From the Institute for General and Experimental
Pathology, Division of Molecular Pathophysiology, University of
Innsbruck Medical School and the § Tyrolean Cancer Research
Institute, A-6020 Innsbruck, Austria
Received for publication, September 7, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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The cyclin-dependent kinase inhibitor
p16INK4A is frequently inactivated in childhood
T-cell acute lymphoblastic leukemia. To investigate possible
consequences of this genetic alteration for tumor development, we
conditionally expressed p16INK4A in the T-cell acute
lymphoblastic leukemia line CCRF-CEM, which carries a homozygous
deletion of this gene. In agreement with its reported function,
p16INK4A expression was associated with hypophosphorylation
of the retinoblastoma protein pRB and stable cell cycle arrest in
G0/G1, documenting that the pRB/E2F pathway is
functional in these cells. Unexpectedly, p16INK4A
expression increased the sensitivity threshold for glucocorticoid (GC)-induced apoptosis from therapeutic to physiologic levels. As a
possible explanation for this phenomenon, we found that
p16INK4A-arrested cells had elevated GC receptor expression
associated with enhanced GC-mediated transcriptional activity and
increased responsiveness of the GC-regulated cyclin D3 gene. These data are supported by our previous findings that GC receptor levels critically influence GC sensitivity and imply that p16INK4A
inactivation, in addition to allowing unrestricted proliferation, represents a mechanism by which lymphoid tumor cells might escape cell
death triggered by endogenous GC.
The INK4A gene locus is located at chromosome
9p21 and encodes two different cell cycle inhibitors, namely
p16INK4A and p19ARF (p14ARF),
transcribed from alternative exons of the same gene, but dissimilar in
their protein structures (1, 2). Inactivation of this gene locus is
observed in up to 80% of primary T-cell acute lymphoblastic leukemias
(T-ALLs),1 thereby being the
most consistent genetic alteration in this disease (3-6). The high
frequency in T-ALLs suggests that the tumor suppressor genes encoded by
this locus might play an important role in the development of this
malignancy. Ink4a knockout mice develop spontaneous
lymphomas with high frequency, further suggesting that
p16INK4A might be involved in negative regulation of
proliferation in the lymphoid lineage (7). p16INK4A acts by
binding to and inhibiting the activity of CDK4 and CDK6 kinases (1, 8).
When complexed to regulatory cyclins of the D-type, these kinases
phosphorylate and inactivate the retinoblastoma protein pRB and its
family members, thereby releasing transcription factors, such as E2F-1,
from inhibition by pRB. This allows transcription of genes essential
for the onset of S phase (9).
To further investigate a possible role of p16INK4A
inactivation in T-ALL tumor development, we used the childhood T-ALL
model CCRF-CEM, which contains a homozygous deletion of the
INK4A locus (10, 11), and the tetracycline-regulated gene
expression system (12) to conditionally express transgenic
p16INK4A.
p16INK4A expression was associated with a stable arrest in
the G0/G1 phase of the cell cycle.
Surprisingly, p16INK4A arrest dramatically increased the
sensitivity of these cells to glucocorticoid (GC), a hormone that is
routinely used for its apoptosis-inducing property in the therapy of
this malignancy. Since p16INK4A increased the sensitivity
of these leukemia cells to physiologic levels of the hormone, we
speculate that p16INK4A inactivation (or a functionally
equivalent mechanism) might be required for lymphoid malignancies to
escape an as yet unrecognized tumor surveillance for lymphoid lineage,
i.e. GC-induced apoptosis.
Cell Lines, Culture Conditions, and Reagents--
CEM-C7H2 (13)
is a highly GC-sensitive subclone of the CCRF-CEM-C7 cell line (14).
The generation and analysis of the CEM-C7H2 subclone C7H2-2C8, which
contains the p Plasmids and cDNAs--
Full-length p16INK4A
cDNA was subcloned from pSK-p16INK4A (1) into the
EcoRI-XbaI sites of the pUHD10-3 vector
for tetracycline-regulated expression (16), generating plasmid
pUHD10-3-p16INK4A. pKS-tkHyg is a pBluescript II-KS vector
(Stratagene, La Jolla, CA) containing the hygromycin B resistance gene
under the control of a thymidine kinase promoter and a SV40
polyadenylation site (17). Transactivation reporter assays were
performed using an MMTV-luciferase construct (18) cotransfected with
the pRL-SV40 plasmid (Promega, Madison, WI) as a transfection control.
Stable Transfections--
For stable introduction of
p16INK4A, C7H2-2C8 cells were transfected by
electroporation as previously described (19). Briefly, ~1 × 107 mid-log phase cells in 800 µl of PBS containing 30 µg of linearized pUHD10-3-p16INK4A plasmid and 25 µg of
pKS-tkHyg plasmid were electroporated at 300 V and 500 microfarads
using a Bio-Rad Gene Pulsar. The cells were cultured in two 96-well
plates for 2 days and then exposed to 0.25 mg/ml hygromycin B. Resistant transformants were selected and analyzed for conditional
p16INK4A expression by Northern and Western blot analyses.
Northern Blot Analysis--
Total RNA was extracted from 5 × 106 cells with TriReagentTM (LPS
Industries, Moonachie, NJ). Approximately 10 µg of RNA were separated by electrophoresis on a denaturing 1% agarose gel containing formaldehyde in MOPS buffer and blotted overnight onto
ZetabindTM nylon membranes (Cuno, Inc., Meriden, CO)
according to standard protocols. RNA was cross-linked to the membranes
by UV light. Filters were then prehybridized in phosphate blocking
buffer containing SDS and bovine serum albumin at 65 °C for 3 h
and hybridized for 12 h to a [32P]dATP-labeled,
heat-denatured, full-length p16INK4A probe. After
hybridization, blots were washed in 1× saline/sodium phosphate/EDTA
and 0.1% SDS at 65 °C and in 0.1× saline/sodium phosphate/EDTA and
0.1% SDS at 65 °C and exposed to Agfa Curix x-ray films with an
amplifying screen at Determination of Apoptosis--
For quantification of apoptosis,
nuclear staining with propidium iodide in concert with forward/sideward
scatter analysis was used (20). Briefly, cells were centrifuged, and
the pellets were resuspended in 0.7 ml of hypotonic propidium iodide
solution. The tubes were kept at 4 °C in the dark overnight. Nuclear
fluorescence intensity and forward/sideward scatter were analyzed with
a Becton Dickinson FACScan. Cell debris and small particles were
excluded from analysis, and nuclei in the sub-G1 marker
window were considered to represent apoptotic cells.
Immunoblotting--
Cells were washed in PBS and lysed for 30 min on ice in PBS lysis buffer containing 1% Nonidet P-40 and 10 mM sodium fluoride, to which 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml
leupeptin, and 1 µg/ml pepstatin were added just before use. Cell
lysates were cleared by centrifugation. An equal amount of 2× SDS
sample buffer containing 10% Flow Cytometric Analysis of Glucocorticoid Receptor (GR)
Expression--
Cultured cells were washed twice in PBS and 1% bovine
serum albumin. Aliquots of 1 × 106 cells were fixed
in 1% paraformaldehyde at room temperature for 30 min, washed, and
permeabilized with 0.1% Triton X-100 in 0.1% citrate buffer for 5 min
on ice. Cells were washed twice and incubated at room temperature for
70 min in the presence of either FITC-conjugated anti-GR antibody 5E4
(a kind gift from Dr. A. Falus) (21) or an FITC-conjugated isotype
control (IgG1, Pharmingen). Finally, cells were washed
three times, incubated with propidium iodide, resuspended in PBS and
1% bovine serum albumin, and analyzed on a FACScan.
Radioligand Binding Assay--
For determination of ligand
binding by the GR, whole cell ligand binding assays were performed as
described previously (22). Briefly, ~5 × 106 cells
were incubated in triplicate with increasing amounts of [3H]triamcinolone acetonide (PerkinElmer Life Sciences)
in the presence or absence of a 500-fold molar excess of unlabeled
triamcinolone acetonide at 37 °C for 1 h, washed three times,
and resuspended in scintillation mixture (Packard Instrument Co.,
Groningen, The Netherlands). The samples were counted in a
scintillation counter.
Luciferase Reporter Assays--
For transient MMTV reporter
transfections, 5 × 106 mid-log phase cells were
washed in PBS and resuspended in 4 ml of RPMI 1640 medium containing
10% fetal calf serum. In each experiment, 5 µg of pMMTV-luc and 1 µg of pRL-SV40 DNA were transfected using SuperfectTM
(QIAGEN Inc., Valencia, CA) according to the manufacturer's
instructions. After a 3-h incubation, 15 ml of medium were added, and
cells were split and incubated in the presence or absence of 200 ng/ml doxycycline for another 18 h. Cultures were then split again, and
100 nM dexamethasone was added for another 10 h to
study specific induction of the MMTV reporter. Thereafter, the cells
were harvested washed once in PBS, and cell pellets were lysed in 25 µl of reporter lysis buffer for 15 min at room temperature and
centrifuged. The supernatant of each sample was analyzed using the
dual-luciferase reporter system (Promega) according to the
manufacturer's instructions.
Ectopic Expression of p16INK4A in CCRF-CEM Leukemia
Cells Causes G0/G1 Arrest--
To determine
whether the CDK4/6 inhibitor p16INK4A can induce cell cycle
arrest in CCRF-CEM cells, we generated stably transfected CCRF-CEM
derivatives with tetracycline-inducible p16INK4A
expression. For this purpose, C7H2-2C8, a CEM-C7H2 subclone with constitutive reverse tetracycline-responsive transactivator expression (12), was cotransfected with a plasmid expressing p16INK4A
from a reverse tetracycline-transactivator-responsive promoter and a
hygromycin resistance plasmid. Three hygromycin-resistant clones,
referred to as 6E2/p16, 1D2/p16, and 1E10/p16, showed high levels of
p16INK4A mRNA (Fig.
1A) and protein expression
(Fig. 1B) upon addition of the tetracycline analog
doxycycline, but essentially no p16INK4A expression in its
absence. The p16INK4A protein expression level was
comparable to that found in aged human fibroblasts (data not shown).
Two of these subclones, 6E2/p16 and 1D2/p16, were selected for further
analysis. Induction of p16INK4A increased the
electrophoretic mobility of pRB (Fig. 2),
presumably by prevention of pRB phosphorylation. Cyclin D3, which forms
a functional complex with CDK4 and CDK6, remained at unchanged levels. Twenty-four hours after addition of doxycycline,
p16INK4A-expressing cells were completely arrested in the
G0/G1 phase of the cell cycle, as demonstrated
by FACS cell cycle analysis (Fig.
3), suggesting that the downstream
components of the p16INK4A/pRB pathway are intact.
p16INK4A-mediated cell cycle arrest was maintained over
72 h without signs of reduced viability.
p16INK4A-induced G0/G1 Arrest
Sensitizes Cells to GC-induced Apoptosis--
Since GCs are central
components in the therapy of childhood T-ALL, we investigated
whether p16INK4A expression might exert any effects on the
sensitivity of these leukemia cells to GC-induced apoptosis. To
this end, 6E2/p16, 1D2/p16, and parental C7H2-2C8 control cells were
cultured for 24 h in the presence or absence of doxycycline and
subsequently exposed to a 10 or 100 nM concentration of the
therapeutic GC analog dexamethasone for another 24, 36, and 48 h.
As already shown in Fig. 3, exposure to doxycycline induced cell cycle
arrest in 6E2/p16 and 1D2/p16, but had no effect upon the parental
C7H2-2C8 control line. When G0/G1-arrested
cells, i.e. doxycycline-treated 6E2/p16 and 1D2/p16 cells,
were treated with 100 nM dexamethasone, they exhibited
accelerated apoptosis compared with cycling cells, starting within
24 h and reaching a plateau during the following 12 h. In
contrast, doxycycline had no effect on the kinetics of cell death
induced by 100 nM dexamethasone in parental C7H2-2C8 control cells (Fig. 4, left
panels). Moreover, 10 nM dexamethasone (Fig. 4,
right panels), which had no apoptotic effect on
proliferating cells, was almost as efficient as 100 nM
dexamethasone in cell death induction in
G0/G1-arrested 6E2/p16 and 1D2/p16 cells.
p16INK4A Expression Sensitizes Cortisol-resistant CEM
Cells to Physiologic Concentrations of Cortisol--
We next studied
the sensitivity of these cells to the physiologic GC, cortisol. The
parental 2C8 control line and its p16INK4A-transfected
subclones (in the absence of doxycycline) were highly resistant to
cortisol at concentrations up to 5000 nM, which is ~100-fold higher than free cortisol in healthy humans (Fig.
5A). Surprisingly, massive
apoptosis induction was observed in p16INK4A-expressing
cells treated with as little as 50 nM cortisol (Fig. 5A). Apoptosis induction by physiologic cortisol in
G0/G1-arrested cells was prevented by addition
of RU486, suggesting that the observed effect was mediated by the GR
(Fig. 5B). Thus, p16INK4A expression sensitized
the cells to physiologic concentrations of cortisol.
Reintroduction of p16INK4A Increases Expression of the
GR--
To investigate possible mechanisms for the increased GC
sensitivity of p16INK4A-expressing cells, GR protein levels
were determined in p16INK4A-expressing and nonexpressing
cells by flow cytometry using an FITC-labeled anti-human GR antibody.
As indicated by mean fluorescence intensity, GR expression in
doxycycline-treated, and hence G0/G1-arrested, 6E2/p16 and 1D2/p16 cells exceeded that in untreated controls by
32-38%, whereas no alteration was found in the parental control cell
line, C7H2-2C8 (Fig. 6, left
panels). Increased GR expression, measured by antibody detection,
was paralleled by whole cell radioligand binding assays that showed
30-50% increases in binding of the radiolabeled GC analog
triamcinolone in p16INK4A-expressing cells compared with
their corresponding controls. Again, ligand binding of parental
C7H2-2C8 cells was not significantly influenced by addition of
doxycycline (Fig. 6, right panels).
p16INK4A Expression Increases the Activity of the
GR--
The increased expression and ligand-binding activity of the GR
led to a 2-4-fold increase in transcriptional activity, as shown in
transient transfection experiments with a GC-responsive MMTV-luciferase
reporter construct, thereby proving that the increased levels of the GR
were functional (Fig. 7A).
Next, we investigated whether the sensitivity of endogenous genes to
regulation by GC might also be increased in
p16INK4A-arrested cells. For these experiments, we studied
cyclin D3, which is repressed by GC treatment in mouse lymphoma cells
(23) and seems to be critical in the induction of GC-mediated cell cycle arrest in CEM cells.2
Exposure to 10 nM dexamethasone had no detectable effect
upon cyclin D3 expression in C7H2-2C8 control cells. It did, however, lead to its down-regulation in G0/G1-arrested
6E2/p16 cells (Fig. 7B). The combined data clearly showed
that cells arrested in G0/G1 by
p16INK4A had elevated levels of functional GR, which might
explain, at least in part, the increased sensitivity to GC-induced cell
death.
Increased GR Expression Is Not Secondary to Accumulation of Cells
in G0/G1--
To investigate whether the
observed up-regulation of the GR was simply a consequence of cell
accumulation in a cell cycle phase with high GR expression, the cells
were double-stained with propidium iodide and anti-GR antibody, and
cell cycle phase-specific GR protein levels were determined. In cycling
cells, endogenous GR levels were decreased by 33% in
G0/G1 phase compared with cells in
S/G2/M (Fig. 8A).
In contrast, p16INK4A-arrested
G0/G1 cells showed a 55% increase in receptor
expression compared with cycling cells in G0/G1
(Fig. 8B), suggesting that the increased GR expression is
not the result of enriching cells in G1, but of a specific
induction in p16INK4A-expressing cells.
Deletion of the INK4 gene locus is the most
frequent genetic alteration in T-ALLs (3, 5, 24), suggesting a critical role of p16INK4A and/or p19ARF in the
development of these tumors. Using the T-ALL model CCRF-CEM, we show
that p16INK4A expression alone is sufficient to mediate
G0/G1 arrest and dramatically sensitizes these
cells for apoptosis induced by GC, a hormone routinely used in the
therapy of malignant lymphoproliferative disorders. The former was in
good agreement with numerous studies on various malignancies that
suggested a tumor suppressor role of p16INK4A due to its
known inhibitory effect upon cell cycle progression (1, 7, 25). The
latter observation was, however, unexpected and raises questions
regarding its underlying mechanism, possible significance, and implications.
As to possible mechanisms for the increased GC sensitivity, we observed
up-regulation of functional GR, as evidenced by flow cytometry, radio
receptor assays, transient transfection studies with a GC-responsive
reporter construct, and down-regulation of an endogenous GC-responsive
gene. We (26) and others (27) have shown previously that GR expression
levels are critical parameters for sensitivity to GC-induced apoptosis.
Thus, although GR up-regulation may not be the sole cause of the
dramatic increase in GC sensitivity, it may well contribute to this
phenomenon. Interestingly, the observed increase in GR expression was
not simply due to cell accumulation in the G1 phase of the
cell cycle since proliferating cells going through G1 had
reduced GR levels. Rather, it appeared specific for the p16-induced
cell cycle arrest.
Another issue is whether the observed increase in GC sensitivity bears
any significance for tumor development. Given that lymphocytes at
certain stages of differentiation are sensitive to apoptosis induction
by endogenous GC (28), malignant transformation of such cells might
require inactivation of this lymphocyte-specific form of tumor
surveillance. Whether p16INK4A inactivation contributes to
this escape mechanism and, if so, how this might happen is unclear. In
circulating cells, p16INK4A is not expressed and therefore
cannot influence GC sensitivity. Stimulated lymphocytes have only a
limited life span, and leukemogenesis has to invoke a program to extend
the life span of proliferating T-cells, e.g. by suppressing
apoptotic pathways. In addition, leukemia cells have to evolve ways
to overcome replicative senescence, and inactivation of
p16INK4A might be one of those means. The dramatically
increased sensitivity to GC associated with induction of
p16INK4A suggests a novel role for GC in the regulation of
lymphocyte homeostasis, i.e. the induction of cell death in
p16INK4A-expressing lymphocytes. Loss of
p16INK4A might contribute to leukemogenesis by reducing GC
sensitivity and causing inability to undergo replicative senescence. In
addition, the dramatic increase in GC sensitivity in
p16INK4A-expressing cells may have implications for the
therapy of malignant lymphoproliferative disorders. Thus, combination
of GC with substances that mimic p16INK4A function, such as
CDK4/6-inhibiting peptides or specific kinase inhibitors, might improve
T-ALL therapy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
rtTA plasmid coding for the reverse
tetracycline-responsive transactivator (15), have been published (12).
All cells were maintained in RPMI 1640 medium containing 10% fetal
calf serum (Life Technologies, Paisley, United Kingdom), 100 units/ml
penicillin, 100 µg/ml streptomycin, and 2 mM
L-glutamine (Life Technologies) at 5% CO2 and
37 °C in saturated humidity. Dexamethasone and cortisol were stored
as 10 mM stock solutions in 100% ethanol; doxycycline was
kept as a 10 mM solution dissolved in phosphate-buffered
saline (PBS). All reagents were from Sigma (Vienna, Austria), unless
indicated otherwise. For each experiment, mid-log phase cultures
(2.5-5 × 105 cells/ml) were centrifuged and
resuspended in fresh medium at a concentration of ~2.5 × 105 cells/ml.
90 °C for several hours to days. After each
hybridization, the blots were stripped by boiling in 0.1% SDS and
rehybridized with a full-length glyceraldehyde-3-phosphate dehydrogenase probe.
-mercaptoethanol was added, and
proteins were denatured by boiling for 2 min. Samples were separated by
SDS-polyacrylamide gel electrophoresis on 7.5-15% polyacrylamide
gels. Proteins were then transferred to nitrocellulose membranes by a
Bio-Rad semidry transfer apparatus and stained using Ponceau red. The
membranes were incubated with Tris-buffered saline blocking buffer
containing 1% Tween 20 and 5% nonfat dry milk for 30 min, followed by
an overnight incubation at 4 °C with primary antibody diluted in
blocking buffer. Blots were probed with monoclonal or polyclonal
antibodies against human cyclin D3, p16INK4A (Pharmingen,
Hamburg, Germany), pRB, and
-tubulin (Oncogene Research, Cambridge,
MA). Membranes were washed in Tris-buffered saline and incubated with a
horseradish peroxidase-conjugated anti-mouse secondary antibody
(Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) diluted
in blocking buffer. Finally, the blots were developed by the enhanced
chemiluminescence substrate ECL (Amersham Pharmacia Biotech) according
to the manufacturer's instructions and exposed to Agfa Curix x-ray
films from seconds to several minutes. Stripping and reprobing were
performed as described by the manufacturer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Stably transfected CCRF-CEM subclones with
doxycycline-regulated expression of p16INK4A. C7H2-2C8
subclones 6E2/p16, 1D2/p16, and 1E10/p16 were cultured in the presence
or absence of 200 ng/ml doxycycline for the times indicated. The
samples were subjected to Northern blot analysis with
32P-labeled cDNA probes to p16INK4A and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(A) and to immunoblot analysis of cytoplasmic extracts with
anti-p16INK4A and anti- -tubulin antibodies
(B).
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Fig. 2.
p16INK4A expression in stably
transfected CCRF-CEM leukemia cells is associated with pRb
hypophosphorylation. Parental C7H2-2C8 control
(2C8/Ctr) and p16INK4A-transfected 6E2/p16 and
1D2/p16 cells were cultured in the presence or absence of 200 ng/ml
doxycycline (Dox) for 24 h. Cytoplasmic extracts were
analyzed by immunoblotting using monoclonal antibodies directed against
p16INK4A, cyclin D3 (Cyc D3), pRB, and
-tubulin (
-Tub).
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Fig. 3.
p16INK4A expression in stably
transfected CCRF-CEM leukemia cells is followed by a stable arrest in
G0/G1. C7H2-2C8 control
(2C8/Ctr) and p16INK4A-transfected 6E2/p16 and
1D2/p16 cells were cultured in the presence (+Dox) or
absence ( Dox) of 200 ng/ml doxycycline for
36 h. The cells were subjected to cell cycle determination by FACS
analysis of propidium iodide-stained nuclei.
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Fig. 4.
p16INK4A expression sensitizes
CCRF-CEM cells to GC-induced apoptosis. Parental C7H2-2C8 control
(2C8/Ctr.) cells and sublines 6E2/p16 and 1D2/p16 were
maintained for 24 h in the presence ( and
) or absence (
and
) of 200 ng/ml doxycycline (Dox). Subsequently, the
cell populations were split and cultured either in the presence (
and
) or absence (
and
) of 10 nM (right
panels) or 100 nM (left panels)
dexamethasone (Dex) for the times indicated. The cells were
then subjected to apoptosis detection by FACS analysis of propidium
iodide-stained nuclei. Each panel represents the mean ± S.D. from
three independent experiments.
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Fig. 5.
p16INK4A-expressing CEM cells are
sensitive to physiologic concentrations of cortisol. A,
C7H2-2C8 cells and their subclones 6E2/p16 and 1D2/p16 were cultured
for 24 h in the presence or absence of 200 ng/ml doxycycline
(Dox) and then for another 36 h in the presence of
various concentrations of cortisol and finally subjected to apoptosis
determination by FACS analysis of propidium iodide-stained nuclei. The
results obtained with the two p16INK4A-transfected cell
lines were very similar and have been combined (for better
readability). Shown is the mean ± S.D. of two independent
experiments. B, 6E2/p16 and 1D2/p16 cells were cultured for
24 h in the presence or absence of 200 ng/ml doxycycline and then
for an additional 24 and 48 h in the presence of 50 nM
cortisol with or without 500 nM RU486. Apoptosis was
determined by FACS analysis. Shown is the mean ± S.D. of the data
derived from both cell lines and two independent experiments.
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Fig. 6.
p16INK4A increases expression of
the GR. A, C7H2-2C8 control (2C8/Ctr.),
6E2/p16, and 1D2/p16 cells were cultured for 36 h in the presence
(solid lines) or absence (dotted lines) of 200 ng
of doxycycline (Dox), fixed, stained with either an
FITC-conjugated anti-GR antibody (solid and dotted
lines) or an FITC-labeled isotype control (dashed
lines), and subjected to FACS analysis. B, C7H2-2C8
control cells and conditional p16INK4A-expressing sublines
cultured for 36 h with ( ) or without (
) 200 ng/ml
doxycycline were subjected to whole cell ligand binding assays with the
indicated amounts of labeled triamcinolone. A representative experiment
performed in triplicate is shown.
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Fig. 7.
p16INK4A expression increases the
activity of the GR. A, C7H2-2C8 control
(2C8/Ctr.), 6E2/p16, and 1D2/p16 cells were transfected with
a GC-responsive MMTV-firefly luciferase vector and an SV40-driven
Renilla luciferase control vector, cultured in the presence
(black bars) or absence (white bars) of 200 ng/ml
doxycycline (Dox) for 18 h, and thereafter treated with
100 nM dexamethasone for another 10 h to detect
GC-mediated transcriptional activity. Specific induction of the MMTV
reporter is expressed as a percentage of the untreated controls
(adjusted to the SV40-driven Renilla luciferase transfection
control). Shown is the mean of two independent transient transfection
experiments. B, C7H2-2C8 cells and
p16INK4A-expressing 6E2/p16 cells were maintained for
24 h in the presence of 200 ng/ml doxycycline and subsequently
treated with 10 nM dexamethasone for the times indicated.
Thereafter, lysates were analyzed by immunoblotting using specific
antibodies directed against cyclin D3 (Cyc D3) and
-tubulin (
-Tub).
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Fig. 8.
Increased GR expression is not secondary to
accumulation of cells in G0/G1. 1D2/p16
cells were cultured for 36 h in the presence or absence of 200 ng/ml doxycycline, fixed, stained with an FITC-conjugated anti-GR
antibody and propidium iodide, and subjected to FACS analysis. Shown
are the GR fluorescence intensity of cycling cells in
G0/G1 phase (dotted line)
versus S/G2/M phase (solid line)
(A) and the GR staining of proliferating cells in
G0/G1 phase (dotted line)
versus p16INK4A-arrested
G0/G1 cells (solid line)
(B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. A. Helmberg and S. Schwarz for valuable discussion; Drs. D. Beach, H. Gossen, and B. Auer for donating vectors; S. Lobenwein for excellent technical assistance; and M. Kat Occhipinti for editing the manuscript.
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FOOTNOTES |
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* This work was supported by Austrian Science Fund Grants SFB-F002, P11964-Med, and P11306-Med; Austrian National Bank Project 6156; and the Krebshilfe/Krebsgesellschaft Tirol.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Molecular Pathophysiology, Inst. for General and Experimental Pathology, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria. Tel.: 43-512-507-3102; Fax: 43-512-507-2867; E-mail: Reinhard.Kofler@uibk.ac.at.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M008188200
2 M. J. Ausserlechner, P. Obexer, S. Geley, and R. Kofler, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: T-ALLs, T-cell acute lymphoblastic leukemias; CDK, cyclin-dependent kinase; GC, glucocorticoid; GR, glucocorticoid receptor; PBS, phosphate-buffered saline; MMTV, murine mammary tumor virus; MOPS, 4-morpholinopropanesulfonic acid; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter.
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