From the Departments of Pathology,
§ Medicine, and ¶ Microbiology and Immunology, State
University of New York Health Science Center, College of Medicine,
Syracuse, New York 13210
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ABSTRACT |
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Dysregulated apoptosis may underlie the etiology of T cell depletion by human immunodeficiency virus type 1 (HIV-1). We show that HIV-induced apoptosis is preceded by an exponential increase in reactive oxygen intermediates (ROIs) produced in mitochondria. This leads to caspase-3 activation, phosphatidylserine (PS) externalization, and GSH depletion. Since mitochondrial ROI levels are regulated by the supply of NADPH from the pentose phosphate pathway (PPP), the effect of transaldolase (TAL), a key enzyme of PPP, was investigated. Jurkat and H9 human CD4+ T cells were transfected with TAL expression vectors oriented in the sense or antisense direction. TAL overexpression down-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. Alternatively, decreased TAL expression up-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. HIV-induced 1) mitochondrial ROI production, 2) caspase-3 activation, 3) proteolysis of poly(ADP-ribose) polymerase, and 4) PS externalization were accelerated in cells overexpressing TAL. In contrast, suppression of TAL abrogated these four activities. Thus, susceptibility to HIV-induced apoptosis can be regulated by TAL through controlling the balance between mitochondrial ROI production and the metabolic supply of reducing equivalents by the PPP. The dominant effect of TAL expression on oxidative stress, caspase activation, PS externalization, and cell death suggests that this balance plays a pivotal role in HIV-induced apoptosis.
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INTRODUCTION |
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The ability of the cell to undergo or resist apoptosis, a form of programmed cell death (PCD)1 in response to viral infection is crucial in determining the outcome of virus infection at both the cellular and organismal levels (1). Recent studies have suggested that increased apoptosis may underlie the etiology of depletion of lymphocytes, primarily of the CD4+ T cell subset, during infection by HIV-1 (2). Various mechanisms that are not mutually exclusive have been proposed to trigger the decline of CD4+ T cells. While HIV infection was previously associated with decreased GSH levels (3), involvement of ROI production in diminishing intracellular GSH has not been directly addressed. Decline of GSH levels in HIV-infected cells was associated with cysteine deficiency, which served as a rationale for treatment with N-acetylcysteine (4). More recently, decreased GSH was correlated with increased levels of GSSG in HIV-infected CD4+ T cells, suggesting that a lack of reducing equivalents rather than decreased GSSG synthesis is responsible for GSH deficiency (5, 6). These data may explain the failure of N-acetylcysteine treatment to increase GSH in lymphocytes of patients with AIDS (7).
Signaling through the APO-1/Fas/CD95 antigen (8) and the structurally
related cell surface receptor for tumor necrosis factor (TNF) (9, 10)
has been implicated in HIV-induced apoptosis. Deletion studies of
the cytoplasmic death domains shared by Fas and the type I TNF receptor
(11, 12) suggested that both receptors may mediate cell death via a
similar mechanism, which involves the assembly of Fas and TNF
receptor-associated death-inducing signaling complex with
interleukin-1-converting enzyme/caspase-1-like activity (13-17).
Activation of interleukin-1
-converting enzyme and related cysteine
proteases results in the proteolysis of several cellular substrates,
which in turn leads to the characteristic morphologic and biochemical
changes of apoptosis (16). However, HIV-induced PCD was found to be
decreased by cysteine protease/caspase inhibitors but not by
Fas/CD95/TNF receptor-1 antagonists (18, 19). Activation of the
apoptotic protease cascade is dependent on cytochrome c,
which is normally present exclusively in mitochondria (20). Production
of ROIs was also demonstrated in TNF (21-23) and Fas-mediated cell
death (24, 25) and associated with a disruption of the mitochondrial
membrane potential (25-27). Thus, changes in mitochondrial membrane
integrity leading to cytochrome c release appear to be the
point of no return in the effector phase of PCD (20). Mitochondrial
membrane permeability is subject to regulation by an
oxidation-reduction equilibrium of ROIs, pyridine nucleotides (NADH/NAD
plus NADPH/NADP), and GSH levels (28). bcl-2, the prototype
of a novel family of protooncogenes, has antioxidant behavior (29, 30)
and inhibits apoptosis by interference with the release of cytochrome
c from mitochondria (31, 32).
ROIs have long been considered as toxic by-products of aerobic existence; however, evidence is now accumulating that controlled levels of ROIs modulate various aspects of cellular function and are necessary for signal transduction pathways, including those mediating apoptosis (33-39). Because apoptosis and bcl-2 protection were demonstrated in very low oxygen pressure, ROI may not be absolutely required for PCD (40). A cell may normally generate 1011 ROI molecules/day (41). ROI production during apoptosis may be controllable by increased synthesis of reducing equivalents (42). A normal reducing atmosphere, required for cellular integrity, is maintained by GSH, which protects the cell from damage by excess ROIs (43). Synthesis of GSH from its oxidized form, GSSG, depends on NADPH produced by the pentose phosphate pathway (PPP) (43). In fact, a fundamental function of PPP is to maintain glutathione in a reduced state, thereby protecting sulfhydryl groups and cellular integrity from emerging oxygen radicals. The PPP has been recognized as playing a significant role in many developmental and disease processes, including embryogenesis (44), neurulation (45), myelination (46), inflammation (47-50), lymphocyte activation (51, 52), phagocytosis (48, 50), cardiac arrhythmias (53), and resistance to radiation of malignant tumors (54). All of the foregoing processes are associated with PCD (55).
The PPP comprises two separate, oxidative and nonoxidative, phases.
Reactions in the oxidative phase are irreversible, whereas all
reactions of the nonoxidative phase are fully reversible. The two
phases are functionally connected. The nonoxidative phase converts
ribose 5-phosphate to glucose 6-phosphate for utilization by the
oxidative phase and thus, indirectly, contributes to generation of
NADPH. Different enzymes are rate-limiting in the two phases. The
oxidative phase primarily depends on glucose 6-phosphate dehydrogenase (G6PD) (56), whereas transaldolase (TAL) is the rate-limiting enzyme
for the nonoxidative phase (57). TAL catalyzes the transfer of a
3-carbon fragment (corresponding to dihydroxyacetone) to D-glyceraldehyde 3-phosphate, D-erythrose
4-phosphate, and a variety of other acceptor aldehydes (58). TAL
expression and enzymatic activity is regulated in a tissue-specific
(57, 59, 60) and development-specific manner (45). TAL overexpression
lowers G6PD and 6-phosphogluconate dehydrogenase activities and NADPH and GSH levels and renders the cell highly susceptible to apoptosis induced by serum deprivation, hydrogen peroxide, nitric oxide, TNF,
and anti-Fas monoclonal antibody. When TAL levels are reduced, G6PD and
6-phosphogluconate dehydrogenase activities and GSH levels are
increased and apoptosis is inhibited. TAL activity profoundly impacts
the balance between the two branches of PPP and the ultimate output of
NADPH and GSH (24). These findings are consistent with a dominant role
for TAL within the metabolic network that controls the propagation of
biochemical signals (61). This is particularly important because
depletion of CD4+ T cells, immunosuppression, and disease development
in HIV-infected patients are associated with wasting (62, 63) and
abnormal glucose metabolism (64, 65). The present study provides
evidence that levels of TAL expression can determine the extent of
increased mitochondrial ROI production and subsequent caspase
activation, PS externalization, and cell death during HIV infection.
Overexpression of TAL accelerated HIV-induced oxidative stress,
protease activation, PS externalization, and cell death in two human
CD4+ T cell lines. In contrast, suppression of TAL activity abrogated
these effects and blocked HIV-induced PCD. The results show that
increased mitochondrial ROI production is a defining step in
HIV-induced apoptosis and identify TAL as a possible target in the
development of new therapeutics against HIV disease.
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MATERIALS AND METHODS |
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Construction and Transfection of Eukaryotic Expression Vectors-- TAL cDNA clone 4/2-4/1 (58) was inserted into the HpaI site of the metallothionein promoter-driven pMAXRHneo-1 vector (66) following removal of the coding sequence of the p40tax protein of HTLV-I by cleavage with HpaI. Clones containing the TAL cDNA in the sense (pL26-3) and antisense orientation (pL18-3) were selected. 20 µg of plasmid DNA linearized with KpnI were used to stably transfect Jurkat cells by electroporation as described (66). Transfected cells were grown in the presence of G418 (750 µg/ml for Jurkat and 1 mg/ml for H9 cells) and cloned by limiting dilution. Levels of transaldolase expression were measured by enzyme assay and Western blot analyses in the absence and presence of 5 µM CdCl2. Jurkat cell lines, producing increased (L26-3/1 and L26-3/2D1) and suppressed levels of transaldolase (L18-3/1 and L18-3/1D9), were earlier described (24). In comparison with control H9 cells and in the absence of CdCl2, L26/1D3 and L26/3H6 cells displayed higher transaldolase levels, whereas transaldolase activity was decreased by in L18-3/B and L18-3/4C4 cells based on four independent enzyme activity measurements (Table I). Transfection of the pMAXRHneo-1 vector alone had no effect on TAL activity (not shown). While overexpression and suppression were doubled by incubating the cells with 5 µM CdCl2, CdCl2 was not utilized in the following experiments to preclude possible interference with apoptotic pathways (34).
Apoptosis Assays-- Cells were cultured in RPMI 1640 medium, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 international units/ml penicillin, and 100 µg/ml gentamicin at 37 °C in a humidified atmosphere with 5% CO2. 24 h prior to assays, cells were fed with fresh medium and seeded at a density of 2 × 105 cells/ml. CD95/Fas/Apo-1-mediated cell death was induced with 50 or 100 ng/ml anti-Fas monoclonal antibody CH-11 (MBL, Watertown, MA) (67-69). Anti-Fas antibody refers to IgM antibody CH-11, unless indicated otherwise. Anti-Fas monoclonal IgG1 antibody ZB4 was obtained from Upstate Biotechnology (Lake Placid, NY) and utilized to inhibit IgM Fas antibody-induced apoptosis. An anti-human TNF receptor (p80) monoclonal antibody, IgG2b was obtained from Genzyme (Cambridge, MA). Apoptosis was monitored by observing cell shrinkage and nuclear fragmentation and quantified by trypan blue exclusion (69). DNA fragmentation during apoptosis was monitored by agarose gel electrophoresis (24). Apoptosis was also measured by flow cytometry after concurrent staining with fluorescein-conjugated annexin V (annexin V-FITC; R & D Systems, Minneapolis, MN) (FL1) and propidium iodide (FL2) as earlier described (70, 71). Staining with phycoerythrin-conjugated annexin V (annexin V-PE; R & D Systems) was used to monitor PS externalization (FL2) in parallel with measurement of ROI levels by dihydrorhodamine 123 (DHR) fluorescence (FL1; see below). Staining with annexin V alone or in combination with DHR was carried out in 10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2.
Flow Cytometric Analysis of Mitochondrial ROI Production and
Membrane Integrity--
The production of ROIs was estimated
fluorometrically using oxidation-sensitive fluorescent probes
5,6-carboxy-2',7'-dichlorofluorescein-diacetate (DCFH-DA) and DHR
(Molecular Probes, Eugene, OR) as earlier described (24, 72, 73).
Following apoptosis assay, cells were washed three times in 5 mM Hepes-buffered saline, pH 7.4, incubated in Hepes-buffered saline with 0.1 µM DCFH-DA or DHR for 2 min, and samples were analyzed using a Becton Dickinson FACStar Plus
flow cytometer equipped with an argon ion laser delivering 200 megawatts of power at 488 nm. Fluorescence emission from
5,6-carboxy-2',7'-dichlorofluorescein (DCF; green) or DHR (green) was
detected at a wavelength of 530 ± 30 nm. Dead cells and debris
were excluded from the analysis by electronic gating of forward and
side scatter measurements. ROI levels as determined by fluorescence of
control cells served as a base line for assessment of increased ROI
levels in response to HIV infection or Fas stimulation. While R123, the
fluorescent product of DHR oxidation, binds selectively to the inner
mitochondrial membrane, DCF, the fluorescent product of DCFH oxidation,
remains in the cytosol of living cells (73). In agreement with earlier data (24), DHR was significantly more sensitive than DCFH in detecting
early increases in ROI levels in response to Fas stimulation. Upon
infection by HIV-1, DHR fluorescence was enhanced significantly more
than DCF fluorescence. Selective increase of DHR fluorescence indicated
that increased mitochondrial ROI production occurred with maintenance
of mitochondrial transmembrane potential and membrane integrity (73).
DHR fluorescence decreased in annexin V-positive cells with diminished
size possibly reflecting a decline of negative internal mitochondrial
membrane potential and leakage of ROI at later stages of the apoptotic
process. Mitochondrial transmembrane potential (m)
was also estimated by staining with 40-nm 3,3'-dihexyloxacarbocyanine
iodide (DiOC6, Molecular Probes), a cationic lipophilic dye
(25, 74, 75), for 15 min at 37 °C before flow cytometry (excitation,
488 nm; emission, 525 nm recorded in FL1). Fluorescence of
DiOC6 is oxidation-independent and correlates with
mitochondrial transmembrane potential (75). Co-treatment with a
protonophore, 5 µM carbonyl cyanide
m-chlorophenylhydrazone (Sigma), for 15 min at 37 °C
resulted in decreased DHR and DiOC6 fluorescence and served
as a positive control for disruption of mitochondrial transmembrane
potential (75).
Transaldolase and G6PD Activities and Glutathione
Levels--
Transaldolase enzyme activity was tested in the presence
of 3.2 mM D-fructose 6-phosphate, 0.2 mM erythrose 4-phosphate, 0.1 mM NADH, 10 µg
of -glycerophosphate dehydrogenase/triosephosphate isomerase at a
1:6 ratio at room temperature by continuous absorbance reading at 340 nm for 20 min (76). The enzyme assays were conducted in the activity
range of 0.001-0.01 units/ml. G6PD was measured in the presence of 120 mM Tris, pH 8.4, 10 mM MgCl2, 2 mM glucose 6-phosphate, 0.9 mM NADP, and 0.1 unit/ml 6-phosphogluconate dehydrogenase (77). Total glutathione
content was determined by the enzymatic recycling procedure essentially
as described by Tietze (78). 106 cells were resuspended in
100 µl of 4.5% 5-sulfosalicylic acid. The acid-precipitated protein
was pelleted by centrifugation at 4 °C for 10 min at 15,000 × g. The total protein content of each sample was determined
using the Lowry assay (79). GSH content of the aliquot assayed was
determined in comparison with reference curves generated with known
amounts of GSH (24).
Infection with HIV-1--
Jurkat-tat cells were transfected with
HIV-1 DNA clone 4803 (80) by electroporation at 600 microfarads/250
V/72 ohms. Infectious stock of the strain HIV-14803 was
harvested from 24-h supernatants of freshly reinfected Jurkat-tat
cells, and infectious titer was determined by an in situ
infectivity (MAGI) assay (81). Supernatants with titers of 2.1 × 105 infectious units/ml were filtered through a 0.45-µ
filter, and aliquots were stored at 70 °C. Infections were
standardized by incubating 107 Jurkat, Jurkat-tat, or H9
cells with cell-free virus supernatants containing 100 ng of p24 core
protein as measured by an enzyme-linked immunosorbent assay following
the manufacturer's recommendations (NEK-060; DuPont). After virus
infection, cells were washed and resuspended in RPMI 1640 medium
containing 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 2 mM L-glutamine. Noninfected
control cells were cultured under identical conditions. Transmission of
HIV-1 was monitored by production of gag p24. Recombinant HIV-1 gag p24
(82) was utilized as a control antigen. As positive control sera, HIV-1
gag p24-specific polyclonal sheep antibody (83) and a monoclonal
antibody to p24 (84) were utilized. Expression of CD4 antigen was
assessed using mAb SIM.2 (85). Expression of fusin/CXCR4 was monitored
with mAb 12G5 (86). Viral reagents were obtained from the National
Institutes of Health AIDS Research and Reference Program.
Caspase-3/CPP32 Enzyme Assay and Protease Inhibitors--
CPP32
activity was measured by incubating cell extracts in 50 µl of 80 µM DEVD-7-amino-4-trifluoromethyl-coumarin (DEVD-AFC; Calbiochem), 250 mM sucrose, 20 mM HEPES-KOH,
pH 7.5, 50 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol for 15 min at 37 °C (32, 87). Protein
content of cell extracts was determined by the Lowry assay (79).
Fluorescence (400-505 nm) after the addition of 1 ml of ice-cold water
was compared with a standard curve of AFC (Sigma). Specificity of the
enzymatic reaction was tested by using caspase-3/CPP32 inhibitor
DEVD-CHO and caspase-1/interleukin-1-converting enzyme inhibitor
YVAD-CMK (Bachem, King of Prussia, PA) at a concentration range of
50-300 µM (87).
Western Blot Analysis--
40 µg of total cell lysate in 10 µl/well was separated by SDS-polyacrylamide gel electrophoresis and
electroblotted to nitrocellulose (88). For visualization of
poly(ADP-ribose) polymerase (PARP), cells were lysed in 62.5 mM Tris-HCl, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.00125% bromphenol blue, and 5% -mercaptoethanol,
sonicated for 15 s, and boiled for 5 min (89). Nitrocellulose
strips were incubated in 100 mM Tris, pH 7.5, 0.9% NaCl,
0.1% Tween 20, and 5% skim milk with the primary antibodies, HIV-1
gag p24-specific polyclonal sheep antibody (83) or anti-PARP monoclonal
antibody (89), at a 1000-fold dilution at room temperature overnight. After washing, the blots were incubated with biotinylated goat anti-mouse or rabbit anti-sheep IgG and subsequently with horseradish peroxidase-conjugated avidin (Jackson Laboratories, West Grove, PA).
Between the incubations, the strips were vigorously washed in 0.1%
Tween 20, 100 mM Tris, pH 7.5, and 0.9% NaCl. The blots were developed with a substrate composed of 1 mg/ml 4-chloronaphthol and 0.003% hydrogen peroxide.
Statistics-- Alterations in cell survival, ROI levels, caspase-3 and PPP enzyme activities, and GSH levels were analyzed by Student's t test. Changes were considered significant at p < 0.05.
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RESULTS |
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Production of ROI during HIV-induced Apoptosis-- HIV-induced apoptosis was investigated by infection of Jurkat-tat cells with HIV-1 strain 4803 (80). Jurkat-tat cells are stably transfected with the tat transactivator gene of HIV-1 (90), which increases replication and cytopathicity of HIV-1 (80). Viral infection was monitored by detection of HIV-1 gag p24 protein in Jurkat-tat cell lysates using Western blot analysis (Fig. 1A). In accordance with previous observations (3), HIV-induced apoptosis was associated with a progressive depletion of intracellular GSH levels (Fig. 1B). While HIV infection was previously associated with decreased GSH contents, involvement of ROI production in diminishing intracellular GSH has not been directly addressed. To assess changes in intracellular ROI levels, we used oxidation-sensitive fluorescent probes DHR and DCFH-DA (73, 91). DHR is nonfluorescent, uncharged, and readily taken up by cells, whereas R123, the product of DHR oxidation, is fluorescent, positively charged, and binds selectively to the inner mitochondrial membrane of living cells (73). Fluorescence of this dye is an indicator of mitochondrial ROI production and membrane integrity. DCFH-DA is also readily taken up by cells and, after deacetylation to DCFH, is oxidized to its fluorescent derivative, DCF, and remains in the cytosol (73). We evaluated the rates of increase in fluorescence of cells treated with 50 ng/ml anti-Fas mAb or infected by HIV in comparison with untreated control cell populations. In agreement with earlier data (24), DHR fluorescence was significantly more enhanced than DCF fluorescence in response to Fas stimulation. Upon infection by HIV-1, DHR was more sensitive than DCFH in detecting early increases in ROI production (data not shown). Relative to Fas stimulation, a slower but more robust increase in ROI levels was detected in HIV-infected cells (Fig. 2). Significant increases of ROI levels were observed 2 days after infection with HIV. ROI production as measured by DHR fluorescence continued to increase 5-fold or more over the base line on a logarithmic scale (DHR fluorescence was 7.17 ± 0.9 in control and 64 ± 8.9 in HIV-infected H9 cells (8.9-fold increase); 7.8 ± 0.8 in control and 40.8 ± 4.2 in HIV-infected Jurkat-tat cells (5.2-fold increase); Fig. 2, C and D). ROI levels increased 2 days after (p < 0.001, Fig. 2) while significant depletion of GSH levels occurred 7 days after infection by HIV-1 (p < 0.01, Fig. 1).
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Oxidative Stress Precedes Activation of Caspase-3 and PS Externalization during HIV-induced Apoptosis-- Activation of CPP32 (caspase-3/apopain) was measured in HIV-infected cells using a fluorimetric assay with the substrate DEVD-AFC. In accordance with previous findings (92), maximal DEVD-AFC cleavage activity was observed 2-4 h after stimulation with CH-11 anti-Fas antibody (data not shown). A kinetic analysis of extracts of HIV-1-infected cells revealed a significant increase of CPP32 activity 6-8 days after exposure to virus (p < 0.001; Fig. 2, E and F). Increased caspase-3 activities occurred with a precipitous acceleration of cell death and were delayed with respect to ROI production (Fig. 2, E and F).
PS, which is normally confined to the inner leaflet of the plasma membrane, is exported to the outer plasma membrane leaflet during apoptosis. PS externalization is an early event of PCD that may serve as a flag allowing phagocytes to recognize and engulf these apoptotic cells before they become leaky and rupture (70, 71). To assess the timing of PS externalization with respect to oxidative stress, cells undergoing HIV-induced apoptosis were analyzed by concurrent staining with annexin V-PE and DHR. As shown in Fig. 3, DHR fluorescence increased in annexin V-negative cells, suggesting that HIV-induced oxidative stress occurred before PS externalization both in H9 and Jurkat-tat cells. ROI levels remained elevated in annexin V-positive cells until they underwent apoptotic shrinking as determined by forward angle light scattering as a direct measure of particle size (data not shown). With a precipitous decline of cell viability and size, 7-8 days after HIV infection, DHR fluorescence returned toward base-line levels, in correlation with a decrease in DiOC6 fluorescence (data not shown), possibly reflecting leakage of ROI secondary to damage of mitochondrial and cellular membranes (73-75). Similar to HIV-treated cells, Fas-induced oxidative stress preceded PS externalization. Therefore, as presented in Fig. 2, both Fas and HIV-induced increase of ROI levels occurred in annexin V-negative cells.
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Effect of Fas Antagonists and Inhibitors of Cysteine Proteases on
HIV-induced Apoptosis--
While ROI levels were increased, cell
viability of Jurkat-tat or H9 cells was not significantly affected 2 days after infection with HIV-1. In agreement with previous findings
(8), following stimulation with Fas antibody CH11, cell death was
accelerated in HIV-infected H9 (p < 0.001) or
Jurkat-tat cells (p < 0.001, Fig.
4). We utilized an IgG1 anti-Fas mAb,
ZB4, to block apoptosis triggered by stimulation of the Fas antigen
with IgM mAb CH-11 (93). While anti-Fas antibody ZB4 had no effect on
viability of Jurkat or Jurkat-tat cells, mAb CH-11-induced apoptosis
was dose-dependently abrogated by pretreatment with mAb ZB4
(Fig. 4B). By contrast, HIV-induced death of Jurkat-tat
cells was not inhibited but rather accelerated by mAb ZB4
(p < 0.01, Fig. 4C). Antibody ZB4
had no significant effect on apoptosis of HIV-infected H9 cells. In
correlation with recent findings (18, 19), antibody to the TNF receptor
had no effect on HIV-induced cell death of Jurkat-tat or H9 cells (data
not shown). The addition of anti-Fas ligand (NOK1) antibody also failed
to inhibit HIV-induced apoptosis of Jurkat-tat or H9 cells (Fig.
5, A and B). To
assess the role of cysteine proteases, interleukin-1-converting
enzyme-inhibitor YVAD and CPP32 inhibitor DEVD peptides were added to
HIV-infected cells. While YVAD had no significant influence, DEVD
partially inhibited apoptosis of HIV-infected Jurkat-tat
(p < 0.05, Fig. 5A) and H9 cells
(p < 0.01, Fig. 5B). The
apoptosis-inhibitory effect of DEVD was slightly enhanced by concurrent
NOK1 antibody treatment of Jurkat-tat (p < 0.05) but not of H9 cells, suggesting that Fas-mediated signaling may
contribute to HIV-induced cell death in Jurkat-tat but not in H9 cells.
Delayed apoptosis of DEVD-treated cells was accompanied by a reduction
of oxidative stress in both Jurkat-tat and H9 cells
(p < 0.01, Figs. 5, C and D). The inhibitory effect of DEVD treatment on cell death
and ROI levels did not become apparent until 6 or more days after HIV
infection (Fig. 5). ROI levels of DEVD-treated cells remained significantly elevated, more than 3-fold on a logarithmic scale, as
compared with uninfected control cells (p < 0.001, Fig. 5, C and D).
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HIV-induced Oxidative Stress, Activation of Cysteine Proteases, and
Cell Death Are Regulated by Transaldolase--
Previous studies raised
the possibility that a lack of reducing equivalents, decreased GSH, and
increased GSSG in CD4+ T cells (5, 6) contribute to oxidative stress
and may explain the failure of N-acetylcysteine
supplementation to increase GSH in lymphocytes of patients with AIDS
(7). Synthesis of GSH from its oxidized form, GSSG, is solely dependent
on NADPH produced by the PPP (43). Since the overall NADPH output of
PPP, GSH levels, and sensitivity to apoptosis in response to serum
deprivation, hydrogen peroxide, nitric oxide, TNF, and anti-Fas
monoclonal antibody are regulated by TAL, we investigated the effect of
changes in TAL activity on HIV-induced cell death. Infection of Jurkat L26-3/4 and L26-3/2D1 cells with increased TAL activity and decreased NADPH, NADH, and GSH levels (24) resulted in accelerated cell death and
increased ROI production in comparison with control Jurkat cells (Fig.
6). Along the same line, HIV-induced cell
death and oxidative stress was inhibited in Jurkat L18-3/1 and
L18-3/1D9 cells with decreased TAL activity and increased GSH content
(Fig. 6A). Changes in cell survival and ROI levels were
statistically significant in L26-3/4 and L18-3/1D9 cells
(p < 0.02). Altered susceptibility to
HIV-induced cell death in Jurkat L26-3/4 and L18-3/1D9 cells
suggested that availability of reducing equivalents from the PPP
influences this apoptotic process. In accordance with earlier findings
(80, 90), in comparison with Jurkat cells, cytopathicity of HIV-1 was
significantly accelerated in H9 or Jurkat-tat cells. To assess whether
TAL can influence survival of cells highly susceptible to cytopathicity
of HIV-1, H9 cells were stably transfected with eukaryotic expression
vectors containing full-length transaldolase cDNA in the sense
(pL26-3) or antisense orientation (pL18-3). Several cell lines with
altered TAL enzymatic activities were selected for further studies. In
agreement with earlier observations, increased TAL activities were
associated with diminished intracellular GSH levels in L26/1D3 and
L26/3H6 cells, whereas decreased TAL activities occurred with an
increase of G6PD activities and GSH levels in L18-3/B and L18-3/4C4
cells (Table I). Expression of CD4 (85)
and fusin (86), which mediate binding of HIV to the T cell surface
(94), was not significantly changed in cells with altered TAL activity
levels (data not shown). By day 6 after infection with HIV-1, cell
death was markedly accelerated in TAL-overproducing L26/1D3 and L26/3H6
cells in comparison with control H9 cells (p < 0.001; Fig. 6B). Suppressed TAL activities inhibited cell
death in L18-3/B and L18-3/4C4 cells (p < 0.001; Fig. 6B). Cell survival inversely correlated with ROI
production as regulated by TAL activities. As shown in Fig.
7A, HIV-induced oxidative
stress was accelerated in L26/1D3 and L26/3H6 cells and profoundly
suppressed in L18-3/B and L18-3/4C4 cells, in comparison with control
H9 cells.
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DISCUSSION |
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Despite intensive efforts, the mechanism by which HIV infection depletes CD4+ helper T cells and leads to the development of AIDS is incompletely understood. Recent studies have suggested that dysregulation of apoptosis may underlie the etiology of this T cell depletion (2). Three general mechanisms have been suggested. Observations of decreased intracellular GSH suggested a role for oxidative stress (3); however, production of ROI has not been directly demonstrated. Fas-mediated (8) and TNF-mediated signaling (9, 10) were shown to accelerate HIV-induced PCD. More recently, the inhibitory effect of caspase antagonists suggested that activation of the caspase family of cysteine proteases may mediate apoptosis triggered by HIV (18). The present study provides evidence that HIV-induced apoptosis is accompanied by increased mitochondrial ROI production, activation of caspase-3, and PS externalization. ROI production preceded activation of caspase 3, PS externalization, and depletion of intracellular GSH.
Sensitivity to HIV-induced cell death was effectively controlled by regulating activity of TAL, a key enzyme of the PPP. TAL overexpression in Jurkat or H9 human T cells down-regulated G6PD and 6-phosphogluconate dehydrogenase activities and decreased NADPH, NADH, and GSH levels. Alternatively, decreased TAL expression up-regulated G6PD and 6-phosphogluconate dehydrogenase activities and increased GSH. TAL overexpression increased sensitivity, while suppression of TAL decreased sensitivity of two human CD4+ T cell lines to HIV-induced apoptosis. These findings are consistent with a pivotal role of ROI in HIV-induced apoptosis. Hence, TAL expression has a dominant effect on oxidative stress, protease activation, PS externalization, and, ultimately, cell death, through regulation of the PPP, thereby controlling the levels of intracellular reducing equivalents. Our results are consistent with previous findings that a lack of reducing equivalents rather than decreased GSSG synthesis is responsible for GSH deficiency (5, 6). This may also explain why N-acetylcysteine supplementation fails to increase GSH in lymphocytes of AIDS patients (7).
In the present studies, ROI production was assessed by using oxidation-sensitive fluorescent probes. In living cells, R123, the fluorescent product of DHR oxidation, binds selectively to the inner mitochondrial membrane, whereas DCF, the fluorescent product of DCFH oxidation, remains in the cytosol (73). DHR was significantly more sensitive than DCFH in detecting early increases in ROI levels in response to Fas stimulation or infection by HIV-1, suggesting that HIV-induced ROI production was confined to mitochondria. Mitochondrial ROI production increased exponentially as early as 2 days after HIV infection, i.e. prior to activation of caspases, PS externalization, and a precipitous decline of cell viability. The selective increase of DHR fluorescence indicated that increased mitochondrial ROI production occurred, while mitochondrial transmembrane potential and membrane integrity were maintained (73). DHR and DiOC6 fluorescence decreased in annexin V-positive cells with diminished size, possibly reflecting a decline of negative internal mitochondrial membrane potential and leakage of ROI at later stages of the apoptotic process (73-75). Fas-dependent signaling also triggered an early increase of DHR fluorescence in annexin V-negative cells (24), which was followed by a decline of DHR and DiOC6 fluorescence at later stages of PCD. A similar pattern of early increase in DHR fluorescence was recently suggested as a key event preceding cytochrome c release and membrane depolarization during PCD (27). DHR fluorescence depends on oxidation of its nonfluorescent precursor and its accumulation within a negative inner mitochondrial membrane, both of which processes are subject to regulation by the redox equilibrium of pyridine nucleotides (NADH/NAD plus NADPH/NADP) and of GSH levels (28). Thus, the profound effect of transaldolase on HIV-induced ROI production and apoptosis is consistent with a pivotal role for this enzyme in the PPP and for NADPH and GSH production (24, 61).
Protease activation was involved in HIV-induced apoptosis, as demonstrated by increased caspase-3 proteolytic activity. Partial inhibition of cell death by the CPP32 antagonist peptide DEVD was observed 6 days after infection. Delayed apoptosis of DEVD-treated cells (both Jurkat-tat and H9) was accompanied by a reduction of oxidative stress, suggesting that caspase activation contributes to ROI production and serves as a positive feedback loop at later stages of the apoptotic process. Nevertheless, ROI levels of DEVD-treated cells remained significantly elevated, more than 3-fold (on a log scale), as compared with control uninfected cells (Fig. 5, C and D). Increased ROI production preceded caspase activation. Further, activation of caspase-3 and PARP cleavage was accelerated in cells with increased TAL expression and completely abrogated in cells with suppressed TAL expression. The dominant effect of TAL expression on oxidative stress and caspase activation suggested that provision of reducing equivalents by the PPP is a key factor in the effector phase of HIV-induced PCD.
Signaling through the APO-1/Fas/CD95 antigen (8) and the TNF receptor (9, 10) has been implicated in HIV-induced apoptosis. We also noted that Fas-induced cell death was accelerated in HIV-infected Jurkat-tat or H9 cells, a finding that may be explained by an additive effect of Fas-dependent signaling on caspase activation (13-15) and ROI production (20, 24, 25). HIV-induced oxidative stress may also accelerate Fas-dependent signaling by inducing overexpression of the Fas receptor/CD95 (95, 96) and its ligand (8, 97). Nevertheless, in contrast to Fas-mediated signaling, Fas antagonist antibodies did not inhibit HIV-induced apoptosis. These findings are concordant with recent reports that apoptosis related to HIV infection does not depend on signaling through the Fas/TNF receptor (18, 19). While Fas antagonist IgG antibody ZB4 dose-dependently abrogated PCD triggered by the cross-linking Fas-stimulatory IgM antibody (mAb CH-11) in control Jurkat or Jurkat-tat cells, apoptosis of HIV-infected Jurkat-tat cells was increased rather than inhibited by mAb ZB4. This result may be due to oxidative stress-induced structural changes in functionally important cysteine-rich repeats of the Fas receptor of HIV-infected Jurkat-tat cells (98).
The present results demonstrate that TAL can regulate key biochemical events of apoptosis: mitochondrial ROI formation and activation of caspases as well as the shortened lifespan of HIV-infected T cells. This effect of TAL is consistent with its central position in regulating the balance between the two branches of the PPP (24). This pathway is an essential biochemical mechanism that generates NADPH for the synthesis of GSH, which, in turn, protects cellular integrity from oxygen radicals. The profound effect of TAL activity on apoptotic signaling (24) may be related to an overwhelming influence of TAL-catalyzed dihydroxyacetone transfer reactions on the distribution of flux between the PPP and nucleotide metabolism, which determines overall propagation of biochemical signals in the context of on a metabolic network (61). Reversibility of the TAL reaction has been proposed as a control mechanism for the entire PPP in yeast (99). In cells overexpressing TAL, glucose 6-phosphate is profoundly depleted (24), which may be directly responsible for diminished G6PD activities and GSH levels and increased sensitivity to apoptosis. Alternatively, increased G6PD activities and GSH levels of cells with suppressed TAL activity may result from increased availability of glucose 6-phosphate for generation of NADPH. Interestingly, disease development and depletion of CD4+ T cells in HIV-infected patients was associated with cachexia (62, 63) and abnormal glucose metabolism (64, 65). Infection of H9 cells with HIV is accompanied by increased glucose transporter expression and enhanced glucose uptake (100). Augmented glucose utilization may reflect increased metabolic demand needed to combat oxidative stress in the infected cells. The results clearly indicate that TAL may be a dominant regulator of glucose utilization and propagation of apoptotic signals in HIV-infected T cells. Thus, TAL and other endogenous apoptosis regulators are attractive targets for the development of novel pharmacological agents aimed at abrogating mitochondrial ROI production and caspase activation and prolonging the survival of HIV-infected cells.
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ACKNOWLEDGEMENTS |
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We thank Drs. Anthony Martonosi and Sandy Livnat for helpful discussions and Dr. Paul Phillips for continued encouragement and support.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO1 DK 49221, National Multiple Sclerosis Society Grant RG 2466A1/3, the Arthritis Foundation, and the Central New York Community Foundation.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 and reprint requests should be
addressed: SUNY HSC, 750 E. Adams St., Syracuse, NY 13210. Tel.:
315-464-4192; E-mail: perla{at}vax.cs.hscsyr.edu.
1 The abbreviations used are: PCD, programmed cell death; TAL, transaldolase; G6PD, glucose-6-phosphate dehydrogenase; PPP, pentose phosphate pathway; ROI, reactive oxygen intermediate; HIV, human immunodeficiency virus; TNF, tumor necrosis factor; annexin V-FITC, fluorescein-conjugated annexin V; annexin V-PE, phycoerythrin-conjugated annexin V; PS, phosphatidylserine; DHR, dihydrorhodamine 123; DCFH-DA, 5,6-carboxy-2',7'-dichlorofluorescein-diacetate; DCF, 5,6-carboxy-2',7'-dichlorofluorescein; gag, glycoprotein antigen; DEVD, Asp-Glu-Val-Asp; YVAD, Tyr-Val-Ala-Asp; PARP, poly(ADP-ribose) polymerase; mAb, monoclonal antibody; AFC, 7-amino-4-trifluoromethyl-coumarin.
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