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Introduction
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AIDS caused by HIV-1 involves the apoptotic destruction of lymphocytes (13). The envelope glycoprotein complex (Env) constitutes one of the major apoptosis inducers encoded by the HIV-1. The soluble Env derivative gp120 can induce apoptosis through interactions with suitable surface receptors (3, 4). HIV-1infected cells that express mature Env (the gp120/gp41 complex) on the surface can also kill uninfected cells expressing the receptor (CD4) and the coreceptor (CXCR4 for lymphotropic Env variants, CCR5 for monocytotropic Env variants). This type of bystander killing is obtained by at least two distinct mechanisms. First, the two interacting cells (one that expresses Env and the other that expresses CD4 plus the coreceptor) may not fuse entirely and simply exchange plasma membrane lipids, after a sort of hemi-fusion process, followed by caspase-independent death (5). Second, the two cells can undergo cytoplasmic fusion (cytogamy), thus forming a syncytium, and undergo nuclear fusion (karyogamy) and apoptosis (6). In syncytia, the cyclin B1dependent kinase 1 (Cdk1) is activated, a process that is required for karyogamy and that culminates in the mammalian target of rapamycin (mTOR)mediated phosphorylation of p53 on serine 15. The transcriptional activation of p53 results in the expression of proapoptotic proteins, including Bax and cell death (7, 8). p53 and Bax have also been found to participate in the death of HIV-1infected primary lymphoblasts (9, 10). Both the syncytium-dependent and -independent Env-induced apoptosis are suppressed by inhibitors of the gp41- and gp120-mediated coreceptor interactions (5, 6). Moreover, both are reduced by transfection with the Bax antagonist Bcl-2 (5, 6). In contrast, only the syncytium-dependent cell death can be blocked by inhibitors of Cdk1 (e.g., roscovitine), mTOR (e.g., rapamycin), or p53 (e.g., cyclic pifithrin-
; references 7, 8).
Some of the characteristics of syncytium-induced apoptosis, namely aberrant cyclin B expression (11), overexpression/activation of mTOR (7), phosphorylation of p53 on serine 15 (7, 11), and destabilization of mitochondrial membranes (6), also affect a subset of lymph node and peripheral blood lymphocytes from HIV-1infected donors, correlating with viral load (7, 1113). This suggests that the proapoptotic signal transduction pathway, which can be studied in Env-elicited syncytia, reflects, to some extent, the HIV-1stimulated cell death, as observable among circulating lymphocytes. Based on this premise, we decided to further explore the proapoptotic signal transduction pathway participating in Env-induced syncytium-dependent cell killing. Therefore, we analyzed Env-elicited changes in the transcriptome and established the preponderant role of nuclear factor
B (NF-
B) as well as of p53 as proapoptotic transcription factors in HIV-1induced cell death. Moreover, we identified Puma as a proapoptotic, p53-inducible protein that is critical for in Env-induced apoptosis. The induction of Puma was not restricted to Env-elicited syncytia but was also found in Env-triggered syncytium-independent cell death in vitro. In addition, Puma expression was induced in HIV-1infected patients.
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Materials and Methods
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Cells and Culture Conditions for Apoptosis Induction.
HeLa cells stably transfected with the Env gene of HIV-1 LAI (HeLa Env) and HeLa cells transfected with CD4 (HeLa CD4) were cocultured at a 1:1 ratio (6) in DMEM supplemented with 10% FCS, L-glutamine, in the absence or presence of 1 µM roscovitin, 1 µM rapamycin (Sigma-Aldrich), 10 µM cyclic pifithrin-
, 5 µM MG132 (Calbiochem), the NF-
B inhibitory peptide SN50, or its negative control SN50M (20 µM; BIOMOL Research Laboratories, Inc.). HeLa/U937 cell cocultures were performed at different ratios (6). U937 or Jurkat cells were cultured with 500 ng/ml recombinant gp120 protein.
Plasmids, Transfection, and Transcription Factor Profiling.
For transcription factor profiling, HeLa CD4 cells were transfected with different luciferase constructs (MercuryTM pathway profiling system from CLONTECH Laboratories, Inc.; 2 µg DNA) using Lipofectamine 2000TM (2 µl; Invitrogen) 24 h before fusion with HeLa Env cells (25 x 104 in 2 ml), and the luciferase activity was measured 24 h later using the luciferase reporter assay kit from CLONTECH Laboratories, Inc. Transfection with pcDNA3.1 vector only, WT, dominant-negative (DN) Cdk1 mutant (14), the I
B super-repressor (IKSR; a gift from L. Schmitz, University of Bern, Bern, Switzerland), p53 DN plasmids (p53H273; a gift from T. Soussi, Institut Curie, Paris, France), or a p53-responsive enhanced green fluorescent protein (GFP) plasmid (a gift from K. Wiman, Karolinska Hospital, Stockholm, Sweden) was performed 24 h before coculture of HeLa CD4 and HeLa Env cells. The frequency of GFP-expressing cells was assessed by cytofluorometry on a FACSVantageTM (Becton Dickinson) equipped with a 100-µm nozzle, allowing for the analysis of relatively large cells. Afterwards, cells were counterstained with 10 µg/ml Hoechst 33324 (15 min), and the FACS® gates were set on syncytia (i.e., cells with a DNA content >4 n) (11).
Immunofluorescence and Cytofluorometry.
Rabbit antisera specific for p53S15P, p53S46P (Cell Signaling Technology), or the NH2 terminus of Bax (N20; Santa Cruz Biotechnology, Inc.) were used on paraformaldehyde (4% wt:vol) and picric acidfixed (0.19% vol:vol) cells (15) and revealed with a goat antirabbit IgG conjugated to Alexa 568 (red) or Alexa 488 (green) fluorochromes obtained from Molecular Probes. Cells were also stained for the detection of the NH2 terminus of Bak (Ab1; Oncogene Research Products), p53S15P (mAb), cyclin B1 (mAb; BD Transduction Laboratories), and I
B
S32/36P (mAb; Cell Signaling Technology) revealed by antimouse IgG Alexa conjugates. Cells were counterstained with Hoechst 33324, which allows discernment of karyogamy and apoptotic chromatin condensation (11).
Immunoblots.
Samples were prepared from HeLa Env and HeLa CD4 single cells mixed at a 1:1 ratio in lysis buffer (single cells control) or from HeLa Env/CD4 syncytia. Protein samples from HeLa Env and U937 mixed at different ratios were also prepared and subjected to immunoblot analysis. Aliquots of protein extracts (40 µg) were subjected to immunoblots using antibodies specific for I
B
S32/36P, p53S15P, p53S46P, p53 (Cell Signaling Technology), Puma (rabbit antihuman Puma antisera; United States Biological or Orbigen; and mouse antihuman Puma antibody; Upstate Biotechnology; all gave similar results), ß-tubulin (mAb; Sigma-Aldrich), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, mAb; Chemicon).
Microarrays, Macroarrays, and Quantitative RT-PCR.
HeLa CD4 and HeLa Env cells were labeled with CellTrackerTM red or CellTrackerTM green (15 µM, 30 min at 37°C), respectively, and washed extensively before coculture. After 18 or 36 h of coculture, in the presence or absence of 10 µM cyclic pifithrin-
(readded once upon 24 h), the cells were subjected to FACS® purification of syncytia (which are double positive) or single cells, as described previously (11). mRNA preparations were obtained with the RNeasy Mini kit (QIAGEN), quality-controlled with a bioanalyzer (model 2100; Agilent Technologies), alternatively labeled with Cy3/Cy5 fluorochromes, mixed for pairwise comparisons, and hybridized to 19-k human arrays. Arrays were scanned with a confocal laser scanner (ScanArray model 4000; PerkinElmer), quantified with software (QuantArray; PerkinElmer) at the Ontario Cancer Institute Microarray Center (http://www.microarrays.ca), and analyzed with GeneTraffic software (Iobion Informatics). All experiments were performed in triplicates, while switching the Cy3/Cy5 labeling (six data points for each pair-wise comparison) and up-regulations by 50% or down-regulations by 30% were listed. Data showing the transcriptional modification of genes with unknown function are listed in Table SI (available at http://www.jem.org/cgi/content/full/jem.20031216/DC1). Macroarrays were performed using a TranSignalTM p53 target gene array (Panomics) and quantified using the National Institutes of Health image software. For quantitative RT-PCR, cDNAs were synthesized from 1 µg of total RNA with reverse transcriptase MuLV (Roche). The TaqMan universal PCR was performed on an ABI PRISM® 7000 Sequence Detection System (Applied Biosystems) following the manufacturer's instructions. The following primer sequences were used: bcl-2, forward, 5'-CATGTGTGTGGAGAGCGTCAA-3', reverse, 5'-CAGGTGTGCAGGTGCCG-3'; bax
, forward, 5'-CCAAGGTGCCGGAACTGAT-3', reverse, 5'-AAGTAGGAGAGGAGGCCGTCC-3'; bax ß, forward, 5'-CCAAGGTGCCGGAACTGAT-3', reverse, 5'-GAGGAGGCTTGAGGAGTCTCA-3'; bak, forward, 5'-ACCGACGCKATGACTCAGAGTTC-3', reverse, 5'-ACACGGCACCAATTGATG-3'; and puma (predeveloped Taqman Assays Reagents; www.appliedbiosystems.com).
Antisense Constructs and RNA Interference.
HPLC-purified antisense phosphorothioate oligonucleotides (antisense Puma, 5'-CATCCTTCTCATACTTTC-3'; and control, CTTTCATACTCTTCCTAC-3'; GeneSet Oligos) (100 nM) were transfected with Oligofectamine (Invitrogen) into HeLa CD4 cells 24 h before coculture with HeLa Env cells. RNA interference of Bax and Bak expression was obtained by means of double-stranded morpholino oligonucleotides (GeneSet Oligos) specific for Bax (sense strand, 5'-rCUrgrgrArCrArgUrArArCrAUrgrgrArgrCTT-3') or Bak (sense strand, 5'-UrgrgUrCrCrCrAUrCrCUrgrArArCrgUrgTT-3'), transfected into both HeLa CD4 and HeLa Env cells 48 h before coculture.
Patients' Samples and In Vitro Infection with HIV-1.
Axillary lymph node biopsies or peripheral blood samples were obtained from healthy and HIV-1infected individuals (all males, mean age = 36 yr), who were naive for highly active antiretroviral therapy (HAART) with a plasma viral load >20,000 copies/ml or were receiving HAART with a viral load <5,000 copies/ml. Plasma HIV-1 RNA levels were determined by the bDNA procedure (Versant HIV RNA 3.0) according to the manufacturer's instructions (Bayer). None among the HIV-1+ patients received interferons, glucocorticoids, was positive for hepatitis B or C, or exhibited signs of autoimmunity. PBMCs were isolated by Ficoll/Hypaque (Amersham Biosciences) centrifugation of heparinized blood from either healthy donors and HIV-seropositive individuals and fixed with 4% paraformaldehyde in PBS, pH 7.2. Biopsies were fixed with 10% formalin, dehydrated, and paraffin embedded. Deparaffinized tissue sections and PBMCs were subjected to immunocytochemistry with antibodies specific for p53S46P, I
B
S32/36P, or PUMA and counterstained with hematoxylin. PBMCs were treated with TRIzol (Invitrogen) to extract total proteins, followed by immunoblot analysis. Alternatively, freshly purified PBMCs were stained with anti-CD4 antibody (FITC-labeled OKT3) and FACS® sorted, followed by protein extraction. Primary CD4+ lymphoblasts (10 x 106) cells (7) were incubated with the HIV-1LAI/IIIB strain or different clinical HIV-1 isolates (500 ng of p24; reference 16) for 4 h at 37°C. After washing out unabsorbed virus, cells (106 cells/ml) were cultured in RPMI 1640 medium containing 20% FCS and 10 U/ml IL-2.
Online Supplemental Material.
The transcripts with unknown function that are modulated by Env-dependent syncytium formation, as compared with control HeLa cells, are listed in Table SI available at http://www.jem.org/cgi/content/full/jem.20031216/DC1.
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Results
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Transcription Factor Profiling of HIV-1 Env-elicited Apoptosis.
In a model culture system of syncytium-dependent cell death, HeLa cells stably transfected with the Env gene from HIV-1LAI/IIIb (HeLa Env) were fused by coculture with CD4/CXCR4-expressing HeLa cells (HeLa CD4; references 6, 7, 17, 18). Syncytium-induced transcriptional effects were monitored by transfecting each of the two cell lines with a series of luciferase reporter gene constructs containing promoter elements responsive to a series of different transcription factors. This approach revealed that, among a panel of 12 different transcription factors, only NF-
B, p53, and AP1 were activated by the Env-dependent syncytium formation (Fig. 1 A). To determine the hierarchy among these transcription factors, cells were cotransfected with the aforementioned luciferase reporter constructs as well as a nonphosphorylable mutant of I
B (IKSR; reference 19) or with a DN mutant of p53 (p53H273; reference 20). Both IKSR and p53H2731 abrogated the increase of NF-
B, p53, and AP1-dependent transcription found in syncytia (Fig. 1 B). These data point to an obligate cooperation between NF-
B and p53, upstream of AP1, in Env-elicited syncytia.