From the Laboratory of Receptor Biology and Gene
Expression and Laboratory of Pathology, NCI, National Institutes of
Health, Bethesda, Maryland 20892 and the § Department of
Microbiology, Howard University, Washington, D. C. 20001
Received for publication, August 20, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Although extensive homology exists between
related genes p53 and p73, recent data suggest
that the family members have divergent roles. We demonstrate that the
differential regulatory roles of p53 family member p73 are highly
cell-context and promoter-specific. Full-length p73 expressed in the
transformed leukemia cell line Jurkat behaves as a specific dominant
negative transcriptional repressor of the cell cycle inhibitor gene
p21 and blocks p53-mediated apoptosis. These findings
provide evidence for a new mechanism in oncogenesis through which the
functional properties of p73 can be altered in an inheritable and
cell-specific fashion independent of transcriptional coding.
The meticulous regulation of cellular functions from proliferation
to programmed cell death in T-cells is instrumental in maintaining
proper immune response. Disruption of T-cell homeostasis can lead to
compromised immunity, autoimmune disorders, and leukemia. T-cell
receptor-activated induced cell death is a vital cellular program necessary for proper T-cell homeostasis. The recently described
role of the p53 family member, p73, in T-cell receptor-activated induced cell death emphasizes its critical role in T-cell immune function (1). The well established role of tumor suppressor p53 in
apoptosis correlates with the high prevalence of p53 gene mutations in 30% of adult T-cell lymphomas (2-5). Unlike p53, numerous studies have failed to demonstrate p73 tumor-associated mutations (6-13). In addition, p73-deficient mice do not present spontaneous tumors, suggesting a limited role of p73 in tumor suppression (14).
Although the phenotypes of p53- and p73-deficient mice differ, the
related gene products share trans-activation function of certain
pro-apoptotic p53 target genes including BAX,
p21, MDM2, and GADD45 (15-17).
However, p73 does not regulate all p53 target genes in the same manner
as p53. For example, pro-apoptotic PIGs and
KILLER/DR5 genes do not respond at all or as well to
exogenously expressed p73 in H1299 cells (16). The primary structure of p73 diverges from that of p53 by the inclusion of an extended carboxyl
terminus greater than 200 amino acids containing a sterile Of particular interest for this study was the role of p73 in the
regulation of p53 target gene p21 in lymphoma cells. As a cell cycle-dependent kinase inhibitor, p21 plays a major
role in cell cycle regulation at the G1/S checkpoint. As
reported recently (36), p21 also functions in T-cell homeostasis
through involvement in CD95-induced apoptosis. Although p53 is mutated
and protein levels are undetectable in the lymphoblastoma T-cell line
Jurkat, p21 expression remains inducible during T-cell
activation (37). Reported herein is the unexpected finding that p73
does not compensate for this p53 deficiency at the p21
promoter. In fact, not only is p73 incapable of inducing the
p21 gene in Jurkat cells, it acts as a dominant negative
repressor of p53-dependent transcription and apoptosis in a
cell type- and promoter-specific manner. Moreover, this cell-specific
divergence in p73 function is associated with cell-restricted
post-translational modifications of p73.
Plasmids--
HA1-p73
( Cell Culture and Transfections--
Jurkat T-cells were
maintained in RPMI 1640 media containing 10% fetal bovine serum
and 100 units/ml penicillin and streptomycin (Gemini, Invitrogen) at
37 °C, 5% CO2 in 2-liter tissue culture roller bottles
with rotation. Transfections of Jurkat T-cells were carried out by
electroporation using the BTX ECM 830 electroporator (Genetronics,
Inc.). Twenty million cells in 400 µl of RPMI 1640 medium were
electroporated in a 0.4-cm gap electroporation cuvette at 260 mV for a
50-ms pulse. For apoptosis studies involving evaluation of active
caspase-3 levels, Jurkat T-cells were transfected using the AMAXA
Biosystems NucleofectorTM device (program A23) following
the manufacturer's instructions for human T-cells. As indicated, 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma) and 720 ng/ml
ionomycin (Calbiochem) were added to cells 1-h post-electroporation.
A2870 ovarian cancer cells were maintained in RPMI 1640 media
containing 10% fetal bovine serum and 0.1 unit/ml insulin at 37 °C,
5% CO2. SaOs-2 osteosarcoma cells were maintained in
McCoy's media (Invitrogen) containing 15% fetal bovine serum at
37 °C, 5% CO2. A2870 and SaOS-2 cells were transfected
using FuGENE 6 transfection reagent following the manufacturer's
instructions (Roche Molecular Biochemicals). The ratio of plasmid DNA
to FuGENE 6 reagent used was 1:3.
Luciferase Assays--
Jurkat T-cells were transferred to Falcon
2052 tubes and centrifuged at 500 × g for 8 min at
room temperature. The medium was aspirated off, and cells were washed
with PBS. After centrifugation, PBS was aspirated and cell pellets were
resuspended in 150 µl of reporter lysis buffer (Promega). Adherent
cells were harvested by aspirating media and washing cells with PBS
three times. 200 µl of reporter lysis buffer were added to the wells,
and the cells were scraped. Cell lysates were transferred to 96 conical
well plates (NUNC) and frozen on dry ice or at Antibodies--
Mouse antibodies against HA epitope were
purchased from Covance. Mouse anti-human p53 antibody, anti-p21
antibody, and anti-caspase-3 antibody were obtained from BD
Biosciences. Secondary anti-mouse antibodies conjugated with
horseradish peroxidase or Alexa Fluor® 594 were purchased from
Amersham Biosciences and Molecular Probes, respectively.
Programmed Nuclear Extract Preparation--
Jurkat T-cells were
transfected with 2 µg of p53, 10 µg of HA-p73 DNA Affinity Precipitation Assays--
For preparation of the
DNA-coated beads, PCR was performed using human genomic DNA as template
and the following oligonucleotide pairs: p21 (3351-3792,
segment C), 5'-[bioTEG]TTCAGGCACAGAAAGGAGGCAAG-3' and
5'-CCTGAACAGAAGAAACCCCTGTGGT-3'; p21 (2693-3244, segment B), 5'-[bioTEG]ATTGAATGTCGTGGTGGTGGTGAGC-3' and
5'-catctcaggctgctcagagtctgga-3'; and p21 (2260-2717, segment A),
5'-[bioTEG]TTGGGCAGCAGGCTGTGGCT-3' and
5'-GCTCACCACCACCACGACATTCA-3'. One microgram of the
resulting PCR products was incubated with 100 µl of Neutravidin bead
slurry (Pierce), which had been previously blocked in blocking buffer (PBS, 1 mg/ml BSA, 1× casein solution (Vector), 0.5% Tween 20) and
then maintained in binding buffer (10 mM HEPES, 60 mM NaCl, 20% glycerol, 4 mM
The indicated amounts of programmed nuclear extracts were mixed with
100 µl of bead slurry in binding buffer containing 1× casein
solution for 2 h with vortexing at room temperature. The bead
suspension was sedimented at 300 × g, and the
supernatant was removed. The beads were washed with 750 µl of binding
buffer and then resuspended in 400 µl of binding buffer. The bead
suspension was overlaid upon a 450-µl sucrose cushion (20% sucrose,
200 mM NaCl, 0.05% Tween 20 in Buffer D). The gradient was
centrifuged at 500 × g for 10 min. The supernatant and
sucrose cushion were carefully aspirated, and the beads were washed
once with binding buffer. SDS-PAGE loading buffer was added to the
beads, and proteins associated with the bead-bound DNA were detected by
Western blot analysis.
Immunofluorescence and Apoptosis Assay--
Cells were
transfected with 2 µg of p53, 10 µg of HA-p73, or both expression
plasmids. Four hours post-electroporation, transfected Jurkat T-cells
were Ficoll-purified and stimulated with 50 ng/ml PMA. After a 15-h
incubation, 5 × 104 cells were isolated onto glass
slides by CytospinTM centrifugation at 500 rpm (Shandon).
The slides were air-dried for 15 min, and then the cells were fixed to
the slides by incubation in 4% paraformaldehyde for 30 min at room
temperature. After three washes in PBS, cells were permeabilized with
ice-cold 0.1% Triton X-100, 0.1% sodium citrate buffer for 2 min.
Slides were washed with PBS containing 1% BSA and 0.05% Triton X-100
and then blocked with PBS containing 1% BSA and 5% normal goat serum
for 1 h at room temperature. Primary antibody was added to the
slides for overnight incubation at 4 °C in a humidity chamber. The
slides were then washed three times with PBS containing 1% BSA, 0.05% Triton X-100, and then anti-mouse antibody conjugated with Alexa Fluor
594 was added to the slides at a 1:200 dilution in blocking buffer.
After a 1-h incubation at room temperature, the slides were washed
twice with PBS containing 1% BSA and 0.05% Triton X-100 and then one
time with PBS. Excess fluid was removed from the slides and TUNEL
reactions were performed using the In Situ cell death kit
following the manufacturer's protocol (Roche Molecular Biochemicals).
Transfection-positive (Red) and TUNEL-positive (fluorescein
isothiocyanate staining) cells were visualized using a Zeiss LSM 510 confocal microscope at ×10 magnification. To best determine cells that
were both positive for transfection and TUNEL, Zeiss LSM Image Examiner
software version 2.5 was used to create a mask of cells only positive
for both green and red fluorescence. Five representative fields (~200
cells/field) were analyzed for each data point.
Apoptosis Evaluation by Active Caspase-3 Expression--
Jurkat
T-cells were transfected with pcDNA3.1+, 1 µg of p53 expression
plasmid, 4 µg of p73 expression plasmid, or both using the AMAXA
Biosystems Nucleofector device and AMAXA Human T-cell Nucleofector kit
following manufacturer's directions using program A23. 1-h
post-transfection, cells were Ficoll-separated and then stimulated with
PMA and ionomycin for 6 h. Following a PBS wash, cells were lysed
in standard radioimmune precipitation assay buffer. Protein
concentrations were determined by BCA protein assay (Pierce) and
samples (75 µg) size-separated by SDS-PAGE followed by immunoblotting with anti-caspase-3 and anti-p53 antibodies.
Regulation of p53 Target Genes by p73 Is Both Promoter and Cell
Type-specific--
Numerous studies demonstrate the ability of p53
family member, p73, to regulate p53 target genes including the cell
cycle regulator p21 gene (15-17). Since lymphoblastoma
Jurkat T-cells are wild type p53-deficient, we had speculated that
perhaps the p21 promoter was transcriptionally activated in
these cells by p53-related protein p73 (37). However, the
overexpression of p73 Antagonism of p53 Is Target- and Cell
Type-specific--
Interestingly, p73 does regulate the expression of
p21, not as a transcription enhancer but as an antagonist of
p53 trans-activation. The co-expression of p53 and p73 in Jurkat
T-cells has an inhibitory effect on the in vivo induction of
p21 (Fig. 3A). As shown in Fig. 3, A and B, the co-expression of p53 and p73
not only repressed p53 activation of the transfected p21
promoter reporter but also inhibited increases in endogenous levels of
p21. Western analysis demonstrated that endogenous p21 protein levels
decreased by 57% upon overexpression of both p53 and p73 compared with
cells overexpressing p53 alone (Fig. 3A). As in all
co-transfection experiments, Western analysis confirmed consistent
expression levels of p53 (Fig. 3A). Similar co-transfection
experiments with the PG13-LUC reporter indicate that this antagonism
occurs at the level of the p53-responsive elements (Fig.
3C).
The ability of p53 to act as either an activator or repressor at target
promoters is well established. Recently reported data (2) demonstrate
that p53 represses IL-2 expression in T-cells. Interestingly, the
antagonism between p73 and p53 is not recapitulated at the
IL-2 promoter. Specific trans-repression of the
IL-2 promoter in Jurkat cells is not an activity shared by
p73, because its expression shows little effect on the IL-2
promoter either in the presence or absence of p53 (Fig.
3D).
Cell-type specificity of p73 dominant negative function was
demonstrated upon co-transfection of p53 and p73 expression plasmids along with p21-LUC into SaOs-2 and A2870 cell lines. Unlike in Jurkat
T-cells, p73 does not repress p53 activation of the p21 promoter in either SaOs-2 or A2870 cells (Fig.
4, A and B).
p73 Binds to and Competes with p53 for Binding at the p21
Promoter--
p73 may act as a transcriptional repressor of the
p21 promoter through multiple mechanisms, many of which
involve the interaction of p73 with p53 DNA response elements. DNA
affinity precipitation experiments were performed to determine whether
or not p73 was capable of binding to segments of the p21
promoter containing p53 response elements in Jurkat T-cells. The
p21 promoter contains three well characterized p53 binding
sites referred to as p53 sites 1, 2, and 3 (Fig.
5A) (41). Biotinlyated duplex
DNA fragments of the p21 promoter-encoding segments
containing p53 site 1 (segment A), p53 sites 2 and 3 (segment B), or
sequence 3' to the p53 binding sites (segment C) were bound to
avidin-coated beads and then incubated with Jurkat nuclear extract
programmed by transfection with p73 expression plasmid. Western
analysis of bead bound protein fractions confirmed that p73 binds
selectively to the p21 promoter fragments containing p53
response elements (Fig. 5B). Therefore, similar to p53, p73
recognizes the p21 promoter in Jurkat cells, although it
lacks the ability to drive transcription of the gene.
DNA affinity precipitation analyses of nuclear extracts programmed with
both p53 and p73 demonstrated that p73 can compete for p53 binding of
p21 promoter elements. The analyses of nuclear extract
programmed by transfection with p53 and p73 in ratios that produced
repression of p53 trans-activity showed reduced binding of p53 to
p21 promoter segments in comparison with extracts programmed
with p53 alone (Fig. 5C). This competition was clearly demonstrated when the DNA fragment containing p53 sites 2 and 3 (segment B) was used as the binding template. Competition by p73 at the
p21 promoter segment encoding the higher binding affinity p53 site 1 (segment A) required higher levels of p73 (41). As shown in
Fig. 5D, when increasing doses of p73 programmed nuclear extract was added to DNA affinity precipitations with constant levels
of p53-programmed nuclear extract, p73 blocked the binding of p53 to
site 1.
p73 Inhibits p53-mediated Apoptosis--
The function
of p73 as an antagonist of p53 target genes is reflected in its ability
to block p53-induced apoptosis. Enforced expression of p73 did not
induce apoptosis in PMA-stimulated Jurkat cells as demonstrated by
TUNEL (Fig. 6A). However, in
cells overexpressing p53, a measurable induction of apoptosis ensued.
This p53-mediated apoptosis is reduced almost 50% by
co-expression of p73 (Fig. 6A). Furthermore, this
anti-apoptotic activity of p73 is associated with decreased amounts of
active caspase-3 in cells overexpressing both p53 and p73. Active
caspase-3 protein levels were reduced nearly 50% in cells
co-expressing p53 and p73 in comparison with cells overexpressing p53
alone (Fig. 6, B and C).
p73 Post-translational Modification Is Cell Type-specific--
The
divergence in p73 function may occur at the level of post-translational
modification. HA-p73 overexpressed in Jurkat and SaOs-2 cells was
analyzed by Western analysis. Interestingly, HA-p73 in Jurkat cell
lysate migrated with slower mobility, indicative of covalent
modification (Fig. 7). The mobility
change is independent of protein load as demonstrated by varying the
relative protein amounts.
The transformation of normal lymphoid cells to cancerous phenotype
is a multi-step process beginning with pre-commitment steps that
progress through a crisis point where massive alterations in genetic
make-up occur by increasing genetic instability. The loss of p53
function occurs as either a cause or result of such genetic
instability. In most lymphoid cancers, somatic mutations of p53 are
rarely associated with lymphoma and leukemia at their initial stages
prior to treatment and or relapse (46). However, recent findings in
human T-cell lymphotrophic virus, type I-infected cells suggest that
p53 and p73 function can be altered in the absence of somatic mutation
by the action of the Tax oncogene (39, 47-50). Data presented herein
suggest a novel epigenetic mechanism whereby p53 function can be
modified by cell-specific alterations of p73 activity that arise in the
absence of alternative splicing or somatic mutation to either the
p73 or p53 gene. These divergent and dominant
negative functions of p73 are closely associated with cell-specific
covalent modifications of p73 protein expressed from a wild type gene.
With high homology in the functional domains of p53 and p73, it is not
surprising that the related proteins trans-activate a similar subset of
genes. The role of p73 in enhancing expression of the p21
gene has been demonstrated in primary T-cells (data not shown) and many
cancer cell lines including lung H1299, ovarian A2870, and osteosarcoma
SaOs-2 and U2OS (15-17, 22, 23). To our knowledge, Jurkat T-cell is
the only cancer cell line studied in which exogenous p73 does not
upregulate p21 expression. Furthermore, although p73 has been shown to
induce apoptosis in lymphocytes, it does not mediate certain apoptotic
pathways in Jurkat cells as does family member p53 (23, 24, 36). The
inability of p73 to potentiate p21 expression and specific
apoptotic pathways in Jurkat T-cells may act in parallel or synergy to
promote the progression of hyper-proliferative lymphoid neoplasms to
the transformed state. Therefore, the mechanism of p73 action described
herein may play a key role in leukemogenesis.
The mechanism of p73 antagonism of p53 gene regulation at the
p21 promoter appears to occur at the level of p53
DNA-responsive elements as demonstrated by p73 attenuation of p53
trans-activity at the synthetic p53-specific response element PG13.
Furthermore, p73 antagonism is specific for functional roles of p53 as
demonstrated by the inability of p73 to reverse p53 repression at the
IL-2 promoter. Finally, the dominant negative functions of
p73 are cell context-specific. Overexpression of both p53 and p73 in
SaOs-2 and A2870 cells did not affect p53 trans-activity of the
p21 promoter. Interestingly, previously published data (22)
suggest that although p73 trans-activates the PG13 response element, it
attenuates p53 activation of the PG13 reporter in
cis-platin-treated A2870 cells. This finding taken in
combination with our current results reinforces the concept that
cell-state and promoter specificity determines p73 antagonism of
p53-dependent gene regulation.
Our study is the first to demonstrate that full-length p73 can
attenuate p53-mediated apoptosis. The ability of p73 to regulate p53-potentiated apoptosis strongly supports the labeling of p73 as an
oncogene, not a tumor suppressor. The cell-specific dominant negative
effect of p73 described here together with reports that p73 protein
levels are increased in numerous cancers suggests a fastidious
oncogenic mechanism that occurs in the absence of somatic mutation of
either gene family member (17, 18, 31-34).
Interestingly, a naturally occurring N terminus deletion p73 isoform
( The cell-dependent modification state of p73 may explicate
the divergent and dominant negative function in lymphoma cells. When
comparing HA-p73 expressed in Jurkat to that produced in SaOs-2 cells,
it appears that Jurkat-derived HA-p73 is covalently altered. This study
is not the first to show that covalent modification of p73 may dictate
its selective function. Recently, a promoter-specific regulatory
function for p73 determined by acetylation status has been reported
(52). Costanzo et al. (52) report that non-acetylatable p73-enhanced p21 transcription but not transcription of
pro-apoptotic gene p53AIP1. Ongoing research concerns
elucidating what specific modifications of p73 are important for the
divergent roles in Jurkat T-cells.
In addition to broadening our knowledge about p73 functional roles in
lymphoid tumor biology, this report highlights the importance of
examining post-genomic events to understand the evolution and progression of cancer. The ability of expression products translated from the identical p73 gene to have completely different
phenotypes that are dependent on cellular context presents a novel
epigenetic mechanism for tumor progression in lymphoid cells. Such
observations may lend insight into how normal lymphoid homeostasis is
subject to deregulation in diseases as diverse as autoimmunity, immune deficiency, and various forms of lymphoid dyscrasias.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-motif
(15, 18, 19). Interactions with other proteins through this
oligomerization domain could result in the variability of trans-regulatory functions. Differences in the ability of p73 to
activate p53 target genes may also rely on cell-specific
post-translational modifications of the protein. Recent reports have
shown that p73 trans-activation of p53 targets genes is cell
type-dependent (15-17, 20, 21). For example, p73 induces
p53-regulated BAX in A2870 ovarian cells but not in H1229
lung cancer cells (16, 22). The ability of p73 to induce apoptosis is
also dependent on cellular context. The p73 gene product is
necessary for T-cell receptor-activated induced cell death in primary
T-cells and pro-apoptotic in SaOs-2 cells, whereas in developing
sympathetic neurons, p73 plays an anti-apoptotic role (1, 23-26). Not
only is the function of p73 cell context-specific, its expression also
varies among cancer types. The silencing of the p73 gene in
various cancers including neuroblastoma, squamous cell carcinoma, lung,
and Burkitt's lymphoma supports the labeling of p73 as a tumor
suppressor (27-29). However, in some cancers including B-cell chronic
lymphocytic leukemia, ovarian, bladder, and breast, p73 gene
expression is elevated in comparison with the respective cells of
origin, suggesting that p73 functions in tumor progression (30-35).
Although, the role of p73 in oncogenesis remains elusive, it is evident
that the complex nature of p73 involvement is cell
context-dependent and varies from that of its family member p53.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
) and p53 expression plasmids were described previously
(38-40). Reporter plasmids p21-LUC, PG13-LUC, IL-2-LUC, and MDM2-LUC
were described previously (41-45). The amount of plasmid DNA added to
transfections was normalized with pcDNA 3.1+ (Invitrogen).
80 °C. Lysates were thawed at room temperature, and then the plate was centrifuged at 4000 rpm in a Sorvall RC5B Plus plate rotor at 4 °C. After a 20-min
centrifugation, 20 µl of supernatant were transferred to an opaque
96-flat bottom well plate (NUNC). One hundred microliters of luciferase
substrate (Promega) were injected into each well, and the fluorochromic
reaction was analyzed by a Microplate Luminometer LB96V (EG+G
Berthold). Aliquots of lysis supernatant were transferred to a standard
96-well plate for protein concentration reading using Bio-Rad protein
dye reagent following manufacturer's instructions. Luciferase values
were normalized to protein concentration.
, or both
expression plasmids. The amount of plasmid DNA added to each
transfection was kept constant by the addition of pcDNA3.1+.
Multiple transfections with the same plasmid DNA were pooled and
incubated for 1 h at 37 °C, and then the cells were centrifuged
at 400 × g for 5 min, the medium was removed, and the
cells were resuspended in fresh medium. The cell suspension was
underlaid with 3 ml of Ficoll and centrifuged at 400 × g for 45 min at room temperature. The interface layer of
live cells was removed, washed once in fresh medium, and resuspended in
RPMI 1640 medium containing 50 ng/ml PMA. After a 7-h incubation, cells were centrifuged at 400 × g and then washed three
times with ice-cold PBS. Cells next were resuspended in 5 volumes of
Buffer A (hypotonic lysis buffer) containing 4 mM
-mercaptoethanol and 1:200 dilution of protease inhibitor
mixture (Sigma). After a 10-min incubation on ice, cells were
homogenized with 16 passes of a Craftsman power drill-driven Teflon
pestle. Homogenate was collected and centrifuged at 1540 × g for 10 min at 4 °C. The supernatant cytosolic fraction was collected, and the nuclear pellet was resuspended in Buffer D (20%
glycerol, 20 mM HEPES, 0.2 mM EDTA) containing
4 mM BME and 1:200 dilution of protease inhibitor
mixture. Ammonium sulfate was added to a final concentration of 0.3 M. The nuclear suspension was incubated with rocking for 45 min at 4 °C and then spun at 100,000 × g for 45 min. The supernatant was immediately recovered and stored at
80 °C. Protein concentrations of extracts were obtained using
Bio-Rad protein assay reagent, and the extracts were tested for
exogenous protein expression by Western blot analysis.
-mercaptoethanol, protease inhibitor mixture). After
vortexing for 2 h at room temperature, the slurry was centrifuged at 300 × g, and the supernatant was removed. To ensure
that beads were saturated with DNA, the supernatant was analyzed for
DNA by agarose gel electrophoresis. The DNA-coated beads were washed twice with binding buffer and then stored as a 50% slurry in binding buffer at 4 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-,
-, or
-p73 did not increase
p21 promoter activity or endogenous p21 levels (Figs.
1A and 3A),
whereas, exogenous p53 readily activated the p21 promoter
(Figs. 1A and 3A). Western analysis demonstrated that HA-tagged p73 was expressed (data not shown). This surprising data
led us to question whether or not p73 regulated other p53 target genes
in Jurkat T-cells. Interestingly, p73
did not trans-activate the
multimer p53 response element PG13 (Fig. 1B). However,
exogenous p73 did trans-activate the p53 target gene mdm2 in
Jurkat T-cells, indicating that its trans-regulatory potential is
promoter-specific (Fig. 1C). This specificity is also cell
context-dependent. As was previously reported, we showed
that exogenous p73 trans-activates the p53-responsive p21
promoter in SaOs-2 osteosarcoma cells and A2870 ovarian cancer cells
(Fig. 2, A and B)
(15-17, 22).
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Fig. 1.
Regulatory roles of p73 are promoter
specific. A, p73 does not trans-activate the
p21 promoter in Jurkat T-cells. In increasing amounts (0.1, 0.5, 3, and 9 µg), -,
-, and
-HA-p73 isoforms or 0.8 µg of
p53 expression plasmid were co-transfected with 4 µg of p21-LUC
reporter into Jurkat cells. Cells were stimulated with PMA and
ionomycin and harvested 7 h later. Fold activations are above
control (unstimulated cells) shown in lane 1. B,
p73 does not trans-activate the PG13-LUC reporter. 0.1, 0.5, 3, and 9 µg of HA-p73
or 0.8 µg of p53 expression plasmid were
co-transfected with 1 µg of PG13-LUC reporter into Jurkat cells.
Cells were stimulated with PMA and harvested 7 h later.
C, p73 trans-activates the mdm2 promoter in
Jurkat cells. 0.2, 0.5, or 2 µg of HA-p73
or 0.8 µg of p53
expression plasmid were co-transfected with 4 µg of MDM2-LUC reporter
into Jurkat T-cells. Cells were stimulated with PMA and harvested
7 h later. Fold activations are over PMA-treated pcDNA control
as shown in lane 1.
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Fig. 2.
Regulatory Roles of p73 are cell
context-specific. A, SaOs-2 cells were transfected with
1 µg of p21-LUC reporter and 0.25 or 1.25 µg of p73 or 0.08 µg
of p53 expression plasmid. B, A2870 cells were transfected
with 1 µg of p21-LUC reporter and 1.6 or 4.5 µg of p73
or 0.4 µg of p53 expression plasmid. Cells were harvested after an 18-h
incubation. Fold activations are over pcDNA control as shown in
lane 1.
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Fig. 3.
p73 functions as a dominant negative
repressor. A, p53-potentiated increase of endogenous
p21 levels is attenuated by p73. p21 (below) and p53
(above) immunoblots of transfected Jurkat cell lysates are
shown. Jurkat cells were transfected with 1 µg of p53, 4 µg of p73,
and pcDNA expression plasmids. Cells were harvested after a 6-h
incubation with PMA and ionomycin. IB, immunoblot.
B, p73 attenuates p53 trans-activity of the p21
promoter in Jurkat cells. Jurkat cells were transfected with 4 µg of
p21-LUC reporter and 1, 3, or 9 µg of p73 with and without 0.4 µg of p53 expression plasmid. C, antagonism between p73
and p53 occurs at the level of p53 target sequences as demonstrated by
activity at the PG13-LUC reporter. Jurkat cells were transfected with 1 µg of PG13-LUC reporter and 1, 3, or 9 µg of p73
with and
without 0.8 µg of p53 expression plasmids. Transfected cells were
stimulated with PMA and harvested 6 h later. D, p73
antagonism is not recapitulated at the IL-2 promoter. Jurkat cells were
transfected with 4 µg of IL-2-LUC reporter and 0.1, 0.5, or 3 µg of
p73
with and without 3 µg of p53 expression plasmids. Transfected
cells were stimulated with PMA/ionomycin and harvested 6 h later.
Fold activations are above pcDNA control as shown in lane
1.
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Fig. 4.
The dominant negative role of p73 is cell
type-specific. p73 does not repress p53 trans-activation of the
p21 promoter in SaOs-2 cells and A2870 cells. A,
SaOs-2 cells were transfected with 1 µg of p21-LUC reporter and 0.25 or 1.25 µg of p73 with and without 0.08 µg of p53. B,
A2870 cells were transfected with 1 µg of p21-LUC reporter and 1.6 or
4.5 µg of p73
with and without 0.4 µg of p53. Cells were
harvested after an 18-h incubation. Fold activations are over pcDNA
control as shown in lane 1.
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Fig. 5.
p73 binds selectively to segments of the
p21 promoter in Jurkat T-cells and competes p53
binding. A, schematic of p53 binding sites in
the p21 promoter. A, B, and
C represent segments of p21 promoter sequence
used as templates in DNA affinity precipitation assays. B,
p73 binds to the p21 promoter. Anti-HA immunoblot of
proteins isolated from HA-p73 programmed nuclear extract using duplex
DNA segments A and C (top) and
B and C (bottom) as template in DNA
affinity precipitation assays. NE, nuclear extract;
IB, immunoblot. C, p73 competes for p53 binding
of response elements within the p21 promoter. Anti-p53
immunoblot of proteins isolated from 150 µg of Jurkat nuclear extract
programmed by transfection with both p53 and p73 or p53 alone using
segments A, B, and C as template in
DNA affinity precipitation assays. D, in a
dose-dependent manner, p73 competes for p53 binding at the
p21 promoter. Proteins isolated from segment A
DNA affinity precipitation assays in which p73-programmed nuclear
extract was added in increasing amounts while keeping the concentration
of p53-programmed nuclear extract constant were analyzed by
immunoblotting with both anti-HA (top) and anti-p53
(bottom). I, denotes input; M, denotes
marker lane.
View larger version (14K):
[in a new window]
Fig. 6.
p73 attenuates p53 induced apoptosis.
A, cells overexpressing p53, p73, or both and undergoing
apoptosis were identified by performing immunofluorescence followed by
TUNEL analysis on PMA-stimulated Jurkat cells. Graph depicts the
percentage of transfected cells that are TUNEL-positive. Transfected
cells were identified by immunofluorescence using HA antibody
(lanes 1 and 3) or anti-p53 (lanes 2 and 4). B, PMA and ionomycin-stimulated Jurkat
cells overexpressing p53, p73, or both were assayed for apoptosis by
evaluating the levels of active caspase-3. Bottom,
anti-caspase-3 immunoblot of programmed lysates; top,
anti-p53 immunoblot. IB, immunoblot. C, density analysis of
17-kDa bands present on anti-caspase-3 immunoblot fold over pcDNA
control.
View larger version (24K):
[in a new window]
Fig. 7.
Jurkat-derived HA-p73 modification state is
cell type-dependent. A and B,
Jurkat-derived HA-p73 migrates slower than HA-p73 expressed in SaOs-2
cells. Jurkat cells and SaOs-2 cells were transfected with p73
expression plasmid, and cell lysates were assayed for HA-p73 expression
by Western blotting. The reduced mobility of HA-p73 in Jurkat cells is
independent of relative protein load and PMA stimulation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N-p73
) has been shown to play an anti-apoptotic role in
developing sympathetic neurons by direct protein interaction with p53
(25). Data presented herein provide a different mechanism for the
antagonistic function of full-length p73, namely direct competition for
p53 DNA binding elements by covalently altered p73. Current studies
focus on determining whether or not p73 associates with a repressor at
the p21 promoter simply competes directly with p53 for
specific response elements or a combination of both. There are numerous
pathways through which the p21 gene can be activated in the
absence of p53, and supra-physiological levels of p73 do inhibit
PMA-mediated expression of the p21 promoter in Jurkat cells.
The mechanism of repression by p73 may be highly selective for p53
induction, and the action of a putative repressor may function through
interactions between p53 and other co-activators such as p300 or the
basal machinery. Noteworthy is the speculation that p53/p73 antagonism
could be caused by the physical interaction and possible sequestration
of the related proteins as demonstrated by
N-p73
/p53 association
in neurons (25). Equally as provocative is the report that attenuation
of p73 trans-activity is caused by the interaction of p73 with mutant
p53 (51). As previously demonstrated in other cell lines,
immunoprecipitation assays designed to determine whether or not wild
type p53 and p73 associate in Jurkat cells have resulted in no
observable direct interactions (see supplemental data) (22, 51).
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ACKNOWLEDGEMENTS |
---|
We thank Drs. K. H. Vousden and G. Melino for the generous gift of p73 expression plasmids.
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FOOTNOTES |
---|
* 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.
The on-line version of this article (available at
http://www.jbc.org) contains supplemental Fig. 1.
¶ To whom correspondence should be addressed: Laboratory of Receptor Biology and Gene Expression and Laboratory of Pathology, DHHS/NCI/CCR, National Institutes of Health, Advanced Technology Center, Rm. 134C, 8717 Grovemont Circle, Bethesda, MD 20892. Tel.: 301-496-1055; Fax: 301-435-7558; E-mail: gardnerk@mail.nih.gov.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M208517200
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ABBREVIATIONS |
---|
The abbreviations used are: HA, hemagglutinin; LUC, luciferase; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TUNEL, terminal dUTP nick-end labeling; IL, interleukin.
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