IFN-alpha and high dose ascorbic acid (AA) have a modest clinical benefit in HAM/TSP (Nakagawa, 1996). We investigated the effect of ex vivo and in vitro AA and IFN-alpha treatment on HAM/TSP PBMC and HTLV-1-infected cell lines, respectively.
We treated cells for 48-72h ex vivo and in vitro with low-high (10-100µg/ml) dose of AA and 1000U/ml of IFN-alpha and quantified lymphoproliferation by [3H]thymidine incorporation, tetraploid DNA content and PCNA expression (flow cytometry). Viral expression was measured at the RNA (tax, LTR) and protein (Tax, p19) level by RT-PCR, Western blot and ELISA, respectively. Apoptosis was quantified by subdiploid DNA content (flow cytometry). Th1/Th2/Th17 cytokines were quantified by cytometric bead array. AA induced a dramatic 95% decrease (control 3689±755 cpm vs. AA 121±52 cpm, p=0.001) in spontaneous, virus-driven lymphoproliferation and a decrease in tax and LTR transcription in HAM/TSP PBMC. In addition, AA decreased the exacerbated ex vivo IFN-gamma production in HAM/TSP PBMC. In HTLV-1 infected cell lines (MT-2 and MT-4), AA induced a dose-dependent increase in DNA fragmentation (p=0.02, p=0.005), paralleled by a decrease in PCNA (p=0.003), as well as p19 (p<0.001) and Tax levels. These effects appear virus-specific, since high-dose (100µg/ml) AA did not exert a significant antiproliferative or pro-apoptotic effect on PBMC of normal donors. On top, AA displayed a superior antiproliferative, antiviral and immunomodulatory effect over IFN-alpha in both cell lines and HAM/TSP PBMC. Considering the selective antiproliferative and antiviral effects of AA ex vivo and in vitro, the therapeutic potential of combination therapy with high dose AA in HAM/TSP should be further explored.
Glucocorticoids (GCs) affect peripheral immune responses by inhibiting T cell immunity at several stages of the activation cascade, causing impaired cytokine production and effector function. The recent demonstration that the thymic epithelium and possibly thymocytes themselves produce steroids suggests that endogenous GCs also play a role in the control of T cell development. As both peripheral responsiveness and thymic differentiation appear to be regulated by the quantity and quality of intracellular signals issued by antigen–major histocompatibility complex-engaged T cell receptor (TCR) complexes, we investigated the effects of GCs on the signaling properties of T cells stimulated by anti-CD3 monoclonal antibodies or agonist peptides. We demonstrate in this work that dexamethasone, a synthetic GC, inhibits the early signaling events initiated upon TCR ligation, such as tyrosine phosphorylation of several TCR-associated substrates including the ζ chain, the ZAP70 kinase, and the transmembrane adapter molecule linker for activation of T cells. Hypophosphorylation was not a consequence of reduced kinase activity of src protein tyrosine kinases, but was correlated with an altered- membrane compartmentalization of these molecules. These observations indicate that in addition to their well-described ability to interfere with the transcription of molecules involved in peripheral responses, GCs inhibit T cell activation by affecting the early phosphorylating events induced after TCR ligation.
ABSTRACTThe human T-cell leukemia virus type 1 Tax protein transforms human T lymphocytes, which can lead to the development of adult T-cell leukemia. Tax transformation is related to its ability to activate gene expression via the ATF/CREB and the NF-κB pathways. Transcriptional activation of these pathways is mediated by the actions of the related coactivators CREB binding protein (CBP) and p300. In this study, immunocytochemistry and confocal microscopy were used to localize CBP and p300 in cells expressing wild-type Tax or Tax mutants that are able to selectively activate gene expression from either the NF-κB or ATF/CREB pathway. Wild-type Tax colocalized with both CBP and p300 in nuclear bodies which also contained ATF-1 and the RelA subunit of NF-κB. However, a Tax mutant that selectively activates gene expression from only the ATF/CREB pathway colocalized with CBP but not p300, while a Tax mutant that selectively activates gene expression from only the NF-κB pathway colocalized with p300 but not CBP. In vitro and in vivo protein interaction studies indicated that the integrity of two independent domains of Tax delineated by these mutants was involved in the direct interaction of Tax with either CBP or p300. These studies are consistent with a model in which activation of either the NF-κB or the ATF/CREB pathway by specific Tax mutants is mediated by distinct interactions with related coactivator proteins. ACKNOWLEDGMENTSWe thank Richard Goodman for providing the CBP clone, David Livingston for providing p300 cDNA and the p300 monoclonal antibody, Joan Steitz and Gunther Schutz for providing the Sm and CREM antibodies, and the NIH AIDS Research and Reagent Program for providing the MT2 cells and the Tax monoclonal antibody. In addition, we acknowledge Caroline Vanhulle for technical assistance and Sharon Johnson and Anthony Cancel for the preparation of the manuscript and the figures.This work was supported by grants from the Belgian Fonds National de la Recherche Scientifique and Télévie, from the Fonds de Financement de la Recherche Cancérologique de la CGER Assurances, from the NIH, and from the Council of Tobacco Research.
The functional analysis of regulatory proteins Tax-1 and Tax-2 can provide useful information to further understand the difference in pathogenicity between HTLV-1 and HTLV-2.
Tax-1 molecules phosphorylated at serine residues 300 and 301, ubiquitinated or sumoylated at lysine residues 280 and 284 and acetylated at lysine 346 have been identified. These modifications control Tax-1 intracellular localization, the formation of Tax-1 nuclear bodies and sequential steps in Tax-1-mediated activation of the NF-κB pathway, a critical event thought to be involved in HTLV-1 transforming capacity [1].
By comparing internally tagged Tax-1 and Tax-2B, we demonstrated that Tax-2B activates gene expression via the NF-κB pathway, is present in nuclear bodies and in the cytoplasm and is modified by ubiquitination and sumoylation similarly to its homologue Tax-1 [2].
In the present study, we constructed a series of Tax-2B mutants with substitutions of specific lysine residues by arginines. The post-translational modifications, subcellular localization and capacity to activate gene expression of these Tax-2B mutants will be compared to those obtained for the corresponding mutants of Tax-1.
We investigated and compared the mechanisms by which two dust mite proteolytic allergens, Der p 1 and Der p 3, and a peptide agonist of proteinase-activated receptor 2 (PAR(2)AP) trigger interleukin (IL)-8 release from human pulmonary epithelial cells (A549). Although all three stimuli tested induced the up-regulation of IL-8 (mRNA and protein), the Der p 1-mediated signaling events did not exactly match those induced by PAR(2)AP and Der p 3. First, Der p 1 was less effective in stimulating IL-8 gene transcriptional activity than PAR(2)AP and Der p 3. Second, Der p 1-mediated IL-8 expression was mainly dependent on NF-kappaB, whereas Der p 3 and PAR(2)AP regulated IL-8 expression through the activation of both NF-kappaB and AP-1. Third, although all three MAP kinases, ERK1/2, p38, and JNK, were activated, Der p 1 induced IL-8 release exclusively via the ERK1/2 signaling pathway, whereas PAR(2)AP and Der p 3 also involved the other kinases. Fourth, in HeLa cells, Der p 1 was able to up-regulate IL-8 secretion independent of PAR(2) expression, and in contrast with PAR(2)AP and Der p 3, Der p 1 was unable to affect calcium signaling via PAR(2) in PAR(2)-expressing KNRK cells. Finally, cleavage by Der p 1 of a synthetic peptide representing the N-terminal activation-cleavage site of PAR(2) did not release a high potency activator of PAR(2) as does Der p 3. We conclude that Der p 1 (but not Der p 3)-induced IL-8 production in A549 epithelial cells is independent of PAR(2) activation.
At concentrations where benzimidazole inhibited partially total protein synthesis in yeast, the drug also inhibited protein synthesis mediated by cytoplasmic ribosomes and by isolated mitochondria in vitro. Except for cytochrome oxidase, the effects of benzimidazole on the anabolism of hemoproteins were also partial and unspecific; the induction by oxygen of the particulate cytochromes b and c 1 , the synthesis of which is determined by the obligate co‐operation of both protein‐synthesizing systems, was not affected more than the induction of catalase, an extramitochondrial hemo‐protein, which is synthesized exclusively in cytoplasm. The drastic inhibition of cytochrome oxidase was the consequence of a specific blocking of cytochrome ( a , a 3 ) production. The delayed adaptation to oxygen, following the exclusion of benzimidazole, was characterized by its exceptionally high rate and correlated with the simultaneous increase of hemo‐protein ( a , a 3 ) content and the development of cytochrome oxidase activity; the latter activity ruled respiration rate. The process was largely unsensitive to the concerted actions of cycloheximide and chloramphenicol; under identical conditions, the synthesis of other hemoproteins was immediately stopped. The reconstitution of cytochrome oxidase in vivo at the expense of the double set of cytoplasmic and mitochondrial adaptation products, which were simultaneously accumulated, was established by reference to the specific effects of cycloheximide and chloramphenicol. As the prevailing effect of benzimidazole was operationally to switch off the assembly of cytochrome ( a , a 3 ), it is inferred that the drug will afford new experimental approaches in further studies of cytochrome oxidase anabolism.
La proteine Tax du virus HTLV-1 a les proprietes d'un oncogene viral et joue un role important dans l'induction de la transformation cellulaire menant a l'ATL. L'activite oncogene de Tax resulte d'effets pleiotropes sur divers mecanismes cellulaires y compris l'activation de l'expression de genes cellulaires specifiques par la voie NF-B et la deregulation de la progression du cycle cellulaire. Dans ce travail, nous avons mis en evidence que Tax est une proteine hautement modifiee dans diverses lignees cellulaires y compris dans les lymphocytes T transformes par HTLV-1. L'ensemble des modifications post-traductionnelles de Tax forment une suite hierarchisee qui controlent la localisation intracellulaire de Tax, sa capacite d'activer les kinases IKK et la voie de signalisation des facteurs NF-B et sa capacite d'induire un arret dans la progression de la mitose. En effet, la phosphorylation de Tax controle son ubiquitination et son passage dans le noyau ou elle est sumoylee et acetylee. L’ubiquitination et la sumoylation de Tax agissent de maniere concertee pour permettre l’activation de l’expression des genes par la voie NF-B tandis que son acetylation permet de lever le blocage induit en fin de mitose. Les effets exerces par les modifications post-traductionnelles sur les fonctions oncogenes de la proteine Tax suggerent que ces modifications pourraient etre des cibles therapeutiques pour le developpement de traitements contre l'ATL.
