p57Kip2, a Glucocorticoid-Induced Inhibitor of Cell Cycle Progression in HeLa Cells
Magnus K. R. Samuelsson,
Ahmad Pazirandeh,
Behrous Davani and
Sam Okret
Department of Medical Nutrition Karolinska Institute
Huddinge University Hospital, Novum F60 SE-141 86 Huddinge,
Sweden
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ABSTRACT
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Glucocorticoids exert antiproliferative effects on
a number of cell types, including the HeLa cervical carcinoma cell
line. However, the mechanism responsible for the antiproliferative
effect is poorly understood. In this report we have investigated the
role of the recently identified cyclin-dependent kinase inhibitor (CDI)
p57Kip2 in the antiproliferative effect
conferred by glucocorticoids. When HeLa cells were treated with the
synthetic glucocorticoid dexamethasone (DEX), the doubling time of
exponentially growing cells increased 2-fold. Within 11 h of DEX
treatment, this was accompanied by an accumulation of cells in the
G1 phase of the cell cycle with a corresponding
decreased proportion of cells in the S phase and decreased CDK2
activity. DEX treatment of the HeLa cells dramatically induced the
protein and mRNA expression of the CDI p57Kip2.
This induction was seen within 4 h of DEX treatment, preceding a
major DEX-induced accumulation of cells in the
G1 phase. DEX-induced mRNA expression of
p57Kip2 did not require de novo
protein synthesis, and the transcription of the
p57Kip2 gene was increased as determined by a
run-on transcription assay. Furthermore, DEX induction of
p57Kip2 was not a consequence of the cell
cycle arrest, since other growth inhibition signals did not result in
strong p57Kip2 induction. Overexpression of
p57Kip2 using HeLa cells stably transfected
with a tetracycline-inducible vector showed that
p57Kip2 is sufficient to reconstitute an
antiproliferative effect similar to that seen in DEX-treated cells.
Selective p57Kip2 expression by the
tetracycline analog doxycycline to levels comparable to those observed
on DEX induction resulted in a 1.7-fold increase in the doubling time
and a shift of HeLa cells to the G1 phase as
well as a decrease in CDK2 activity. Taken together, these results
suggest that glucocorticoid treatment directly induces transcription of
the p57Kip2 gene and that the
p57Kip2 protein is involved in the
glucocorticoid-induced antiproliferative effect.
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INTRODUCTION
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Glucocorticoids, produced in the adrenal cortex, are steroid
hormones that play important roles in metabolism, immune responses, and
cellular proliferation and differentiation (1). They bind to the
intracellular glucocorticoid receptor (GR), a ligand-activated
transcription factor. When liganded, GR interacts with specific DNA
sequences, so-called glucocorticoid response elements (GREs) in target
genes, increasing or decreasing their transcription. GR can also modify
the activity of other transcription factors, e.g. AP-1 and
nuclear factor-
B (NF-
B), through protein-protein
interactions (reviewed in Ref. 2).
Glucocorticoids inhibit cell division in many tissues and cells,
including those of lymphoid, fibroblastic, epithelial, and bone origin
(1). Glucocorticoids have also been shown to inhibit proliferation of
several cell lines, normally by causing an arrest in the
G1-phase of the cell cycle (3, 4, 5, 6, 7). However, the exact
molecular mechanisms responsible for this effect remain unclear.
The propagation of the cell cycle is positively regulated by the action
of a family of serine/threonine kinases called cyclin-dependent kinases
(CDKs). Different CDKs are required in each phase of the cell cycle,
and they are positively regulated after binding to regulatory subunits,
cyclins, that are necessary for CDK activity. There are also several
phosphorylation events that modulate the activity of CDKs (for review,
see Ref. 8). Two families of proteins associate with specific
CDK-cyclin complexes and inhibit their activity, thus acting as CDK
inhibitors (CDIs). The first family, the Ink4 family, consists of four
members (p15, p16, p18, and p19). These proteins specifically inhibit
the activity of G1 phase CDK4 or -6 cyclin D complexes,
preventing entry into the S phase. The second family, the Cip/Kip
family, has three members (p21, p27, and p57). To various extents, the
proteins in this family bind to and inhibit CDK-cyclin complexes in all
phases of the cell cycle (reviewed in Ref. 9).
p21Cip1 and p57Kip2 also inhibit DNA
replication in a more direct way by binding to the proliferating cell
nuclear antigen (10, 11, 12, 13). p21Cip1,
p27Kip1, and p57Kip2 are highly expressed in
differentiated cells and tissues (14, 15, 16, 17). Despite this, mice lacking
p21Cip1 develop normally (18). This is also true for
p27Kip1-deficient mice, which develop essentially normally
except for a general increase in body size, female sterility, and an
increased frequency in pituitary tumorigenesis (19, 20, 21).
p57Kip2-deficient mice, on the other hand, display several
severe and lethal phenotypes including changes in the gastrointestinal
tract, shortened limbs, adrenal cortical hyperplasia, and abdominal
muscle defects (22, 23). Interestingly, it has also been reported that
p57Kip2 expression is reduced in some human malignancies
such as lung tumors and adrenocortical carcinomas (24, 25).
p57Kip2 is thus clearly important in normal development and
may act as a tumor suppressor.
Inhibition of proliferation by several different families of hormones
is mediated or correlated to an induction of CDIs. For instance,
transforming growth factor-ß (TGFß) induces p15Ink4B
and p21Cip1 in human HaCaT keratinocyte cells (26, 27), and
progesterone induces p21Cip1 and p27Kip1 in
human T47D-YB breast cancer cells (28). Vitamin D3 induces
p21Cip1 in human U937 monocyte-like cells (29) and
p27Kip1 in human HL60 leukemia cells (30). In human
SMS-KCNR neuroblastoma cells, retinoic acid increases protein
expression of p27Kip1 (31), while interferon-
induces
p21Cip1 in Daudi and U-266 lymphoid cells and
p15Ink4B in U-266 cells, respectively (32). Although little
is known about the direct mechanism for the glucocorticoid-induced
antiproliferative effects, it has been shown that glucocorticoids
inhibit the expression of cyclin D3 and the protooncogene
c-myc in P1798 murine T lymphoma cells (5, 33). This study
also showed that overexpression of cyclin D3 and c-myc
together was sufficient to overcome this antiproliferative block.
Glucocorticoid-induced cell cycle block may also be due to an increased
level of CDIs. In mouse L929 fibroblastic cells, rat BDS1 epithelial
hepatoma cells, and rat lung alveolar epithelial cells, glucocorticoids
induce the CDK inhibitor p21Cip1 (7, 34, 35, 36). This
regulation has been shown to involve both transcriptional and
posttranscriptional mechanisms. In a study by Rogatsky et
al. (4), the two human osteosarcoma cell lines, U2OS and SAOS2,
were compared for mechanisms responsible for the antiproliferative
effects of glucocorticoids. In SAOS2, a retinoblastoma protein
(Rb)-deficient cell line, glucocorticoids increased the expression of
the CDK inhibitors p21Cip1 and p27Kip1.
However, in U2OS cells, which express Rb, glucocorticoids decreased the
expression of cyclin D3, CDK4, and CDK6, without increasing the
expression of CDIs. The results from this study suggest that the
mechanism responsible for the antiproliferative phenotype induced by
glucocorticoids may vary across different cell types. In most cases, a
direct relation between the changes in CDI expression and the
glucocorticoid-induced antiproliferative response is not clear.
It has previously been shown that glucocorticoids inhibit cellular
proliferation in the human HeLa cervical carcinoma cell line
either by causing an accumulation of cells in the G1 phase
(37, 38) or by prolongation of the G2/M phase (39). In this
study we have focused our investigation on the role of the Cip/Kip
family of CDIs, particularly p57Kip2, in the
glucocorticoid-mediated inhibition of HeLa cell proliferation. The
Cip/Kip family of CDIs has been implicated in inhibition of CDK-cyclin
complexes not only in the G1 phase of the cell cycle, but
also in other cell cycle phases. Furthermore, the action of this family
of CDIs is not dependent on Rb, which is absent in the HeLa
cells (see below). We found that the synthetic glucocorticoid
dexamethasone (DEX) inhibited proliferation of HeLa cells and that this
antiproliferative effect correlated with a decrease in CDK2 activity
and a major increase in both the mRNA and the protein expression of the
CDI p57Kip2. We also showed that p57Kip2, on
its own, was sufficient to partially reconstitute both the
antiproliferative effect and the inhibition of CDK2 activity seen in
glucocorticoid treated-cells.
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RESULTS
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DEX Induces an Accumulation of HeLa Cells in the
G1 Phase and Decreases the CDK2 Activity
After addition of the synthetic glucocorticoid DEX to HeLa cells,
cell proliferation was inhibited (Fig. 1a
). A 2-fold increase in the doubling
time of an exponentially growing cell population after DEX treatment
was observed compared with untreated cells, when a saturating
concentration of DEX (100 nM) was used. The
antiproliferative effect of DEX could be reversed by the glucocorticoid
antagonist RU486, indicating that this is a GR-mediated effect (Fig. 1a
). Administration of RU486 alone revealed no effect on the
proliferation of the HeLa cells (data not shown).

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Figure 1. The Effect of the Glucocorticoid Agonist DEX and
the Antagonist RU486 on the Proliferation of HeLa tk-
Cells
a, Cells were treated with 100 nM DEX in the presence or
absence of 1 µM RU486 and counted in a Bürker
counter at 24-h intervals. Each measuring point is represented by
triplicate samples from which the mean value and SD have
been calculated. b, A representative flow cytometric analysis of
asynchronously growing HeLa tk- cells. The cells were
cultured in the presence or absence of DEX (100 nM) for
5 h and 11 h, respectively.
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To further characterize the DEX effect on cell cycle
distribution in HeLa cells, the cells were subjected to flow
cytometric analysis (Fig. 1b
). As compared with untreated cells,
11 h of DEX treatment increased the number of cells in the
G1 phase from 62% to 75%, indicating a G1
accumulation. This increase in the amount of cells in G1
phase was accompanied by a decrease in the fraction of cells in S phase
from 16% in untreated to 5% in treated HeLa cells. However, a
complete growth arrest was not achieved as a significant number of
cells remained in the S- and the G2/M phases also after
longer DEX treatment (data not shown). The changes in cell cycle
distribution were visible after 11 h of DEX treatment, whereas
after 5 h of treatment only a minor increase of the amount of
cells in G1 (61% in untreated as compared with 64% in
treated) was observed. These results showed that DEX treatment
induces an accumulation of cells in the G1 phase within the
first cell cycle after treatment, explaining the growth retardation
seen in the proliferation assay.
The HeLa cells did not express any Rb as analyzed by Western blotting,
while other human cell lines, e.g. HT29, gave a positive
signal using the same assay conditions (results not shown). Since Rb is
the key substrate for the CDK4/6-cyclinD complexes (40, 41, 42), we instead
focused our investigation on the other positive regulator of
G1 progression, namely the CDK2-cyclin E complex. This was
studied by immunoprecipitating CDK2 from HeLa whole-cell extracts and
performing a Histone H1 kinase assay. The result showed that DEX
treatment (100 nM) decreased the CDK2 activity (Fig. 2
). This effect was already pronounced
after 5 h and reached a maximal inhibition after 11 h,
correlating well with the accumulation of cells in the G1
phase seen after DEX treatment.

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Figure 2. CDK2 Activity in HeLa Cells after Glucocorticoid
Treatment
CDK 2 activity as determined by Histone H1 phosphorylation in HeLa
tk- cells after DEX treatment (100 nM) for
various time periods. As a negative control (neg.), an extract from
untreated cells precipitated in the absence of the CDK 2 antibody was
used.
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The Effect of DEX on Protein Expression of
G1-Acting Cell Cycle Proteins
As shown above, DEX treatment of HeLa cells caused a decrease in
CDK2 activity. This glucocorticoid-mediated effect could either involve
induction of different CDIs of the Cip/Kip family and/or
down-regulation of the positively acting G1 factors, CDK2
or cyclin E. We therefore analyzed the HeLa cells for expression of
these proteins. Cells were treated with DEX for various time periods,
after which they were harvested and whole-cell extracts (WCEs)
prepared. The WCEs were subjected to immunoblot analysis, using
antibodies against cyclin E, CDK2, p21Cip1,
p27Kip1, and p57Kip2. As can be seen in Fig. 3
, DEX dramatically induced the protein
expression of p57Kip2, whereas only minor inductions of the
p21Cip1 and p27Kip1 proteins were observed.
During the same time period the CDK2 level was not significantly
changed, while the cyclin E expression was slightly decreased after
10 h and 24 h. The increase in p57Kip2 level was
seen as early as 4 h after treatment, and the maximal induction
was observed after 810 h of treatment. This early induction of
p57Kip2 by DEX, which precedes any major G1
accumulation (cf. Fig 1b
where only a minor change in cell
cycle distribution is seen after 5 h of DEX treatment), clearly
suggests that the induction is a primary glucocorticoid-mediated effect
and not secondary to a cell cycle block (see also below).

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Figure 3. p57Kip2, p21Cip1,
p27Kip1, CDK2, and Cyclin E Protein Expression in HeLa
tk- Cells after DEX Treatment
The cells were treated with DEX (100 nM) for various time
periods and protein expression was analyzed by Western blotting, using
antibodies directed to the different proteins.
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A small induction of p57Kip2 protein expression was
detectable after treating HeLa cells with 1 nM of DEX for
24 h, while almost maximal induction was observed when the cells
were exposed to 10 nM DEX (Fig. 4a
). The responses seen with the
different DEX concentrations are in line with the reported affinity of
DEX for the GR [dissociation constant (Kd)
3
nM (43)]. Treating the cells with DEX together with a
10-fold excess of the antiglucocorticoid RU486 completely reversed the
DEX-mediated induction of the p57Kip2 expression (Fig. 4b
).
This also showed that RU486 acts as a pure antagonist when it comes to
p57Kip2 induction. The antagonistic effect of RU486 and the
low concentration of DEX required to induce p57Kip2 protein
expression further suggest that this is a GR-mediated effect.

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Figure 4. Western Blot Analysis of p57Kip2
Protein Expression in HeLa tk- after Treatment with
Different Concentrations of DEX
a, The cells were treated for 24 h with the DEX concentrations
indicated in the figure. b, The effect of RU486 on the DEX-mediated
induction of p57Kip2 protein expression. The concentrations
of DEX and RU486 used are indicated in the figure.
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The Effect of Alternative Growth-Inhibiting Signals on
p57Kip2 Expression
To exclude the possibility that induction of
p57Kip2 expression after DEX treatment is a secondary
response to the cell cycle block, HeLa cells were exposed to
alternative antiproliferative signals. Western blot analysis showed
that serum starvation for 48 h only weakly induced
p57Kip2 expression (Fig. 5a
),
while growing HeLa cells to confluency did not induce
p57Kip2 expression (Fig. 5b
). Under both conditions, DEX
could induce p57Kip2 protein expression. HeLa cells were
also blocked in late G1/early S phase, using a double
thymidine block. As a control for cell cycle arrest,
[3H]thymidine incorporation was measured, which showed
that the cells retained only 3% of their DNA synthesis capacity after
the block (result not shown). The double-thymidine block did not affect
the p57Kip2 protein levels as determined by Western
blotting (Fig. 5c
). All these results further support the notion that
the induction of p57Kip2 by DEX is a primary effect.

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Figure 5. The Effect of Serum Deprivation, Confluency,
and Double-Thymidine Block on p57Kip2 Protein Expression in
HeLa tk- Cells
a, The cells were cultured for 48 h in the presence or absence of
serum and DEX (100 nM), as indicated in the figure. b, The
cells were grown to confluency, after which they were cultured in the
presence or absence of DEX (100 nM) for 24 h. c, The
cells were subjected to a double thymidine block, and DEX (100
nM, 24 h)-treated cells were used as a positive
control. p57Kip2 expression was analyzed by Western blot
analysis using 20 µg of protein from WCEs.
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DEX Induces mRNA Expression as Well as Transcription of the
p57Kip2 Gene
The short period of DEX treatment needed for induction of
p57Kip2 protein expression (4 h, cf. Fig 3
)
suggested that the response is directly mediated by GR and does not
require de novo protein synthesis. To test this hypothesis,
we performed a Northern blot analysis on polyA-selected RNA
from HeLa cells treated with DEX in the presence or absence of the
protein synthesis inhibitor cycloheximide (CHX). The result, as shown
in Fig. 6
, revealed a single DEX-induced
p57Kip2 transcript of
1.7 kb both in the presence and
absence of CHX, comparable to the previously reported size for one of
the p57Kip2 transcripts (16). As a control for RNA loading,
the same membrane was hybridized with a probe against
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig. 6
, lower panel). Standardizing the p57Kip2 signal
to the GAPDH signal showed that DEX induced p57Kip2 mRNA
expression 14-fold in the absence of CHX and 9-fold in the presence of
CHX. Under the conditions used, CHX blocked
85% of cellular
protein synthesis as measured by [3H]leucine
incorporation (data not shown). This indicates that the majority of the
DEX-mediated induction of p57Kip2 mRNA occurs in the
absence of de novo protein synthesis, further supporting the
notion of a direct GR-mediated effect.

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Figure 6. The Effect of DEX Treatment on the Expression of
the p57Kip2 mRNA
HeLa tk- cells were treated with or without DEX (1
µM) for 6 h in the presence or absence of CHX (5
µg/ml), after which p57Kip2 mRNA was detected by Northern
blot analysis. The 1.7-kb p57Kip2 transcript is indicated
in the figure (upper panel). As a control for RNA
loading, the membrane was reprobed against GAPDH (lower
panel). In the experiment 1 µg of poly A selected RNA was
used in each lane. In the cases when CHX was used, it was added 1
h before DEX treatment.
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The above results indicate that the p57Kip2 gene is
transcriptionally regulated by GR. To examine this question, we
performed a nuclear run-on transcription assay on nuclei prepared from
HeLa cells treated with or without DEX for 6 h. The result showed
that DEX strongly induced transcription of the p57Kip2 gene
(Fig. 7
), while transcription of the
ß-actin gene, which was used as a control for identical loading, was
unaffected. No binding of radiolabeled RNA was seen to an empty vector
(pcDNA3, result not shown).

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Figure 7. p57Kip2 Transcription in HeLa
tk- Cells Treated with or without DEX
A run-on transcription assay was performed on nuclei prepared from HeLa
tk-cells nontreated or treated for 6 h with 100 nM
DEX. An equal amount of 32P-rUTP radiolabeled RNA (600 000
cpm/ml) from treated or nontreated cells was hybridized with a filter
to which 3 µg of pcDNA3-hp57Kip2 or pGEM3-ß-actin
plasmid had been applied in each slot.
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p57Kip2 Expression on Its Own Is Sufficient
to Induce Growth Inhibition in HeLa Cells
We next asked whether p57Kip2 on its own was
sufficient to cause the antiproliferative phenotype seen in these
cells. To investigate this, full-length human p57Kip2 cDNA
was cloned into the tetracycline-inducible expression vector pTRE and
stably transfected into HeLa Tet-On (CLONTECH Laboratories, Inc., Palo Alto, CA) cells. Several individual clones were
isolated and screened for p57Kip2 transgene protein
expression after stimulation with the tetracycline analog doxycycline
(DOX). Examples of a p57Kip2 transgene-inducible (S9) and a
noninducible (S8) clone are shown in Fig. 8
. Notably, both clones induced
endogenous p57Kip2 protein expression after DEX treatment.
Furthermore, the magnitude of the DOX-mediated induction of transgene
p57Kip2 expression in the S9 clone was similar to the
DEX-mediated induction of endogenous p57Kip2 expression
(Fig. 8
).

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Figure 8. Western Blot Analysis of p57Kip2
Expression in the S8 and the S9 Clones of HeLa Tet-On Cells Stably
Transfected with the pTRE-p57Kip2 Tetracycline-Inducible
Vector
The cells were treated for 15 h as indicated in the figure in the
presence or absence of DEX (100 nM) or DOX (2 µg/ml),
after which a Western blot analysis of p57Kip2 expression
was performed.
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The proliferation of the S8 and the S9 clones was investigated after
DOX or DEX treatment. The doubling time of exponentially growing S9
cells increased 1.7-fold in the presence of DOX (Fig. 9a
) as compared with untreated cells. In
the control clone S8, the rate of proliferation after DOX treatment was
essentially unaffected as compared with untreated cells (Fig. 9b
). In
contrast, DEX treatment increased the doubling time 2-fold in both
clones.


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Figure 9. The Effect of DEX and DOX on Proliferation, Cell
Cycle Distribution, and CDK2 Activity of HeLa Tet-On Cells Stably
Transfected with the pTRE-p57Kip2 Tetracycline-Inducible
Vector
a, The p57Kip2 transgene-inducible clone S9 was subjected
to DEX (100 nM) or DOX (2 µg/ml) treatment for various
time periods. b, The noninducible control clone S8 was subjected to DEX
(100 nM) or DOX (2 µg/ml) treatment for various time
periods. In both cases the amount of cells were counted in a
Bürker chamber. Each measuring point is represented by triplicate
samples from which the mean value and SD have been
calculated. c, Flow cytometric analysis was performed on asynchronously
growing cells from the S8 and the S9 clone, after treatment with DEX
(100 nM) or DOX (2 µg/ml) for 15 h as indicated in
the figure. Shown here is one representative analysis from each clone.
d, CDK2 kinase activity as determined by Histone H1 phosphorylation in
the p57Kip2 transgene inducible clone S9 and the
noninducible clone S8 after treatment with DEX (100 nM) or
DOX (2 µg/ml) for 15 h. As a negative control (neg.) an extract
from untreated S9 cells precipitated without the CDK2 antibody was
used.
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Flow cytometric analysis of cell cycle distribution after DEX
treatment of the S9 clone showed that the percentage of cells in the
G1 phase increased from 51% to 63% and was associated
with a decrease in the amount of cells in G2/M phase from
33% to 18% (Fig. 9c
). Similarly, DOX treatment of the same clone
increased the percentage of cells in G1 phase from 51% to
56% and decreased the fraction of cells in G2/M phase from
33% to 28%. Conversely, the S8 clone was unaffected by DOX treatment,
while DEX treatment of S8 cells produced an increase of the amount of
cells in G1 phase from 48% to 61% (Fig. 9c
). This shows
that elevated p57Kip2 on its own is sufficient to induce
antiproliferative effects and changes in cell cycle distribution
resembling the ones observed in DEX-treated HeLa Tet-On cells. To
mechanistically compare the DEX-induced and the DOX-induced
antiproliferative effect in the S8 and S9 clones, we performed a CDK2
kinase assay on immunoprecipitated CDK2 from cells treated with or
without DEX or DOX. The Histone H1 phosphorylation assay (Fig. 9d
)
showed that DEX inhibited the CDK2 activity both in the S8 and S9
clones, while DOX inhibited the CDK2 activity only in the
transgene-inducible (S9) clone and not in the noninducible (S8) clone.
Transgene induction of p57Kip2 thus inhibits CDK2 activity
in HeLa cells, identically to the DEX-induced effect observed in these
cells. In summary, all the results obtained from induction of the
p57Kip2 transgene strongly support the involvement of this
protein in the antiproliferative effect exerted by glucocorticoids on
HeLa cells.
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DISCUSSION
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Glucocorticoids inhibit cell proliferation in a number of tissues
and cell lines. This antiproliferative effect is most often mediated by
a cell cycle arrest in the G1 phase (3, 4, 5, 6, 7). Different
mechanisms have been reported for this arrest. In some model systems
glucocorticoids decrease the levels of proteins that positively
regulate cell cycle progression, e.g. c-myc,
cyclin D3 (5, 33), and CDK4/6 (4). In other cells, glucocorticoids
increase the levels of the CDIs p21Cip1 (4, 7, 34, 35, 36) and
p27Kip1 (4). Thus, no consensus mechanism for this
antiproliferative effect has been found and cell-specific mechanisms
seem to operate.
We have investigated the role of the newly identified CDI
p57Kip2 in the glucocorticoid-induced antiproliferative
effect in HeLa cells. In our experiments, DEX induced accumulation of
cells in the G1 phase. This effect was clearly visible
after 11 h of DEX treatment. Consistent with an earlier report
(39), this was accompanied by a general enlargement of the cells
together with a more flattened appearance (results not shown).
HeLa cells have been reported to express the human papilloma virus E7
protein (44), which sequesters and inactivates the Rb (for review see
Ref. 45), one of the major tumor suppressors acting in the
G1 phase of the cell cycle. Furthermore, HeLa cells have
been described to lack cyclin D-CDK4 complexes (46) and express little
or no Rb (M. K. R. Samuelsson and S. Okret, result not
shown). Since it is generally accepted that Rb is the key target for
the CDK4/6-cyclinD-Ink4 signaling pathway, this pathway should not play
a major role in the Rb-deficient HeLa cells (40, 41, 42, 47, 48, 49). An
alternative mechanism for the glucocorticoid-induced G1
effect is that glucocorticoids inhibit the activity of the second major
G1/S kinase activity, namely CDK2. CDK2 has been reported
to phosphorylate p27Kip1, triggering its degradation (50, 51), and may also modify other proteins that are involved in origin
firing and DNA replication (52, 53). These mechanisms may be
alternative pathways by which CDK2 operates in Rb-deficient cells.
Indeed, we found that DEX treatment of HeLa cells decreased the
activity of CDK2 and that the time needed for this inhibition is
consistent with the time needed for induction of the G1
accumulation. Thus, we focused our investigation on whether the change
in CDK2 activity could be explained by glucocorticoid-mediated
alterations in expression of CDK2, cyclin E, and/or the Cip/Kip family
of CDIs. The Cip/Kip family of inhibitors interacts with and inhibits
most CDK-cyclin complexes. These proteins can, at least theoretically,
inhibit cell cycle progression by acting in all cell cycle phases,
although overexpression of the Cip/Kip family of CDIs in different cell
lines primarily causes a G1 arrest (for review see Ref.
9).
Our results showed that DEX treatment of HeLa cells dramatically
induced p57Kip2 protein expression and reduced CDK2
activity within a few hours. During the same time period the protein
levels of CDK2 and cyclin E were essentially unchanged, while the
p21Cip1 and p27Kip1 levels were only marginally
elevated. To our knowledge, this is the first demonstration of an
external signal directly regulating p57Kip2 expression,
although a recent publication (54) showed ubiquitin-dependent
proteosomal degradation of p57Kip2 after TGFß1 treatment
of primary rat osteoblastic cells. The induction of p57Kip2
protein was detectable after 4 h of DEX treatment and thus it
preceded the major G1 accumulation seen in these cells. The
induction occurs pretranslationally, since DEX also induces
p57Kip2 mRNA expression. This induction does not require
de novo protein synthesis, and we were able to show, using a
run-on transcription assay, that the p57Kip2 gene is
transcriptionally induced by glucocorticoid treatment. Furthermore, no
DEX-mediated change in the protein stability of the endogenous
p57Kip2 was found, since its rate of degradation was
identical to the rate of degradation of the transgene
p57Kip2 induced by DOX in the tetracyclin-inducible S9
clone (result not shown). In summary, these experiments suggest
that DEX-mediated induction of p57Kip2 expression is a
primary effect caused directly by DEX and not a secondary effect caused
by the cell cycle inhibition itself. This is further supported by the
observation that other antiproliferative signals such as serum
deprivation, confluence, and a double-thymidine block do not induce
p57Kip2 protein expression at all or only to a very small
extent.
By stably transfecting HeLa cells with the human p57Kip2
cDNA under the control of a tetracycline (DOX)-inducible promoter, we
were able to show that p57Kip2 alone could induce an
antiproliferative effect similar to that observed after DEX treatment
of the cells. Furthermore, induction of p57Kip2 with either
DEX or DOX induced an accumulation of cells in the G1 phase
as well as a decrease in the CDK2 activity, supporting an important
role for p57Kip2 in this process. Thus, we were able to
show a functional interaction between p57Kip2 and CDK2 in
HeLa cells, correlating well with earlier studies demonstrating a
direct protein-protein interaction between these proteins (16, 17).
Despite similar phenotype induced by p57Kip2 alone and DEX
treatment, we cannot exclude the involvement of additional factors in
the antiproliferative effect exerted by glucocorticoids on HeLa cells;
for instance, cyclin E expression is slightly decreased, and both
p21Cip1 and p27Kip1 protein levels are slightly
increased after DEX treatment. Furthermore, the morphological changes
of the HeLa cells observed after DEX treatment were not seen when
p57Kip2 was expressed on its own, suggesting another
mechanism for this effect (M. K. R. Samuelsson, result not
shown). Interestingly, a recent paper by Kaltschmidt et al.
(55) showed that I
B-
overexpression in HeLa cells led to cell
cycle inhibition as well as morphological changes similar to those
observed in the DEX-treated cells. This opens up the possibility that
the full antiproliferative response induced by glucocorticoids in HeLa
cells is a combination of transcriptional induction of the
p57Kip2 gene and transrepression of NF-
B by GR (2). The
reason why DEX treatment of the HeLa Tet-On cells results in a smaller
DEX effect on the S phase as compared with the HeLa tk-
cells is unclear, but could be due to the fact that the cells (and
clones), which originate from different sources, are not completely
identical. This is in line with reports from various groups describing
slightly different effects of DEX on the cell cycle phases in
HeLa cells (37, 38, 39).
Although we have shown that p57Kip2 on its own is
sufficient to induce an antiproliferative effect in HeLa cells
mimicking the effect after DEX treatment, it may not be necessary for
this effect. We have attempted to investigate this issue by performing
several experiments aiming at reducing the DEX-mediated induction of
p57Kip2. Techniques such as transfections with antisense
constructs, antisense phosphorthioate DNA oligonucleotides, and stable
transfections with hammerhead ribozymes have been used. In none of
these experiments were we able to efficiently down-regulate the
DEX-mediated induction of the p57Kip2 protein expression
(results not shown). There could be several reasons for this,
i.e. the induction of p57Kip2 is too powerful to
be efficiently counteracted by any antisense expression or degradation
by ribozymes or the high GC content of the p57Kip2
transcript makes it unavailable for the antisense constructs (56).
As mentioned above, DEX increased the level of p57Kip2 mRNA
in HeLa cells, without any requirement for de novo protein
synthesis. We also showed that the p57Kip2 gene is
transcriptionally regulated by glucocorticoids. Several putative GREs
have been identified within the human p57Kip2 promoter when
a computer search was performed (data not shown). It is posssible that
GR exerts its effect by binding to one or several of these elements.
Alternatively, GR may function through binding to other transcription
factors. The latter mechanism has been shown for progesterone receptor
activation of the p21Cip1 promoter, which occurs through a
Sp1 site (57).
Further investigations must be performed to test whether the induction
of p57Kip2 by glucocorticoids is a general mechanism. We
have screened several cell lines known to be growth inhibited by
glucocorticoids and found p57Kip2 induction after DEX
treatment in some, but not all, of them (result not shown). This shows
that the induction of p57Kip2 does not only occur in HeLa
cells, suggesting that this regulation may be a common mechanism in the
antiproliferative action of glucocorticoids.
In summary, we report a novel mechanism for glucocorticoid-mediated
inhibition of cell proliferation, involving transcriptional induction
of the p57Kip2 gene. p57Kip2 has earlier been
shown to be important for embryonic development in mice (22, 23) and
has been proposed to be a tumor suppressor in humans (24, 25). In
connection with this, it is interesting to note that glucocorticoid
treatment can inhibit tumor promotion as in the case of the mouse skin
carcinoma model (58, 59). Further experiments will reveal a possible
connection between glucocorticoids and p57Kip2 in these
processes.
 |
MATERIALS AND METHODS
|
---|
Cell Culture Conditions and Hormone Treatment
The two strains of human cervical carcinoma HeLa cells used in
this study, HeLa tk- (ATCC, Manassas, VA) and
HeLa Tet-On (CLONTECH Laboratories, Inc.), were cultured
in MEM supplemented with MEM nonessential amino acids (1x), sodium
pyruvate (1 mM), penicillin-streptomycin (100 IU/ml),
L-glutamine (2 mM) and FBS (10%). All cell
culture medium was obtained from Life Technologies, Inc.
(Paisley, UK). All cells were grown at 37 C in a humidified
atmosphere of 90% H2O and 5% CO2.
DEX (Sigma Chemical Co., St. Louis, MO) and RU486
(Roussel-UCLAF, Romanville, France) were dissolved in 99.5%
ethanol. In all experiments where DEX or RU486 was added to the cells,
the same final concentration of ethanol was used as a control for
solvent effects.
Proliferation Assay
For cell proliferation assays, cells were seeded in six-well
multidishes (Nunc, Roskilde, Denmark). After hormone treatment,
cells were harvested at 24-h intervals by treatment with Trypsin-EDTA
(Life Technologies, Inc.), single-cell suspensions were
prepared, and cells were counted in a Bürker chamber. Triplicates
from each time point were analyzed. The doubling time for each
condition was calculated from cells in exponential growth phase.
Flow Cytometric Analysis
For flow cytometric analysis 12 x 106 cells
were washed with sample buffer (PBS supplemented with 1 g/liter
glucose), fixed in cold (-20 C) 70% ethanol and stored at -20 C
overnight. The samples were centrifuged and the pellets were
resuspended in sample buffer containing propidium iodide (50 µg/ml)
(Sigma Chemical Co.) and DNase-free RNase A (50 µg/ml)
(Roche Molecular Biochemicals, Mannheim, Germany)
for 1 h at room temperature while the tubes were vigorously agitated on
a shaking platform. Ten thousand cells were analyzed by flow cytometry
in a FACSscan (Becton Dickinson and Co., Franklin Lakes,
NJ). Living cells were gated to exclude debris and clumps.
Immunoprecipitation and Histone H1 Kinase Assay
HeLa cell extracts for immunoprecipitation were prepared by
sonication in the absence of any detergent, and the subsequent
immunoprecipitation and Histone H1 kinase assays were performed
according to the protocol described by Sangfelt et al. (32).
For each reaction, 50 µg protein were immunoprecipitated with a
polyclonal rabbit antihuman CDK2 antibody (15536E,
PharMingen, San Diego, CA) (1:1000). Extracts treated
without the antihuman CDK 2 antibody were used as negative
controls.
Western Blot Analysis
WCEs for Western blot analysis were prepared by lysing the cells
in ice-cold Nonidet P-40 (NP-40) buffer (0.5% NP-40, 50 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA) for
10 min, after which cell debris was removed by centrifugation
(14,000 x g, 10 min, 4 C). An equal volume of 2x SDS
gel-loading buffer (60) was then added and the samples were boiled for
2 min. Protein concentrations in cell extracts were quantitated
spectrophotometrically before addition of the loading buffer with the
Bio-Rad protein assay kit, according to the instructions of the
manufacturer (Bio-Rad Laboratories, Inc., Hercules, CA).
Twenty micrograms of protein from each whole cell extract were
electrophoretically separated on a 9% SDS-polyacrylamide gel and
electroblotted onto a Hybond C-extra membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). To check for equal loading
and transfer, the membranes were stained with Ponceau red (Sigma Chemical Co.). For protein detection, the immunoblots were then probed
either with a rabbit IgG polyclonal anti-p57Kip2 antibody
(C-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
(1:100), a rabbit IgG polyclonal anti-p21Cip1 antibody
(C-19, Santa Cruz Biotechnology, Inc.) (1:100), a mouse
IgG monoclonal anti-p27Kip1 antibody (Transduction
Laboratories, Lexington, KY) (1:2000), a rabbit polyclonal IgG
anticyclin E antibody (M-20, Santa Cruz Biotechnology, Inc.) (1:50), and a rabbit polyclonal IgG anti-CDK2 antibody
(M2, Santa Cruz Biotechnology, Inc.) (1:50). As secondary
antibodies, either a horseradish peroxidase-conjugated antirabbit or an
antimouse IgG antibody (Amersham Pharmacia Biotech)
(1:3000) was used. The membranes were then subjected to enhanced
chemiluminescence (Amersham Pharmacia Biotech, UK) and
autoradiography, according to the instructions of the manufacturer.
Double Thymidine Block of HeLa Cells
HeLa cells were cultured as described before and subsequently
subjected to a double thymidine block as described by Stein et
al. (61). According to the same protocol,
[3H]thymidine incorporation was measured as a control for
cell cycle arrest. This showed that the cells only retained less than
3% of their DNA synthesis capacity after the block (result not
shown).
Northern Blot Analysis
HeLa cells were incubated in the presence or absence of DEX (1
µM) and/or CHX (5 µg/ml) (Sigma Chemical Co.) for 6 h. When CHX was used, it was added 1 h
before the addition of DEX. Poly A selection of cellular RNA was
carried out using the Dynabeads mRNA DIRECT kit
(Dynal, Oslo, Norway) according to the instructions
of the manufacturer. Northern blot analysis was carried out according
to Ref. 62 , using 1 µg of poly A selected RNA. The 0.249.5 kb RNA
ladder from Life Technologies, Inc. was used as a size
marker. The Northern blot filters (Hybond-N+, Amersham Pharmacia Biotech) were hybridized with a 32P-labeled cRNA
probe, first against p57Kip2 and subsequently against
GAPDH. All probes were synthesized by using the Riboprobe in
vitro transcription kit (Promega Corp., Madison, WI),
according to the instructions of the manufacturer. As a template for
the p57Kip2 riboprobe, we used a pBSKSII+ plasmid
(Promega Corp.) with the human p57Kip2 cDNA
cloned into it (kindly provided by Dr. S. J. Elledge, Department
of Human and Molecular Genetics, Baylor College of Medicine, Houston,
TX) and as a template for the GAPDH riboprobe we used a linearized
antisense probe template (Ambion, Inc. Austin, TX).
Quantification of the hybridization signals were made using a BasIII
phosphoimager (Fuji Photo Film Co., Ltd., Tokyo,
Japan).
Run-on Transcription Assay
Preparation of cell nuclei from HeLa cells treated with or
without DEX for 6 h was performed as described in Current
Protocols in Molecular Biology (62A ). The subsequent run-on
transcription assay detecting p57Kip2 and ß-actin
transcripts was performed as follows: 50 µl transcription buffer (50
mM HEPES, 2 mM MgCl2, 2
mM MnCl2, 1 µg/ml BSA, 300 mM
NH4Cl), 5 µl rATP, 5 µl rGTP, 5 µl rCTP (all 10
mM, Promega Corp.), 1 µl Rnasin
(Promega Corp.), 2 µl Heparin (5 mg/ml), 10 µl
32P-rUTP (10 µCi/µl, Amersham Pharmacia Biotech), and 50 µl nuclei were incubated for 20 min at 26 C.
Ten units of DNase (RNase free, Promega Corp.) were added
and incubated for 30 min at 37 C, after which 400 µl Proteinase K
buffer [10 mM Tris (pH 7.5), 100 mM NaCl, 2
mM KCl, 1 mM EDTA, 0.5% SDS] and 10 µl
proteinase K (10 mg/ml) were added and incubated for 30 min at 37 C.
After adding 12 µl 4 M NaCl and 16 µl tRNA (16 mg/ml),
a phenol-chloroform extraction was performed. The radiolabeled RNA was
subsequently precipitated once with trichloroacetic acid (10%) and
once with LiCl (1 M) + ice-cold ethanol (50%). Each filter
was hybridized (Hybond-N+, Amersham Pharmacia Biotech) for
72 h at 45 C with 600,000 cpm/ml of radiolabeled RNA as determined
with a scintillation counter. The filters were washed with 0.2xSSC,
0.1% SDS at 72 C for detection of the p57Kip2 transcript,
and at 60 C for the ß-actin transcript, and analyzed by
autoradiography. The reason for the different washing
temperatures used was that the p57Kip2 transcript
hybridized more strongly than the ß-actin transcript due to a high GC
content in the p57Kip2 cDNA (56). The plasmids used as
probes were the pcDNA3-hp57Kip2 (the human
p57Kip2 cDNA cloned into the EcoRI site of the
pcDNA3 vector (Invitrogen, Carlsbad, CA)) and the
pGEM3-ß-actin vector (63). The plasmids were linearized and
denatured, and 3 µg were added to each slot and blotted on to the
filter by a slot blot apparatus (Src 072/0 Minifold II,
Schleicher & Schuell, Inc., Dassel, Germany).
Transfection Conditions and Selection of Stably Transfected
Cells
The human p57Kip2 cDNA was subcloned into the
EcoRI site of the tetracycline-inducible pTRE vector and
transfected together with the pTK-Hyg selection vector into HeLa Tet-On
cells using the Lipofectin transfection reagent (Life Technologies, Inc.) according to the instructions of the
manufacturer. A 5:1 molar ratio of pTRE-hp57Kip2 to pTK-Hyg
was used. Selection was performed in 200 µg/ml of Hygromycin B
(Life Technologies, Inc.). After selection, individual
clones were isolated and continuously cultured in the presence of
Hygromycin B. To induce transgene p57Kip2 expression, the
cells were treated with 2 µg/ml of doxycycline hydrochloride
(Sigma Chemical Co.).
 |
ACKNOWLEDGMENTS
|
---|
The authors acknowledge Ingalill Rafter for outstanding
technical assistance. We thank Dr. Stephen J. Elledge for kindly
providing the human p57Kip2 cDNA; and Drs. Katarina Alheim,
Sven Erickson, Gary Faulds, Dan Grander, Johan Lidén, and
Nanthakumar Subramaniam for helpful discussions and technical
advice.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Sam Okret, Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Novum F60, SE-141 86 Huddinge, Sweden.
This study was supported by a grant from the Swedish Cancer
Society to S.O. (2039-B98-11XBB).
Received for publication November 23, 1998.
Revision received July 12, 1999.
Accepted for publication August 9, 1999.
 |
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