From the Department of Biomedical Sciences and Biotechnology,
School of Medicine, University of Brescia, 25123 Brescia, Italy, the
§ Department of Experimental and Diagnostic Medicine,
Section of Microbiology, University of Ferrara, 44100, Ferrara, Italy,
the ¶ Institute of Biomedical Sciences, University of Ancona,
60131, Ancona, Italy, the Institute of Structural Biology,
38027, Grenoble, France, the ** International Center for Genetic
Engineering and Biotechnology, 34012 Trieste, Italy, and the
Institute of Anatomy, Histology and
Embryology, University of Bari, 70124, Bari, Italy
Received for publication, November 29, 2000, and in revised form, February 7, 2001
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ABSTRACT |
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HIV-1 Tat protein, released from
HIV-infected cells, may act as a pleiotropic heparin-binding growth
factor. From this observation, extracellular Tat has been implicated in
the pathogenesis of AIDS and of AIDS-associated pathologies. Here we
demonstrate that the heparin analog pentosan polysulfate (PPS) inhibits
the interaction of glutathione S-transferase (GST)-Tat
protein with heparin immobilized to a BIAcore sensor chip. Competition
experiments showed that Tat-PPS interaction occurs with high affinity
(Kd = 9.0 nM). Also, GST·Tat
prevents the binding of [3H]heparin to GST·Tat
immobilized to glutathione-agarose beads. In vitro, PPS
inhibits GST·Tat internalization and, consequently, HIV-1 long
terminal repeat transactivation in HL3T1 cells. Also, PPS
inhibits cell surface interaction and mitogenic activity of GST·Tat
in murine adenocarcinoma T53 Tat-less cells. In all assays, PPS exerts its Tat antagonist activity with an ID50 equal
to ~1.0 nM. In vivo, PPS inhibits the
neovascularization induced by GST·Tat or by Tat-overexpressing T53
cells in the chick embryo chorioallantoic membrane. In conclusion, PPS
binds Tat protein and inhibits its cell surface interaction,
internalization, and biological activity in vitro and
in vivo. PPS may represent a prototypic molecule for
the development of novel Tat antagonists with therapeutic implications
in AIDS and AIDS-associated pathologies, including Kaposi's sarcoma.
Tat protein, the transactivating factor of the human
immunodeficiency virus type 1 (HIV-1),1 is released from
HIV-1 infected cells (1). Extracellular Tat can enter the cell and
nucleus to stimulate the transcriptional activity of HIV-LTR (2).
Moreover, extracellular Tat induces several biological effects on
uninfected target cells (3). Tat has been implicated in pathological
processes associated to AIDS, including Kaposi's sarcoma (KS) (4).
Indeed, KS spindle cell xenografts grow faster in Tat transgenic mice
than in non-transgenic animals (5). Also, Tat-overexpressing T53 cells,
derived from an adenocarcinoma of a BKV/Tat transgenic mouse
(6), are angiogenic and induce highly vascularized tumors in nude mice
that resemble KS (7, 8).
Tat binds the polyanionic glycosaminoglycans (GAGs) heparin and heparan
sulfate via its basic domain (9, 10). The binding of extracellular Tat
to heparan sulfate proteoglycans (HSPGs) mediates cell surface
interaction (11) and accumulation in the extracellular matrix (ECM)
(12). Also, HSPGs are required for Tat internalization and consequent
HIV-LTR transactivation (13). Conversely, the interaction of
extracellular Tat with free GAGs inhibits cellular uptake and HIV-LTR
transactivating activity in HL3T1 and CHO cells (13, 14), affects its
mitogenic and protease-inducing activities in T53 cells (14, 15), and
modulates its angiogenic activity in vivo (11).
Several polyanionic compounds inhibit HIV-1 replication by preventing
the binding of the virus to target cells (16). Some of these compounds,
including unmodified heparin, heparan sulfate, and suramin-like
distamycin A derivatives, although not dextran sulfate,
The heparin analog xylanopolyhydrogensulfate (pentosan polysulfate,
PPS) prevents the interaction of HIV-1 with target cells (19) and
inhibits its reverse transcriptase activity (20). Also, PPS affects
tumorigenicity and angiogenic potential of KS xenografts in nude mice
(21). The capacity of PPS to inhibit the biological activity of various
heparin-binding angiogenic growth factors, including fibroblast growth
factor-2 (FGF-2) (22-25) raises the possibility that PPS may interfere
additionally with extracellular Tat protein. To assess this
possibility, as a part of a long-term project aimed to identify
polyanionic multitarget HIV-Tat antagonists (9, 10, 13-15, 17), we
evaluated the capacity of PPS to bind Tat protein and to inhibit the
biological activity of extracellular Tat in vitro and
in vivo. The results demonstrate that PPS binds Tat and
prevents its interaction with heparin. Accordingly, PPS inhibits cell
surface interaction and internalization of extracellular Tat causing
the inhibition of HIV-LTR-transactivating activity, mitogenic capacity,
and angiogenic potential of the protein. These observations identify
PPS as a prototypic molecule for the design of novel multitarget drugs for the treatment of AIDS and AIDS-associated pathologies.
Reagents--
Recombinant HIV-1 Tat (86-amino acid isoform) was
expressed in Escherichia coli as glutathione
S-transferase (GST) fusion protein (9). GST·Tat was also
fused at its C terminus to the green fluorescent protein (GFP) (14).
The GST and GFP moieties do not interfere with LTR-transactivating
activity and heparin-binding capacity of Tat (13, 14). Pentosan
polysulfate was from Sigma (St Louis, MO). Heparin was from Laboratori
Derivati Organici SpA, (Milan, Italy). BIAcore Binding Assay--
Surface plasmon resonance (SPR) was
used to measure changes in refractive index caused by the capacity of
free PPS to bind GST·Tat and prevent its interaction with heparin
immobilized to a BIAcore sensor chip (26, 27). To this purpose,
size-defined heparin (9 kDa) was biotinylated on its reducing end, and
a flow cell of an F1 sensor chip was activated with streptavidin. Then, biotinylated heparin was allowed to react with the streptavidin-coated sensor chip. GST·Tat alone or in the presence of increasing
concentrations of PPS was then injected over the heparin surface for 5 min to allow the association of the protein with heparin and then
washed until dissociation was observed. The SPR signal was expressed in
terms of resonance units (RU).
Preparation of 3H-labeled Heparin and GST·Tat
Affinity Chromatography--
Heparin was 3H-labeled as
described (28) with a specific radioactivity of 5,000 cpm/nmol. To
assay the capacity of PPS to compete with [3H]heparin for
the binding to immobilized GST·Tat, [3H]heparin was
loaded onto a GST·Tat-glutathione-agarose column in the presence of
increasing concentrations of PPS, Cell Cultures--
HL3T1 cells are derived from HeLa cells and
contain integrated copies of pL3CAT, a plasmid in which the
chloramphenicol acetyltransferase (CAT) bacterial gene is driven by
HIV-1 LTR (29). The T53 cell line was established from adenocarcinoma
of skin adnexa of Tat transgenic mice and secretes biologically active
Tat (6, 7). T53 Tat-less cells, obtained by subcloning T53
cells, do not produce detectable amounts of extracellular Tat but
retain the capacity to proliferate when exposed to exogenous Tat (14).
All cell types were grown in Dulbecco's modified Eagle's medium
(DMEM) with 10% fetal calf serum (DMEM/FCS, Life Technologies, Inc., Grand Island, NY).
LTR-CAT Transactivation Assay--
HL3T1 cells were seeded in
24-well dishes at 20,000 cells/cm2 in DMEM/FCS. After
24 h, cells were incubated for another 24 h period in fresh
DMEM/FCS and 100 µM chloroquine in the absence or in the
presence of 200 ng/ml of GST·Tat and increasing concentrations of the
polysulfated compound that was to be tested. Then, conditioned medium
was removed, and cells were incubated for an additional 24 h in
DMEM/FCS. In some experiments, subconfluent cultures of HL3T1 cells
were lipofectin-transfected with 2 µg/ml of pCEP-Tat expression
vector harboring the HIV-1 Tat cDNA (kindly provided by Dr. A. Gualandris, University of Torino, Italy). Five hours after
transfection, cells were washed and incubated for another 24 h in
DMEM/FCS in the absence or presence of PPS (100 nM). At the
end of the incubation, the amount of CAT protein present in the cell
extracts was determined using the CAT ELISA kit (Roche Molecular
Biochemicals).
T53 Cell Proliferation Assays--
T53 Tat-less cells
were seeded in 96-well dishes at 10,000 cells/cm2 in
DMEM/FCS. After 24 h, cells were incubated for another 24 h
in fresh DMEM/FCS in the absence or presence of 200 ng/ml of GST·Tat
and increasing concentrations of the polysulfated compound being
tested. At the end of the incubation, cells were trypsinized and
counted in a Burker chamber.
Internalization of GST·Tat·GFP in HL3T1 Cells--
HL3T1
cells adherent to glass coverslips were incubated for 6 h at
37 °C in DMEM/FCS containing 400 ng/ml of GST·Tat·GFP in the
absence or presence of increasing concentrations of the polysulfated compound being tested. At the end of the incubation, the cells were
washed and fixed. GST·Tat·GFP internalization was quantified by
computerized image analysis as described (14).
Binding of GST·Tat·GFP to T53 Tat-less Cells--
T53
Tat-less cells adherent to glass coverslips
(20,000/cm2) were incubated for 6 h at 37 °C in
DMEM/FCS and 400 ng/ml of GST·Tat·GFP in the absence or presence of
PPS (1 µM). At the end of the incubation, the medium was
removed, and cells were washed and fixed as described (8). Observations
were carried out in a Nikon photomicroscope equipped for epifluorescence.
Chick Embryo Chorioallantoic Membrane (CAM) Assay--
The CAM
assay was performed as described (30). Briefly, a window was opened in
the egg shell of 3-day-old fertilized chicken eggs. At day 8, gelatin
sponges (Gelfoam; Upjohn Co., Kalamazoo, MI) were implanted on the CAMs
and adsorbed with 10 µl of phosphate-buffered saline alone or
containing GST or GST·Tat (both at 400 ng/sponge) in the absence or
presence of PPS (25 µg/sponge; 5-6 embryos per group). In parallel
experiments, the sponges were adsorbed with 10 µl of DMEM/FCS alone
or containing T53 cells (6 × 104 cells/sponge) in the
absence or presence of 25 µg of PPS (5-6 embryos per group). After 4 days, CAMs were observed in ovo under a Zeiss SR
stereomicroscope, and the angiogenic response was scored by two
investigators without knowledge of the samples tested and graded on an
arbitrary scale of 0-4+, with 0 representing no angiogenic response
and 4+ representing the strongest activity.
PPS Binds Tat Protein--
To assess its ability to interact with
Tat protein, PPS was evaluated for the capacity to prevent GST·Tat
binding to biotinylated heparin immobilized onto a
streptavidin-activated BIAcore sensor chip. Preliminary experiments
were performed to assess the ability of GST·Tat to bind immobilized
heparin. In a typical experiment, increasing concentrations of
GST·Tat were injected over the heparin surface, and a set of
sensograms was obtained (Fig.
1A). An association rate
constant (kon) equal to 4.2 × 104 M
PPS was also evaluated for its capacity to compete with free
[3H]heparin for the binding to GST·Tat immobilized onto
glutathione-agarose beads. As shown in Fig. 2B, PPS inhibits
the binding of [3H]heparin to GST·Tat in a
dose-dependent manner with a potency similar to unlabeled
heparin (ID50 equal to 100 and 300 nM,
respectively). Under the same experimental conditions, the polysulfated
PPS Inhibits Cell Internalization and HIV-LTR Transactivating
Activity of Extracellular Tat--
The capacity of PPS to abolish the
interaction of Tat with immobilized heparin raised the possibility that
PPS may prevent the binding of extracellular Tat to cell-associated
heparin-like HSPGs that are required for its internalization (13). To
assess this hypothesis, we evaluated the capacity of PPS to inhibit
cell internalization of GST·Tat conjugated to fluorescent GFP. As
shown in Fig. 3, PPS inhibits the
internalization of GST·Tat·GFP in HL3T1 cells in a
dose-dependent manner with an ID50 equal to 1.0 nM, close to that observed for heparin (ID50 = 3.0 nM). When tested under the same experimental
conditions, PPS does not affect the uptake of fluorescein-labeled BSA,
as assessed by image analysis of the total fluorescence internalized by
HL3T1 cells incubated with 400 ng/ml of FITC-BSA in the absence or in
the presence of 100 nM PPS (1.21 ± 0.48 and 1.92 ± 0.67 arbitrary units, respectively). It must be pointed out that ~90% of
GST·Tat·GFP or FITC-BSA remained associated to the cells after a
2.0 M NaCl wash of the cell monolayer, thus confirming the
intracellular localization of the fluorescent proteins (data not
shown). The specificity of PPS-Tat interaction was demonstrated further
by the inability of polysulfated
In agreement with its capacity to inhibit Tat internalization, PPS
abolished the LTR-CAT transactivating activity exerted by GST·Tat in
HL3T1 cells whereas
To investigate further the mechanism(s) of action of PPS on
extracellular Tat activity, PPS was administered to HL3T1 cells together with GST·Tat or at different times after the transactivating factor. As shown in Fig. 4C, the LTR-transactivating
activity of Tat is completely abolished when PPS was administered
within the first 2 h following the beginning of GST·Tat
treatment. Addition of PPS to the cell culture medium at later time
points instead causes a progressive decrease of its antagonist activity
that depends on an early interaction with extracellular Tat. Similar results were obtained when PPS was replaced by heparin as Tat antagonist (Fig. 4C).
PPS Inhibits Cell Surface Binding and Mitogenic Activity of
Extracellular Tat--
Endogenous Tat induces cell proliferation in
T53 cells derived from an adenocarcinoma of the skin adnexa of a
tat transgenic mouse (6). We have characterized a subclone
of T53 cells, named T53 Tat-less cells, that does not
produce significant amounts of endogenous Tat but that retains the
capacity to proliferate when exposed to exogenous Tat (14). Also, the
mitogenic activity exerted by Tat in T53 Tat-less cells is
inhibited by heparin (14). On this basis, the Tat antagonist activity
of PPS was evaluated in this model. As shown in Fig.
5A, PPS inhibits the binding
of GST·Tat·GFP to the surface of T53 Tat-less cells.
Approximately 80% of cell-associated fluorescence was removed by a 2.0 M NaCl wash of the cell monolayer, thus confirming the
extracellular nature of GST·Tat·GFP (data not shown). In agreement
with these observations, PPS inhibits the mitogenic activity exerted by
GST·Tat with a potency similar to that shown by heparin
(ID50 equal to 1.0 nM for both compounds) (Fig.
5B). On the contrary, PPS does not affect T53
Tat-less cell proliferation induced by 10% FCS (Fig.
5B).
PPS Inhibits the Angiogenic Activity of Extracellular
Tat--
Extracellular Tat is endowed with angiogenic potential
in vivo (7, 8, 11, 17). Accordingly, GST·Tat induces
neovascularization in the chick embryo chorioallantoic membrane (CAM)
(Fig. 6A). This activity is
specific because GST devoid of the Tat moiety does not exert any effect
when tested under the same experimental conditions (Fig.
6A). In agreement with its in vitro antagonist activity, PPS caused a significant inhibition of GST·Tat-induced neovascularization when tested in vivo at the dose of 25 µg/implant (Fig. 6A). PPS also inhibited the unstimulated,
physiological vascularization of the CAM that is mediated by endogenous
FGF-2 produced by the chick embryo (31).
Tat-overexpressing parental T53 cells exert a significant angiogenic
response when implanted on the top of the CAM (Fig. 6B), a
biological effect because of the release of bioactive Tat protein (7,
8, 17). Again, PPS was able to suppress neovascularization induced by
T53 cells when tested at the dose of 25 µg/implant (Fig. 6,
B and C).
Tat protein has been considered as a target for anti-AIDS
therapies aimed to block its intracellular interaction with RNA target
structures and various intracellular cofactors required for
transactivation of the viral genome (32). More recently, an increasing
body of evidence point to a role for extracellular Tat in the
progression of AIDS and AIDS-associated pathologies (3). This suggests
that the use of extracellular Tat antagonists may be of therapeutic
benefit for AIDS patients. Accordingly, the development of anti-Tat
vaccines has been undertaken (33, 34). Also, in vitro and
in vivo studies have underscored the possibility of using
polysulfated/polysulfonated compounds as extracellular Tat antagonists
(10, 14, 17, 18, 35). Here, we provide experimental evidence that PPS
binds extracellular Tat with high affinity (Kd = 9.0 nM), similar to the physiological ligand heparin (14). This
interaction inhibits the binding of Tat to free and surface-immobilized
heparin. Accordingly, PPS inhibits cell internalization of
extracellular Tat and its biological activity in different experimental
models. However, PPS does not inhibit LTR-CAT transactivation exerted
by endogenous intracellular Tat and does not affect FITC-BSA
internalization. Also, PPS inhibits the mitogenic activity exerted by
extracellular Tat in T53 cells without affecting cell proliferation
induced by serum. Taken together these data support the hypothesis that the antagonist activity of PPS is mainly caused by a specific interaction with extracellular Tat protein.
The interaction of extracellular Tat with cell-associated HSPGs allows
the accumulation of the bioactive protein in the ECM and mediates its
interaction with the cell surface (11, 12), leading to cell
internalization of the protein and LTR-transactivation (13). The
capacity of PPS to prevent the binding of GST·Tat to immobilized
heparin indicates that PPS may hamper HSPG-mediated interaction of
extracellular Tat with target cells. Accordingly, PPS inhibits the
internalization of extracellular Tat in HL3T1 cells and its association
with the surface of T53 Tat-less cells. Consequently, PPS
antagonizes the transactivating and mitogenic activity exerted by
extracellular Tat in the two cell lines, respectively. Association with
free heparin is required for the chemotactic and angiogenic activity of
extracellular Tat (11). Together with its ability to inhibit
FGF-2-mediated angiogenesis, the capacity of PPS to prevent the binding
of Tat to free heparin may contribute to the angiostatic activity
exerted by PPS in vivo.
PPS retains its Tat antagonist activity when added to HL3T1 cells
2 h after the administration of the transactivating factor, when
extracellular Tat is already bound to the cell surface (10). This
appears to be of particular importance when considering that significant amounts of Tat can be avidly sequestered by ECM HSPGs (12),
as indicated by the slow dissociation rate of the Tat-immobilized heparin complex. It should be pointed out that, at variance with PPS,
thrombospondin-1 retains its Tat antagonist activity only when the
transactivating factor is present in the extracellular environment as a
free protein, being ineffective when Tat is bound to the cell surface
(8). It is also interesting to note that the high stability of the
Tat-heparin complex appears to be a unique feature of this
transactivating factor. Indeed, different heparin-binding growth
factors and cytokines, including FGF-2 (36), stromal cell-derived
factor 1 The capacity of sulfated GAGs and polysulfonated compounds to bind
extracellular Tat and to inhibit its biological activity depends on the
backbone structure and, at least in part, on the degree of sulfation
(9, 10). Indeed, a series of distamycin A derivatives structurally
related to suramin but characterized by different chemical structure
and degree of sulfation differ significantly for their Tat antagonist
activity (10). Here, we have demonstrated that PPS binds Tat and
inhibits its biological activity with a potency similar to that shown
by heparin and 1,000 times higher than that of suramin (10, 14). These
data indicate that the backbone structure of PPS presents its sulfate
group(s) to Tat protein under optimal steric conditions, resembling
more closely heparin interaction.
PPS inhibits the receptor binding and mitogenic activity of FGF-1 and
FGF-2, being ineffective on cell proliferation mediated by IGF-1 or
TGF-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin, chemically desulfated or low molecular weight
heparins, also bind extracellular Tat and inhibit its biological activity in vitro (9, 10, 14, 17) and in vivo
(18). Thus, selected polyanionic molecules endowed with the capacity to
affect HIV life cycle at different levels can be exploited for the
development of novel "multitarget" anti-AIDS drugs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cyclodextrin
tetradecasulfate was from Glycores 2000 (Milan, Italy).
Fluorescein-labeled bovine serum albumin (FITC-BSA) was kindly provided
by Prof. S. Marchesini (University of Brescia, Italy).
-cyclodextrin, or unlabeled
heparin. The column was then washed with phosphate-buffered saline and
eluted with 2.0 M NaCl. Radioactivity in the eluate was
measured in a liquid scintillation counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 s
1 and a
slow dissociation rate constant (koff) equal to
2.7 × 10
3 s
1 characterized the
interaction of GST·Tat with immobilized heparin. Thus,
GST·Tat-heparin interaction occurs with high affinity (dissociation constant (Kd) = koff/kon = 64 nM) and results in the formation of highly stable
complexes. This interaction is specific because GST protein devoid of
the Tat moiety does not bind to immobilized heparin, and GST·Tat does
not bind to a streptavidin surface in the absence of immobilized
heparin (Fig. 1B). In some experiments, the association
phase of GST·Tat-heparin interaction was allowed to proceed to
equilibrium, and the data were used to calculate an affinity value
independent of the kinetics of binding. This analysis demonstrates that
GST·Tat-heparin interaction occurs with a Kd equal
to 58 nM, a value consistent with our kinetics analysis and
with previous determinations obtained with different experimental
approaches (14). On this basis, we evaluated the capacity of PPS to
compete with immobilized heparin for the binding to GST·Tat. To this
purpose, increasing concentrations of PPS were preincubated with
GST·Tat and then injected onto the heparin-coated sensor chip. As
shown in Fig. 2A, PPS causes a dose-dependent inhibition of GST·Tat-heparin interaction,
with an ID50 equal to 120 nM. The binding data
allowed the calculation of the relative affinity of PPS-GST·Tat
interaction (Kd is equal to 9.0 nM).
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Fig. 1.
Interaction of GST·Tat with biotinylated
heparin immobilized onto BIAcore sensor chip: A, GST·Tat
was injected at (from top curve to bottom) 20, 13.3, 8.9, 5.9, 3.9, 2.6, and 0 µg/ml for 5 min over a BIAcore sensor chip
containing streptavidin plus 120 resonance units (RU) of
immobilized biotinylated heparin. B, GST·Tat (solid
line) or GST alone (dotted line) (both at 5 µg/ml)
were injected for 5 min over a streptavidin surface or a
streptavidin-heparin surface, respectively. In both experiments, the
responses (in RU) were recorded as a function of time.
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Fig. 2.
Interaction of PPS with GST·Tat.
Competition binding assays: A, surface plasmon resonance,
GST·Tat (5 µg/ml) alone or in the presence of increasing
concentrations of PPS was injected over a flow cell of a BIAcore sensor
chip containing streptavidin plus 120 RU of immobilized biotinylated
heparin. The responses (in RU) were plotted as a function of PPS
concentration. Inset, overlay of sensograms showing the
binding of GST·Tat to immobilized heparin in the presence of
increasing concentrations of PPS (0, 0.085, 0.13, 0.2, 0.3, 0.45, 0.66, 1.0 µg/ml; from top curve to bottom).
B, GST·Tat glutathione-agarose affinity chromatography,
GST·Tat-glutathione-agarose columns (80 µl) were loaded with 300 µl samples containing 25 µg of [3H]heparin alone or
added with increasing concentrations of unlabeled heparin ( ), PPS
(
), or polysulfated
-cyclodextrin (
). Radioactivity in the 2.0 M NaCl eluate was measured in a liquid scintillation
counter. Nonspecific binding, measured in the presence of an excess of
unlabeled heparin (1.0 mM) was subtracted from all the
values. Each point is the mean of 2-4 determinations performed in
duplicate. The S.E. never exceeded 6% of the mean value.
-cyclodextrin, here used as a negative control (10), did not prevent
the binding of [3H]heparin to immobilized GST·Tat even
when tested at doses as high as 300 µM (Fig.
2B). Taken together, the data demonstrate that PPS binds Tat
in a specific manner with an affinity comparable with that of
Tat-heparin interaction.
-cyclodextrin to affect Tat
internalization (Fig. 3A).
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Fig. 3.
Effect of PPS on GST·Tat·GFP
internalization in HL3T1 cells. A, subconfluent cell
cultures were treated with GST·Tat·GFP (400 ng/ml) in the absence
or presence of increasing concentrations of PPS ( ), heparin (
),
or polysulfated
-cyclodextrin (
). Then, the amount of
GST·Tat·GFP internalized was evaluated by image analysis. Data are
expressed as percent of GST·Tat·GFP internalized in the absence of
polysulfated molecules. Each point is the mean of 3-6 determinations
performed in duplicate. The S.E. never exceeded 9-16% of the mean
value. B, microphotographs (original magnification × 600) of HL3T1 cells treated with GST·Tat·GFP alone (a)
or in the presence (b) of PPS (100 nM).
-cyclodextrin was ineffective (Fig.
4A). It should be noted that
100 nM PPS did not affect the basal levels of LTR-CAT
transactivation measured in HL3T1 cells incubated in the absence of
GST·Tat (0.166 ± 0.036 and 0.170 ± 0.034 A405 nm units for PPS-untreated and -treated
cells, respectively). Also, no inhibition of LTR-CAT transactivation was observed when Tat was expressed as an intracellular protein in
PPS-treated HL3T1 cells following transient transfection with an
expression vector harboring the HIV-1 Tat cDNA (Fig.
4B). These data demonstrate that the antagonist effect of
PPS on GST·Tat-mediated LTR-CAT transactivation is specific and
depends on an extracellular interaction with Tat protein.
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Fig. 4.
Effect of PPS on the transactivating activity
of GST·Tat in HL3T1 cells. A, subconfluent cell cultures
were treated with GST·Tat (200 ng/ml) in the presence of increasing
concentrations of PPS ( ), heparin (
), or polysulfated
-cyclodextrin (
). Then, cell extracts were assayed for levels of
CAT antigen by ELISA, and data were expressed as percent of the
transactivating activity measured in cultures treated with GST·Tat
alone. Each point is the mean ± S.E. of 2-8 determinations in
duplicate. B, subconfluent cell cultures were transiently
transfected with the pCEP-Tat expression vector devoid (a)
or harboring the HIV-1 Tat cDNA (b-d). After
transfection, cells were incubated in fresh medium in the absence
(a and b) or in the presence of PPS
(c) or heparin (d), both at 100 nM.
Then, cell extracts were assayed for the levels of CAT antigen by
ELISA. Each point is the mean ± S.E. of three determinations in
duplicate. C, subconfluent cell cultures were treated with
200 ng/ml of GST·Tat. At the indicated periods of time after the
beginning of GST·Tat treatment, cells were administered with PPS
(
) or heparin (
) (both at 100 nM). Forty-eight hours
after the beginning of GST·Tat treatment, cell extracts were assayed
for levels of CAT antigen by ELISA, and data were expressed as percent
of the transactivating activity measured in cultures treated with
GST·Tat alone. Each point is the mean ± S.E. of three
determinations in duplicate.
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Fig. 5.
Effect of PPS on cell surface binding and
mitogenic activity of Tat in T53 Tat-less cells.
A, subconfluent cell cultures were treated with
GST·Tat·GFP (400 ng/ml) in the absence (a) or in the
presence (b) of 1 µM PPS. Then, cells were
fixed and microphotographed (original magnification × 600).
B, subconfluent cell cultures were treated with GST·Tat
(200 ng/ml) in the presence of increasing concentrations of PPS ( )
or heparin (
). Parallel cultures were treated with DMEM/FCS alone in
the absence or presence (
) of 100 nM PPS. Then, cells
were trypsinized and counted in a Burker chamber. In a typical
experiment, cells incubated with DMEM/FCS alone or added with GST·Tat
undergo 0.8 and 1.6 cell population doublings, respectively. Cell
proliferation was expressed as percent of the cell number measured in
PPS-untreated cell cultures. Each point is the mean ± S.E. of
three determinations in duplicate.
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Fig. 6.
PPS inhibits Tat-induced angiogenesis in the
chick embryo CAM. A, CAMs were implanted with gelatin
sponges adsorbed with vehicle in the absence (a) or presence
(b) of PPS (25 µg), with 400 ng of GST protein
(c), or with 400 ng of GST·Tat in the absence
(d) or presence (e) of PPS (25 µg).
B, CAMs were implanted with gelatin sponges adsorbed with
vehicle (a) or with 6 × 104 T53 cells in
the absence (b) or presence (c) of PPS (25 µg).
Angiogenic response was graded on an arbitrary scale of 0-4+, with 0 representing no angiogenic response and 4+ representing the strongest
activity. Each bar represents the mean ± S.E. of two
independent experiments. C, representative CAMs implanted
with gelatin sponges adsorbed with 6 × 104 parental
T53 cells in the absence (a) or presence (b) of
PPS were fixed by a 2-h incubation with Bouin fluid, removed from the
egg, and placed on a diascope. The images were input via TV camera
mounted onto the stereomicroscope and digitalized by the Image Pro-Plus
analysis system (original magnification × 5).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(SDF-1
) (27), and
RANTES,2 dissociate rapidly
from the GAG.
. In contrast, suramin exerts a nonselective inhibitory effect
at doses 100 times higher than that of PPS (23). Previous observations
had shown that PPS binds also HIV gp120, thus preventing its
interaction with cellular CD4 (19), and inhibits HIV-induced syncytium
formation (37) and lymphocyte-to-epithelial transmission of HIV-1 (38).
Moreover, PPS inhibits reverse transcriptase activity (20). Here we
demonstrate that PPS is a potent extracellular Tat antagonist at doses
significantly lower than those required to inhibit blood coagulation
(21). Accordingly, phase I and II clinical trials have shown that PPS
is well tolerated and may cause stabilization of the disease in
AIDS-related KS patients (39-41). Taken together, the data indicate
that PPS may represent a prototypic molecule for the design of novel
multitarget extracellular Tat antagonists to be used for the treatment
of AIDS and AIDS-associated pathologies. In particular, the potency and
specificity shown by PPS in inhibiting the biological activity of Tat
and FGF-2 may be an advantage for the treatment of AIDS-related KS
where both proteins appear to contribute to the progression of the
disease (42).
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ACKNOWLEDGEMENTS |
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We thank A. Corallini for parental T53 cells and B. Musulin and A. Bugatti for expert technical assistance.
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FOOTNOTES |
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* This work was supported in part by Grant QLK3-CT-1999-00536 from the European Community, Istituto Superiore di Sanità (AIDS Project), Associazione Italiana per la Ricerca sul Cancro, National Research Council (Target Project on Biotechnology) (to M. P.), from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica 2000 (to M. P. and M. R.), and from the Agence Nationale de la recherche sur le SIDA (to H. L-J.).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.
Recipient of a fellowship from the AIDS Project.
§§ To whom correspondence should be addressed: Dept. of Biomedical Sciences and Biotechnology, Via Valsabbina 19, 25123 Brescia, Italy. E-mail: presta@med.unibs.it.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M010779200
2 H. Lortat-Jacob, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CAT, chloramphenicol acetyltransferase; CAM, chick embryo chorioallantoic membrane; DMEM, Dulbecco's modified minimal essential medium; FCS, fetal calf serum; FGF-2, fibroblast growth factor-2; GAG, glycosaminoglycan; GFP, green fluorescent protein; GST, glutathione S-transferase; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; LTR, long terminal repeats; KS, Kaposi's sarcoma; PPS, pentosan polysulfate; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; RU, resonance units; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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