Biomedical subjects

S Chouaib

Publications and source records attributed to S Chouaib.

At least 55 records · Page 3Linked to original sources

PubMed1998

Regulation of major histocompatibility complex class I expression by NF-kappaB-related proteins in breast cancer cells.

Downregulation of MHC Class I antigens has been observed in many cancers and usually results from a decreased gene transcription. A reporter CAT gene dependent on the MHC Class I kappaB site or on a longer promoter is transactivated by NF-kappaB complexes containing p65 or RelB. p100 as well as IkappaB-alpha are potent inhibitors of this transcription and p100 sequesters RelB and p65 complexes in the cytoplasm of breast cancer cells. However, although p100 is highly expressed in a number of breast cancer cell lines, MHC Class I antigen expression was observed on all the cell lines we analysed and could be further induced by stimulation with the cytokines IFN-gamma or TNF-alpha. Stable transfection of a unresponsive mutated IkappaB-alpha Ser 32-36 expression vector showed that TNF-alpha induced MHC Cl I expression in an NF-kappaB-dependent way while IFN-gamma did it independently of any NF-kappaB activation.

Antineoplastic Agents↗

PubMed1998

Isolation of tumor-specific cytotoxic CD4+ and CD4+CD8dim+ T-cell clones infiltrating a cutaneous T-cell lymphoma.

We have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4(+)CD8dim+ and CD4(+)CD8(-) phenotype. Both clones mediated a specific MHC class I-restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vbeta gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vbeta7/Jbeta2.3, Vbeta13/Jbeta2.5, and Vbeta22/Jbeta2.5 rearrangements. Phenotypic analysis using specific anti-Vbeta monoclonal antibodies indicated that only Vbeta13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vbeta gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vbeta transcript, corresponding, respectively, to Vbeta5/Jbeta2.3 and Vbeta17/Jbeta2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vbeta TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.

Aged↗

PubMed1998

Accumulation of the p53 protein allows recognition by human CTL of a wild-type p53 epitope presented by breast carcinomas and melanomas.

The p53 protein is accumulated in tumor cells of many human cancers and can elicit in vivo humoral and proliferative responses. Rare reports about p53-mediated tumor recognition by CTLs have remained questioned. We therefore studied a panel of breast tumor and melanoma cell lines that we assayed for the presence of accumulated p53 and surface HLA-A2 and for the presentation of p53 epitopes. From PBMC of a healthy donor, we have generated a CTL line, D5/L9V, directed against HLA-A2-restricted peptide 264-272 from wild-type p53. It efficiently lysed breast adenocarcinomas MCF-7, MCF7/RA1, and MDA-MB-231, and melanoma M8, which all accumulate the p53 protein. Using competition assays, we made sure that tumor lysis by D5/L9V was due to recognition of endogenously produced p53 peptide 264-272 associated with the HLA-A2.1 molecule on the surface of these tumor cells. Cells with undetectable levels of wild-type p53, such as lymphoblastoid cells and melanoma M74, were not recognized by D5/L9V. Neither were breast tumor cell line MCF7/ADR nor melanoma line M44 because of HLA loss. This study therefore shows that it is possible to obtain in vitro CTL lines that specifically recognize a p53 epitope spontaneously presented by a variety of HLA-A2+ transformed cell lines provided they display abnormal patterns of p53 expression. This work points out that breast tumors and melanomas share a p53 epitope, and raises hopes for future immunotherapeutic approaches.

Antigen-Presenting Cells↗

PubMed1998

NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions.

In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56+ CD7 and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class I- target K562. The CD56+ CD16+ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor, but no IFN-gamma. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.

ADP-ribosyl Cyclase↗

PubMed1998

Failure of TGF beta1 and IL-12 to regulate human FasL and mTNF alloreactive cytotoxic T-cell pathways.

The effect of TGFbeta1 and IL-12 on calcium-independent cytotoxic pathways was investigated. We have previously demonstrated that the regulatory effect of TGFbeta1 and IL-12 on human alloreative CTL activity was associated with regulation of perforin and granzyme B gene expression. To determine the effect of both cytokines on the alternative cytotoxic pathway involving FasL and mTNF, we first investigated the expression of both molecules on human primary alloactivated T cells. Our results show that human allostimulated T lymphocytes express FasL. Cell lysis experiments demonstrate that the FasL cytotoxic pathway is involved in the killing of specific target cells mediated by human alloreactive CTL. In addition, allogeneic stimulation induced significant mTNF expression on both CD4+ and CD8+ responder T cells. Using TNF-sensitive target cells, we also demonstrate that the mTNF-mediated cytotoxic pathway is involved in the cytotoxic activity of human primary allostimulated T lymphocytes. Neither TGFbeta1 nor IL-12 had an effect on FasL or mTNF expression. Furthermore, addition of TGFbeta1 or IL-12 at the initiation of the MLR had no significant effect on Fas- and mTNF-mediated cytotoxicity. Taken together, our results provide a novel insight into the differences between regulation by cytokines of perforin-dependent and -independent cytotoxic mechanisms. Unlike their role in the perforin/granzyme B pathway, TGFbeta1 and IL-12 do not appear to mediate any regulatory effect on FasL and mTNF cytotoxic pathways used by human alloreactive primary CTL.

Adult↗

PubMed1997

Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function.

We have investigated the relationship between the development of tumor resistance towards the cytotoxic action of tumor necrosis factor-alpha (TNF) and p53 function, using the TNF-sensitive MCF7 human breast adenocarcinoma cell line and two TNF-resistant sublines, MCF7/R-A1 and MCF7/Adr. Use of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing shows that MCF7 has a wild-type p53 gene, whereas both TNF-resistant sublines exhibit mutant p53. This includes a point mutation R280K in MCF7/R-A1 cells, and a point mutation at the splicing acceptor site on the upstream border of exon 5 resulting in a 21 pb deletion in MCF7/Adr cells. These mutations result in loss of p53 capacity to transactivate FASAY (functional assay in yeast). In contrast to what is observed for parental MCF7 cells, treatment of resistant sublines with TNF or gamma-irradiation fails neither to induce the expression of the p53-regulated gene products p21waf1/CIP1 and MDM2, nor to arrest the cells in the G1 phase of the cell cycle. Disruption of p53 wild-type function in MCF7 cells by transfection with human papillomavirus type-16 E6 gene, leads to abrogation of the cytotoxic, but not the cytostatic activity of TNF. Altogether, our results strongly suggest that wild-type p53 is involved in cytotoxic action of TNF, and point out that loss of p53 function contributes to resistance of tumor cell to TNF-induced killing.

Apoptosis↗

PubMed1997

IL-15 enhances immune functions during HIV infection.

IL-15, a new cytokine primarily produced by macrophages, has been shown to exhibit several functional properties shared with IL-2. Treatment of PBMC from HIV-infected patients with IL-15 resulted in an increase in NK cell cytotoxicity to levels similar to those of untreated PBMC from healthy donors. This effect is independent of several well-characterized regulatory cytokines, as it is not prevented by Abs that neutralize IFNs, TNF-alpha, IL-2, or IL-12. Enhanced cytotoxicity was accompanied by a significant increase in expression of cytotoxic granules. IL-15 enhanced the proliferative ability in both controls and HIV-seropositive in response to mitogen and recall Ags. Although the addition of IL-15 has a preventive effect on the appearance of spontaneous cell death, this effect was not seen during mitogen-induced apoptosis. The production of IL-15 by PBMC from patients in response to Staphylococcus aureus Cowan strain 1 appeared heterogeneous and was not negatively regulated by cytokines that inhibited IL-12 production. No correlation was found between in vitro HIV infection and IL-15 production, as viral infection had no effect on the ability of monocytes to produce IL-15 in response to S. aureus. Interestingly IL-15 restored the deficient production of IL-12 by PBMC from HIV+ people and had no major effect on modulating viral expression in latently infected cell lines or PBMC from naturally infected people. Taken together, these results suggest a potent immunoregulatory role of IL-15 during HIV infection.

Adult↗

PubMed1997

Quantitative analysis of T helper 1, T helper 2, and inflammatory cytokine expression in patients after allogeneic bone marrow transplantation: relationship with the occurrence of acute graft-versus-host disease.

BACKGROUND: To further delineate the cytokine involvement in human acute graft-versus-host disease (GVHD), we analyzed cytokine expression in peripheral blood mononuclear cells (PBMC) from patients who developed acute GVHD after allogeneic bone marrow transplantation and from those who did not. METHODS: We used a highly quantitative and sensitive polymerase chain reaction assay based on the coamplification of an internal standard, with the cDNA derived from the mRNA of interest. Results are expressed in copy numbers, after normalization to a fixed amount of actin, allowing comparison between different samples. After a myeloablative regimen, 22 patients with hematological diseases received an unmanipulated allograft from a matched sibling. They were subsequently submitted to prophylactic immunosuppression. We examined the transcription of genes encoding cytokines in PBMC and skin biopsies. We selected T helper 1 (interferon ([IFN]gamma, interleukin [IL]-2), T helper 2 (IL-4, IL-10), and inflammatory (IL-1, IL-6) cytokines. RESULTS: Four weeks after bone marrow transplantation, the bulk of the PBMC population exhibited an increased expression of IL-1 and IL-6, with no major difference between GVHD+ and GVHD- patients. In addition, although IL-2 expression was not detected, increased levels of IFNgamma mRNA were observed in allografted patients, with higher levels in GVHD+ patients. In skin biopsies sampled at the beginning of GVHD, although low expression of IL-1 and IL-6 could be observed, neither type 1 (IL-2, IFNgamma) nor type 2 (IL-4, IL-10) cytokines could be detected. CONCLUSIONS: These studies suggest that the occurrence of human GVHD does not seem to be clearly associated with a T helper 1-type cytokine pattern.

Acute Disease↗

PubMed1997

Sphingosine-1-phosphate mobilizes intracellular calcium and activates transcription factor NF-kappa B in U937 cells.

Sphingosine-1-phosphate (SPP), a metabolite of sphingolipids, has been implicated as a second messenger in cell growth regulation and signal transduction via calcium mobilization from internal stores. This study shows that SPP mobilizes intracellular calcium in U937 cells and demonstrates for the first time the ability of SPP to activate the transcription factor NF-kappa B in these cells. Furthermore, calcium release from the internal stores by thapsigargin (TG), an inhibitor of the endoplasmic reticulum Ca2+ pump, was associated with activation of NF-kappa B. Moreover, we have shown that while an intracellular calcium chelator BAPTA/AM was able to inhibit both SPP- and TG-induced NF-kappa B activation, it had no effect on TNF-induced NF-kappa B activation. In addition, SPP-induced NF-kappa B activation was blocked both by cyclosporin A, known to inhibit calcineurin phosphatase activity, and by the antioxidant butylated hydroxyanisole. These observations suggest that intracellular calcium mobilization is required for SPP-induced NF-kappa B activation, which may involve calcineurin- and redox-dependent mechanisms.

Antioxidants↗

PubMed1997

Alteration of the sphingomyelin/ceramide pathway is associated with resistance of human breast carcinoma MCF7 cells to tumor necrosis factor-alpha-mediated cytotoxicity.

The interference of tumor necrosis factor-alpha (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-kappaB (NF-kappaB) activation. However, the transfected cells remained resistant to TNF-induced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.

Breast Neoplasms↗

PubMed1997

IkappaB alpha overexpression in human breast carcinoma MCF7 cells inhibits nuclear factor-kappaB activation but not tumor necrosis factor-alpha-induced apoptosis.

Nuclear factor-kappaB (NF-kappaB) is one of major component induced by tumor necrosis factor-alpha (TNF), and its role in the signaling of TNF-induced cell death remains controversial. In order to delineate whether the involvement of NF-kappaB activation is required for triggering of the apoptotic signal of TNF, we inhibited the nuclear translocation of this transcription factor in TNF-sensitive MCF7 cells by introducing a human MAD-3 mutant cDNA coding for a mutated IkappaB alpha that is resistant to both phosphorylation and proteolytic degradation and that behaves as a potent dominant negative IkappaB alpha protein. Our results demonstrated that the mutated IkappaB alpha was stably expressed in the transfected MCF7 cells and blocked the TNF-induced NF-kappaB nuclear translocation. Indeed, TNF treatment of these cells induced the proteolysis of only the endogenous IkappaB alpha but not the mutated IkappaB alpha. The nuclear NF-kappaB released from the endogenous IkappaB alpha within 30 min of TNF treatment was rapidly inhibited by the mutated IkappaB alpha. There was no significant difference either in cell viability or in the kinetics of cell death between control cells and the mutated IkappaB alpha transfected cells. Furthermore, electron microscopic analysis showed that the cell death induced by TNF in both control and mutated IkappaB alpha transfected cells was apoptotic. The inhibition of NF-kappaB translocation in mutated IkappaB alpha-transfected cells persisted throughout the same time course that apoptosis was occurring. Our data provide direct evidence that the inhibition of NF-kappaB did not alter TNF-induced apoptosis in MCF7 cells and support the view that TNF-mediated apoptosis is NF-kappaB independent.

Apoptosis↗

PubMed1997

Functional interaction between TGF-beta and IL-12 in human primary allogeneic cytotoxicity and proliferative response.

IL-12 is an important cytokine in the control of cell-mediated immunity. We have investigated the functional interaction of IL-12 with TGF-beta1, a cytokine involved in the regulation of growth and differentiation of immunocompetent cells, during human allogeneic response development. Using primary MLR, our data show that addition of exogenous TGF-beta at the sensitizing phase of the primary MLR resulted in the inhibition of both allogeneic cytotoxic and proliferative responses. The inhibitory effect of TGF-beta on allogeneic response involves an abrogation of IL-12/p70 production upon allostimulation. In contrast to its effect on IL-12 production, TGF-beta did not alter the expression of IL-12R beta1-chain (IL-12R beta) in T cells induced upon allogeneic activation. Addition of exogenous IL-12 or IFN-gamma in the MLR cultures in the presence of TGF-beta did not result in reversal of CTL generation and T cell proliferation. Interestingly, TGF-beta was efficient in down-regulating IL-12 responsiveness of alloactivated T cells as well as TCR-alphabeta or TCR-gammadelta alloreactive T cell clones. These studies suggest that the inhibitory effect of TGF-beta on the development of human allogeneic proliferation and cytotoxic responses involves an additional mechanism associated with an interference with IL-12 pathway.

Adult↗

PubMed1997

Partial purification and characterization of a tumor necrosis factor-alpha converting activity.

Tumor necrosis factor (TNF)-alpha is initially synthesized as an extracellular membrane-associated 26-kDa protein that is further cleaved at Ala76-Val77 to yield the soluble 17-kDa form. Recently, peptide-hydroxamate metalloproteinase inhibitors have been reported to block the proteolytic processing of TNF-alpha, thus suggesting that the putative TNF-alpha converting enzyme (TACE) is a zinc-dependent metalloendopeptidase. In this report, we characterize a TNF-alpha converting activity (TACA) that cleaves in vitro the human 26-kDa TNF-alpha at the physiological processing site. The chromatography steps followed for purification and the use of a panel of proteinase inhibitors indicate that the enzyme responsible for TACA is a membrane glycosylated metalloendopeptidase which is most likely different from the matrix-degrading metalloproteinases. The failure of TACA to process a Val77-->Gly77 precursor mutant emphasizes the importance of hydrophobic residue at P1' position. In addition, TACA is not able to cleave the mouse pro-TNF-alpha and does not catalyze in vitro the processing of other transmembrane proteins susceptible to metalloproteinase-mediated shedding, such as interleukin-6 or TNF receptors. These studies suggest the existence of an enzyme specific for TNF-alpha within the metalloproteinases involved in the processing/shedding of a number of cytokines and cytokine receptors.

ADAM Proteins↗

PubMed1997

A metalloproteinase inhibitor blocks the shedding of soluble cytokine receptors and processing of transmembrane cytokine precursors in human monocytic cells.

A number of membrane-anchored cytokines and cytokine receptors are susceptible to yield soluble counterparts. Recently, peptide-hydroxamate metalloproteinase inhibitors have been reported to block the proteolytic processing of tumour necrosis factor (TNF)-alpha 55- and 75-kDa TNF receptors (TNF-R55 and TNF-R75), and interleukin (IL)-6R. In this report the authors studied the effect of an hydroxamate metalloproteinase inhibitor on the secretion of cytokines and the generation of cytokine soluble receptors by human myelomonoycytic cell lines and purified monocytes. Whereas secretion of cytokines lacking a transmembrane domain precursor (IL-1 alpha, IL-1 beta, IL-6 or IL-10) is either unaffected or augmented, shedding/secretion of transmembrane domain-containing cytokines and cytokine receptors [TNF-alpha, macrophage colony-stimulating factor (M-CSF), transforming growth factor (TGF)-alpha, stem cell factor (SCF), TNF-R55, TNF-R75, and IL-6R] was dramatically decreased in the presence of the metalloproteinase inhibitor. The diversity of sequences in the cleavage site of these proteins and differences found in the inhibitory concentration values suggest the existence of a metalloproteinase family displaying different substrate specificity. These results emphasize the important role of metalloproteinases as regulators of membrane expression and secretion of cytokines and cytokine receptors.

Antigens, CD↗

PubMed1997

The host-tumor immune conflict: from immunosuppression to resistance and destruction.

A successful immune response against a tumor is dependent on the cytokine repertoire present at the tumor site. Salem Chouaib and colleagues discuss evidence that, to escape the immune system, tumor cells not only produce immunosuppressive cytokines but also employ strategies involving altered susceptibility to tumor necrosis factor and Fas cytotoxic pathways and, in some circumstances, use of the Fas ligand to neutralize effector cells.

Animals↗

PubMed1997

Cloning of human IL-12 p40 and p35 DNA into the Semliki Forest virus vector: expression of IL-12 in human tumor cells.

IL-12 can enhance the development of effective immune responses against tumors as well as against certain infectious agents. It is therefore a potential candidate for therapeutic use in cancer therapy and in the design of vaccines against several infectious diseases. Several studies have demonstrated that IL-12 could efficiently induce tumor regression in animal models. To investigate the antitumor effect of direct gene transfer of human IL-12 into tumors, human IL-12 p35 and p40 cDNAs were cloned into the Semliki Forest virus (SFV) vector pSFV1. In order to express the two subunits from the same vector, the p35 and the p40 cDNAs were cloned into pSFV1, each under the control of a subgenomic SFV promoter. Recombinant RNA produced by in vitro transcription of SFV-IL-12 construct, was packaged into SFV viral particles with the use of a non-packageable helper RNA. We show that human tumor cell lines infected in vitro in vivo with recombinant SFV-IL-12 viral particles secrete high levels of biologically active heterodimeric p35/p40 IL-12, as demonstrated using ELISA and biological assays.

Gene Expression↗

PubMed1997

Critical stages of tumor growth regulation in transgenic mice harboring a hepatocellular carcinoma revealed by distinct patterns of tumor necrosis factor-alpha and transforming growth factor-beta mRNA production.

There is now good evidence that cytokines contribute to the regulation of tumor growth. The cytokine-driven modulation of tumor growth was investigated during the progression of a hepatocellular carcinoma (HCC) in SV40 large T tumor antigen transgenic mice. In vivo, an increased rate of liver growth correlated with increased transforming growth factor (TGF)-beta 1 mRNA expression, while the greatest amounts of tumor necrosis factor (TNF)-alpha mRNA were detected earlier during tumor development. Conversely, no particular alteration of IL-1 alpha, IL-1 beta, IL-6, IL-2, IL-4 and IFN-gamma mRNA production could be reported. In vitro, hepatocyte-like tumor cell lines established at two stages, either before or after HCC differentiation, were characterized. The early-stage-derived cell line produced TNF-alpha mRNA, but had barely detectable expression of TGF-beta 1 mRNA, while later-stage-derived cell lines showed the reciprocal pattern. All cell lines displayed a lack of sensitivity to TNF-alpha, although some degree of sensitivity to TNF-alpha could be observed in the presence of actinomycin-D or after treatment with IFN-gamma. The early-stage-derived cell line was sensitive to the growth inhibitory effects of TGF-beta 1, but late-stage-derived tumor cell lines displayed a loss of sensitivity to TGF-beta 1 which correlated with the increased expression of TGF-beta 1 mRNA. Altogether, this suggests that tumor cells contribute to the discrete TNF-alpha and TGF-beta 1 expression patterns during HCC progression. This model of HCC could be of valuable interest to assess the impact of various immunotherapeutic strategies on modulation of tumor growth.

Animals↗

PubMed1997

Iron chelation decreases human immunodeficiency virus-1 Tat potentiated tumor necrosis factor-induced NF-kappa B activation in Jurkat cells.

TNF-alpha stimulates HIV-1 replication via activation of the transcription factor NF-kappa B. TNF-mediated activation of NF-kappa B is known to involve the intracellular formation of reactive oxygen intermediates (ROIs). We recently demonstrated that HIV-1 Tat protein potentiates TNF-induced NF-kappa B activation by downregulation of manganese-dependent superoxide dismutase (MnSOD), shifting the cellular redox state towards pro-oxidative conditions. This study shows that treatment of Jurkat cells with iron chelator deferoxamine (DFO) strongly decreases HIV-1 Tat-potentiated TNF-induced NF-kappa B activation but does not modify NF-kappa B activation by TNF-alpha. The ability of iron chelators to reduce Tat-potentiated TNF-induced NF-kappa B binding activity suggests that iron and intracellular hydroxyl radicals (OH.) are required for Tat effect. Moreover, we have shown that exogenously generated OH. markedly enhanced TNF-induced NF-kappa B activation in a dose-dependent manner while was not sufficient to trigger activation of NF-kappa B by itself. In addition, iron chelators had no effect either on MnSOD activity or on the decrease of this activity by Tat. Iron chelators had also no effect on the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), but could elevate the GSH:GSSG ratio decreased by Tat protein. These observations suggest that the formation of intracellular OH. in the presence of iron ions play a major role in HIV-1 Tat enhancement of TNF-induced NF-kappa B activation and that iron chelation may protect Jurkat T cells, at least in part, against oxidative stress induced by Tat.

Down-Regulation↗