From the Cancer Research Campaign Molecular
Pharmacology Group, School of Biological Sciences, University of
Manchester, G38 Stopford Building, Oxford Road, Manchester M13 9PT,
¶ Glaxo Wellcome Research & Development, Medicines Research
Centre, Gunnels Wood Road, Stevenage, Hertsfordshire SG1 2NY,
the
Leukaemia Research Fund Cellular Development Unit,
Department of Biomolecular Sciences, University of Manchester
Institute of Science and Technology, Sackville Street, Manchester M60
1QD, and the ** Cancer Research Campaign Laboratory, Biomedical
Research Centre, Ninewells Medical School, University of Dundee,
Dundee DD1 95Y, United Kingdom
Received for publication, August 4, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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v-Abl protein-tyrosine kinase (PTK) promotes cell
survival without cell proliferation in interleukin (IL)-3-deprived
IC.DP premast cells (1). We now show that in these conditions
v-Abl PTK transcriptionally up-regulated the
cyclin-dependent kinase inhibitor (CDKI)
p21WAF-1 and inhibited CDK2 and CDK4. When readdition
of IL-3 stimulated cell proliferation, p21WAF-1 was
inactivated as a CDKI despite maintenance of elevated protein level.
p21WAF-1 was also up-regulated yet was nonfunctional as a
CDKI when v-Abl PTK was activated in cells maintained in IL-3, but this
occurred without increased p21WAF-1 transcription. Using a
C-terminal epitope-specific p21WAF-1 antibody, v-Abl
PTK-mediated increase in p21WAF-1 could be detected in
intact cells only in the presence of IL-3. This indicated different
binding partners of p21WAF-1 and/or protein conformation in
nondividing or proliferating cells, respectively. The binding of CDK2,
CDK4, or proliferating cell nuclear antigen to
p21WAF-1 and its subcellular localization were unchanged in
the presence or absence of IL-3. However, two-dimensional analysis
revealed different forms of up-regulated p21WAF-1 in
IL-3-deprived, nondividing cells compared with IL-3-stimulated proliferating cells. These data demonstrate that elevation of the CDKI
p21WAF-1 is not always sufficient for cell cycle arrest and
indicate an IL-3-sensitive pathway for the inactivation of
p21WAF-1 function as a CDKI.
One mechanism by which cell cycle progression is regulated is via
the balance between cyclin-dependent kinase
(CDK)1 activation and
inhibition (2). The CDK inhibitor (CDKI) p21WAF-1, the
prototype of the Cip/Kip family of CDKIs, was initially identified in a
coimmunoprecipitate with cyclin D (3, 4). p21WAF-1 was
shown to bind to and inhibit cyclin-CDK complexes (5), incorporating
PCNA into a quaternary complex (3) and thereby regulating the cell
cycle machinery. Initial observations by Xiong et al. (6)
described the primary role for p21WAF-1 as an inhibitor of
CDK activity. However, subsequent investigations suggested that
p21WAF-1 may also function to assemble specifically
CDK4-cyclin D complexes and to target them to the nucleus (7). This has
recently been substantiated in murine fibroblasts lacking
p21WAF-1, wherein CDK4 and cyclin D cannot associate (8).
The generally accepted theory surrounding p21WAF-1 function
therefore suggests that p21WAF-1 is involved in the
assembly of CDK4 complexes but inhibits the activity of CDK2. However,
p21WAF-1 nullizygous mice are developmentally normal (9)
and display normal CDK4 activity, thereby questioning the physiological
role for p21WAF-1 in the assembly specifically of CDK4.
The putative C-terminal nuclear localization signal in
p21WAF-1 may help target the quaternary complex to the
nucleus where it may exert its effect on CDKs. At the onset of mitosis,
p21WAF-1 up-regulation caused the nuclear accumulation of
CDK4 associated with cyclins A and B1 (10). p21WAF-1 also
directs PCNA to the replicative machinery of the cell (11), and cell
cycle arrest caused by p21WAF-1 involves inhibition of PCNA function(s).
p21WAF-1 is required for cell cycle arrest at
G1 phase of the cell cycle; HCT116 colon carcinoma cells
containing wild-type p21WAF-1 undergo growth arrest after
DNA damage, whereas their p21WAF-1 null counterparts do not
(12). Moreover, cells derived from p21WAF-1 null mice have
defective G1 checkpoints (9). p21WAF-1 also
functions at other phases of the cell cycle. During S phase p21WAF-1 prevented replication of DNA possibly because of
the inhibitory effect on PCNA (11) or because of p21WAF-1
mediated inhibition of CDK activity (14). p21WAF-1
overexpression was linked with G1 and G2 arrest
and prevented entry into S phase, although no delay in S phase
progression was observed (15). Conversely, down-regulation of
p21WAF-1 allowed DNA synthesis and entry into mitosis,
thereby reversing growth arrest (16).
We noted previously that the activation of a temperature-sensitive
mutant of v-Abl protein-tyrosine kinase (PTK), a leukemogenic oncogene
promoted cell survival without cell proliferation after the withdrawal
of IL-3 (1). Cell survival was associated with the up-regulation of
Bcl-xL (17). Down-regulation of Bcl-xL using an
antisense approach restored an apoptotic response to IL-3 deprivation
with v-Abl PTK active.2
However, the mechanism whereby v-Abl PTK promoted growth arrest remained unknown. Here we describe the transcriptional up-regulation of
p21WAF-1 3 h after IL-3 withdrawal in cells with
active v-Abl PTK. We also report that readdition of IL-3 to cells with
v-Abl PTK active prevented the inhibition of CDK2 and CDK4, allowing
proliferation despite the maintained high levels of
p21WAF-1 expression.
Reagents--
For Western blot analyses, p21WAF-1
was detected with either monoclonal anti-p21WAF-1 Ab-4 or
polyclonal anti-p21WAF-1 Ab-5 (Calbiochem, Cambridge, MA).
PCNA was detected with monoclonal antibody PC10, CDK2 with polyclonal
antibody C22, and CDK4 with polyclonal antibody M2 (Santa Cruz
Biotechnology Inc., Santa Cruz, CA). Actin was detected with monoclonal
anti-actin antibody (Sigma). All primary antibodies were detected with
horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup,
Denmark) and visualized using Enhanced ChemiLuminescence (PerkinElmer
Life Sciences). For flow cytometric analyses, p21WAF-1
was detected using monoclonal anti-p21WAF-1 antibodies
Ab-4, Ab-6 (Calbiochem), or WA-1 (kindly provided by Dr. Borek
Vojlesek). The irrelevant antibody control was IgG1 mouse
anti-Aspergillus niger glucose oxidase antibody. Primary antibodies were detected using a fluorescein isothiocyanate-conjugated secondary antibody (Dako). For confocal microscopy, monoclonal anti-p21WAF-1 SX118 (Calbiochem) or polyclonal
anti-p21WAF-1 was used followed by Alexa fluor
488-conjugated secondary antibody (Molecular Probes, Eugene, OR).
Unless otherwise stated, all other reagents were obtained from Sigma.
Cell Culture--
The IC.DP cell line was derived from
IL-3-dependent murine mast progenitor cell line IC2.9 by
stable transfection of a temperature-sensitive mutant of the v-Abl PTK
(18). Cells were routinely maintained in Fischer's medium supplemented
with horse serum (10%v/v) (both from Life Technologies, Inc.) and IL-3
conditioned medium (3% v/v) (19). For IL-3 withdrawal experiments,
v-Abl PTK was activated or inactivated by incubation of cells at 32 or
39 °C respectively, for 2 h prior to washing cells in
Fischer's medium (1). Cells were then cultured for up to 96 h at
either temperature. Where appropriate, IL-3 was restored to the culture
medium of IC.DP cells with v-Abl PTK active for 48 h, after the
initial IL-3 withdrawal, or cells were maintained in the presence of
IL-3 throughout the experiment. Cells were treated with actinomycin D
(1 µg/ml), cycloheximide (10 µg/ml), or etoposide (10 µM) as appropriate. Parental IC2.9 cells were used as a
control for the effect of culture temperature.
Cell Cycle Analysis by Flow Cytometry--
Cells were fixed in
ice-cold methanol (70% v/v) and incubated with propidium iodide
(Molecular Probes; 10 µg/ml) for 5 min prior to analysis of DNA
content by flow cytometry. Red fluorescence (DNA-bound propidium,
linear scale, 630 ± 22 nm) was analyzed by a Becton Dickinson
FACSVantage cytometer (Becton Dickinson, San Jose, CA) using the 488-nm
line of the Enterprise laser, (Coherent, Palo Alto, CA) set to excite
at 250 mW. Cell debris was excluded by electronic gating of forward and
orthogonal light scatter profiles, prior to analysis of DNA content.
Immunostaining for p21WAF-1 by Flow Cytometry and
Confocal Microscopy--
For flow cytometric analyses of
p21WAF-1, cells were harvested and fixed in formaldehyde
(1%v/v in phosphate-buffered saline). Immunostaining for
p21WAF-1 was carried out using a panel of monoclonal
anti-p21WAF-1 antibodies (Ab-4, Ab-6, and WA-1). The
majority of experiments were conducted using WA-1 (1:100 in
phosphate-buffered saline containing 500 µg/ml digitonin) and
secondary antibody goat anti-mouse conjugated to fluorescein
isothiocyante (1:40). Cell debris was excluded, and forward and
orthogonal light scatter were measured simultaneously with green
fluorescence (fluorescein isothiocyanate-conjugated antibody; 530 ± 30 nm, log scale) as described above. Data were analyzed using
CellQuest and CellFit BD software. Statistical analysis was by the
two-tailed Student's t test at the p = 0.05 level of significance. Analysis was performed using the Microsoft Excel
package (Microsoft Corporation, Seattle, WA).
For confocal microscopy, cells were harvested and cytospun onto glass
slides before fixing in ice-cold methanol/acetone (1:1 v/v) for 5 min.
Cells were incubated with blocking/permeablilization buffer (0.15 M NaCl, 5 mM KCl, 0.3 mM
Na2HPO4, and 25 mM Trizma base)
containing bovine serum albumin (0.1%w/v), Triton X (0.1% v/v), and
fetal calf serum (1% v/v)) for 30 min. p21WAF-1 was
detected using SX118 at 1 µg/ml or polyclonal
anti-p21WAF-1 at 5 µg/ml in diluent (blocking buffer
containing bovine serum albumin (0.1% w/v), Triton X (0.05% v/v), and
fetal calf serum (1% v/v)) for 1 h, followed by anti-mouse 488 Alexa antibody (1:100 in diluent) for 30 min. Nuclei were stained with
propidium iodide (1 µg/ml containing 100 µg/ml RNase) by incubation
at 37 °C for 30 min. Fluorescence was detected with excitation
wavelengths of 488 or 568 nm using a Leica TCS-4D confocal microscope.
Analysis of S Phase Traverse by BrdU Incorporation--
Cells
were pulse-labeled with BrdU (100 µM) for 1 h at
32 °C and fixed in 1 ml formaldehyde (1% w/v in phosphate-buffered saline). DNA was partially denatured by treatment with 1 ml of 2 N HCl for 30 min at room temperature. Incorporated BrdU was detected by flow cytometry after incubation of cells with
monoclonal anti-BrdU antibody (1:20 in phosphate-buffered saline
containing 500 µg/ml digitonin) for 1 h at room temperature
followed by incubation with goat anti-mouse fluorescein isothiocyanate
secondary antibody (1:40) for 30 min. DNA was counterstained with
propidium iodide, and red fluorescence was analyzed as described above.
Western Blotting--
Western blotting was carried out according
to a standard protocol (20). In brief, 30 µg of protein from cell
lysates were subjected to SDS-PAGE on 14% gels and transferred onto
polyvinylidene difluoride membranes. Immunodetection of
p21WAF-1 was conducted using Ab-4 (1 µg/ml) and goat
anti-mouse horseradish peroxidase (1:3000), followed by detection by
enhanced chemiluminescence (see above). Equal protein loading was
verified by measurement of actin levels. Protein level was assessed
using an imaging densitometer and Molecular Analyst software
(Bio-Rad).
RT-PCR--
RNA was extracted using RNAzol (Biogenesis, Poole,
UK) according to the manufacturer's instructions. cDNA was
synthesized using a reverse transcription-polymerase chain reaction kit
(Stratagene Ltd, Cambridge, UK). PCR was then performed using murine
specific p21WAF-1 primers (Sigma-Genosys Ltd., Pampisford,
UK). Sense primer was 5'-CATTCAGAGCCACAGGCACC-3' and antisense primer
and 5'-CTCCTGACCCACAGCAGAAG-3'.
Assays for CDK2 and CDK4--
CDK2 and CDK4 activities were
analyzed as previously described (21). In brief, 150 µg of protein
from cell lysates was immunoprecipitated with 1 µg of polyclonal
anti-CDK2 or CDK4 antibody. Immunoprecipitates were washed twice in 2×
kinase buffer: 100 mM Hepes, pH 7.4, 20 mM
MgCl2, 5 mM EGTA, 20 mM
Immunoprecipitation--
Cell lysates were prepared using Triton
X lysis buffer: 0.4% (v/v) Triton X-100, 150 mM KCl, 25 mM Hepes, pH 7.6, 5 mM dithiothreitol, 50 mM NaF, and protease inhibitor mixture. 150 µg of protein
from cell lysates was precleared with protein A-Sepharose (Amersham Pharmacia Biotech), followed by immunoprecipitation using polyclonal anti-p21WAF-1 Ab-5. Immune complexes were harvested with
protein A, and immunoprecipitated proteins were analyzed by SDS-PAGE as
above. Immunodetection was carried out using monoclonal
anti-p21WAF-1 Ab-4 (1 µg/ml), monoclonal anti-PCNA (2 µg/ml), polyclonal anti-CDK2 (1 µg/ml), or polyclonal anti-CDK4 (1 µg/ml).
Two-dimensional Gel Electrophoresis--
IgPhor two-dimensional
gel equipment and reagents were obtained from Amersham Pharmacia
Biotech. Ten million cells were lysed directly into rehydration/lysis
buffer containing 8 M urea, 2 M thiourea, 2%
(w/v) CHAPS, 65 mM dithiothreitol, and 0.5% (v/v) immobilized pH gradient ampholites. For the first dimension,
lysates were subjected to isoelectric focusing for 90 KVh on a precast pH gradient gel (pH 3-10 linear gradient). Second dimensional electrophoresis was carried out by standard SDS-PAGE on 15% gels, and
p21WAF-1 was detected by Western blotting using polyclonal
anti-p21WAF-1 (1 µg/ml) as described above.
Cell Cycle Arrest Follows IL-3 Withdrawal in Cells with Activated
v-Abl PTK--
Fig. 1 confirms our
previous studies that in the presence of active v-Abl PTK (at 32 °C)
there was no change in viable IC.DP cell number following the
withdrawal of IL-3 (17). In contrast, in IC.DP cells with inactive
v-Abl PTK (at 39 °C) and parental IC2.9 cells (with no v-Abl PTK),
there was a decrease in viable cell number with time (Fig. 1 and Ref.
1). Analysis of cell cycle phase distribution in IC.DP cells after IL-3
withdrawal with v-Abl PTK active demonstrated that by 24 h there
was a 10% increase of cells in G1 phase and a
corresponding decrease in cells in G2 phase of the cell
cycle, indicating that v-Abl allows the completion of ongoing rounds of
DNA synthesis but no reinitiation from G1 (Table
I).
v-Abl PTK Up-regulates p21WAF-1 Protein
Level--
v-Abl PTK activation in the absence of IL-3 resulted in the
accumulation of cells at the G1/S checkpoint. So we
examined the effects of IL-3 withdrawal and/or v-Abl PTK activation on
the cellular levels of the CDKI p21WAF-1. The DNA sequence
of p21WAF-1 in IC.DP cells was confirmed as being wild type
(data not shown). Fig. 2A
shows that v-Abl PTK activation results in the up-regulation of
p21WAF-1 as early as 3 h after IL-3 withdrawal, with a
5-fold increase observed by 24 h (mean, n = 3).
Conversely, when v-Abl PTK was inactivated in IL-3-deprived IC.DP cells
and parental IC.9 cells (no v-Abl PTK) at both 32 °C and 39 °C,
there was no up-regulation of p21WAF-1; instead its level
decreased as cells died. The readdition of IL-3 to IC2.9 cells that had
been deprived of this cytokine for 2 h did not increase
p21WAF-1 levels above basal levels (data not shown). Taken
together these data show that the removal of the mitogenic stimulus of
IL-3 per se did not cause a 5-fold increase in
p21WAF-1 but that this resulted from v-Abl PTK
signaling.
IL-3 Drives Re-entry into Cell Cycle Despite Maintained High Levels
of p21WAF-1--
The most notable effect of v-Abl PTK
shown in Fig. 2 was the suprastimulation of p21WAF-1
levels. Does this mean that the readdition of IL-3 would fail to
stimulate stationary cells with v-Abl PTK active to re-enter the cell
cycle? Would IL-3 decrease p21WAF-1 levels returning them
to basal levels? To investigate this, cell population growth was
monitored by measurement of viable cell number (Fig.
3A) and by S phase traverse
following pulse labeling with BrdU (Fig. 3B). Viable cell
number doubled 48 h after the readdition of IL-3 to cells with
v-Abl PTK activity maintained. Immediately prior to IL-3 withdrawal
there were 12% (12 ± 3%, n = 3) cells in S
phase, 48 h after IL-3 withdrawal the percentage S phase cells was
reduced to 6% (6 ± 2%, n = 3), but 24 h
after the readdition of IL-3 to this stationary cell population, the percentage of cells moving through S phase was increased to 17% (17 ± 4%, n = 3). Fig. 3C
demonstrates that despite this increase in cell cycle traverse, the
v-Abl PTK-mediated up-regulation of p21WAF-1 was
maintained. When v-Abl PTK was activated in cells continually cultured
in the presence of IL-3, p21WAF-1 levels were elevated
6-fold (mean n = 3) despite continued cell proliferation (Fig. 3E). Parallel experiments with parental
2.9 cells (no v-Abl PTK) demonstrated that there were no
temperature-dependent changes in the levels of
p21WAF-1 (data not shown).
The levels and activities of CDK2 (which acts at both G1/S
and G2/M boundaries) and CDK4 (which acts only at
G1/S) were assessed for cells deprived of IL-3 for 48 h and protected by v-Abl PTK and compared with those from cells that
were restimulated with IL-3 for 48 h that had re-entered the cell
cycle. CDK2 or CDK 4 were immunoprecipitated, and the levels
coimmunoprecipitated p21WAF-1 were analyzed by Western
blotting. The levels of p21WAF-1 associated with CDK2 and
CDK4 when v-Abl PTK was activated was regardless of the presence or
absence of IL-3 (Fig. 3D). Although p21WAF-1
levels were elevated in the protein complex, the protein expression levels of CDK2 and CDK4 were the same with or without IL-3 (Fig. 3D). The activity of CDK2 or CDK4 in these
immunoprecipitates was assessed in vitro by measurement of
substrate phosphorylation using histone H1. Predictably, CDK activities
were high in the presence of IL-3 and lowered when the cytokine was
absent (Fig. 3D). These data prove that increased levels of
p21WAF-1 bound to CDK2 or CDK4 do not always inhibit their
activity. This finding necessarily implicates an IL-3-sensitive
mechanism for inactivation of the activity of p21WAF-1 as a
CDKI.
p21WAF-1 Is Differentially Regulated in
Nonproliferating and Proliferating IC.DP Cells with Active v-Abl
PTK--
To probe the mechanism of up-regulation of
p21WAF-1 by v-Abl PTK and the effects of IL-3 upon this, we
examined p21WAF-1 mRNA levels using RT-PCR. An increase
in p21WAF-1 transcript was observed in IC.DP cells with
v-Abl PTK active in nonproliferating cells in the absence of IL-3, when
high levels of p21WAF-1 protein were detected (Fig.
2A). However, in proliferating IC.DP cells in the presence
of IL-3 with v-Abl PTK active and up-regulated p21WAF-1
protein levels (Fig. 3E), there was no increase in
p21WAF-1 transcript (Fig.
4A). Treatment of
IL-3-stimulated IC.DP cells with the topoisomerase II inhibitor
etoposide resulted in the stabilization of p53 and up-regulation of
p21WAF-1 in the presence or absence of v-Abl PTK activity
(data not shown). Here the elevation of p21WAF-1 protein is
mediated by transcription after DNA damage, but in this case,
p21WAF-1 transcription is not prevented by the IL-3 signal
(Fig. 4A, lane E).
When transcription was inhibited with actinomycin D, the up-regulation
of p21WAF-1 protein was abolished in cells with v-Abl PTK
active in the absence of IL-3 but not in the presence of IL-3 at 3 h (Fig. 4B, panel i). When translation was
inhibited with cycloheximide, the up-regulation of p21WAF-1
protein was abolished in cells both in the presence and absence of IL-3
(Fig. 4B, panels i and ii). Taken
together these data implicate that both transcriptional and
translational mechanisms drive the up-regulation of
p21WAF-1 by active v-Abl PTK in IL-3-deprived cells.
However, inhibition of transcription by actinomycin D had no effect on
the initial up-regulation of p21WAF-1 by v-Abl PTK in the
presence of IL-3. Thus, the increase in p21WAF-1 protein
levels at 3 h must be mediated via continued translation of
existing p21WAF-1 transcript. These data illustrate two
distinct mechanisms that both lead to the elevation of
p21WAF-1 protein level in IC.DP cells but that result in
different functional forms of p21WAF-1:
p21WAF-1 active as a CDKI in nonproliferating cells but
inactive in proliferating cells.
Different Conformational Forms of p21WAF-1 Are Seen in
Nonproliferating Compared with Proliferating IC.DP Cells--
To
characterize further the mechanism whereby v-Abl PTK up-regulated
p21WAF-1 and the effects of IL-3 upon this, flow cytometric
analysis of p21WAF-1 was performed in intact, fixed cells
using a panel of anti-p21WAF-1 monoclonal antibodies (Fig.
5A). Of the three antibodies
that detected p21WAF-1 in IC.DP cells, only WA-1 revealed
changes in p21WAF-1 immunoreactivity after v-Abl PTK
activation, and this was dependent on the presence of IL-3. Fig.
5B (panel i) illustrates that the increase in
p21WAF-1 protein observed by Western blot was not detected
using the C-terminal specific antibody WA-1 and flow cytometry in cell
cycle-arrested IC.DP cells. The basal level of p21WAF-1 was
still detectable, suggesting that the newly transcribed and newly
synthesized p21WAF-1 protein had been modified to mask this
C-terminal epitope. However, when IL-3 was present, a significant
increase in p21WAF-1 associated immunofluorescence was
detected using WA-1 (p < 0.01) in agreement with
Western blot results (Figs. 3E and 5B,
panel ii); in this cellular context the C terminus of
p21WAF-1 was exposed on the newly synthesized protein. When
v-Abl PTK was inactive, both in the presence and absence of IL-3 (Fig.
5B, panels iii and iv), no increase in
p21WAF-1 associated fluorescence was detected, and a
subpopulation of cells were observed in both cases that had no
detectable p21WAF-1-associated fluorescence.
In addition, confocal microscopy was employed to confirm the
IL-3-dependent changes in the availability for antibody
binding to the p21WAF-1 C terminus observed by flow
cytometry. Fig. 5C (panel i) demonstrates that in
the absence of IL-3, the C terminus of p21WAF-1 was
inaccessible to another C-terminal epitope-specific
p21WAF-1 antibody SX118 despite v-Abl PTK-mediated
up-regulation of p21WAF-1. However, in the presence of
IL-3, the p21WAF-1 C-terminal epitope for SX118 binding is
available (Fig. 5C, panel ii), and the
up-regulated p21WAF-1 protein was detectable. Together
these results indicate that in IC.DP cells with active v-Abl PTK, the C
terminus of p21WAF-1 is occluded under conditions of cell
cycle arrest and exposed during cell proliferation. This may reflect a
difference in p21WAF-1 protein conformation or a difference
in p21WAF-1 protein-protein interactions in the presence of
IL-3 to reveal the C terminus.
To investigate the existence of different forms of p21WAF-1
in cell cycle arrested and proliferating IC.DP cells with active v-Abl PTK, cell lysates were subjected to two-dimensional gel
electrophoresis. Fig. 5D illustrates that multiple forms of
p21WAF-1 do indeed exist in IC.DP cells. Additionally, a
different profile of isoforms of p21WAF-1 was observed when
comparing lysates from cells stimulated by IL-3 to proliferate with
those deprived of IL-3 and cell cycle arrested. Specifically, the
presence of IL-3 consistently resulted in the loss of a basic isoform,
spot 6, and the appearance of an additional acidic isoform, spot 1, and
a neutral isoform, spot 3 when compared with the isoform profile of
IL-3-deprived cells.
There Are No Differences in Nonproliferating Compared with
Proliferating IC.DP Cells with Respect to Subcellular Localization of
p21WAF-1 or Its Binding to CDKs or
PCNA--
p21WAF-1 has a putative nuclear localization
signal at its C terminus and has been reported typically to be a
nuclear protein (22). However, there are also reports that
p21WAF-1 can be detected as a cytosolic protein (23). One
explanation for the inactivity of high levels of p21WAF-1
in IL-3-stimulated IC.DP cells with v-Abl PTK active could be that it
is sequestered in the cytosol. The subcellular localization of
p21WAF-1 was examined in conditions of cell cycle arrest
and proliferation in IC.DP cells with active v-Abl PTK using a
polyclonal antibody to ensure the detection of different forms of
p21WAF-1. Fig. 6 indicates
that p21WAF-1 was detected exclusively in the nuclei of
IC.DP cells both in conditions of cell cycle arrest and
proliferation.
The binding of p21WAF-1 to CDKs and to PCNA is important
for p21WAF-1 mediated inhibition of cell cycle progression
(11, 14). Therefore, we investigated the binding of
p21WAF-1 to PCNA, CDK2, and CDK4 by coimmunoprecipitation
from IC.DP cells with v-Abl PTK active. Fig. 7 illustrates that both in
the presence and absence of IL-3, no differences in
p21WAF-1 binding of PCNA, CDK2, and CDK4 were detected.
These results were also confirmed by immunofluorescence and confocal
microscopy (data not shown).
We set out to determine how cells deprived of IL-3 with v-Abl PTK
activated accumulated in G1 phase of the cell cycle (Fig. 1
and Table I). We show that this cell cycle arrest is associated with
v-Abl PTK-mediated up-regulation of the "universal" CDKI, p21WAF-1 (Fig. 2A). After IL-3 withdrawal in the
absence of v-Abl PTK activity, there was no up-regulation of
p21WAF-1 at any time before the onset of cell death (Fig.
2). Thus, the activation of v-Abl PTK rather than the withdrawal of the
mitogen per se caused the observed increase in this
CDKI.
p21WAF-1 has been shown to have a dual role in the
regulation of CDK4 kinase activity: Transfection of
p21WAF-1 into U20S cells resulted in the assembly of kinase
active CDK4/cyclin D1 complexes (7). Here we show that in the presence
of v-Abl PTK and IL-3 cells proliferate but the up-regulated
p21WAF-1 fails to inactivate the kinase activity of both
CDK4 and CDK2 (both of which remain at basal expression levels).
p21WAF-1 function may therefore vary depending on the
incoming signal(s): when it is up-regulated by v-Abl PTK signaling in
the absence of IL-3, p21WAF-1 functions as a CDKI, whereas
in the presence of IL-3 it has a distinct function possibly as an
assembly factor and/or subcellular localization cue for CDK/cyclins,
although we found no evidence of any difference in the subcellular
distribution of p21WAF-1 itself in the presence and absence
of IL-3 (Fig. 6).
We sought to determine at a molecular level how IL-3 signaling bypasses
the cell cycle arrest associated with the up-regulation of
p21WAF-1 and inhibition of CDK2 and CDK4. v-Abl PTK
activation resulted in the transcriptional up-regulation of
p21WAF-1 mRNA and protein level (Figs. 2A,
3E, and 4A). Initially p21WAF-1 was
identified as the main transcriptional target of p53 (4). Protein
stabilization of p53 follows DNA damage in IC.DP cells (data not
shown), but this did not occur after IL-3 withdrawal with v-Abl active.
Nevertheless, we cannot rule out a p53-dependent mechanism
for v-Abl PTK-mediated transcriptional up-regulation of
p21WAF-1. There are p53-independent mechanisms to
transcriptionally up-regulate p21WAF-1 via a diverse group
of transcription factors including the progesterone receptor, E2F, and
several STATs (STAT1, STAT3, and STAT5; reviewed in Ref. 24). We do not
yet know which transcriptional activator(s) drive p21WAF-1
up-regulation downstream of v-Abl PTK signaling; however, because STATs
are activated downstream of v-Abl PTK signaling (25), these seem likely candidates.
v-Abl PTK-mediated up-regulation of p21WAF-1 protein level
was further increased by the readdition of IL-3, and this was
concomitant with entry to the cell cycle (Fig. 3, A and
C). In the presence of this mitogen, p21WAF-1
up-regulation was not transcriptional (Fig. 4, A and
B, panel ii). Several transcription independent
mechanisms to up-regulate p21WAF-1 have been reported.
Increased stabilization of p21WAF-1 mRNA has led to
up-regulated p21WAF-1 protein, for example, by binding of
the Elav-like protein HuD to the transcript (26). Increased protein
stability contributed to increased levels of p21WAF-1 by
direct inhibition of the proteosome (27) or binding and abrogation of
PCNA function, thus preventing progression through the proteosome (28).
However, neither of these mechanism(s) are consistent with our
observations in IL-3 replete IC.DP cells with v-Abl PTK activated,
implying another route to p21WAF-1, possibly by increase
translation of existing mRNA, although further work is required to
confirm this. IL-3 does not suppress the transcription of
p21WAF-1 in every cellular context as shown in Fig.
4A (lane E) where the etoposide damage signal
maintains p21WAF-1 transcription in the presence of
IL-3.
One of the striking questions posed by our studies is how IC.DP cells
proliferate at the same rate with either basal levels of
p21WAF-1 (with IL-3 but inactive v-Abl PTK) or with 6-fold
up-regulated levels of p21WAF-1 (with IL-3 and active v-Abl
PTK), where in the latter case p21WAF-1, although
associated in increased amount with CDK4 and CDK2 (Fig. 3D),
does not function as a CDKI. We showed that the p21WAF-1
protein up-regulated in proliferating IC.DP cells exists in a different
conformation to that in cell cycle arrested cells, reflected by
different C-terminal epitope availability (Fig. 5, B and
C). This suggests either different conformational forms of
p21WAF-1 and/or a change in protein-protein interactions.
Our studies using two-dimensional gel electrophoresis demonstrate that
there are several isoforms of p21WAF-1 and that the isoform
profile is different in IC.DP cells with v-Abl PTK active in the
presence or absence of IL-3 (Fig. 5D). We are currently
pursuing the identity of these isoforms. Post-translational modification of p21WAF-1 at its C terminus can occur;
phosphorylation in the PCNA binding domain of p21WAF-1 has
been recently reported (29), and post-translational modification might
affect its subcellular location and/or the binding of
p21WAF-1 to other proteins. However, we have not detected
any difference in the subcellular location or the binding of several
established binding partners of p21WAF-1 (Fig. 6, 7), and
these parameters could not therefore account for the changes in epitope
availability monitored by flow cytometry and confocal microscopy (Fig.
5, B and C). Other p21WAF-1 binding
proteins have been identified including GADD45 (30), stress-activated
protein kinases (31), and apoptosis signaling kinase 1 (23), but the
effects of these binding partners on p21WAF-1 function as a
CDKI are currently unknown. Interestingly, the protein phosphatase
inhibitor SET binds the C terminus of p21WAF-1 (in the
region of the WA-1 and SX118 binding sites), abrogating its function as
an inhibitor of CDK2 specifically bound to cyclin E (32). We were
unable to demonstrate SET binding to p21WAF-1 in IC.DP
cells cultured with or without IL-3 (data not shown).
In summary, we propose a model whereby v-Abl PTK reinforces cell cycle
arrest in the absence of IL-3 that is associated with the
transcriptional up-regulation of p21WAF-1 protein, in a
form bound to PCNA and CDK2 or CDK4 that functions as a CDKI (Fig.
8). In conditions of IL-3-driven cell
proliferation, the transcriptional up-regulation of
p21WAF-1 by v-Abl PTK is abrogated, yet
p21WAF-1 protein is maintained at elevated levels. However,
these high levels of p21WAF-1 molecules in proliferating
cells exist in a different form that is functionally inactive with
respect to CDK inhibition. We are currently investigating the mechanism
of inactivation of p21WAF-1 by IL-3 with respect to CDKI
function in proliferating cells expressing elevated levels of this cell
cycle regulator.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerphosphate, 2 mM NaF, 2 mM
dithiothreitol, and 20 µM cAMP-dependent
kinase inhibitor protein kinase inhibitor. Kinase assays were
prepared with 2× kinase buffer and calf thymus histone H1 (1 mg/ml).
Reactions were started by addition of [32P]ATP (1:100 in
0.5 mM cold ATP in 10 mM Hepes, pH 7.4),
incubated at 30 °C for 30 min and terminated by heating to 95 °C
in SDS-PAGE sample buffer, followed by SDS-PAGE analysis and autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cell death kinetics following IL-3 withdrawal
from IC.DP and IC2.9 murine premast cells. Viable cell number was
assessed following the withdrawal of IL-3. IC.DP at 32 °C (v-Abl PTK
active; ); IC.DP at 39 °C (v-Abl PTK inactive;
); IC2.9 at
32 °C (no v-Abl PTK;
); IC2.9 at 39 °C (no v-Abl PTK;
).
Percentage of cell cycle distribution up to 72 h after IL-3
withdrawal of IC.DP cells containing activated v-Abl PTK
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Fig. 2.
Up-regulation of p21WAF-1 by
v-Abl PTK activation after withdrawal of IL-3. Western blot
analysis of p21WAF-1 protein expression following IL-3
withdrawal from IC.DP cells (A) and IC2.9 cells
(B). Also indicated is the percentage of cell death
occurring in the cell samples from which the lysates were made. The
data shown are representative of three independent repeat
experiments.
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Fig. 3.
IL-3 promotes proliferation of IC.DP cells
with maintained high levels of p21WAF-1. A,
kinetics of the increase in viable cell number following readdition of
IL-3 to cell cycle-arrested cells. Results shown are presented as the
means ± S.E. (n = 3). B, S phase
traverse in IL-3-restimulated IC.DP cells expressing active v-Abl PTK.
IC.DP cells with active v-Abl PTK were deprived of IL-3 for 48 h
before readdition of the cytokine. Data shown are representative of
three independent repeat experiments. C,
p21WAF-1 protein levels in IL-3-restimulated IC.DP cells
containing active v-Abl PTK. Western blot analysis of
p21WAF-1 protein expression following IL-3 readdition after
48 h IL-3 withdrawal from IC.DP cells with v-Abl PTK. The data
shown are representative of three independent repeat experiments.
E, p21WAF-1 protein expression in IC.DP cells
with v-Abl TK activation maintained in the presence of IL-3. Western
blot analysis of p21WAF-1 protein expression in IC.DP cells
with v-Abl PTK maintained in IL-3. The data shown are representative of
three independent repeat experiments. D, association of CDK2
or CDK4 with p21WAF-1 and resultant CDK activity in IC.DP
cells with v-Abl PTK active in the presence or absence of IL-3. Western
blots of CDK2 (panel i), CDK4 (panel ii), and
p21WAF-1 (panels iii and
iv) in coimmunoprecipitates from lysates of IC.DP
cells cultured for up to 48 without IL-3 and from cells cultured up to
48 h after the readdition of IL-3. E, panel
v, shows the autoradiographs of 32P-labeled histone H1
incubated with either CDK2 or CDK4 immunoprecipitate. Data shown are
representative of three independent repeat experiments.
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Fig. 4.
Differential regulation of
p21WAF-1 in cell cycle arrested and proliferating IC.DP
cells containing activated v-Abl PTK. A, RT-PCR
analysis of p21WAF-1 mRNA levels after v-Abl PTK
activation in the presence of absence of IL-3. mRNA levels of
p21WAF-1 in IC.DP with v-Abl PTK activated and incubated at
the times shown in the presence of absence of IL-3 or in the presence
of IL-3 after exposure to the topoisomerase II inhibitor etoposide
(lane E). RT-PCR was performed using primers specific for
-actin and p21WAF-1 (see methods). Data shown are
representative of four independent repeat experiments. B,
effects of inhibition of transcription and translation on
p21WAF-1 protein levels. IC.DP cells with active v-Abl PTK
were treated either with actinomycin D or with cycloheximide (see
"Experimental Procedures") in the absence (panel i) or
presence (panel ii) of IL-3. Western blot analysis of
p21WAF-1 protein expression was performed using cell
lysates prepared after cell culture for the times shown. The data shown
are representative of four independent repeat experiments.
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Fig. 5.
Analyses of different forms of
p21WAF-1 protein in IC.DP cells containing activated v-Abl
PTK in the presence and absence of IL-3. A, epitope map
for p21WAF-1 monoclonal antibodies. B, flow
cytometric frequency histograms from IC.DP cells containing activated
v-Abl PTK. Cells were cultured for 0 or 24 h either in the absence
(panel i) or presence (panel ii) of IL-3. Cells
were fixed and stained with WA-1, a monoclonal antibody raised to a
C-terminal epitope on p21WAF-1 (Vojlesek and Ball, personal
communication). The filled histogram is from an irrelevant antibody
control. In panel i the open histograms from 0 and 24 h
are superimposed. Data shown are representative of three independent
repeat experiments. C, immunofluorescence for
p21WAF-1 in IC.DP cells containing activated v-Abl PTK.
Cells were cultured for 0 or 48 h either in the absence
(panel i) or presence (panel ii) of IL-3. Cells
were fixed and stained with SX118, a monoclonal antibody raised to a
C-terminal epitope on p21WAF-1. p21WAF-1 was
detected with a Alexa 488-conjugated secondary antibody
(green), and nuclei were stained with propidium iodide
(red). Data shown are representative of three independent
repeat experiments. The white bar delineates 10 µm.
D, two-dimensional electrophoresis of p21WAF-1
in IC.DP cells with active v-Abl PTK. Whole cell lysates from IC.DP
cells cultured for 24 h either in the absence (panel i)
or presence (panel ii) of IL-3 were analyzed. Different
isoforms of p21WAF-1 are numbered 1-6. Arrows
represent differences observed in the absence and presence of
IL-3. Data shown are representative of three independent
repeat experiments.
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Fig. 6.
Subcellular location of
p21WAF-1. Immunofluorescence for p21WAF-1
was carried out on IC.DP cells with active v-Abl PTK 48 h after
IL-3 withdrawal (A) or in continuous culture with IL-3
(B), using polyclonal anti-p21WAF-1, Ab-5.
p21WAF-1 was detected with a Alexa 488-conjugated secondary
antibody (green), and nuclei were stained with propidium
(red). Data shown are representative of three independent
repeat experiments. The white bar delineates 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Analysis of p21WAF-1 binding to
PCNA, CDK2, and CDK4. p21WAF-1 coimmunoprecipitated
proteins were analyzed by SDS-PAGE and immunoblotting for
p21WAF-1, PCNA, CDK2, and CDK4. The Ab-5 polyclonal
anti-p21WAF-1 antibody was used to immunoprecipitate
(IP) p21WAF-1. Data are representative of three
independent repeat experiments.
Although it may seem counter-intuitive that an oncogenic tyrosine
kinase reinforces cell cycle arrest, we speculate that there are
circumstances in which this might be advantageous for a tumor cell
(e.g. when it is located in a hostile microenvironment with limiting growth/survival factors). Entry to cell cycle in the absence
of appropriate mitogenic stimuli promotes apoptosis (33). Therefore,
the rapid imposition of p21WAF-1 driven G1
arrest may be important in the mechanism whereby v-Abl PTK maintains
cell viability during the period prior to the up-regulation of the
anti-apoptotic protein Bcl-xL (17). When
p21WAF-1 up-regulation was abrogated in HCT116 colon
carcinoma cells using an antisense approach, instead of growth arrest
after irradiation cells committed to apoptosis (34). If we can
understand how growth arrest associated with up-regulated levels of
p21WAF-1 is overridden by incoming signals from mitogens,
this information might be exploitable therapeutically to modulate the
cellular decision between growth arrest and
apoptosis. In situations where tumor cells survive after drug-induced damage because they can undergo
p21WAF-1-mediated cell cycle arrest and buy time to repair
the damage, they may instead be pushed through the cell cycle via
inactivation of p21WAF-1 function and commit to apoptosis
as they attempt replication on a damage DNA template.
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ACKNOWLEDGEMENTS |
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We thank Boris Vojlesek for the gift of the WA-1 antibody and Duncan Smith for guidance with two-dimensional gels. We thank John Hickman, Andy Koff, Nic Jones, Guy Makin, and Bernard Corfe for constructive criticism of this manuscript.
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FOOTNOTES |
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* 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.
§ Supported by a Biotechnology and Biological Sciences Research Council case studentship with GlaxoWellcome.
Supported by the Lister Institute of Preventive Medicine. To
whom correspondence should be addressed. Tel.: 44-161-275-5495; Fax: 44-161-275-5600; E-mail: cdive@man.ac.uk.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M007073200
2 Q. Chen and C. Dive, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; CDKI, CDK inhibitor; PCNA, proliferating cell nuclear antigen; PTK, protein-tyrosine kinase; IL, interleukin; BrdU, bromodeoxyuridine; PAGE, polyacrylamide gel electrophoresis; RT, reverse transcription; PCR, polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Evans, C. A., Owen-Lynch, P. J., Whetton, A. D., and Dive, C. (1993) Cancer Res. 53, 1735-1738[Abstract] |
2. | Morgan, D. O. (1995) Nature 374, 131-134[CrossRef][Medline] [Order article via Infotrieve] |
3. | Xiong, Y., Zhang, H., and Beach, D. (1992) Cell 71, 505-514[Medline] [Order article via Infotrieve] |
4. | El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) Cell 75, 817-825[Medline] [Order article via Infotrieve] |
5. | Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) Cell 75, 805-816[Medline] [Order article via Infotrieve] |
6. | Xiong, Y., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. (1993) Nature 366, 701-704[CrossRef][Medline] [Order article via Infotrieve] |
7. | LaBaer, J., Garrett, M. D., Stevenson, L. F., Slingerland, J. M., Sandhu, C., Chou, H. S., Fattaey, A., and Harlow, E. (1997) Genes Dev. 11, 847-862[Abstract] |
8. |
Cheng, M.,
Olivier, P.,
Diehl, J. A.,
Fero, M.,
Roussel, M. F.,
Roberts, J. M.,
and Sherr, C. J.
(1999)
EMBO J.
18,
1571-1583 |
9. | Deng, C., Zhang, P., Harper, J. W., Elledge, S. J., and Leder, P. (1995) Cell 82, 675-684[Medline] [Order article via Infotrieve] |
10. |
Dulic, V.,
Stein, G. H.,
Far, D. F.,
and Reed, S. I.
(1998)
Mol. Cell. Biol.
18,
546-557 |
11. | Cayrol, C., Knibiehler, M., and Ducommun, B. (1998) Oncogene 16, 311-320[CrossRef][Medline] [Order article via Infotrieve] |
12. | Waldman, T., Lengauer, C., Kinzler, K. W., and Volgelstein, B. (1996) Nature 381, 713-716[CrossRef][Medline] [Order article via Infotrieve] |
13. | Deleted in proof |
14. | Ogryzko, V. Y., Wong, P., and Howard, B. H. (1997) Mol. Cell. Biol. 17, 4877-4882[Abstract] |
15. | Medema, R. H., Klompmaker, R., Smits, V. A. J., and Rijksen, G. (1998) Oncogene 16, 431-441[CrossRef][Medline] [Order article via Infotrieve] |
16. | Nakanishi, N., Adami, G. R., Robetorye, R. S., Noda, A., Venable, S. F., Dimitrov, D., Pererira-Smith, O. M., and Smith, J. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4352-4356[Abstract] |
17. | Chen, Q., Turner, J., Watson, A. J. M., and Dive, C. (1997) Oncogene 15, 2249-2254[CrossRef][Medline] [Order article via Infotrieve] |
18. | Kipreos, E. T., Lee, G. J., and Wang, J. Y. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1345-1349[Abstract] |
19. | Karasuyama, H., and Melchers, F. (1988) Eur. J. Immunol. 18, 97-104[Medline] [Order article via Infotrieve] |
20. | Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , pp. 481-501, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
21. | Dulic, V., Lees, E., and Reed, S. I. (1992) Science 257, 1958-1961[Medline] [Order article via Infotrieve] |
22. | Duttaroy, A., Qian, J. F., Smith, J. S., and Wang, E. (1997) J. Cell. Biochem. 64, 434-446[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Asada, A.,
Yamada, T.,
Ichijo, H.,
Delia, D.,
Miyazono, K.,
Fukumuro, K.,
and Mizutani, S.
(1999)
EMBO J.
18,
1223-1234 |
24. | Gartel, A. L., and Tyner, A. L. (1999) Exp. Cell. Res. 246, 280-289[CrossRef][Medline] [Order article via Infotrieve] |
25. | Danial, N. N., Pernis, A., and Rothman, P. B. (1995) Science 269, 1875-1877[Medline] [Order article via Infotrieve] |
26. |
Joseph, B.,
Orlian, M.,
and Furneaux, H.
(1998)
J. Biol. Chem.
273,
20511-20516 |
27. | Blagosklovy, M. V., Wu, G. S., Omura, S., and El-Deiry, W. S. (1996) Biochem. Biophys. Res. Commun. 227, 564-569[CrossRef][Medline] [Order article via Infotrieve] |
28. | Cayrol, C., and Ducommun, B. (1998) Oncogene 17, 2437-2444[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Scott, M. T.,
Morrice, N.,
and Ball, K. L.
(2000)
J. Biol. Chem.
275,
11529-11537 |
30. | Kearsey, J, M., Coates, P. J., Precott, A. R., Warbrick, E., and Hall, P. A. (1995) Oncogene 11, 1675-1683[Medline] [Order article via Infotrieve] |
31. | Shim, J., Lee, H., Park, J., Kim, H., and Choi, E.-J. (1996) Nature 381, 804-807[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Estanyol, J. M.,
Jaumot, M.,
Casanovas, O.,
Rodriguez-Vilarrupla, A.,
Agell, N.,
and Bachs, O.
(1999)
J. Biol. Chem.
274,
33161-33165 |
33. | Colombel, M., Olsson, C. A., Ng, P. Y., and Buttyan, R. (1992) Cancer Res. 16, 4313-4319 |
34. |
Tian, H.,
Wittmack, E. K.,
and Jorgensen, T. J.
(2000)
Cancer Res.
60,
679-684 |