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IMMUNOBIOLOGY
From the Ottawa Hospital Research Institute, University
of Ottawa; and the Division of Infectious Diseases, Ottawa
Hospital-General Campus, Ottawa, ON, Canada.
Treatment of cells with the HIV drugs ritonavir, saquinavir, or
nelfinavir (Nfv) inhibits apoptosis induced by a variety of stimuli. Because these drugs are protease inhibitors, they have been
postulated to inhibit apoptosis by blocking caspase activity. This
study shows that Nfv has no effect on caspase activity or on the
transcription or synthesis of a variety of apoptosis regulatory molecules. Instead, Nfv inhibits mitochondrial transmembrane potential loss ( Current therapies used in the treatment of
HIV-infected patients include combinations of inhibitors of reverse
transcriptase and HIV protease. Multidrug therapy has reduced morbidity
and mortality among patients infected with HIV1-3 and has
led to quantitative and qualitative improvements in host immune
function.4-8 Although increases in CD4 T-cell counts
typically occur in parallel with a reduction in viral replication,
these effects may be seen before significant changes in plasma RNA
levels9 or in the absence of antiviral
effect.10-13 These and other findings suggest that the
protease inhibitor (PI) class of drugs may influence T-cell turnover in
a manner that is independent of their role in viral suppression.
Nonviral effects of HIV PIs have been evaluated recently. PIs influence
proteasome activity, antigen presentation, cytotoxic T-lymphocyte
activity,14 lipoprotein metabolism,15
adipocyte differentiation,16,17 cytochrome P450
activity,18 and adenosine triphosphate (ATP)-dependent
drug export mechanisms.19 Furthermore, PIs may affect the
ability of cells to undergo both spontaneous apoptosis and apoptosis
induced by a variety of stimuli.9,20-23 These data are
consistent with the in vivo observation that apoptosis in both
peripheral blood24 and in lymphatic tissues25
is rapidly decreased following initiation of PI-based therapy. In this
study, we have evaluated the mechanism by which PIs inhibit apoptosis.
Cells
Drugs
Induction and measurement of apoptosis and mitochondrial membrane permeability changes Jurkat or H9 cells were cultured in the presence of AZT, Nfv, or diluent at indicated concentrations for periods of time ranging from 1 to 5 days before induction of apoptosis. Apoptosis was induced with actinomycin D (ActD; 10 µg/mL; Sigma, Oakville, ON, Canada), camptothecin (CPT; 10 µM; Sigma), an agonistic anti-Fas antibody (CH11; 0.5 µg/mL; Beckman Coulter, Mississauga, ON, Canada), or recombinant synthetic Vpr peptide. For Vpr (Genemed Systems, San Francisco, CA) stimulation, cells were incubated in isotonic buffer with 2.5 µM synthetic Vpr peptide (residues 52-96) for 30 minutes, followed by culture in complete media. After treatment, apoptosis was measured by annexin-V and propidium iodide staining26; mitochondrial membrane permeability changes (  m) were
determined with the use of 3,3'-dihexyloxacarbocyanine iodide
(DiOC6; 40 mM; Molecular Probes, Eugene,
OR).27 Samples were analyzed using an Epics ELITE flow
cytometer (Beckman Coulter, Missisauga, ON, Canada), and 10 000 events
were acquired for each sample. Where indicated, protein synthesis was
inhibited by a 1-hour pretreatment with cycloheximide (CHX; 10 µg/mL;
Sigma). Protein synthesis was evaluated by biosynthetic labeling with
35S-labeled methionine and cysteine. 35S
incorporation was measured with the 1450 microbeta liquid scintillation counter (Wallac, Turku, Finland).
Ribonuclease protection assays Total RNA was isolated with an RNeasy mini kit (Qiagen, Mississauga, ON, Canada) from cells treated with methanol, AZT, or Nfv for 3 days and evaluated by ribonucleotide protection assay (RPA; Pharmingen, Mississauga, ON, Canada). We probed 40 µg RNA with the use of 2 custom templates containing probes for caspase-3, -8, and -9; DCR-1; death receptor 4 (DR-4) and -5; BID; Bfl-1; tumor necrosis factor (TNF)-related apoptosis-inducing factor (TRAIL); TNF receptor (TNFRp55); X-linked inhibitor of apoptosis protein (XIAP); neuronal apoptosis inhibitory protein (NAIP); FAS-associated phosphatase (FAP); cellular inhibitor of apoptosis protein 1 (cIAP-1) and -2; Bcl-2; BclXL; and BclXS. Following gel resolution, band intensities were quantified using the ImageQuant software and normalized to the band intensity of glyceraldehyde-3-phosphate dehydrogenase.Immunoblotting Following indicated treatments, protein expression of pro-caspases-3 and -8 was assessed by washing cells in cold phosphate-buffered saline (PBS) and lysing them in buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, leupeptin, and pepstatin, 1 mM Na3VO4, 1 mM NaF) before determining the protein concentration (Pierce, Rockford, IL). Subsequently, 40 µg of protein was subjected to 4% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Millipore, Mississauga, ON, Canada). Membranes were blocked overnight with 5% nonfat dry milk powder in TBST (50 mM Tris-HCl, pH 7.4,150 mM NaCl, 0.1% Tween 20) and immunoblotted for 1 hour at room temperature using antibodies specific for caspase-3 (1:1000; Transduction Laboratories, Lexington, KY) or caspase-8 (1:15; gift from Dr P. Krammer).28 Membranes were washed 5 times with TBST for 5 minutes and exposed to various dilutions of anti-IgG-horseradish peroxidase conjugate (Amersham, Pharmacia Biotech, Oakville, ON, Canada) for 1 hour at room temperature. Proteins were visualized by enhanced chemiluminescence (Amersham, Pharmacia Biotech). The loading and transfer of equal amounts of protein were confirmed by blotting with an anti-proliferating cell nuclear antigen (PCNA) antibody (PC-10, 1:1000; Santa-Cruz Biotechnologies, Santa Cruz, CA).Cytochrome-c release assay Jurkat cells were treated overnight with methanol, AZT, or Nfv before being stimulated to undergo apoptosis with CPT. After induction of apoptosis, cells were washed twice with ice-cold PBS, pH 7.4, and pelleted by centrifugation at 200g for 5 minutes. The pellet was resuspended in caspase buffer (100 mM HEPES, pH 7.5, 250 mM sucrose [wt/vol], 0.5 mM EDTA, and 10 mM dithiothreitol [DTT] and PIs) before being lysed in a dounce homogenizer (Kontes Glass, Vineland, NJ). Cytosolic and mitochondrial fractions were separated by centrifugation at 14 000g for 15 minutes29 and blotted for cytochrome c (1:500; Pharmingen). To ensure that the cytosolic fraction contained only cytochrome c that was released from mitochondria, we blotted both cytosolic and mitochondrial fractions for the mitochondrial protein Hsp 70.30Determination of caspase-1, -3, -6, -7, and -8 activity The effect of Nfv on the activity of recombinant active caspase-1, -3, -6, and -8 (Biomol, Plymouth Meeting, PA) was assayed with the use of 7-amino-4-trifluoromethyl coumarin (AFC)-conjugated substrates, and the activity of recombinant active caspase-7 was assessed using MCA (7-methoxy coumarin-4 acetyl)-conjugated substrate. Nfv, methanol alone, or caspase inhibitor (100 µm zVAD-FMK for caspase-1, -6, and -7; 100 µm DEVD-FMK for caspase-3; and 100 µm IETD-FMK for caspase-8; Enzyme System Products, Livermore, CA) was added to caspase buffer containing 50 U recombinant enzyme and fluorogenic caspase-specific substrate (WEHD-AFC for caspase-1, DEVD-AFC for caspase-3, VEID-AFC for caspase-6, IETD-AFC for caspase-8, Enzyme System Products; and MCA-VDQVDGW[K-DNP] for caspase-7, Calbiochem, San Diego, CA). Caspase activity was determined after excitation at 400 nm or 325 nm by measuring the fluorescence emission at 505 nm or 392 nm for AFC or DNP conjugates, respectively, using a Cytofluor 2300 Series (Millipore). Results are shown as relative fluorescence relative to controls. As a control for Nfv activity, the ability of Nfv to inhibit cleavage of a fluorogenic gag-pol substrate by HIV protease was also assayed. Recombinant HIV-1 protease (50 U; Bachem, King of Prussia, PA) was added to protease buffer (50 mM NaOAc, pH 4.9, 200 mM NaCl, 5 mM DTT, and 10% glycerol) containing the gag-pol substrate and either Nfv or methanol.Isolation of Jurkat T-cell mitochondria Mitochondria from Jurkat T cells were isolated as described previously.31,32 Briefly, Jurkat T cells were resuspended in isolation buffer (0.32 M sucrose, 1.0 mM EDTA, 10 mM Tris-HCl, pH 7.4) and homogenized by hand in a glass dounce homogenizer using 4 up-and-down strokes with the A pestle (0.12 mm clearance) and 8 strokes with the B pestle (0.05 mm clearance). Homogenates were centrifuged at 4000g for 3 minutes, and supernatants were poured into a clean centrifuge tube. The pellet was resuspended in isolation buffer and spun at 4000g for 3 minutes. Supernatants were combined and spun at 16 000g for 10 minutes. The pellet was resuspended in 15% Percoll and layered on a discontinuous density gradient (bottom 40%, middle 23%). Tubes were centrifuged at 19 000g for 5 minutes, and the material at the interface of the lower 2 Percoll layers was diluted 1:4 with isolation buffer and centrifuged at 14 000g for 10 minutes. The pellet was then resuspended in incubation buffer (100 mM KCl, 75 mM mannitol, 25 mM sucrose, 5 mM phosphate [adjusted to pH 7.4 with Tris], 0.05 mM EDTA, and 10 mM Tris-HCl, pH 7.4). All steps were performed at 4°C or on ice.Statistics For comparisons of apoptosis between treatment groups, Student t tests were performed. Data presented represent means of experiments with SDs.
Nfv inhibits T-cell apoptosis in a time- and dose-dependent manner We and others have previously determined that both ritonavir9,20-23 and saquinavir9 have potent antiapoptotic effects in different cellular systems. To test whether these are a generalized effect of this class of drugs, we assessed the antiapoptotic effects of Nfv, an HIV PI whose chemical structure differs from those of ritonavir and saquinavir.33-35 Jurkat T cells were incubated with methanol (vehicle control), AZT (non-PI antiviral drug control), ritonavir, saquinavir, or Nfv for 3 days at doses up to 10 µM, and cells were stimulated to undergo apoptosis through Fas receptor ligation. All 3 PIs tested demonstrated a dose-dependent inhibition of apoptosis (data not shown), indicating that the antiapoptotic effects of PIs are common to these members of the PI class.Next we analyzed the effect of Nfv34,35 on apoptosis
induced by a variety of other stimuli. Jurkat T cells were pretreated with methanol, AZT, or Nfv, and the percentage of annexin-V-positive, propidium iodide-negative cells was determined daily for 5 days in
aliquots of treated cells that were stimulated to undergo apoptosis with ActD, CPT, or an agonistic anti-Fas antibody (clone CH11). Inhibition of CPT-induced apoptosis by Nfv was observed as early as day
1, whereas methanol or AZT had no effect (Figure
1A). Similar results were seen with ActD
and CH11 (data not shown). Strikingly, resistance to apoptosis was seen
within 1 hour of treatment with Nfv (Figure 1B) at a minimal dose of
3.5 µM, and concentrations of 7 µM and 10 µM caused significant
inhibition of apoptosis (Figure 1C). Nfv concentrations of 7 µM are
similar to trough levels of Nfv achieved in patients receiving
treatment36,37; consequently, we chose to use this dose
for the remainder of our experiments.
Nfv inhibition of apoptosis is independent of protein and RNA synthesis Inhibition of apoptosis within 1 hour of Nfv treatment suggests that it acts through a mechanism that is independent of transcription or protein synthesis. To confirm this, we performed 3 different sets of experiments. First, we determined a dose of CHX (10 µg/mL) that inhibits protein synthesis in Jurkat T cells (35S incorporation in Nfv-treated cells without CHX 125 554 CPM, 35S incorporation in Nfv-treated cells with 10 fg/mL CHX 64 777 CPM; 51% of control). Cells coincubated with CHX and Nfv showed the same degree of apoptosis inhibition as cells incubated with Nfv alone (Figure 2A), demonstrating that Nfv inhibition of apoptosis is independent of protein synthesis. Next, we assessed 2 caspase family members, pro-caspase-8 (full length 55 kd) and pro-caspase-3 (full length 32 kd), which are necessary for apoptosis after Fas ligation. As shown in Figure 2B, pro-caspase-8 and pro-caspase-3 levels were not altered after incubation with Nfv compared with control cells. Finally, RPAs failed to show differences at the mRNA level for a cross-section of antiapoptotic proteins and proapoptotic proteins in cells pretreated with methanol, AZT, or Nfv for 3 days (Figure 2C). Data from 3 independent experiments (groups 1, 2, 3) failed to show differences between treatments in the amount of mRNA present for caspase-3, -8, or -9; DCR-1; DR-4 or -5; BID; Bfl-1; TRAIL; TNFRp55; XIAP; NAIP; FAP; cIAP-1 or -2; Bcl-2; BclXL; or BclXS.
Nfv does not inhibit caspase activity Because PIs inhibit the proteolytic activity of the viral protease, it has been postulated that they might have a similar effect on cellular proteases.20,21 Because caspases (cysteine-specific aspartyl proteases) are key regulators of the apoptotic cascade, we chose to assess whether Nfv could inhibit caspase activity. Recombinant active caspase-1, -3, -6, -7, or -8 was incubated in the presence or absence of Nfv and fluorogenic tetrapeptide substrates specific for each respective caspase. Following cleavage of the fluorogenic substrate by active caspases, fluorescence is released, allowing assessment of caspase activity as a function of time. As shown in Figure 2D, Nfv did not inhibit caspase-3 activity (top panel); similarly, Nfv had no effect on the activity of caspase-1, -6, -7, or -8 (data not shown). To confirm the activity of Nfv, we demonstrated that Nfv inhibited HIV protease cleavage of a fluorogenic gag/pol consensus site (bottom panel).Nfv blocks loss of mitochondrial transmembrane potential Mitochondrial transmembrane potential loss (m)
is seen in many forms of apoptosis and serves to link activation of
early initiator caspases with activation of downstream effector
proteases and nucleases. We therefore analyzed whether Nfv-induced
inhibition of apoptosis occurs above or below the level of
mitochondrial permeability changes. Jurkat T cells were incubated in
the presence of methanol, AZT, or Nfv and stimulated to undergo
apoptosis with ActD, CPT, or CH11. Apoptosis was determined using
annexin-V and propidium iodide, and m was assessed
using the cationic lipophilic dye DiOC6.27,38
DiOC6 stains mitochondria with intact mitochondrial transmembrane potential, whereas mitochondria that have lost
transmembrane potential lose their ability to retain the dye
(DiOC6 low). In Nfv-treated samples, the apoptotic fraction
(annexin-V positive, propidium iodide negative) had a decreased
proportion of DiOC6-low cells (Figure
3A,B), indicating that Nfv-treated cells
that became apoptotic had less loss of transmembrane potential than
untreated cells. Together our data suggest that Nfv may inhibit
apoptosis by virtue of inhibiting mitochondrial
m.
Nfv inhibits the release of apoptotic mediators from mitochondria To determine whether Nfv affects the mitochondrial events of apoptosis, we examined cytochrome-c release in cells induced to undergo apoptosis, with or without prior Nfv treatment. CPT induces apoptosis via a direct effect on mitochondria, resulting in permeability transition pore complex (PTPC) opening, loss ofm,
and subsequent release of cytochrome c and apoptosis-inducing factor
(AIF), resulting in postmitochondrial activation of caspase-9, -3, and -7.39-41 Cytosolic and mitochondrial fractions of
Jurkat T cells stimulated with CPT were blotted for cytochrome c.
Nfv-pretreated cells demonstrated a decrease in cytochrome-c release
from the mitochondria as compared with control cells (Figure 3C,
bottom). This suggests that Nfv inhibits apoptosis at or above the
level of loss of mitochondrial m.
Nfv inhibits mitochondrial PTPC opening To confirm the postulate that Nfv inhibits apoptosis at the mitochondrial level, we analyzed the effects of Nfv on apoptosis induced by factors that directly open the mitochondrial PTPC. The PTPC is a multiprotein channel that is believed to consist of a voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), cyclophilin D, and peripheral benzodiazepine receptor.38,42 PTPC opening results in loss of mitochondrialm and the release of cytochrome c into
the cytosol.38 Cytochrome c, together with Apaf-1 and in
the presence of dATP, forms the apoptosome,43 which
initiates apoptotic signaling downstream of the mitochondria by
mediating autocatalysis of pro-caspase-9.44 The HIV
accessory molecule Vpr, when added to intact cells or isolated
mitochondria, causes m and consequent release of
cytochrome c and AIF,45 possibly mediated by binding of
Vpr to VDAC or ANT. These effects of Vpr are inhibited by both Bcl and
bongkrekic acid (BA), which is a ligand for ANT and inhibits PTPC
opening.46
To determine whether Nfv inhibits PTPC opening, we incubated intact
Jurkat cells with recombinant Vpr (peptides 52-96) in the presence or
absence of Nfv. If Nfv functions to prevent PTPC opening, it should
block Vpr-induced cell death. Wild-type Vpr at a concentration of 2 µM induced significant apoptosis that was inhibited by Nfv (Figure
4A). Furthermore, Nfv treatment resulted in a decrease in the percentage of cells that exhibited a
DiOC6-low phenotype (Figure 4B), indicating that Nfv
inhibited loss of mitochondrial transmembrane potential in these cells.
Western blot analyses confirmed that cells coincubated with Vpr and Nfv
had less release of cytochrome c and less pro-caspase-8 cleavage than
cells treated with Vpr alone (Figure 4C).
To characterize further the effect of Nfv on mitochondrial PTPC
opening, we induced PTPC opening in isolated mitochondria. Atractyloside (ATR) interacts with ANT and directly causes loss of
Effect of Nfv on type-I Fas signaling If PIs block apoptosis through inhibition of PTPC opening, then apoptosis that does not depend on mitochondrial activation should be unaffected by PI pretreatment. Fas receptor ligation may activate one of 2 different pathways that lead to apoptosis.47-49 Cells with a type-II Fas signaling pathway recruit Fas-associated death domain (FADD) to the trimerized Fas receptor, causing caspase-8 activation and cleavage of BID, which in turn leads to PTPC opening and loss of mitochondrialm. This results in
release of cytochrome c; activation of caspase-9, -7, and -3; and
finally DNA fragmentation and cell death.39-41 By
contrast, in cells with a type-I Fas signaling pathway, caspase-8
activation immediately causes caspase-3 activation, thereby bypassing
the requirement for loss of mitochondrial m. Jurkat T
cells use a type-II pathway in response to Fas receptor ligation,
whereas H9 cells use a type-I signal transduction
pathway.47-49 We therefore evaluated the effect of Nfv on
H9 cells treated with either Fas, which induces apoptosis independent
of changes in mitochondrial m, or with CPT, which
induces apoptosis via loss of mitochondrial
m   50 (Figure
5). Whereas Nfv-pretreated H9 cells
underwent the same amount of apoptosis as control cells in response to
Fas ligation, Nfv inhibited CPT-induced apoptosis in H9 cells. Thus,
Nfv inhibits apoptotic pathways that require mitochondrial PTPC
opening, but does not inhibit pathways that are independent of
mitochondria.
HIV principally infects CD4+ lymphocytes and cells of the monocyte/macrophage lineage.51,52 The genome of HIV codes for several propeptides that must undergo posttranslational proteolytic cleavage to yield functional proteins. This is accomplished by a virally encoded aspartyl protease. Inhibition of HIV protease blocks this process and, consequently, has become a major therapeutic strategy. Several factors make HIV protease an ideal therapeutic target, including the observation that structural and enzymatic proteins generated after proteolytic cleavage of the propeptides are necessary for viral maturation and infectivity53 and that HIV protease is not present in uninfected cells.54 Also, current HIV PIs demonstrate specificity and do not significantly inhibit the human aspartyl proteases pepsin, renin, gastricin, and cathepsins E and D.55 This study demonstrates that the HIV PI Nfv inhibits Jurkat T-cell apoptosis induced by a variety of different stimuli, including ActD (a chemotherapeutic agent),56 CPT (a topoisomerase I inhibitor),50 and an agonistic anti-Fas antibody (clone CH11).57 We further show that inhibition of apoptotic death is not related to alterations at the mRNA expression level of a variety of pro- and antiapoptotic factors, as determined by RPA, and does not depend on protein synthesis. Whereas others have reported that PIs may affect the generation of active caspase-1 (ICE) or caspase-3 (CPP32),20,21 our experiments show that Nfv has no effect on the ability of recombinant caspase-1, -3, -6, -7, and -8 to cleave their tetrapeptide consensus cleavage sequences. The mitochondrial transmembrane potential that results from the
asymmetric distribution of ions on both sides of the inner mitochondrial membrane is normally maintained by the mitochondrial membrane PTPC.58,59 Soon after receiving an apoptotic
signal, the mitochondrial PTPC opens, disrupting transmembrane
potential46,60-62 and releasing apoptogenic factors
including cytochrome c,63 pro-caspase-9,64
and AIF.46,65 We analyzed Nfv-treated Jurkat cells
following CPT induction of apoptosis and monitored To confirm that Nfv inhibits apoptosis by influencing mitochondria, we next assessed the effects of Nfv on Vpr-induced apoptosis. Vpr is cell permeable and directly interacts with the PTPC, resulting in its opening and the subsequent release of apoptogenic factors45 from mitochondria. Vpr-induced apoptosis was inhibited by Nfv, therefore supporting the hypothesis that Nfv inhibits apoptosis by preventing PTPC opening. Indeed, these data are further confirmed by experiments using isolated mitochondria, in which Nfv inhibited PTPC opening induced by both ATR and Vpr, to degrees greater than that seen by BA, a known inhibitor of PTPC opening. Finally, we treated H9 cells with apoptotic stimuli that either require (CPT) or bypass (Fas ligation) mitochondrial PTPC opening. Whereas Nfv inhibited CPT-induced apoptosis in H9 cells, it did not inhibit apoptosis induced by Fas ligation, therefore confirming that Nfv inhibits apoptosis via an effect on mitochondrial PTPC opening. The clinical significance of antiapoptotic effects of PIs remains to be fully defined; however, both the antiapoptotic effect on CD4+ T cells9,20-23 and putative antiapoptotic effects on CD34+ progenitor cells22 may serve to increase CD4+ cell number in HIV-infected patients independent of the drug's effects on viral replication. Whether this effect is significant in vivo has been disputed,66 yet it is supported by several distinct lines of evidence: (1) enhanced CD4+ T-cell levels with PI-containing versus -sparing regimens that achieve equivalent suppression of viral replication,10,67 (2) increases in CD4+ T-cell number despite no observable antiviral effect,11-13 (3) greater increases in CD8+ T-cell number after initiation of PI therapy than non-PI therapy,68 and (4) dramatic decreases in CD4+ T-cell number after discontinuation of PI-containing therapy, even when PI therapies were unsuccessful in achieving viral suppression.11,69 Together with in vitro observations of antiapoptotic effects, these observations argue in favor of a clinically significant effect of PI-containing therapy on CD4+ T-cell number as compared with PI-sparing therapies. However, until results from ongoing prospective trials of PI-containing versus -sparing therapies of equally high potency are available, these effects remain speculative. Our cumulative data are consistent with previous reports9,20-23 in which Nfv as well as ritonavir and saquinavir have been shown to inhibit apoptosis; however, in contrast to previous reports suggesting that HIV PIs inhibit the enzymatic activity of caspases,20,21 we demonstrate that Nfv inhibits apoptosis via an effect on mitochondrial PTPC opening. These results have implications not only for the care of patients infected with HIV, but potentially for the therapy of other diseases associated with enhanced levels of apoptosis.
We gratefully acknowledge the expert administrative assistance of Ms A. Carisse, as well as helpful discussions and careful manuscript review by Dr D. Lynch, Dr R. Ogden, Dr B. Badley, and the entire Badley lab. B.N.P. and J.J.L. have received Studentship Awards from the OHTN.
Submitted January 26, 2001; accepted April 4, 2001.
Supported by the Medical Research Council of Canada, the Canadian Foundation for AIDS Research (CANFAR), the AIDS Program Committee of Ontario, and the Doris Duke Foundation (A.D.B.). A.D.B. has received a Career Scientist Award from the Ontario HIV Treatment Network (OHTN).
A.D.B. has received consultant fees from Agouron, Roche, and Abbott and unrestricted educational grants from Abbott and Agouron for studies unrelated to this paper.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Andrew D. Badley, Division of Infectious Diseases, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada; e-mail: abadley{at}ottawahospital.on.ca.
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Top
Abstract
Introduction
), 10 µM
AZT (non-PI antiviral control; 
), or 7 µM Nfv (
). Apoptosis was
induced with 10 µM CPT every day for 5 days and assessed after
annexin-V and propidium iodide staining by flow cytometry. Apoptosis
was significantly inhibited by Nfv as early as day 1 (P = .01). (Day 2, P = .04; day 3, P = .005; day 4, P = .001; day 5, P = .03; n = 3.) Similar results were also obtained
after stimulation with ActD (10 µg/mL) and CH11 (0.5 µg/mL) (data
not shown). (B) Jurkat cells were treated with MeOH (
), 7 µM Nfv (
),
or MeOH (
), and the kinetics were assessed over 30 minutes (top
panel). Similar results were also obtained with active human
recombinant caspase-1, -6, -7, and -8 (data not shown). Data are
representative of at least 3 independent experiments for each caspase
tested. HIV-1 protease cleavage of gag/pol fluorogenic
substrate was assessed alone (
) before being stimulated with 2.5 µM Vpr or 5 mM ATR. Loss of 