosphorylation
status of pRB family proteins. Exponentially growing A375S2 cells
were treated at time 0 with 1.0 ng/ml IL-1. At intervals thereafter,
whole cell lysates were prepared, subjected to SDS-PAGE, and
immunoblotted with antibodies to either pRb (9032), p107 (C-18), or
p130 (R27020). The positions of hypophosphorylated proteins
(pRb, p107, and p130) and
hyperphosphorylated proteins (ppRb, pp107, and
pp130) are indicated. Representative data from two
independent experiments are shown.
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DISCUSSION |
This study represents an extensive analysis of the potential roles
of the cell cycle machinery in IL-1-induced growth arrest of A375S2
human melanoma cells. The first important observation in this study is
that IL-1 arrested A375S2 cells at both G1 and G2 phases of the cell cycle, suggesting that IL-1 targets a
molecule(s) critical for progression through G1 (or
G1-S) and G2 (or G2-M). In the
previous study (14), in some contrast with our data, A375 cells, which
are less sensitive to IL-1 than A375S2 cells (33), were shown to be
arrested by IL-1 at G1 phase but not at G2
phase, although IL-1 did retard progression of A375 cells through
G2-M. Another A375-derived IL-1-sensitive clone named A375-C6 has been reported to be arrested by IL-1 at
G0/G1 phase, but no evidence for blockage or
retardation at G2 phase was obtained (15). It therefore
appears likely that the type of arrest induced by IL-1 varies with the
clone of the A375 cell line. The cell cycle analysis of A375S2 cells
also showed that IL-1 started to brake cell cycle progression at
G1 and G2 phases within the first 6-8 h of
treatment, and the blockage at the two phases was nearly completed by
24 h. To elucidate the molecular mechanism of the IL-1 action, it
is of importance to distinguish the induced changes in cell
cycle-regulating molecules causing growth arrest from those that are a
consequence of the cell cycle arrest. We therefore focused on the
changes and/or modulations of cell cycle-regulating molecules observed
during the early phase (<6-8 h) of IL-1 treatment before any changes
in cell cycle distribution could be detected. We found that the kinase
activities of cyclin E-Cdk2 and cyclin B-Cdc2 complexes both started to
decrease as early as 4 h after treatment with IL-1, suggesting
that these changes were causes rather than consequences of the
inhibition of cell cycle progression. Since cyclin E-Cdk2 and cyclin
B1-Cdc2 complexes have been shown to have crucial roles in
G1-S and G2-M transition, respectively, it is
likely that the IL-1-induced rapid down-regulation of cyclin E-Cdk2 and
cyclin B1-Cdc2 activities is a mechanism of braking cell cycle
progression at their respective points. On the other hand, the kinase
activity of cyclin A-Cdk2, whose function is essential in S phase,
showed no change during the first 8 h of IL-1 treatment.
Consistent with this, no evidence for blockage in S phase was obtained
during the early phase of IL-1 treatment. After 24 h, when the
vast majority of cells have exited the cell cycle, the kinase activity
of cyclin A-Cdk2, as well as that of cyclin E-Cdk2 and cyclin B1-Cdc2,
disappeared completely. The complete loss of these cyclin-CDK
activities after 24 h may be a secondary response reflecting the
cessation of cell cycle and maintenance of cells at G1 and
G2 phases. The kinase activity of cyclin-CDK complexes can
be regulated in a number of ways (see Ref. 42 and references therein).
In this study, examination of potential mechanisms for the IL-1-induced
rapid down-regulation of cyclin E-Cdk2 and cyclin B1-Cdc2 activities
revealed that the CDK inhibitor p21 is likely to be responsible for the
inhibition of cyclin E-Cdk2 activity, whereas an increase in tyrosine
phosphorylation of Cdc2 may be involved in the inhibition of cyclin
B1-Cdc2 activity.
p21 has been shown to inhibit a wide variety of cyclin-CDK complexes
including G1 cyclin/CDK and has been implicated in
G1 arrest following DNA damage (37, 43) in response to
negative growth factors, such as transforming growth factor-
(44,
45) or interferon-
(46), and in maintenance of terminally
differentiated cells in a nonproliferative state (47, 48). Our results
provide the following evidence to support a possible role for p21 in
IL-1-mediated inhibition of cyclin E-Cdk2 activity causing cell cycle
arrest at G1. First, p21 induction is the first change to
be observed, occurring well before the start of cell cycle arrest. In
contrast with p21, the p21-related CDK inhibitor p27, which was
expressed at very low levels in proliferating A375S2 cells, was induced only after 24 h of IL-1 treatment. Other CDK inhibitors examined, including p57, p15, p16, p18, and p19, were not induced at all by IL-1.
Second, the increase in p21 expression levels is paralleled by an
increased binding of p21 to cyclin E-Cdk2 complex, coinciding with the
decrease of kinase activity of the complex. Although the binding of p21
to cyclin A-Cdk2 and cyclin B1-Cdc2 complexes also increased in
parallel with the increase in p21 expression levels, immunodepletion
experiments revealed that the vast majority of the cyclin A-Cdk2 and
cyclin B1-Cdc2 complexes were still free from p21 after IL-1 treatment,
indicating that the increased binding of p21 to these cyclin-CDK
complexes after IL-1 treatment is not sufficient in quantity to inhibit
the activity of these complexes. In contrast, all of the cyclin E-Cdk2
complex was found to be complexed with p21 after and even before IL-1
treatment. At first glance, this observation appears to be in conflict
with the fact that cyclin E-Cdk2 complex is active in growing cells and
that the relative amount of p21 bound to the complex increased still further following IL-1 treatment. The apparent paradox, however, is
resolved by proposing that p21 can associate with cyclin E-Cdk2 complex
in a noninhibitory mode and that multiple molecules of p21 can
associate with the complex. This is fully consistent with the idea that
the first molecule of p21 that associates with a cyclin-CDK complex
does not inhibit its activity and that the binding of a second p21
molecule is required to inhibit its kinase activity (35, 36). Based on
this idea, it has been proposed that cyclin-CDK complexes become
maximally sensitive to increases in p21 levels by the binding of one
p21 molecule to each complex (35). This is consistent with our
observation that the decrease of cyclin E-Cdk2 activity occurred
quickly, coinciding with the rapid increase in p21 expression levels
following IL-1 treatment. A similar observation has been reported in
normal human fibroblasts, where all of the active cyclin E-Cdk2 complex
is associated with p21, and the active complex containing p21 can be
inhibited by up-regulated p21 following UV-induced DNA damage (37).
This phenomenon was also explained based on the stoichiometric
inhibitory action of p21.
Although p21 is known as a universal CDK inhibitor, recent reports
provide evidence that prime targets of p21 for inhibition are cyclin
E/A-Cdk2 complexes in vivo (37, 49, 50). Consistent with
this, our observations suggest that IL-1-induced p21 is a potent
inhibitor of cyclin E-Cdk2 but not effective for cyclin D-Cdk6 or
cyclin B1-Cdc2. However, our observations also suggest that the induced
p21 has little inhibitory effect even on cyclin A-Cdk2. This appears to
be associated with the finding that all of the cyclin E-Cdk2 complex,
but only a minor portion of the cyclin A-Cdk2 complex, was already
associated with p21 before treatment with IL-1. Such a physical
association of p21 with active cyclin E-Cdk2 but not with cyclin A-Cdk2
in growing cells is also observed in normal human fibroblasts (37) and
in certain cell lines (51, 52). These observations suggest that at
least in these cells and cell lines, p21 binds to cyclin E-Cdk2 complex with much higher affinity than to cyclin A-Cdk2 complex, and thus p21
is a more effective inhibitor of cyclin E-Cdk2 than of cyclin A-Cdk2
in vivo. It remains to be clarified, however, whether the relative affinity of p21 to cyclin E- and cyclin A-Cdk2 complexes varies with the cell type.
While this work was in progress, Nalca and Rangnekar (53), using
another A375-derived IL-1-sensitive clone, A375-C6, showed that IL-1
caused rapid induction of p21 at the mRNA and protein level.
However, they have proposed that p21 does not play an important role in
the growth-arresting effect of IL-1, since they found that inhibition
of p21 expression by the antisense construct resulted in only a
marginal rescue from the growth-arresting action of IL-1. This view
stands in contrast with the present results suggesting that the rapid
induction of p21 is of major importance in the action of IL-1 in A375S2
cells. This discrepancy may be attributable to a difference in the
steadiness of the IL-1-induced increase in p21 protein levels between
the two clones of A375 cells used. In A375-C6 cells, Nalca and
Rangnekar (53) observed a rapid but transient increase (peaked at
3 h) in p21 protein expression levels following IL-1 treatment, in
contrast to our data showing that IL-1 caused a rapid and sustained
increase (up to 48 h) in p21 levels in A375S2 cells. Thus, in
A375-C6 cells, the transient induction of p21 may not be sufficient to
cause cell cycle arrest, and other mechanisms that control cell
proliferation may be responsible for the arrest. Alternatively, it is
conceivable that compensatory regulation mechanisms become operative in
the absence of p21. In addition, it is also possible that the
regulatory functions of p21 are redundant.
An unexpected finding of this study was the increase in cyclin D-Cdk6
activity during the early phase of IL-1 treatment. The increase was
paralleled by the increase in the relative amount of p21-bound cyclin
D-Cdk6 complex, coinciding with the increase in cellular p21 levels. A
possible interpretation of these findings is that p21 facilitates the
association of the complex formation between cyclin D and Cdk6 without
hindering the function of the complex. This is consistent with the idea
that besides simply inhibiting kinase activity, members of the p21
family can promote the association of CDKs with cyclins and thus
stabilize cyclin-CDK complexes (36, 40). There is increasing evidence
that such a role of p21 is crucial during assembly of cyclin-CDK
complexes. For instance, recent data suggest that p21 promotes the
association of D-type cyclins with CDKs by counteracting the effects of
the Ink4 family of CDK inhibitors (54, 55). Thus, it may be concluded that the increase in cyclin D-Cdk6 activity during the early phase of
IL-1 treatment mirrored the increased assembly of the active cyclin
D-Cdk6-p21 complexes driven by the increase in cellular p21 levels. In
this context, the possibility that this novel function of p21 also
contributes to the formation of active cyclin E-Cdk2-p21 ternary
complexes in growing A375S2 cells may be admitted.
In addition to the inhibition by CDK inhibitors, phosphorylation of
conserved threonine and tyrosine residues near the ATP binding sites of
CDKs (Thr-14 and Tyr-15 on both Cdk2 and Cdc2) is also an important
mechanism employed to keep the CDKs inactive (34, 41). Our data showed
that there was an increase in phosphotyrosine content in Cdc2 but not
in Cdk2 during the early phase of IL-1 treatment. Thus, it is
conceivable that the increase in Tyr-15 phosphorylation is a mechanism
that inhibits the cyclin B1-Cdc2 but is unrelated to the decrease in
cyclin E-Cdk2 activity during the early phase of IL-1 treatment.
Phosphorylation of Tyr-15 on Cdc2 is controlled by the opposing
activities of the Wee1/Myt1 kinases (56-58) and Cdc25 phosphatase
(59). Thus, the IL-1-induced increase in Tyr-15 phosphorylation on Cdc2
could be due to an increase in Wee1/Myt1 kinase activity and/or due to
a decrease in Cdc25 phosphatase activity. Both Myt1 and Cdc25 also
control phosphorylation of Thr-14 on Cdc2. It seems possible,
therefore, that IL-1 causes an increase in phosphorylation of Thr-14,
as well as Tyr-15, on Cdc2, thereby inhibiting cyclin B-Cdc2 activity. Furthermore, we do not exclude the possibility that other
post-translational modifications, such as a decrease in Thr-161
phosphorylation on Cdc2, are also involved in the down-regulation of
cyclin B1-Cdc2 activity during the early phase of IL-1 treatment.
Recently, it has been reported that growth inhibition of A375-C6 human
melanoma cells by IL-1 is mediated at least in part by the suppression
of pRb phosphorylation to retain pRb in an unphosphorylated,
growth-inhibitory state (15). In the present study using A375S2 cells,
however, a shift in electrophoretic mobility of pRb to a
hypophosphorylated form was not observed until 24 h after IL-1
treatment, when cell cycle arrest had already been completed. This
finding suggests that the observed suppression of pRb phosphorylation
at the 24-h time point is the result of IL-1-induced growth arrest, and
thus pRb is not a functional mediator of IL-1 action in A375S2 cells.
However, since the kinase activity of cyclin E-Cdk2, which is one of
the candidates for pRb kinases in vivo, was inhibited in the
early phase (<8 h) of IL-1 treatment, we cannot fully rule out the
possibility that the early inhibition of cyclin E-Cdk2 activity caused
dephosphorylation of a few phosphorylation sites in pRb that could not
appreciably alter the electrophoretic mobility of pRb. In respect to
the substrate of cyclin E-Cdk2 kinase, several lines of evidence
indicate that cyclin E-Cdk2 catalyzes events that are rate-limiting for
the G1/S transition that are independent of pRb
phosphorylation. For example, cyclin E, but not cyclin D, is essential
for entry into S phase in mammalian cells that lack a functional pRb
(60-62). In vitro phosphorylation by Cdk2 kinases had
little effect on pRb function in a microinjection-based in
vivo cell cycle assay (63). Thus, it is suggested that cyclin E-Cdk2 phosphorylates key substrates other than pRb. Consistent with
this hypothesis, here we found that phosphorylation of the pRb-related
protein p107 was suppressed from 4 h following IL-1 treatment,
coincident with the inhibition of cyclin E-Cdk2 kinase activity,
suggesting that the suppression of p107 phosphorylation is mediated
through inhibition of cyclin E-Cdk2 activity by IL-1. It is possible,
therefore, that p107 acts as a downstream target of the IL-1-induced
arrest pathway. We also found that the total protein level of another
pRb-related protein, p130, gradually increased in the early phase of
IL-1 treatment, whereas the ratio between the hyper- and
hypophosphorylated forms of p130 was constant. This finding suggests an
additional function of IL-1, up-regulation of the p130 protein level,
which also plays an important role in IL-1-induced G1 arrest.
Both p107 and p130, like pRb, can regulate cell proliferation, and
there are a number of cell systems where the retinoblastoma family
members have been involved in eliciting cell cycle arrest. The growth
arrest mediated by the three pocket proteins are however not identical
(64, 65), and their relative importance varied with the
antiproliferative agent and the cell type employed in the arrest
system. For example, it has been shown that pRb plays a central role in
cell cycle arrest after DNA damage, whereas p107 and p130 are
dispensable for this process (66). Although all of the pRb family
members have been reported to be associated with transforming growth
factor-
-induced G1 arrest (67-69), recent experiments
suggest that p130 is the major downstream target in transforming growth
factor-
-regulated growth arrest in gastric-carcinoma cells (45).
Muthukkumar et al. (15) have suggested that growth arrest of
A375-C6 melanoma cells by tumor necrosis factor-
is mediated by a
pRb-independent pathway, whereas that by IL-1 is dependent on pRb
function. Koudssi et al. (70) have also shown that pRb is
involved in IL-1-induced G1/S arrest of rat cardiac fibroblasts, although they have not addressed the involvement of p107
or p130. Our data, while in contrast, suggest that IL-1 causes cell
cycle arrest not through pRb but by regulating the phosphorylation
state of p107 and the abundance of p130. The discrepancy implies that
the pRb family proteins involved in IL-1-induced cell cycle arrest
could vary with cell lines examined. Although these pRb family proteins
share considerable sequence homology, each protein has been shown to
have a different temporal profile of interaction with different E2F
family members. The p107 and p130 proteins bind specifically to E2F4
and E2F5 (71-73), whereas pRb interacts with each of the E2F family
members (74, 75). pRb seems to bind preferentially to E2F in middle to
late G1 and S phases, and p107 forms complexes with E2F
predominantly in late G1 and S phases, while
p130-E2F complexes accumulate when cells exit from the cell
cycle. Of note, p130 has been suggested to function as the major pocket
protein during various differentiation programs (76-78), and the
protein is expressed at high levels in terminally differentiated cells,
such as neurons and skeletal muscle (79). It is tempting, therefore, to
speculate that the p130 protein induced by IL-1 plays a crucial role in
initiation and maintenance of the growth-arrested state. Although the
precise role of each individual pRb family protein is not addressed
here, the data presented provide a new clue that should help in future investigations of the molecular mechanisms underlying the
antiproliferative effect of IL-1.
In conclusion, our findings demonstrate that IL-1 inhibits the growth
of A375S2 cells by arresting them at G1 and G2
phases of the cell cycle, and the arrests are preceded by a rapid
decrease in cyclin E-Cdk2 and cyclin B1-Cdc2 kinase activities. Thus,
the rapid down-regulation of these two cyclin-CDK activities is likely to be the mechanism responsible for the cell cycle arrest at
G1 and G2 phases, respectively. The CDK
inhibitor p21 is likely to be responsible for the inhibition of cyclin
E-Cdk2 activity, whereas an increase in tyrosine phosphorylation of
Cdc2 may be involved in the inhibition of cyclin B1-Cdc2 activity.
Furthermore, we found that IL-1 causes rapid dephosphorylation of p107,
but not of pRb or p130, while the total protein levels of p130 are
increased. Thus, IL-1 may exert its growth-arresting effects not
through pRb but by regulating the phosphorylation state of p107 and the abundance of p130. It will be important in our future studies to define
the upstream signal transduction pathway mediating the IL-1 action.