From the Laboratory of Molecular Virology,
Department of Biological Sciences, Southern Methodist University,
Dallas, Texas 75275-0376, the ¶ Basic Research Laboratory, Center
for Cancer Research, NCI, National Institutes of Health, Bethesda,
Maryland 20892-0001, the
Université Libre de
Bruxelles, Institut de Biologie et de Medecine, Moleculaires,
Service de Chimie Biologique, Laboratoire de Virologie, Moleculaire,
6041 Gosselies, Belgium, and the
Laboratory
of Receptor Biology and Gene Expression, Fluorescence, Imaging
Facility, Center for Cancer Research, NCI, National Institutes of
Health, Bethesda, Maryland 20892-0001
Received for publication, October 31, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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Patients with AIDS are
at increased risk for developing various neoplasms, including
Hodgkin's and non-Hodgkin's lymphomas, Kaposi's sarcomas, and
anal-rectal carcinomas, suggestive that human immunodeficiency virus
type-1 infection might promote establishment of AIDS-related cancers.
Tat, the viral trans-activator, can be endocytosed by
uninfected cells and has been shown to inhibit p53 functions, providing
a candidate mechanism through which the human immunodeficiency virus
type-1 might contribute to malignant transformation. Because Tat has
been shown to interact with histone acetyltransferase domains of
p300/cAMP-responsive element-binding protein (CREB)-binding protein and
p300/CREB-binding protein-associated factor, we have investigated
whether Tat might alter p53 acetylation and tumor suppressor-responsive
transcription. Here, we demonstrate that both Tat and p53 co-localize
with p300/CREB-binding protein-associated factor and p300 in nuclei of
IMR-32 human neuroblastoma cells and in PC-12 pheochromocytoma cells.
Further, p53 trans-activation of the 14-3-3 Although the most frequent malignancies observed in AIDS patients
are non-Hodgkin's lymphomas, central nervous system non-Hodgkin's lymphomas, and Kaposi's sarcomas, compartmentalization of human immunodeficiency virus, type-1
(HIV-1)1 in the central
nervous system might be associated with the recent increase of rare
intracranial tumors, such as glioblastomas, anaplastic astrocytomas,
and subependymomas (1-3). The viral trans-activator, Tat,
can be endocytosed by surrounding uninfected cells and has been
demonstrated to inhibit the G1/S arrest-inducing functions of p53, providing a candidate mechanism through which HIV-1 might contribute to malignant transformation in the central nervous system
(4-7). Tat is a 82-101-amino acid peptide that contains an
arginine-rich motif required for binding a uracil-containing bulge in
the Tat-associated region (TAR) of HIV-1 transcripts (8). Interactions
between Tat/TAR-RNA stabilize viral mRNAs; thus, Tat principally
acts as an elongation factor to enhance long terminal repeat
trans-activation. The arginine-rich motif of Tat interacts
with the catalytic histone acetyltransferase (HAT) domains of
transcriptional co-activators, p300/CREB-binding protein (CBP) and
p300/CBP-associated factor (P/CAF):hGCN5 (9-15), and Tat is acetylated
by p300 on lysine residues Lys50/Lys51
(Lys51 is only weakly acetylated) and by P/CAF on
Lys28 (12, 16-18). Acetylation of Tat on Lys50
diminishes its binding affinity for TAR-RNA, and acetylation on
Lys28 enhances Tat binding to the Tat-associated kinase
complex containing cdk9 and human cyclin T1 (16, 17). Importantly,
Mujtaba et al. (19) demonstrated that acetylated Tat peptide
interacts with the bromodomain of P/CAF, and this interaction could
play an important role in dissociation of Tat/TAR-RNA complexes
(20-22). Tat/co-activator interactions are essential for HIV-1 long
terminal repeat trans-activation (9, 12-18). Because the
HAT domains of p300/CBP and P/CAF also target p53 for acetylation on
residues Lys373/Lys382 and Lys320,
respectively, we hypothesized that Tat-HAT interactions might competitively interfere with p53 acetylation and, consequently, tumor
suppressor-responsive transcription functions (23-25).
Cell Culture, Immunofluorescence Laser Confocal Microscopy, and
FACS--
IMR-32 human neuroblastoma cells (ATCC number CCL-127) were
cultured in ATCC 2003, Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin-sulfate (Invitrogen). PC-12 rodent pheochromocytoma cells
(ATCC number CRL-1721) were cultured in ATCC F-12K medium supplemented
with 5% fetal bovine serum, 15% horse serum, 100 units/ml penicillin,
and 100 µg/ml streptomycin-sulfate. All of the cells were grown
either in tissue culture dishes or eight-chamber slides (Nalge Nunc
International) coated with mouse type IV collagen (Invitrogen) and were
incubated under 10% CO2 at 37 °C. The transfections were performed using a calcium-phosphate system as recommended by the
manufacturer (Invitrogen). Calu-6 carcinoma cells (ATCC number HTB-56)
were cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin-sulfate and were transfected using LipofectAMINE reagent
(Invitrogen) as recommended in the manufacturer's protocol. Molt-4
CD4+ lymphoblastic leukemia cells were grown at 37 °C
under 10% CO2 in RPMI medium supplemented with 20% human
donor serum (Sigma), 100 units/ml penicillin, and 100 µg/ml
streptomycin-sulfate. The protein extracts were prepared by rapid
freeze-thawing followed by centrifugation at 14,000 rpm at 4 °C and
quantified using the Bradford microassay and spectrophotometric
analyses at 595 nm; 20 µl from each sample was measured using a
luciferase assay kit (Promega Corp.) and a Lumat model LB 9501 luminometer (Berthold, Inc.). All of the experiments were carried out
as dose responses in two different cell-lines (IMR-32 and PC-12) or in
duplicates for single-point analyses (error bars representative of
standard deviations are shown). Immunofluorescence laser confocal
microscopy was performed on IMR-32, PC-12, or HIV-1-infected Molt-4
cells using a Leica TCS spectrophotometric confocal microscope
equipped with krypton and argon lasers, controlled by a Windows
NT-based work station. Relative fluorescence intensities were
quantified using TCS linear quantification software. Briefly, the cells
were fixed in 0.2% glutaraldehyde and 1% formaldehyde in PBS, and
nonspecific antigens were blocked by incubation with 3% (w/v) bovine
serum albumin, 0.5% (v/v) Tween 20 in PBS. HIV-1 Tat was detected
using the rabbit primary antibody, C-2145, and p53 was detected using either a monoclonal anti-p53 antibody (BP53-12; Upstate Biotechnology, Inc.) or an anti-p53 rabbit polyclonal antibody (Santa Cruz
Biotechnologies, Inc.). p300 was detected using a monoclonal anti-p300
CT antibody (RW-128; Upstate Biotechnology, Inc.) or a rabbit
polyclonal antibody (N-15; Santa Cruz Biotechnology, Inc.); P/CAF was
detected using a goat polyclonal antibody (C-16; Santa Cruz
Biotechnology, Inc.). Fluorescent secondary antibodies were used in
appropriate combinations: rhodamine red-conjugated donkey anti-mouse
IgG, fluorescein isothiocyanate- or Cy-3-conjugated donkey anti-goat
IgG, fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG, or
rhodamine red-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch
Laboratories, Inc.). For apoptosis and cell cycle analyses, transfected
IMR-32 cells were treated with an arrest-inducing concentration of
adriamycin (100 µM) and incubated for 24 h at
37 °C. The cells were harvested, washed twice with PBS, and either
resuspended in 1× annexin-buffer and stained for annexin-V surface
expression for 10 min (Pharmingen, Inc.) or fixed in 3.7%
formaldehyde/PBS for 10 min, washed, and permeabilized in 0.1% IGEPAL
CA-630 (Sigma)/PBS containing 5 µg/ml RNase and incubated on ice for
15 min. The samples were then stained using acridine orange (Molecular
Probes, Inc.) at 4 °C for 30 min. FACS analyses were performed using
a Becton Dickinson, FACSCalibur flow cytometer. The cell cycle analyses
were gated to exclude aggregates and fragments.
Protein Purification and in Vitro Acetylation--
GST-Tat,
GST-TatK28A/K50A, and GST-p53 fusion proteins were
expressed in Escherichia coli, strain DH5 Effects of HIV-1 Infection upon UV-responsive p53
Acetylation--
To assess effects of HIV-1 infection upon p53
acetylation in response to UV irradiation in vivo, we
infected 3 × 106 Molt-4 CD4+
lymphoblastic leukemia cells with 150 µl of an infectious stock of
HIV-1, HXB2IIIB (HXB2IIIB virus stock and
Molt-4 cells were generously provided by Dr. G. Roderiquez, Food and
Drug Administration, Center for Biologics Evaluation and
Research, National Institutes of Health, Bethesda, MD), titered at
~2 × 105 pg/ml of p24gag protein determined
by a standard anti-p24gag enzyme-linked immunosorbent assay
method. Aliquots were taken from HIV-1-infected samples on consecutive
days (4-6 days post-infection), and half of the volume of infected
cells were UV-irradiated for 1.5 min (Fisher UV-Crosslinker at 120 mJ/cm2 energy level), before culturing for an additional
3 h at 37 °C under 10% CO2. Induction of p53
protein by UV treatment was confirmed by immunofluorescence laser
confocal microscopy and immunoblotting using uninfected or
HIV-1-infected cells and a p53-specific monoclonal antibody (Upstate
Biotechnology, Inc). Tat expression was measured in lysates prepared
from infected cells by immunoblotting using a rabbit polyclonal
anti-Tat antibody or by direct visualization by immunofluorescence
laser confocal microscopy. Co-localization of Tat and p53 in
UV-irradiated cells was determined and quantified by immunofluorescence
laser confocal microscopy. The status of p53 acetylation on lysine
residues Lys320 and Lys373 was detected by
immunoprecipitating total intracellular p53 from untreated or
UV-irradiated, HIV-infected Molt-4 cells. The protein G-agarose immune
complexes were washed, and the bound products were resolved by 12.5%
SDS-PAGE and immunoblotting using rabbit polyclonal antibodies that
specifically recognize Lys320-acetylated p53 or
Lys373-acetylated p53 (Upstate Biotechnology, Inc.).
Inhibition of p53-dependent Transcriptional Activation
by HIV-1 Tat--
We first assayed whether Tat-HAT interactions might
interfere with neuronal p53-responsive transcription in human IMR-32
neuroblastoma cells and rodent PC-12 pheochromocytoma cells. Because
p53 regulates G2/M cellular arrest by driving 14-3-3 Ectopic P/CAF and p300 Counter Inhibitory Effects of
HIV-1 Tat upon 14-3-3 Nuclear Localization of p53, HIV-1 Tat, and
TatK28A/K50A in Transfected IMR-32
Cells--
HIV-1 Tat and p53 were expressed in nuclei of transfected
IMR-32 neuroblastoma cells, as determined by immunofluorescence laser
confocal microscopy, and both proteins displayed varied levels of
co-localization with the transcriptional co-activator/acetyltransferase p300 (Fig. 3, A and
B). The subcellular distribution of Tat was observed to be
both nucleoplasmic and nucleolar by immunostaining (Fig.
3B). Importantly, the Tat-derived mutant,
TatK28A/K50A, was present in the nuclei of IMR-32 cells
(Fig. 3C). We examined the localization of
TatK28A/K50A because the K50A mutation is targeted within
the nuclear localization sequence of Tat, and it was therefore
necessary to establish whether the mutant protein is properly expressed
in the nuclei. In addition, we observed that nuclear P/CAF levels
appeared to be somewhat limiting in IMR-32 neuroblastoma cells, as
revealed by comparatively weak, P/CAF-specific nuclear immunostaining
(Fig. 3, A-C, lower panels).
HIV-1 Tat and Tat Synthetic Peptides Inhibit p53 Acetylation by
P/CAF and p300--
Direct interactions of co-activator
HATs with Tat, p53, and E1A 12S provide a plausible basis for
competitive inhibition of factor acetylation (Fig.
4A and Refs. 17, 23, 25, and
30). In IMR-32 cells co-transfected with p53 and either Tat or various Tat mutants, the wild-type Tat caused markedly diminished
p53Lys-320 acetylation by P/CAF in vivo, whereas
no significant difference was observed for p300-dependent,
p53Lys-373 acetylation (Fig. 4B). The
Tat-derived mutants did not affect p53 acetylation, despite having
partially repressive effects upon p53-responsive transcription. These
discrepancies may derive from weakened co-activator-binding affinities
for the mutants or could suggest that alternate signaling interactions
contribute to Tat-repression of p53 transcription. Overexpression of
p53 did not result in increased levels of p53-acetylated forms (compare
lanes 1 and 2 of Fig. 4B), supporting
reports that nuclear co-activator concentrations are limiting (32, 33).
That no apparent difference was observed for
p300-dependent, p53Lys-373 acetylation suggests
that the restoring effect of ectopic p300 upon 14-3-3
We next tested whether GST-Tat or GST-TatK28A/K50A proteins
or various Tat-derived synthetic peptides (Biosynthesis, Inc.) might
influence the acetylation of GST-p53 in vitro by recombinant P/CAF or p300 (Upstate Biotechnology, Inc.). Amino acid sequences of
full-length Tat and peptide derivatives (wild type and mutants) used in
these analyses are provided in Fig. 4D. Relative input levels of GST-Tat, GST-TatK28A/K50A, and GST-p53 are shown
in Fig. 4E. GST-Tat inhibited p53 acetylation by P/CAF and
p300 in a dosage-dependent manner; in contrast,
GST-TatK28A/K50A had no significant effect upon
acetyltransferase activities (Fig. 4, E and F).
Synthetic peptides spanning residues 23-43 (Tat23-43) and
41-61 (Tat41-61) of Tat inhibited p53 acetylation by P/CAF and p300, respectively, whereas mutant derivatives of these peptides (TatK/A23-43 and
TatKK/AA41-61) did not (Fig. 4, E and
F). Interestingly, the Tat41-61 peptide did not alter p53 acetylation by P/CAF, whereas p53 acetylation by p300 was
slightly inhibited by Tat23-43. These results may reflect differences in substrate affinities between p300 and P/CAF.
The wild-type Tat peptides (residues 23-43 or 41-61) were weakly
acetylated in our in vitro assays (data not shown); we did
not observe GST-Tat acetylation under the conditions promoting p53
acetylation. In addition, Tat-HAT binding inhibited acetylation of
histones H3/H4 in vitro (data not shown). We infer, based
upon these results, that the release (or "turnover") kinetics of
Tat acetylation by p300 and P/CAF are comparatively slow
versus p53/histone acetylation consistent with the
acetyltransferase inhibitory functions of Tat.
Because others have previously demonstrated that HIV-1 Tat directly
interacts with p53 (6), we attempted to determine whether observed
differences in co-activator-mediated p53 acetylation between wild-type
Tat and the TatK28A/K50A mutant might be attributed to
differences in p53 binding. The results from co-immunoprecipitation experiments in Fig. 5A
indicate that both purified recombinant GST-HIV-1 Tat and
GST-TatK28A/K50A interact with GST-p53 in vitro with similar apparent affinities. In addition, we have also tested whether Tat-P/CAF acetyltransferase domain interactions might interfere
with binding of p53 to the catalytic acetyltransferase site of the
co-activator and thereby competitively inhibit p53 acetylation. To
address this question, we expressed p53 in the background of p53-null,
Calu-6 carcinoma cells and used equivalent amounts of total cellular
proteins from prepared extracts in GST pull-down experiments.
Increasing amounts of synthetic wild-type Tat23-43 or
TatK/A23-43 mutant peptides were added to binding
reactions in the presence of purified recombinant
GST-P/CAF352-382, comprising the minimal acetyltransferase
domain of P/CAF. As shown in Fig. 5B, the
p53-P/CAF352-382 interaction was significantly diminished
in a dosage-dependent manner in the presence of wild-type Tat23-43 peptide but was unaffected by the
TatK/A23-43 mutant peptide. Indeed, because
Tat23-43 peptides lack amino acid residues that are
essential for p53 binding by Tat, we infer that HIV-1 Tat competes
against p53 for binding to the minimal acetyltransferase domain of
P/CAF in our in vitro assays.
Wild-type HIV-1 Tat, but Not TatK28A/K50A,
Facilitates Bypass of 22/M Cellular
Arrest Induced by Adriamycin in IMR-32 Cells--
Because 14-3-3 HIV-1 Infection Interferes with p53 Acetylation in Response to UV
Irradiation--
Finally, we examined the influence of HIV-1 infection
upon acetylation of the p53 tumor suppressor in response to genotoxic stress caused by UV irradiation (24, 25, 34). CD4+ Molt-4
lymphoblastic leukemia cells were infected with HIV-1, HXB2IIIB, over a 6-day period, and consecutive aliquots
were removed and exposed to UV irradiation. The cells were cultured for
an additional 3 h before extracts were prepared for
immunoprecipitation and immunoblot analyses. HIV-1 Tat protein
expression was significantly detectable by the fourth day of infection
and continued to increase through the sixth day (Fig.
7A). Ultraviolet irradiation
is known to induce increased intracellular p53 levels (35, 36), and indeed, p53 expression was detectable in uninfected UV-treated cells
(Fig. 7B, upper left panel). Uninfected control
cells or HIV-1-infected Molt-4 cells that were not exposed to UV
irradiation did not contain high p53 protein levels as determined by
immunofluorescence laser confocal microscopy (Fig. 7B,
upper middle and right panels) and immunoblotting
using an anti-p53 monoclonal antibody (Fig. 7D). Nuclear Tat
and p53 co-localized in HIV-1-infected cells, consistent with reports
by others that Tat directly interacts with the p53 tumor suppressor
(6); relative fluorescence intensities and signal overlaps for both
proteins were measured by linear quantification and are shown
below the micrographs in Fig. 7C. To assess the status of
p53 acetylation in response to UV irradiation in HIV-1-infected cells,
we prepared whole cell extracts and examined the levels of
intracellular p53 (Fig. 7D). Actin is shown as an input
control. Immunoprecipitations were performed, and the acetylated forms
of p53 were identified by immunoblotting using antibodies that
discriminate between Lys320-acetylated p53 or
Lys373-acetylated p53 (Fig. 7D). As demonstrated
in Fig. 7D, there was slight diminishment of
p53Lys-320 acetylation in HIV-1-infected cells on the fifth
day of infection in the absence of UV treatment. More apparent,
however, was the decrease in both p53Lys-373 and
p53Lys-320 acetylation induced by UV irradiation on the
fifth and sixth days of HIV-1 infection. Interestingly, the observed
reduction in UV-responsive p53 acetylation did not correlate with
intracellular levels of Tat, suggesting that other virally encoded
proteins or host factors might influence p53 acetylation and responses to genotoxic stress (Fig. 7A). These results indicate that
HIV-1 infection significantly prevents acetylation of p53 by
co-activators/acetyltransferases in response to UV irradiation, and our
in vitro evidence suggests that Tat could play an important
role in mediating this inhibitory effect.
The tumor suppressor p53 induces the arrest of cell cycle
progression in G1/S and G2/M under conditions
of genotoxic stress (26, 27, 37, 38). Because AIDS-affected individuals
demonstrate increased risks for developing various neoplasms,
inhibition of p53Lys-320 acetylation/transcription by
Tat-HAT binding could promote the acquisition of deleterious mutations
by subverting p53-regulated checkpoint defenses. p53
trans-activation functions and G2/M cell cycle
control are regulated by post-translational modifications, e.g. acetylation and phosphorylation, and destabilization of
p53 by Mdm2 is dependent upon p400/TRRAP-associated
chromatin-remodeling complexes (39-46). Mdm2 and adenoviral E1A 12S
and E1B proteins have been shown to inhibit p53 acetylation by P/CAF,
and therefore, the P/CAF co-activator/acetyltransferase may be a key
modulator of p53 tumor suppressor-associated activities (47-49).
Recently, others have demonstrated that Tat inhibits acetyltransferase
activities of the co-activators Tip60 and TAFII250, thereby
causing repression of cellular transcription (50, 51). Extracellular
Tat produced by surrounding, infected cells might also enter and target
p53 in nuclei of adjacent cells to create a local "by-stander"
effect that might allow Tat to cooperate with oncogenic factors encoded
by promoter was
markedly repressed by Tat-histone acetyltransferase interactions, and
p53 acetylation by p300/CREB-binding protein-associated factor on
residue Lys320 was diminished as a result of
Tat-histone acetyltransferase binding in vivo and in
vitro. Tat also inhibited p53 acetylation by p300 in a
dosage-dependent manner in vitro. Finally,
HIV-1-infected Molt-4 cells displayed reduced p53 acetylation on
lysines 320 and 373 in response to UV irradiation. Our results allude
to a mechanism whereby the human immunodeficiency virus type-1
trans-activator might impair tumor suppressor functions in
immune/neuronal-derived cells, thus favoring the establishment of
neoplasia during AIDS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, by induction
with 100 µM
isopropyl-
-D-thiogalactopyranoside (Invitrogen) in LB broth containing 100 µg/ml ampicillin at 37 °C overnight. The cells were pelleted at 5000 rpm, washed twice with PBS, centrifuged, and resuspended in 5 ml of cold PBS containing the protease inhibitors antipain-dihydrochloride, bestatin, chymostatin, leupeptin, and pepstatin (Roche Molecular Biochemicals) at 50 ng/ml each. Bacteria were lysed by sonication over an ice bath using a Bronson sonic dismembrator equipped with a microtip and operated at 70% duty cycle.
The lysates were clarified by centrifugation at 14,000 rpm in a Sorvall
SS-34 rotor (DuPont/Sorvall) for 30 min at 4 °C, and the
supernatants were incubated for 2 h at 4 °C with 500 µl of a
50% mixture of equilibrated glutathione-Sepharose 4B (Amersham Biosciences). Following incubation, the matrices were pelleted by
centrifugation at 2500 rpm at 4 °C and then washed twice with PBS
(with protease inhibitors). Bound GST fusion proteins were eluted in
four 200-µl fractions of 10 mM reduced glutathione
(Sigma) in PBS (with protease inhibitors). The eluted fractions were
analyzed by electrophoresis through a 12.5% SDS-polyacrylamide gel;
the proteins were visualized by Coomassie staining. Purified proteins were dialyzed overnight at 4 °C against 25 mM Hepes, pH
7.9, 5 mM KCl, 0.5 mM MgCl2, 0.5 mM EDTA, 0.25 mM dithiothreitol, and 10%
glycerol; the fractions were stored in 20-µl aliquots at
80 °C.
For in vitro acetylation, 100 ng of purified GST-p53 was
mixed either with 5 units of purified p300 or P/CAF (Upstate
Biotechnology, Inc.) in acetylation buffer containing 50 mM
Tris-HCl, pH 8.0, 1 mM dithiothreitol, 0.1 mM
EDTA, 50 µg/ml bovine serum albumin, 10% glycerol, and 5 µl of
[acetyl-1-14C]acetyl coenzyme A (51.60 mCi/mmol, NEC-313;
PerkinElmer Life Sciences) in the presence or absence of increasing
concentrations of GST-Tat or GST-TatK28A/K50A (25, 50, or
100 ng) or Tat synthetic peptides (Tat23-43,
TatK/A23-43, Tat41-61, or TatKK/AA41-61: 50, 100, or 200 ng; Biosynthesis, Inc.). The samples were incubated at 37 °C for 30 min, then the reactions were quenched by the addition of 6 µl of 5× SDS-PAGE loading buffer, and the acetylated products were resolved through 4-20% Tris-glycine gels (Invitrogen, Inc.) and visualized by autoradiography/fluorography using ENHANCE reagent (PerkinElmer Life Sciences). Kodak XAR scientific imaging film was exposed for 72 h at
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
promoter trans-activation, we investigated the effects of
HIV-1 Tat expression upon a 14-3-3
promoter luciferase reporter
construct containing three p53-responsive elements (other regulatory
elements are deleted in this plasmid. Refs. 26 and 27). As shown in
Fig. 1, HIV-1 Tat effectively inhibited
p53 trans-activation from the 14-3-3
promoter in IMR-32 and PC-12 cells, respectively, whereas neither the
TatK28A/K50A mutant nor green fluorescent protein
significantly influenced p53 transcription. Consistently, Tat
expression did not seem to alter p53 protein levels, suggesting that
Tat causes functional impairment of p53 (Fig. 1A).
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Fig. 1.
HIV-1 Tat inhibits p53-responsive
transcription functions. A, IMR-32 neuroblastoma cells
were co-transfected with 1 µg of 14-3-3 luciferase and 1 µg of
CMV-p53 in the presence of 0.5, 1.5, or 3 µg of RSV-Tat,
RSV-TatK28A/K50A, or pcDNA 3.1 green fluorescent
protein (GFP) (Invitrogen). Relative protein levels of p53,
Tat, TatK28A/K50A, and actin were detected by standard
immunoblotting techniques using 30 µg of total proteins.
B, PC-12 cells were co-transfected and assayed as in
A.
Promoter trans-Activation--
To evaluate
the relative contributions of Tat-p300 or Tat-P/CAF interactions toward
the inhibition of p53 transcription, we analyzed a panel of previously
characterized Tat mutants; the TatK28A mutant is defective
for acetylation by P/CAF, and TatK50A is impaired for
acetylation by p300/CBP (17). The results in Fig.
2A indicate that
TatK28A and TatK50A mutants are partially impaired in their abilities to repress p53-responsive 14-3-3
transcription as compared with wild-type Tat. The differences in
biological activities were not due to fluctuations in protein expression because these mutants were detected at levels comparable with wild-type Tat (Fig. 2A, lower panel). The
trans-dominant-negative p53 mutant, R175H, which is
defective for p53-responsive element DNA binding, was used as a control
to verify the p53 dependence of 14-3-3
promoter luciferase reporter
gene activity (Fig. 2A and Ref. 26). Because the adenoviral
E1A 12S factor also interacts with the CH3/HAT domains of both p300/CBP
and P/CAF, inhibiting their acetyltransferase activities, we compared
the effects of E1A 12S upon p53-responsive, 14-3-3
transcription in
PC-12 and IMR-32 cells (28-30). In both cell-types, E1A 12S inhibited
p53 trans-activation in a dosage-dependent
manner that resembled HIV-1 Tat (Fig. 2, B and
C). An amino-terminal deletion mutant, E1A 12S
N,
defective for interactions with the HAT domains of these co-activators
did not affect p53 transcription, which is consistent with the notion
that p53 trans-activation functions require co-activator acetyltransferase activities (Fig. 2, B and C,
and Refs. 23-25, 30, and 31). Indeed, co-expression of increasing
amounts of either P/CAF or p300 significantly countered the repressive
effects of Tat upon 14-3-3
transcription, although ectopic p300
restored p53 trans-activation somewhat better than P/CAF
(Fig. 2D).
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Fig. 2.
HIV-1 Tat and adenoviral E1A 12S proteins
repress p53-responsive transcription to similar degrees.
A, IMR-32 cells were co-transfected with expression
constructs for Tat acetylation-defective mutants (3 µg),
RSV-TatK28A and RSV-TatK50A, and 1 µg of
CMV-p53 (or CMV-p53R175H) with 1 µg of 14-3-3
luciferase. The protein levels are shown for p53, p53R175H,
Tat, TatK28A, TatK50A, and actin for extracts
(30 µg of total proteins) used in A. B and
C, PC-12 cells (B) and IMR-32 cells
(C) were co-transfected with 14-3-3
luciferase and
CMV-p53 in the presence of increasing amounts of CMV-E1A 12S or CMV-E1A
12S
N (0.5, 1.5, or 3 µg). D, IMR-32 cells were
co-transfected with 14-3-3
luciferase, CMV-p53, and 3 µg of
RSV-Tat, either in the presence of increasing amounts of CMV-P/CAF or
CMV-p300 (1.5, 2.5, or 5 µg). WT, wild type.
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Fig. 3.
Nuclear expression of p53, HIV-1 Tat, and
TatK28A/K50A in transfected IMR-32 cells. IMR-32
neuroblastoma cells were transfected with 0.5 µg of CMV-p53
(A), RSV-HIV-1 Tat (B), or
RSV- TatK28A/K50A (C) and immunostained for
laser confocal microscopy using a rabbit polyclonal antibody against
p53 (Santa Cruz Biotechnologies, Inc.) and fluorescein
isothiocyanate-conjugated anti-rabbit secondary antibody. HIV-1 Tat and
TatK28A/K50A proteins were visualized using an
anti-HIV-1IIIB Tat, rabbit polyclonal antibody (C-2145),
and fluorescein isothiocyanate-conjugated anti-rabbit secondary
antibody. Endogenous p300 was detected using a monoclonal antibody
against the carboxyl terminus of p300 (Upstate Biotechnology, Inc.) and
rhodamine red-conjugated anti-mouse secondary antibody. Endogenous
P/CAF was detected using a goat polyclonal antibody against P/CAF
(Santa Cruz Biotechnology, Inc.) and Cy-3-conjugated anti-goat
secondary antibody. All of the fluorescence-conjugated secondary
antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc.
The nuclei were stained with DAPI (Molecular Probes, Inc.) and are
shown for reference. The merged images are at the right of
each panel. Immunofluorescence laser confocal microscopy was performed
using a 63× objective in combination with 13× digital zoom.
transcription
may have resulted from enhanced factor recruitment
(e.g. P/CAF) to p53-responsive elements (Fig.
2E). The results from electrophoretic mobility shift/DNA
binding assays using a radiolabeled, p53-responsive oligonucleotide
probe revealed that decreased p53-specific DNA binding in nuclear
extracts prepared from IMR-32 cells, expressing p53 in the presence of
Tat or various Tat mutants, coincided with diminished
p53Lys-320 acetylation (Fig. 4C, lower
panel). The DNA binding activities of IMR-32 nuclear extracts were
normalized to yield similar binding to a radiolabeled, consensus cyclic
AMP-responsive element probe (Fig. 4C, top
panel); the same amounts of nuclear extracts were then used to
quantify p53-responsive element binding.
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Fig. 4.
Tat inhibits p53 acetylation in
vivo and in vitro. A, a
diagram of CBP (p300 synologue) and P/CAF depicting functionally
overlapping binding sites for HIV-1 Tat, E1A 12S, and p53 in
acetyltransferase domains. B, IMR-32 cells were
co-transfected with 1 µg of CMV-p53 and 3 µg of RSV-Tat, or various
Tat mutants (RSV-TatK28A, RSV-TatK50A, or
RSV-TatK28A/K50A), extracts were prepared in RIPA buffer,
and immunoblotting was performed using 30 µg of total proteins to
detect p53, acetyl-p53Lys-320,
acetyl-p53Lys-373, and actin. C, nuclear
extracts were prepared from 3 × 106 IMR-32 cells
co-transfected with 1 µg of CMV-p53 and either 3 µg of RSV-Tat,
RSV-TatK28A, RSV-TatK50A, or
RSV-TatK28A/K50A and used to detect binding to
32P-radiolabeled, consensus cyclic AMP-responsive element
(5'-CTAGGATCTTGATGACGCAATACGCCATGGTCGA-3') and p53-responsive
(5'-TACAGAACATGTCTAAGCATGCTGGGG-3') oligonucleotide probes.
D, amino acid sequences of wild-type Tat and Tat-derived
synthetic peptides (Tat23-43, Tat K/A23-43,
Tat41-61, and TatKK/AA41-61) used in in
vitro acetylation assays. E and F,
acetylation of GST-p53 by recombinant P/CAF (E) or p300
(F) in the presence of increasing amounts of GST-Tat,
GST-TatK28A/K50A, or Tat-derived peptides was performed as
described under "Materials and Methods." GST fusion proteins
(GST-p53, GST-Tat, and GST-TatK28A/K50A) were detected
using a goat anti-GST antibody (Amersham Biosciences).
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Fig. 5.
The HIV-1 Tat23-43 peptide
competes against p53 for binding to the minimal acetyltransferase
domain of P/CAF. A, purified recombinant GST-p53 (500 ng) was mixed with recombinant GST-Tat or GST-TatK28A/K50A
proteins (125, 250, and 500 ng), and the reactions were
immunoprecipitated overnight at 4 °C using an anti-p53 monoclonal
antibody (Upstate Biotechnology, Inc.) and protein G-agarose. Affinity
matrices were washed, the samples were electrophoresed through a
4-20% SDS-PAGE precast gel (Invitrogen, Inc.), and bound HIV-1 Tat
was detected using purified anti-HIV-1 Tat polyclonal antibody.
B, p53-null, Calu-6 carcinoma cells were transfected with 3 µg of CMV-p53 or CMV-vector control, the extracts were prepared in
RIPA buffer, and equivalent amounts of total cellular proteins were
used in in vitro binding assays. Increasing amounts of
synthetic HIV-1 Tat23-43 or TatK/A23-43
peptides (250 ng, 500 ng, and 1 µg) were mixed with Calu-6 extracts
expressing p53, and 15 units of purified recombinant
GST-P/CAF352-382 were added to each sample. 30 µl of
50% glutathione-Sepharose 4B in PBS was added to each reaction, and
GST-pull-down assays were carried out overnight at 4 °C. Affinity
matrices were washed twice with PBS, the samples were resolved by 12%
SDS-PAGE, and bound p53 was detected using an anti-p53 monoclonal
antibody. Input levels of synthetic Tat23-43 and
TatK/A23-43 peptides were detected by electrophoresis
through a 4-20% SDS-PAGE precast gel and Coomassie staining.
regulates p53-induced G2/M arrest under conditions of
genotoxic stress, we addressed whether inhibition of p53 functions as a
consequence of Tat-HAT binding might weaken the G2/M
checkpoint in the presence of adriamycin (100 µM; Refs. 26 and 27). IMR-32 cells were transfected with Tat or various Tat
mutants; after 48 h, the cells were treated with adriamycin and
incubated for an additional 24 h prior to FACS analyses to evaluate apoptosis and cellular arrest using annexin-V (Pharmingen Corp.) and acridine orange (Molecular Probes, Inc.) staining methods. As shown in Fig. 6A, neither
adriamycin treatment nor Tat alone caused apoptosis in IMR-32 cells.
However, cells expressing Tat in the presence of adriamycin exhibited
considerable programmed cell death (62.8%) as determined by annexin-V
staining (Fig. 6, A and B). Of the Tat mutants,
only TatK50A was associated with significant apoptosis
(43.3%), suggesting that P/CAF-binding by Tat might be an important
contributing factor for the bypass of G2/M arrest. The
results from this "mitotic trap" assay suggest that Tat-expressing
cells not arrested in G2/M enter mitosis (M phase) and are
killed through the action of adriamycin. Acridine orange staining/cell
cycle analyses revealed that IMR-32 cells were arrested in
G2/M (G1, 27.5%; G2/M, 47.0%) as
a result of adriamycin treatment (Fig. 6C). Expression of
various Tat mutants had no effect upon cellular arrest in
G2/M (G1, ~24%; G2/M, 50% for
each). The wild-type Tat significantly prevented cellular arrest in the
presence of adriamycin (G1, 43.7%; G2/M,
34.4%) coincident with increased apoptosis (Fig. 6C; see
also Fig. 6, A and B). A minor population of
IMR-32 neuroblastoma cells that contained greater than G2/M
DNA content were reproducibly detected in each sample, and these cells
presumably possess inherent growth-regulatory defects resulting in
deregulation of cytokinesis and S phase entry (Fig. 6). This aberrant
cellular population did not appear to be affected by expression of
HIV-1 Tat or Tat-derived mutants.
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Fig. 6.
HIV-1 Tat causes bypass of G2/M
arrest induced by adriamycin. A, IMR-32 cells were
transfected with RSV-Tat, RSV-TatK28A/K50A,
RSV-TatK28A, or RSV-TatK50A, and certain
samples were treated with 100 µM adriamycin
(Adr) and stained for annexin-V surface expression.
B, overlays of FACS profiles for annexin-V staining in the
presence of adriamycin and either wild-type (WT) Tat or
TatK28A/K50A. C, IMR-32 cells were transfected
and treated with adriamycin as in A and were stained to
determine nuclear contents using acridine orange as described under
"Materials and Methods."
View larger version (41K):
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Fig. 7.
HIV-1 infection inhibits p53 acetylation in
response to UV irradiation in Molt-4 cells. A, Molt-4
cells were infected with HIV-1, HXB2IIIB, and Tat
expression was monitored on consecutive days by immunoblotting using an
anti-Tat rabbit polyclonal antibody as described. B,
induction of p53 protein expression by UV irradiation was verified by
immunofluorescence laser confocal microscopy in uninfected or
HIV-1-infected cells in the presence or absence of UV treatment.
C, nuclear co-localization of Tat and p53 in UV-irradiated,
HIV-1-infected Molt-4 cells was detected by immunofluorescence laser
confocal microscopy as described. Relative fluorescence intensities and
signal overlaps were quantified and are shown below the micrographs.
D, actin and p53 protein levels were determined by
immunoblotting whole cell extracts prepared from uninfected or
HIV-1-infected Molt-4 cells in the presence or absence of UV
irradiation (top panels). Total intracellular p53 was
immunoprecipitated (IP), and immunoblots were performed
using antibodies that specifically recognize
Lys373-acetylated p53 or Lys320-acetylated p53
(bottom panels).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-herpesviruses, such as Epstein Barr Virus and Kaposi's Sarcoma
herpes-like virus, during co-infections frequently observed in
AIDS patients (4, 5, 52, 53). The mechanism by which extracellular Tat
enters surrounding cells is largely unknown but requires cell surface
heparan sulfate proteoglycans (54). In this study, we have shown that
Tat-HAT interactions inhibit p53Lys-320 acetylation as well
as p53 DNA binding and transcription in neuronally derived cells. Both
Tat and adenoviral E1A 12S repressed p53 trans-activation to
similar degrees through co-activator/HAT binding. The unusually high
percentages of cells affected by Tat in our cell cycle and apoptosis
analyses are consistent with observations by others that Tat produced
in transfected cells enters nuclei of surrounding untransfected cells,
inhibiting p53 transcription functions. Our subsequent
immunofluorescence microscopy experiments using CMV-HIV-1 Tat-transfected IMR-32 neuroblastoma cells also support this finding (data not shown). Col et al. (10) have reported that
inhibition of CBP-associated acetyltransferase activity by HIV-1 Tat
prevents histone acetylation but not p53 or MyoD acetylation; however, that study dealt exclusively with basal level p53 acetylation, whereas
our present work summarizes effects of Tat-co-activator interactions
upon basal level as well as UV-induced p53 acetylation (24, 25, 34).
Our findings indicate that p53 acetylation on lysines 320 and 373 induced by UV irradiation is significantly diminished in HIV-1-infected
Molt-4 cells in vivo (24, 25, 34). Inhibition of p53
acetylation did not strictly correlate with levels of intracellular
Tat, and it is likely that other virally encoded factors, such as Nef,
that also interact with p53 may influence the p53 response under
conditions of genotoxic stress (24, 25, 34, 55). Alternatively, host
cellular factors induced by HIV-1 or the viral
trans-activator, Tat, could antagonize Tat/co-activator
interactions during certain stages of the infection process.
Interestingly, the majority of viral factors that interact with
p300/CBP are derived from oncogenic viruses. Indeed, Tat has been
demonstrated to transform primary B-cells and induce lymphomas in
transgenic mice, suggesting that Tat might contribute to certain
hematological malignancies (56). Seo et al. (57) have
reported that p300 and P/CAF acetyltransferases are inhibited by a
cellular complex, INHAT (inhibitor of
acetyltransferases), associated with the
Set/TAF-I
oncoprotein involved in myeloid leukemia, indicative that
inhibition of co-activator acetyltransferases might be generally linked
to neoplastic transformation. Our results allude to a mechanism whereby
HIV-1 Tat might impair tumor suppressor functions in
immune/neuro-immune cells of the central nervous system, thus
supporting the establishment of AIDS-related cancers.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. B. Vogelstein for generously
providing CMV-p53, CMV-R175H, and 14-3-3 (BDS-2, 3X) promoter
luciferase constructs and Dr. J. Brady for the GST-p53 expression
construct. We also thank Dr. K. T. Jeang for providing CMV-Tat
that was used in immunoconfocal microscopy experiments, Dr. Y. Nakatani
for permission to use CMV-P/CAF, CMV-E1A 12S, and CMV-E1A 12S
N
expression constructs, and Dr. B. C. Nair for purified anti-Tat
antibody, C-2145.
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FOOTNOTES |
---|
* This work was supported by the NCI, National Institutes of Health, and in part by the Department of Biological Sciences of Southern Methodist University.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: Lab. of Molecular Virology, Dept. of Biological Sciences, Southern Methodist University, 334-DLS, 6501 Airline Dr., Dallas, TX 75275-0376. Tel.: 214-768-3864; Fax: 214-768-3955; E-mail: rharrod@mail.smu.edu.
** Maître de Recherches from the Fonds National de la Recherche Scientifique (Belgium). Supported by the Fonds National de la Recherche Scientifique, Televie, Free University of Brussels (ARC), Internationale Brachet Stiftung, CGRI-INSERM cooperation, the Theyskens-Mineur Foundation, Region Wallone-Commission Europeenne FEDER, and the Agence Nationale de Recherches sur le SIDA (ANRS, France).
Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M211167200
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ABBREVIATIONS |
---|
The abbreviations used are: HIV-1, human immunodeficiency virus, type-1; TAR, Tat-associated region; CBP, CREB-binding protein; P/CAF, p300/CBP-associated factor; HAT, histone acetyltransferase; CREB, cAMP-responsive element-binding protein; FACS, fluorescence-activated cell sorting; PBS, phoshphate-buffered saline; GST, glutathione S-transferase; CMV, cytomegalovirus; RSV, Rous sarcoma virus.
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REFERENCES |
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