RESUMO
AIMS/HYPOTHESIS: Experimental studies have suggested that apoptosis is involved in diabetic embryopathy through oxidative stress. However, the precise mechanism of diabetic embryopathy is not yet clear. Thioredoxin (TRX) is a small, ubiquitous, multifunctional protein, which has recently been shown to protect cells from oxidative stress and apoptosis. Using transgenic mice that overproduce human TRX-1 (TRX-Tg mice), we examined whether oxidative stress is involved in fetal dysmorphogenesis in diabetic pregnancies. METHODS: Non-diabetic and streptozotocin-induced diabetic (DM) female mice were mated with male TRX-Tg mice. Pregnant mice were killed either at day 10 or day 17 of gestation, and viable fetuses and their placentas were recovered, weighed and assessed for gross and histological morphology, biochemical markers and gene expression. RESULTS: In both wild-type (WT) and transgenic (Tg) groups, fetal and placental weights in the diabetic group were significantly decreased compared with the non-diabetic group. The incidence of malformation was higher in the diabetic group, and was significantly decreased in the TRX-Tg group (DM-WT vs DM-Tg; 28.6% vs 10.4%). Oxidative stress markers such as thiobarbituric acid reactive substances and 8-hydroxy-2'-deoxyguanosine were increased in DM-WT group fetuses but were decreased in fetuses from the DM-Tg group. Furthermore, immunohistochemically assayed apoptosis and cleaved caspase-3 production in embryonic neuroepithelial cells was significantly increased in the DM-WT group, and was significantly decreased in the DM-Tg group. CONCLUSIONS/INTERPRETATION: These results indicate that oxidative stress is involved in diabetic embryopathy, and that the antioxidative protein TRX at least partially prevents diabetic embryopathy via suppression of apoptosis.
Assuntos
Apoptose/fisiologia , Doenças Fetais/metabolismo , Doenças Fetais/prevenção & controle , Gravidez em Diabéticas/metabolismo , Gravidez em Diabéticas/prevenção & controle , Tiorredoxinas/metabolismo , Animais , Apoptose/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Feminino , Doenças Fetais/genética , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Células Neuroepiteliais/citologia , Reação em Cadeia da Polimerase , Gravidez , Gravidez em Diabéticas/genética , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Tiorredoxinas/genéticaRESUMO
Using cloned lines with the morphology of large granular lymphocytes (LGL) from BALB/c mice, we studied the exact requirements for proliferation and their functional characteristics, as well as their regulation. Although these cloned LGL lines were interleukin 2 (IL-2) dependent for growth, experiments using human recombinant IL-2 (rIL-2), known to be active on murine cells, indicated that IL-2 was a necessary but not sufficient factor. Coexistance of normal macrophages in addition to rIL-2 was found to support continuous proliferation of cloned LGL in vitro. This role of macrophages could be replaced by partially purified IL-1 derived from macrophage-conditioned medium. An IL-2 binding assay using 125I-rIL-2 suggested that the role of normal macrophages was to selectively induce and/or maintain high affinity IL-2 receptors (IL-2R) (Kd, 0.2-0.5 nM) without affecting low affinity ones (Kd, 10-30 nM). Functional studies indicated that most of the LGL clones killed various combinations of representative groups of natural killer (NK)-susceptible target cells, including leukemic cells (YAC-1, RL male 1), virus-infected cells (HeLa-measles, HeLa-herpes simplex virus), and normal bone marrow cells (BMC), whereas none of them affected any of NK-resistant target cells, including uninfected HeLa cells. Some of these clones also suppressed in vitro hematopoiesis. Such characteristic cytotoxic spectra, as well as serological phenotypes (Thy-1+, Lyt-1-2-, asialo GM1-positive, T200+, TdT-, Fc receptor-positive) indicated that these LGL clones exactly represent endogenous NK cells, rather than a variety of anomalous killer cells generated in various culture conditions. Although there was significant heterogeneity of cytotoxic spectrum among LGL clones, no clonotypic distribution of specificities was observed. Normal macrophages were found to modulate the functional expression of LGL clones. They augmented the cytotoxic potential of the clones against leukemic and virus-infected targets, but suppressed intrinsic reactivity against normal BMC. Similarly, LGL clones maintained with macrophages showed much less suppressive effect on in vitro hematopoiesis. The present observations on the interaction of cloned LGL and normal macrophages provide a basic explanation for the mechanisms by which the immediate responsiveness to IL-2 of the NK effector system, without exogenous stimulation, and the functional selectivity toward abnormal rather than normal cells, are actively maintained in vivo.
Assuntos
Linfócitos/fisiologia , Macrófagos/fisiologia , Animais , Divisão Celular , Linhagem Celular , Células Clonais , Citotoxicidade Imunológica , Feminino , Hematopoese , Técnicas In Vitro , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores Imunológicos/análise , Receptores de Interleucina-2RESUMO
The cytotoxicity of reactive oxygen intermediates (ROIs) has been implicated in the destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM). Thioredoxin (TRX), a redox (reduction/oxidation)-active protein, has recently been shown to protect cells from oxidative stress and apoptosis. To elucidate the roles of oxidative stress in the development of autoimmune diabetes in vivo, we produced nonobese diabetic transgenic mice that overexpress TRX in their pancreatic beta cells. In these transgenic mice, the incidence of diabetes was markedly reduced, whereas the development of insulitis was not prevented. Moreover, induction of diabetes by streptozotocin, an ROI-generating agent, was also attenuated by TRX overexpression in beta cells. This is the first direct demonstration that an antioxidative and antiapoptotic protein protects beta cells in vivo against both autoimmune and drug-induced diabetes. Our results strongly suggest that oxidative stress plays an essential role in the destruction of beta cells by infiltrating inflammatory cells in IDDM.
Assuntos
Antioxidantes/metabolismo , Apoptose , Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 1/prevenção & controle , Ilhotas Pancreáticas/metabolismo , Tiorredoxinas/metabolismo , Animais , Diabetes Mellitus Tipo 1/patologia , Feminino , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Estresse Oxidativo , Coelhos , EstreptozocinaRESUMO
IL-2 binds to high- and low-affinity receptors on activated T cells. The high-affinity receptor was hypothesized to consist of the noncovalent association between the alpha chain (IL-2-R-alpha, p55) and a beta chain (IL-2-R-beta, p70), whereas the low-affinity receptor consists of p55 without p70. We now directly identify p70 as a 65-77-kD glycoprotein doublet. Preparative quantities of the IL-2/p70 complex have been isolated. Further, we demonstrate that p70 is the principal IL-2 binding protein on both resting CD4+ and CD8+ T cells and that both p70 and p55 can be induced on normal B cells and monocytes.
Assuntos
Antígenos de Superfície/isolamento & purificação , Interleucina-2/metabolismo , Leucócitos Mononucleares/análise , Receptores Imunológicos/isolamento & purificação , Antígenos de Superfície/genética , Linfócitos B/análise , Regulação da Expressão Gênica , Glicoproteínas/genética , Humanos , Leucócitos Mononucleares/classificação , Ativação Linfocitária , Peso Molecular , Receptores Imunológicos/genética , Receptores de Interleucina-2 , Linfócitos T/análise , Linfócitos T/classificação , Células Tumorais Cultivadas , Membro 7 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Using cloned murine large granular lymphocyte (LGL) lines, the expression and the rearrangement of the alpha, beta, and gamma chain genes of the T cell receptor (TCR) were analyzed. Morphological, phenotypical, as well as functional studies indicated that the LGL lines were identical to normal, endogenous NK cells. Northern blot hybridization analysis indicated that the full-length transcripts of all the alpha, beta, and gamma chain genes were expressed in most of the LGL lines, including two lines derived from athymic nude mice. In one line, SPB, however, no transcript of the gamma chain gene was detected, whereas the alpha and beta chain genes were clearly expressed. In every LGL line studied, all of the alpha, beta, and gamma chain genes were rearranged. Conforming to the results of Northern blot hybridization study, the gamma chain gene of the SPB line was aberrantly rearranged, whereas those of all the other lines were productively rearranged. The results clearly revealed that NK cells represented a population of lymphocytes genetically committed to the T cell lineage. It was also suggested that the expression and rearrangement of the TCR genes of NK cells might occur in a thymus-independent fashion. An SPB line without expression of the gamma chain gene could exhibit NK activity indistinguishable from other NK lines. Furthermore, the rearrangement patterns of the beta chain gene did not correlate with the specificity of the cytotoxic activity. These results strongly suggested that the cytotoxic activity in NK cells was not directly mediated by TCR on them. We particularly noted that the beta chain gene of most independently established LGL lines showed identical patterns of rearrangement, indicating that they used the same V beta and J beta gene segments. The significance of the restricted pattern of rearrangement of the beta chain gene in LGL lines, as well as the possible functional roles of TCR on NK cells, was discussed.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Recombinação Genética , Animais , Linhagem Celular , Células Clonais/metabolismo , Testes Imunológicos de Citotoxicidade , DNA/metabolismo , Feminino , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Hibridização de Ácido Nucleico , Transcrição GênicaRESUMO
Using the clone-specific rearrangement of the T cell receptor gene as the genetic marker of the clonotype, we analyzed the clonal origin of the interleukin 2 (IL-2)-dependent human T-lymphotrophic virus I (HTLV-I)-positive T cell lines established from various adult T cell leukemia (ATL) patients. From a patient with chronic ATL, whose leukemic cells proliferated in vitro in response to IL-2, we repeatedly established leukemic T cell clones having the same rearrangement profile of the T beta chain gene as the leukemic cells. By contrast, established cell lines from acute ATL patients had different beta chain gene rearrangements from those of the leukemic cells. These HTLV-I+ T cell lines might not be the direct progeny of the leukemic cells, but that of T cells infected either in vivo or in vitro. These IL-2-reactive nonleukemic T cells might have been selected in vitro, because their leukemic cells failed to respond to IL-2, despite the expression of IL-2 receptor. The analysis of the T cell receptor gene rearrangement may give a new approach for the elucidation of the mechanism of leukemogenesis and the origin of the HTLV-I+ T cell lines in ATL.
Assuntos
Infecções por Deltaretrovirus/imunologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismo , Doença Aguda , Adulto , Linhagem Celular , Doença Crônica , Células Clonais/imunologia , Infecções por Deltaretrovirus/metabolismo , Humanos , Interleucina-2/fisiologia , Ativação Linfocitária , Masculino , Linfócitos T/imunologiaRESUMO
Three rat lymphoid cell lines (TARS-1, TARL-2, and TART-1) (12) transformed by human T cell leukemia/lymphoma virus I (HTLV-I) had rearrangement of the beta chain gene of the T cell antigen receptor, and had integrated proviral DNA from HTLV-I in their genomes. As is the case with adult T cell leukemia (ATL)-derived human T cell lines transformed by HTLV-I, these rat cell lines unequivocally expressed interleukin 2 (IL-2) receptor, as determined by radiolabeled IL-2 binding. By Scatchard plot analysis, one of the cell lines, TART-1, proved to have high affinity receptors (Ka = 1.3 X 10(11)/M and 8.8 X 10(9)/M). Rat IL-2 receptor, not human IL-2 receptor, was expressed on HTLV+ rat cell lines, as demonstrated by the fact that they expressed antigens reactive with monoclonal antibodies (ART-18) against rat IL-2 receptor, but not with anti-Tac antibodies. The collective evidence indicates that the endogenous IL-2 receptor gene is activated in human and rat lymphoid cell lines with HTLV-I production. The mechanism of abnormal IL-2 receptor expression in HTLV infection is discussed.
Assuntos
Deltaretrovirus , Interleucina-2/metabolismo , Receptores Imunológicos/biossíntese , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais , Sítios de Ligação , Linhagem Celular , Transformação Celular Viral , Deltaretrovirus/genética , Deltaretrovirus/imunologia , Eletroforese em Gel de Poliacrilamida , Genes Virais , Humanos , Ativação Linfocitária , Ratos , Ratos Endogâmicos , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T/genética , Receptores Imunológicos/análise , Receptores Imunológicos/imunologia , Receptores de Interleucina-2 , Linfócitos T/imunologiaRESUMO
Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.
Assuntos
Fatores Quimiotáticos/farmacologia , Fatores Quimiotáticos/fisiologia , Infecções/fisiopatologia , Inflamação/fisiopatologia , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacologia , Animais , Linhagem Celular , Quimiotaxia de Leucócito/fisiologia , Infecções por HTLV-I/fisiopatologia , Humanos , Técnicas In Vitro , Camundongos , Monócitos/fisiologia , Neutrófilos/fisiologia , Oxirredução , Linfócitos T/fisiologiaRESUMO
BACKGROUND: Human thioredoxin (TRX) is one of redox-active proteins that regulate reactive oxidative metabolisms. In recent study, we found that serum levels of TRX were elevated in asthmatic patients with exacerbation; however, few details are known about the physiological role of TRX in allergic inflammation, involving eosinophil infiltration. OBJECTIVE: In the present study, we examined whether TRX modulated C-C chemokine-induced chemotaxis of human eosinophils. METHODS: Eosinophils were isolated from subjects with mild eosinophilia by modified CD16 negative selection. After incubation with or without recombinant TRX, chemotaxis of human eosinophils was measured using Boyden chamber. RESULTS: Preincubation with TRX suppressed eotaxin- and regulated on activation, normal T-cell expressed and secreted (RANTES)-induced chemotaxis of eosinophils. Although, TRX had no effect on the expression of C-C chemokine receptor 3, which is a receptor of eotaxin and RANTES, we demonstrated that the activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases, which play an important role in eosinophil migration, was attenuated by the treatment with TRX. CONCLUSION: Our results suggest that the elicited TRX is beneficial to reduce allergic inflammation through negative regulation of eosinophil functions and has potential in the treatment of allergic diseases, such as asthma.
Assuntos
Asma/imunologia , Quimiocinas CC/imunologia , Quimiotaxia de Leucócito/imunologia , Eosinófilos/imunologia , Tiorredoxinas/imunologia , Asma/tratamento farmacológico , Quimiotaxia de Leucócito/efeitos dos fármacos , Eosinofilia/imunologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteínas Recombinantes/farmacologia , Tiorredoxinas/farmacologia , Tiorredoxinas/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
The transition from interleukin-2 (IL-2)-dependent to IL-2-independent growth is considered one of the key steps in the transformation of human T-cell leukemia virus type-I (HTLV-I)-infected T cells. The expression of thioredoxin-binding protein-2 (TBP-2) is lost during the transition of HTLV-I-infected T-cell lines. Here, we analysed the mechanism of loss of TBP-2 expression and the role of TBP-2 in IL-2-dependent growth in the in vitro model to investigate multistep transformation of HTLV-I. CpGs in the TBP-2 gene are methylated in IL-2-independent but not in IL-2-dependent cells. Sequential treatment with 5-aza-2'-deoxycytidine and a histone deacetylase inhibitor augmented histone acetylation and TBP-2 expression, suggesting that loss of TBP-2 expression is due to DNA methylation and histone deacetylation. In IL-2-dependent cells, a basal level of TBP-2 expression was maintained by IL-2 associated with cellular growth, whereas TBP-2 expression was upregulated on deprivation of IL-2 associated with growth suppression. Overexpression of TBP-2 in IL-2-independent cells suppressed the growth and partially restored responsiveness to IL-2. Knockdown of TBP-2 caused the IL-2-dependent cells to show partial growth without IL-2. These results suggested that epigenetic silencing of the TBP-2 gene results in a loss of responsiveness to IL-2, contributing to uncontrolled IL-2-independent growth in HTLV-I-infected T-cell lines.
Assuntos
Proteínas de Transporte/genética , Inativação Gênica , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Interleucina-2/genética , Linfócitos T/virologia , Tiorredoxinas/genética , Acetilação , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Transformação Celular Viral , Imunoprecipitação da Cromatina , Ilhas de CpG , Metilação de DNA , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Inibidores Enzimáticos/farmacologia , Regulação Viral da Expressão Gênica , Inibidores de Histona Desacetilases , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Interleucina-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T/metabolismo , Tiorredoxinas/metabolismo , Transcrição Gênica , VorinostatRESUMO
Adaptation to stress evokes a variety of biological responses, including activation of the hypothalamic-pituitary-adrenal (HPA) axis and synthesis of a panel of stress-response proteins at cellular levels: for example, expression of thioredoxin (TRX) is significantly induced under oxidative conditions. Glucocorticoids, as a peripheral effector of the HPA axis, exert their actions via interaction with a ligand-inducible transcription factor glucocorticoid receptor (GR). However, how these stress responses coordinately regulate cellular metabolism is still unknown. In this study, we demonstrated that either antisense TRX expression or cellular treatment with H2O2 negatively modulates GR function and decreases glucocorticoid-inducible gene expression. Impaired cellular response to glucocorticoids is rescued by overexpression of TRX, most possibly through the functional replenishment of the GR. Moreover, not only the ligand binding domain but the DNA binding domain of the GR is also suggested to be a direct target of TRX. Together, we here present evidence showing that cellular glucocorticoid responsiveness is coordinately modulated by redox state and TRX level and propose that cross talk between neuroendocrine control of stress responses and cellular antioxidant systems may be essential for mammalian adaptation processes.
Assuntos
Antioxidantes/metabolismo , Dexametasona/farmacologia , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo , Receptores de Glucocorticoides/fisiologia , Tiorredoxinas/biossíntese , Transcrição Gênica/efeitos dos fármacos , Animais , Northern Blotting , Células CHO , Células COS , Cricetinae , Citocinas/farmacologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/fisiologia , Diamida/farmacologia , Genes Reporter , Células HeLa , Humanos , Cinética , Mamíferos , Proteínas de Neoplasias/farmacologia , Oxirredução , Reação em Cadeia da Polimerase , Receptores de Glucocorticoides/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
We studied the expression of the interleukin-2 (IL-2) receptor and the proliferative response to exogenous IL-2 of peripheral blood leukemic cells from patients with adult T cell leukemia (ATL) in order to see whether IL-2 receptor expressed on ATL cells is different from normal IL-2 receptor and whether it plays a role in the neoplastic growth in ATL. Peripheral blood leukemic cells from 42 patients with ATL examined expressed IL-2 receptors that were detected by anti-Tac monoclonal antibody when examined immediately after the separation of cells or after the culture for 24 or 48 h. The number of anti-Tac binding sites ranged from 3,100 to 11,400 in fresh cells and from 3,600 to 96,000/cell in short-term cultured leukemic cells, whereas phytohemagglutinin-P (PHA-P)-stimulated normal T cells exhibited 6,900-35,000 anti-Tac binding sites per cell. ATL-derived and human T cell leukemia/lymphoma virus, type I (HTLV-I)-infected cell lines such as MT-1 and Hut102 expressed a much higher number of anti-Tac binding sites. Leukemic cells from 15 patients with ATL examined showed no or very poor proliferative response to various concentrations of immunoaffinity-purified IL-2, although they expressed Tac antigen (Ag). Radiolabeled IL-2 binding experiments demonstrated that ATL leukemic cells could bind IL-2, and they expressed both high and low affinity IL-2 receptors, although the number of high affinity IL-2 receptor was much less than that of low affinity IL-2 receptor and that of anti-Tac binding sites. In contrast, leukemic T cells from a patient with T cell chronic lymphocytic leukemia (CLL), in whom HTLV-I infection was not demonstrated, responded as well as PHA-P-stimulated normal T cells, and their IL-2 receptors, unlike ATL cells, were modulated (down regulated) by anti-Tac antibody. No differences were noted between ATL cells and normal activated T cells in one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the IL-2 receptor. Thus, leukemic cells in ATL spontaneously and continuously express IL-2 receptor, which appears to be abnormally regulated and unresponsive to IL-2. These results, taken together with those on normal IL-2 receptors on HTLV-I-negative T-CLL cells, suggest that abnormal expression of the IL-2 receptor in ATL is closely associated with HTLV-I infection and may play a role in the neoplastic growth of ATL cells.
Assuntos
Antígenos de Superfície/biossíntese , Leucemia/metabolismo , Adulto , Idoso , Anticorpos/imunologia , Antígenos de Superfície/imunologia , Sítios de Ligação , Divisão Celular , Deltaretrovirus , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Humanos , Interleucina-2/metabolismo , Cinética , Masculino , Pessoa de Meia-Idade , Peso Molecular , Fito-Hemaglutininas/farmacologia , Linfócitos T , Membro 7 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Thioredoxin is a small ubiquitous protein with multiple biological functions, including cellular defense mechanisms against oxidative stress. In the present study, we investigated the role of human thioredoxin (hTRX) in the acquisition of cellular resistance to cis-diamminedichloroplatinum (II) (CDDP). The expression and activity of hTRX in Jurkat T cells was dose-dependently enhanced by exposure to CDDP, as determined by immunoblot analysis and insulin reducing assay. Furthermore, chloramphenicol acetyltransferase analysis using the hTRX promoter-reporter gene construct revealed that treatment of Jurkat cells with CDDP caused transcriptional activation of the hTRX gene, which might be mediated through increased generation of intracellular reactive oxygen intermediates. To examine the biological significance of hTRX induction, we established hTRX-overexpressing derivatives of L929 fibrosarcoma cells by stable transfection with the hTRX cDNA. The clones, which constitutively expressed the exogenous hTRX, displayed increased resistance to CDDP-induced cytotoxicity, compared with the control clones. After exposure to CDDP, the control cells showed a significant increase in the intracellular accumulation of peroxides, whereas the hTRX-transfected cells did not. Taken together, these results suggest that overexpressed hTRX is responsible for the development of cellular resistance to CDDP, possibly by scavenging intracellular toxic oxidants generated by this anticancer agent.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Tiorredoxinas/metabolismo , Animais , Linhagem Celular , Replicação do DNA , Resistência a Medicamentos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Espécies Reativas de OxigênioRESUMO
Human thioredoxin (TRX) was first identified in human T-cell leukemia virus type I (HTLV-I)-positive T-cell lines and is associated with the pathophysiology of retroviral infections. TRX is a vital component of the thiol-reducing system and regulates various cellular function (redox regulation). Members of the TRX system regulate apoptosis through a wide variety of mechanisms. A family of thioredoxin-dependent peroxidases (peroxiredoxins) protects against apoptosis by scavenging hydrogen peroxide. Thioredoxin 2 is a critical regulator of cytochrome c release and mitochondrial apoptosis; transmembrane thioredoxin-related molecule (TMX) has a protective role in endoplasmic reticulum (ER) stress-induced apoptosis. TRX interacts with apoptosis signal-regulating kinase 1 (ASK1) and is a sensor of oxidative stress. Thioredoxin binding protein-2/vitamin D(3) upregulated protein 1 is a growth suppressor and its expression is suppressed in HTLV-I-transformed cells. Studies of these molecules of the TRX system provide novel insights into the apoptosis associated with retroviral diseases.
Assuntos
Apoptose , Estresse Oxidativo , Infecções por Retroviridae/metabolismo , Tiorredoxinas/metabolismo , Animais , Glutationa/metabolismo , Infecções por HIV/metabolismo , Infecções por HTLV-I/metabolismo , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Proteínas de Membrana/metabolismo , Peroxidases/metabolismo , Peroxirredoxinas , Infecções por Retroviridae/enzimologia , Infecções por Retroviridae/patologiaRESUMO
Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/farmacologia , MAP Quinase Quinase Quinase 5/metabolismo , Neurônios/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Doença de Alzheimer/etiologia , Animais , Morte Celular/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinase 5/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Óxidos de Nitrogênio/metabolismo , Células PC12 , Fragmentos de Peptídeos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , eIF-2 Quinase/metabolismoRESUMO
We have shown that cell cycle progression of human T-cell leukemia virus type I (HTLV-I)-transformed T-cell lines was inhibited by 13-cis-retinoic acid (13cRA). In the present study, we report that 13cRA inhibited proliferation and induced cell death of peripheral blood mononuclear cells obtained from four patients with acute adult T-cell leukemia but not of mitogen- or interleukin 2-activated peripheral blood mononuclear cells from HTLV-I-negative healthy donors. Because HTLV-I-infected lymphocytes are susceptible to oxidative stress, we examined the role of the intracellular redox state in 13cRA-induced cell death using a HTLV-I-positive T-cell line, ATL2, as a model. 13cRA induced apoptosis in ATL2 cells within 48 h in a dose-dependent manner. The ability of 13cRA to induce apoptosis was more potent than that of all-trans-retinoic acid. Apoptosis induction by 13cRA was significantly enhanced by buthionine sulfoximine (BSO), which decreased the levels of intracellular reduced glutathione, although 13cRA by itself did not alter them, suggesting that intracellular reduced glutathione may modulate 13cRA-induced apoptosis. In addition, flow cytometric analysis revealed that 13cRA increased intracellular peroxides in 24 h and that the addition of BSO further enhanced them. Although N-acetylcysteine had only a marginal effect, pretreatment with catalase markedly inhibited 13cRA-induced apoptosis. These results suggest that peroxide generation, ie., oxidative stress, may play a crucial role in the induction of apoptosis by 13cRA and further demonstrate that combined treatment with 13cRA and BSO induces apoptosis of HTLV-I-positive lymphocytes even more potently.
Assuntos
Apoptose/efeitos dos fármacos , Isotretinoína/farmacologia , Ceratolíticos/farmacologia , Leucemia-Linfoma de Células T do Adulto/fisiopatologia , Estresse Oxidativo/fisiologia , Adulto , Butionina Sulfoximina/farmacologia , Catalase/farmacologia , Divisão Celular/efeitos dos fármacos , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Oxirredução , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2RESUMO
Thioredoxin, a cellular thiol, functions as a self-defense mechanism in response to environmental stimuli, including oxidative stress. We first determined cellular levels of thioredoxin in several human bladder and prostatic cancer cell lines resistant to cis-diamminedichloroplatinum(II) (cisplatin). All cisplatin-resistant cell lines had much higher levels of thioredoxin than those in their drug-sensitive parental counterpart. We then, by introducing thioredoxin antisense expression plasmids into human bladder cancer T24 cells, established two bladder cancer cell lines that had decreased levels of thioredoxin. These thioredoxin antisense transfectants showed increased sensitivity to cisplatin and also to other superoxide-generating agents, i.e., doxorubicin, mitomycin C, etoposide, and hydrogen peroxide, as well as to UV irradiation, but not to the tubulin-targeting agents, vincristine, and colchicine. Cellular levels of thioredoxin thus appear to limit sensitivity to various superoxide-generating anticancer drugs in cancer cells.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Mitomicina/farmacologia , Tiorredoxinas/metabolismo , DNA/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Humanos , Células Tumorais CultivadasRESUMO
Thioredoxin (TRX) is a cytoplasmic, redox-sensitive signaling factor believed to participate in the regulation of nuclear transcription factors mediating cellular responses to environmental stress. Activation of the activator protein (AP)-1 transcription factor is thought to be mediated in part by redox-sensitive interactions between the nuclear signaling protein redox factor-1 (Ref-1) and TRX. In this study, the role of TRX and Ref-1 in the activation of the AP-1 complex was examined in HeLa and Jurkat cell lines exposed to ionizing radiation (IR). After exposure to IR, nuclear levels of immunoreactive TRX increased, accompanied by an increase in AP-1 DNA binding activity. It was shown that a physical interaction between Ref-1 and TRX occurs within the nucleus and is enhanced after exposure to IR. Furthermore, TRX immunoprecipitated from irradiated cells was capable of activating AP-1 DNA binding activity in nonirradiated nuclear extracts. In addition, immunodepletion of Ref-1 from nuclear extracts demonstrated that the increase in AP-1 DNA binding activity after IR was also dependent upon the presence of Ref-1 from irradiated cells. Finally, the ability of both TRX and Ref-1 from irradiated cells to stimulate AP-1 DNA binding in nonirradiated nuclear extracts was abolished by chemical oxidation and restored by chemical reduction. These results indicate that, in response to IR, TRX and Ref-1 undergo changes in redox state that contribute to the activation of AP-1 DNA binding activity. These experiments suggest that a redox-sensitive signaling pathway leading from TRX to Ref-1 to the AP-1 complex participates in the up-regulation of DNA binding activity in response to ionizing radiation.
Assuntos
Carbono-Oxigênio Liases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Transdução de Sinais/efeitos da radiação , Tiorredoxinas/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Anticorpos/farmacologia , Células COS , Carbono-Oxigênio Liases/imunologia , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Chlorocebus aethiops , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , DNA/metabolismo , Células HeLa , Humanos , Oxirredução/efeitos da radiaçãoRESUMO
Protein tyrosine phosphorylation was examined after T cells were exposed to oxidative stress in vitro to investigate the possible involvement of redox regulation in T-cell signaling. Oxidative reagents such as hydrogen peroxide (H2O2) and diamide, which oxidize the free sulfhydryl groups in the cells, markedly induced tyrosine phosphorylation of multiple cellular proteins, especially a 55-kDa protein, of cultured peripheral blood T lymphocytes (PBL blasts). The 55-kDa molecule phosphorylated by diamide turned out to be a src family protein tyrosine kinase, p56lck. The immune complex kinase assay showed that the kinase activity of p56lck of diamide-treated PBL blasts was enhanced. The tryptic peptide mapping of p56lck demonstrated that diamide induced the phosphorylation both at Tyr-394 (autophosphorylation site) and at Tyr-505 (negative regulatory site). Taken together, the tyrosine phosphorylation and presumably kinase activity of p56lck were swiftly enhanced by oxidative stress, indicating that T cells have a redox-sensitive signaling mechanism, which is partly mediated by the lymphocyte-specific protein tyrosine kinase p56lck.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Linfócitos T/enzimologia , Células Cultivadas , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Oxirredução , Fosforilação , Compostos de Sulfidrila/metabolismo , Tirosina/metabolismoRESUMO
Interferons (IFNs) and retinoids are potent tumor growth suppressors. We have shown earlier that the IFN-beta and all-trans retinoic acid combination, but not the single agents, induces death in several tumor cell lines. Employing a genetic approach we have recently identified several Genes associated with Retinoid-IFN induced Mortality (GRIM) that mediate the cell death effect of IFN/RA combination. One of the GRIMs, GRIM-12, was identical to human thioredoxin reductase (TR), an enzyme that controls intracellular redox state. To define the participants of TR mediated death pathway we have examined the role of thioredoxin (Trx), its downstream substrate, and its influence on IFN/RA-induced death regulation. Inhibition of the thioredoxin expression by antisense RNA suppressed cell death. Similarly, a mutant Trx1 lacking the critical cysteine residues blocked cell death. In contrast, overexpression of wildtype thioredoxin augmented cell death. This effect of Trx1 was in part due to its ability to augment cell death via caspase-8. The redox inactive Trx1 mutant inhibits the cell death induced by caspase-8 but not caspase-3. These studies identify a novel mechanism of cell death regulation by IFN/RA combination involving redox enzymes.