Biomedical subjects

S Yonehara

Publications and source records attributed to S Yonehara.

At least 37 records · Page 2Linked to original sources

PubMed2000

Oncogenic K-Ras and basic fibroblast growth factor prevent Fas-mediated apoptosis in fibroblasts through activation of mitogen-activated protein kinase.

By an expression cloning method using Fas-transgenic Balb3T3 cells, we tried to obtain inhibitory genes against Fas-mediated apoptosis and identified proto-oncogene c-K-ras. Transient expression of K-Ras mutants revealed that oncogenic mutant K-Ras (RasV12) strongly inhibited, whereas dominant-inhibitory mutant K-Ras (RasN17) enhanced, Fas-mediated apoptosis by inhibiting Fas-triggered activation of caspases without affecting an expression level of Fas. Among the target molecules of Ras, including Raf (mitogen-activated protein kinase kinase kinase [MAPKKK]), phosphatidylinositol 3 (PI-3) kinase, and Ral guanine nucleotide exchange factor (RalGDS), only the constitutively active form of Raf (Raf-CAAX) could inhibit Fas-mediated apoptosis. In addition, the constitutively active form of MAPKK (SDSE-MAPKK) suppressed Fas-mediated apoptosis, and MKP-1, a phosphatase specific for classical MAPK, canceled the protective activity of oncogenic K-Ras (K-RasV12), Raf-CAAX, and SDSE-MAPKK. Furthermore, physiological activation of Ras by basic fibroblast growth factor (bFGF) protected Fas-transgenic Balb3T3 cells from Fas-mediated apoptosis. bFGF protection was also dependent on the activation of the MAPK pathway through Ras. All the results indicate that the activation of MAPK through Ras inhibits Fas-mediated apoptosis in Balb3T3 cells, which may play a role in oncogenesis.

3T3 Cells↗

PubMed2000

Role of apoptosis induction in both peripheral lymph nodes and thymus in progressive loss of CD4+ cells in SHIV-infected macaques.

To investigate the role of apoptosis in the progressive loss of CD4+ lymphocytes in HIV infection, we have used macaques infected with SHIV, a hybrid virus of HIV and simian immunodeficiency virus (SIV). In the present study, we sequentially analyzed apoptosis induction in the acute phase of SHIV infection. Four macaques infected with a pathogenic SHIV, SHIV89.6P, and four macaques infected with a nonpathogenic SHIV, NM-3rN, were analyzed during the first 2 or 4 weeks postinfection. In the 89.6P-infected macaques the number of peripheral CD4+ cells sharply decreased at 2 weeks postinfection and was maintained below 50/microl until 4 weeks postinfection, while in the NM-3rN-infected macques the number of the CD4+ cells did not change significantly. Plasma viral loads peaked at 2 and 2-3 weeks postinfection, and the peak values were about 1 x 10(9) and 10(6)-10(7) copies/ml in the 89.6P- and the NM-3rN-infected macaques, respectively. In the 89.6P-infected macaques, Fas antigen expression and the extent of apoptosis in PBMCs and peripheral lymph nodes increased at 1-2 weeks postinfection. A high number of apoptotic cells was also observed in thymus sections 2 and 4 weeks postinfection. On the other hand, apoptosis was scarcely induced in the NM-3rN-infected macaques. These results suggest that the extent of apoptosis induction is closely correlated with the pathogenicity of SHIV and that the apoptosis induction in peripheral lymphoid tissues and thymus, where T cell maturation occurs, may play an important role in the depletion of CD4+ lymphocytes in 89.6P infection.

Acquired Immunodeficiency Syndrome↗

PubMed2000

Gelatin beads as platforms for targeting molecule and anti-Fas antibody: two major properties of cytotoxic T lymphocytes.

OBJECTIVE: Cytotoxic T lymphocytes (CTL) initially recognize target cells using the T-cell receptor (TCR), then strongly adhere to these cells by accessory molecules, and finally induce apoptosis by Fas ligand (FasL)/Fas or lyse by the granzyme/perforin system. We describe the development of gelatin beads carrying anti-tumor monoclonal antibody (mAb) and anti-Fas mAb mimicking the TCR and FasL, respectively. We hypothesized that these antibody-coated beads can be therapeutically utilized for the elimination of tumor cells. MATERIALS AND METHODS: We evaluated the cytotoxic activity of gelatin beads bearing CH11 (anti-Fas mAb) after incubation with several human leukemia cell lines. Cytotoxic activities were measured using colorimetric DNA fragmentation assay and lactate dehydrogenase (LDH) release assay. RESULTS: We demonstrated that the cytotoxic effects of anti-Fas mAb were markedly enhanced by fixation on gelatin beads. Microscopic examination showed that the beads attached to the target cells and induced their apoptosis. These effects were enhanced further by adding tumor-specific mAb. These in vitro properties of the beads were well reconstituted in the peritoneal cavity of mice. CONCLUSION: Although antibody-coated gelatin beads lack several important properties of natural CTL, such as differentiation, proliferation, and the functions of adhesion molecules, they mimic well the targeting and cytotoxic functions of natural CTL. Our findings suggest that antibody-carrying gelatin beads may be the first step toward the development of artificial CTL and can be applied, for example, to artificial dendritic and stroma cells for the development of novel biotherapeutic approaches.

Animals↗

PubMed2000

A novel murine anti-human Fas mAb which mitigates lymphadenopathy without hepatotoxicity.

Defects in Fas-mediated apoptosis are implicated in autoimmune diseases including rheumatoid arthritis (RA). Although induction of Fas-mediated apoptosis could have therapeutic effects on these diseases, it might cause deleterious effects in liver as Fas ligand or an agonistic anti-murine Fas antibody Jo2 causes severe hepatic injury in mice. We report here on the interesting characteristics of the newly obtained anti-Fas mAb, HFE7A, which cross-reacts with the Fas molecules of various species ranging from human to mouse and mitigates autoimmune symptoms without hepatotoxicity in mice. The administration of HFE7A to mice induced apoptosis in the thymocytes, although administration of HFE7A to mice or to marmosets did not induce any sign of hepatitis. The effect of HFE7A on liver is different from that of anti-murine Fas antibody Jo2, which causes acute and lethal hepatic injury to mice. Administration of HFE7A reduced lymphadenopathy and abnormal T cells in MRL-gld/gld mice. HFE7A induced apoptosis in synovial cells prepared from RA patients. Surprisingly, HFE7A protected mice from fulminant hepatitis induced by Jo2. Therefore, HFE7A is a potential therapeutic antibody not only for autoimmune diseases including RA but also for fulminant hepatitis.

Animals↗

PubMed2000

Reduction of thymocyte numbers in transgenic mice expressing viral FLICE-inhibitory protein in a Fas-independent manner.

A viral FLIP (FLICE/caspase-8-Inhibitory Protein), equine herpesvirus type 2 E8 protein, has been shown to inhibit Death receptor-induced apoptosis by suppressing the activation of FLICE/caspase-8. We generated transgenic mice specifically expressing E8 in thymocytes under the control of lck-proximal promoter. Although E8-expressing thymocytes were resistant to Fas-mediated apoptosis, the total number of thymocytes in 4-8-week-old E8 transgenic mice was more than 3-fold less than that in control littermates. This reduction was also observed in E8 transgenic mice with a Fas-/- background suggesting the reduction to be independent of Fas. The thymocytes of the transgenic mice, however, could similarly respond to CD3-mediated stimulation, indicating that the reduction of thymocyte numbers might be independent of T cell receptor complex-mediated stimulation. Thus, the Death receptor-mediated signaling pathway is too complex to be regarded as only an executor for apoptosis.

Animals↗

PubMed2000

Significance of Fas antigen-mediated apoptosis in human fulminant hepatic failure.

OBJECTIVE: The aim of this study was to elucidate the role of apoptosis in human fulminant hepatic failure. We studied the expression of Fas antigen on liver tissues, Fas ligand in lymphocytes, and soluble Fas ligand in patients' serum. METHODS: On finding apoptotic cells in fulminant hepatic failure liver, we first examined them using the TUNEL method. Subsequently, the expression of Fas was studied by immunostaining. Simultaneously, Fas ligand presenting on both liver-infiltrated cells and peripheral lymphocytes was studied by reverse transcription-polymerase chain reaction, and soluble Fas ligand in sera was measured by ELISA. RESULTS: By using the TUNEL method, we first demonstrated that many apoptotic cells existed in fulminant hepatic failure but not in normal ones. Our immunohistochemistry study showed that many hepatocytes in fulminant hepatic failure strongly expressed Fas. In addition, Fas ligand on both liver-infiltrating lymphocytes and peripheral lymphocytes in fulminant hepatic failure patients was detected. The serum level of soluble Fas ligand was significantly increased in fulminant hepatic failure (mean value, 2.91 ng/ml in fulminant hepatic failure [n = 10], 1.62 ng/ml in acute hepatitis [n = 10], and 0.27 ng/ml in healthy controls [n = 10]). Furthermore, this serum level of sFas ligand was significantly associated with prothrombin time both in acute hepatitis and fulminant hepatic failure. CONCLUSIONS: The present results indicate that Fas-mediated apoptosis may be one of the triggers for the induction of fulminant hepatic failure.

Adult↗

PubMed2000

Spontaneous and cytokine regulated c-fos gene expression in rheumatoid synovial cells: resistance to cytokine stimulation when the c-fos gene is overexpressed.

OBJECTIVE: To study the effect of cytokines on the transactivation of the c-fos gene in relation to the contribution of overexpression of c-fos/AP-1 in rheumatoid joint destruction. METHODS: The promoter region (-447 to +109) of the human c-fos gene was integrated upstream of the chloramphenicol acetyltransferase (CAT) reporter gene, and the effect of cytokines on the expression of the c-fos gene was studied in the rheumatoid synovial cells of early (3-4) or late (14-18) passages, in the presence or absence of cytokines, by the transient transfection assay. RESULTS: Expression of c-fos gene was enhanced by tumour necrosis factor alpha (TNF alpha) and interleukin 6 (IL6) in the synovial cells of early passage, whereas it was not enhanced in the synovial cells of late passage. The c-fos gene expression was also enhanced by 13-O-tetradecanoyl phorbol-13-acetate (TPA) in early passage but was somewhat suppressed in the late passage. It was found that the c-fos gene and c-Fos protein were both increased in the synovial cells of late passage. Similarly, c-fos gene expression was also not increased by TPA or cytokine stimulation in the stable c-fos transformants (fos-pH8) or H-ras transformed NIH3T3 cells (NIH H-ras cells) that constitutively expressed c-fos genes. CONCLUSIONS: Although TNF alpha and IL6 augmented c-fos gene expression of rheumatoid synovial cells, transactivation of c-fos gene became resistant against cytokine stimulation under prolonged expression of c-fos gene, which may impart a tumour-like characteristic to rheumatoid synovial cells.

Arthritis, Rheumatoid↗

PubMed2000

Cloning and expression of a novel murine anti-human Fas antibody.

Agonistic anti-human Fas antibodies that can induce apoptosis are thought to have therapeutic effects for various diseases resulting from an abnormality of the Fas/FasL system. However, some anti-Fas antibodies show toxicity, and it is difficult to investigate their therapeutic and toxicological effect using animals because of their species specificity. We previously obtained a murine anti-human Fas mAb, HFE7A. HFE7A reacted with both human and murine Fas, and mitigated lymphadenopathy without any sign of hepatotoxicity in MRLgld/gld mice. It is suggested that humanized HFE7A would be a therapeutic treatment for various diseases resulting from an abnormality of the Fas/FasL system. Here we isolated the cDNAs that code for the heavy and light chains of HFE7A and identified the corresponding nucleotide sequences. The recombinant HFE7A was indistinguishable in binding and apoptosis-inducing activity to that from a hybridoma cell line. These data provide essential information for the humanization and clinical application of the humanized HFE7A.

Amino Acid Sequence↗

PubMed1999

Cdc42 and Rac1 regulate the interaction of IQGAP1 with beta-catenin.

IQGAP1, a target of Cdc42 and Rac1 small GTPases, directly interacts with beta-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating alpha-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. (1998) Science 281, 832-835). Here we investigated how Cdc42 and Rac1 regulate the IQGAP1 function. IQGAP1 interacted with the amino-terminal region (amino acids 1-183) of beta-catenin, which contains the alpha-catenin-binding domain. IQGAP1 dissociated alpha-catenin from the beta-catenin-alpha-catenin complex in a dose-dependent manner in vitro. Guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).glutathione S-transferase (GST)-Cdc42 and GTPgammaS. GST-Rac1 inhibited the binding of IQGAP1 to beta-catenin in a dose-dependent manner in vitro, whereas neither GDP.GST-Cdc42, GDP. GST-Rac1, nor GTPgammaS.GST-RhoA did. The coexpression of dominant active Cdc42 with IQGAP1 suppressed the dissociation of alpha-catenin from the cadherin-catenin complex induced by the overexpression of IQGAP1 in L cells expressing E-cadherin (EL cells). Consistent with this, the overexpression of either dominant negative Cdc42 or Rac1 resulted in the reduction of E-cadherin-mediated cell adhesive activity in EL cells. These results indicate that Cdc42 and Rac1 negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with beta-catenin, leading to stabilization of the cadherin-catenin complex.

Carrier Proteins↗

PubMed1999

Sequential analysis of apoptosis induction in peripheral blood mononuclear cells and lymph nodes in the early phase of pathogenic and nonpathogenic SIVmac infection.

To investigate the role of apoptosis in the early phase of HIV infection, we used macaques infected with simian immunodeficiency virus strain mac (SIVmac) as a primate model and examined sequentially the characteristics of apoptosis of lymphocytes in peripheral blood mononuclear cells (PBMCs) and lymph nodes in the early phase of SIVmac infection. Five macaques infected with a pathogenic strain of SIV, SIVmac239, were analyzed during the first 4 weeks after infection. Peripheral CD4+ and CD8+ cells transiently decreased at 1 week postinfection. The percentage of apoptotic cells in cultured PBMCs increased from about 2 weeks postinfection. The number of apoptotic cells in lymph node sections was higher on days 13 and 28 postinfection than before infection and on day 5 postinfection. Fas antigen expression on peripheral lymphocytes was upregulated from day 8 postinfection. These results indicate that apoptosis is induced about 2 weeks after SIVmac239 infection, following the upregulation of Fas antigen expression on lymphocytes. Since apoptosis was induced about 1 week after the decrease in peripheral CD4+ and CD8+ cell counts, it appears that the apoptosis induction does not play an important role in the transient lymphopenia in the early phase of SIVmac infection. In macaques infected with a nonpathogenic derivative of SIVmac239, SIVmac delta nef, apoptosis of lymphocytes was induced as it was in SIVmac239-infected macaques, but to a lesser degree, suggesting a correlation between the extent of apoptosis induction in lymphocytes in the early phase of SIVmac infection and the pathogenicity of SIVmac.

Animals↗

PubMed1999

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis.

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production.

Tumor necrosis factor-alpha (TNF-alpha) exerts two separate effects on neutrophils, stimulating effector functions while simultaneously inducing apoptosis. We examined here the involvement of caspases in neutrophil apoptosis and the effect of TNF-alpha-induced apoptosis on reactive oxygen production. Immunoblotting and affinity labeling showed activation of caspase-8, caspase-3, and a caspase with a large subunit of 18 kD (T18) in TNF-alpha-treated neutrophils. Active caspase-6 and -7 were not detectable in this cell type. Caspase-8 activated caspase-3 and T18 in neutrophil cytoplasmic extracts. zVAD-fmk blocked neutrophil apoptosis, in parallel with the inhibition of caspase activation. TNF-alpha-induced caspase activation was accompanied by a decrease in the ability of neutrophils to release superoxide anion. Conversely, TNF-alpha treatment in the presence of zVAD-fmk caused a prolonged augmentation of superoxide release. Granulocyte-macrophage colony-stimulating factor inhibited TNF-alpha-induced caspase activation and apoptosis, while reversing the diminution in superoxide release. These observations not only suggest that a caspase cascade mediates apoptotic events and downregulates oxygen radical production in TNF-alpha-treated neutrophils, but also raise the possibility that suppression of caspase activation with enhanced proinflammatory actions of TNF-alpha may underlie the pathogenesis of inflammatory diseases.

Adult↗

PubMed1999

Requirement of cooperative functions of two repeated death effector domains in caspase-8 and in MC159 for induction and inhibition of apoptosis, respectively.

BACKGROUND: The death effector domain (DED), which functions as a domain for a homophilic protein interaction, plays a role in death receptor-mediated apoptosis. Two tandemly repeated DEDs in the prodomain of caspase-8 (Casp8NC-DED) and those in MC159 (viral FLIP) have been shown to positively and negatively regulate apoptosis, respectively, by binding to caspase-8 and/or Fas-associated death domain (FADD). However, characteristics of each DED in Casp8NC-DED and those in MC159 have not been well examined. RESULTS: We analysed deletion and chimera mutants of DEDs derived from Casp8NC-DED and MC159, and found that MC159 and Casp8NC-DED require the combined effects of the two repeated DEDs to exert their binding and biological activities. The carboxy-terminal DED of Casp8NC-DED (Casp8C-DED) has the potential to induce apoptosis, and the amino-terminal DED of MC159 showed a dominant inhibitory effect on apoptosis when combined with Casp8C-DED. In addition, the two repeated DEDs in Casp8NC-DED and MC159 were shown to regulate the activities of caspase differently from the caspase recruitment domain (CARD) in the prodomains of caspase-2, -9 and Apaf-1. CONCLUSIONS: Although each of the DEDs in Casp8NC-DED and MC159 has the potential to stimulate or inhibit apoptosis, the combination of the two-repeated DEDs is necessary for the DED-containing proteins to stimulate or inhibit apoptosis.

Animals↗

PubMed1999

Severe impairment of B cell function in lpr/lpr mice expressing transgenic Fas selectively on B cells.

Transgenic lpr/lpr mice expressing functional Fas selectively on B cells were produced in an attempt to elucidate the role of Fas on B cells in the regulation of autoantibody production. The homozygous lpr/lpr mice carrying the transgene did not produce anti-double-stranded DNA antibodies throughout their lives, whereas the development of abnormal lpr T cells (double negative, B220(+)) was not suppressed. Further analyses, however, revealed that the expression of the transgenic Fas on B cells of lpr/lpr homozygous mice resulted in severe impairment of the B cell function. The defect was characterized by a decrease in the number of mature peripheral B cells, a reduction in the serum Ig level and the total failure of B cells to mount antibody responses to stimulations of T-dependent as well as T-independent antigens. Such a defect was prominent only when the transgene was expressed on the lpr/lpr homozygous background. On the contrary, B cells of the transgenic lpr/lpr mice were shown to be capable of producing Ig when stimulated with anti-CD40 in the presence of IL-4 and IL-5. Furthermore, lpr/lpr T cells showed enhanced non-specific cytolytic activity. These observations suggested that the observed B cell defect was probably attributable to the destruction of activated B cells expressing transgenic Fas by aggressive lpr/lpr T cells.

Animals↗

PubMed1999

Fas-induced in vivo apoptosis in bone marrow: anti-Fas mAb-induced elimination and successive proliferation of Fas-expressing cells especially those of myeloid lineage.

A single administration of agonistic anti-Fas mAb RK8 into mice decreased the number of bone marrow cells especially Mac1+ and Gr1+ cells of myeloid lineage. These cells, which were shown to be Fas-positive in normal bone marrow, were directly eliminated in vivo by Fas-mediated apoptosis. After the elimination of Fas-positive bone marrow cells, bone marrow was reconstituted by successive increase of numbers of Gr1(low) and Mac1(low) myeloid precursor cells expressing high levels of Fas, which are minor constituents in normal bone marrow. The increased cells consisted at least two components, Gr1(dull) Mac1+ cKit+ cells and Gr1(intermediate) Mac1+ cKit- cells, both of which were shown to be sensitive to Fas-induced apoptosis in vivo. Thus, Fas is functional in normal bone marrow and Fas-induced apoptosis in bone marrow enhances marked proliferation of Fas-expressing myeloid precursor cells in vivo.

Animals↗

PubMed1999

Effects of anti-Fas antibodies on lymphocytes and other organs: preparation of original and new monoclonal antibodies and amelioration of systemic autoimmune disease.

Fifteen and ten years have passed since original anti-human Fas mAb CH-11 with associated apoptosis-inducing activity was prepared in 1984 and reported in 1989, respectively. Fas antigen (FS-7 cell-associated cell surface antigen) has been shown to be a cell surface receptor molecule (Fas) which can introduce apoptosis-inducing signals into Fas-bearing cells. Studies on lpr (lymphoproliferation) and gld (generalized lymphoproliferative disease) mice, which are loss-of-function mutant mice of Fas and Fas ligand (FasL), respectively, show that these mutations are responsible for the early onset of systemic autoimmune disease in MRL mice, suggesting that autoreactive immunocytes are eliminated by the function of Fas/FasL system ("physiological function" of Fas). Fas/FasL system, however, plays an important role in not only prevention but also aggravation of autoimmune disease. Fas/FasL was shown to be involved in the mechanisms responsible for tissue disruption in autoimmune diseases ("pathological function" of Fas). Studies on the in vivo administration of anti-mouse Fas mAbs, Jo2 and RK-8, emphasized pathological and physiological functions of Fas, respectively: Jo2 induces fulminant hepatitis and RK-8 ameliorates systemic autoimmune disease without the induction of fulminant hepatitis. The different in vivo effects of Jo2 and RK-8 coincide well with different target cell specificity of these mAbs in vitro. Similarly, I observed different target cell specificity on anti-human Fas mAbs. In this review, I propose new strategy of therapeutic use of anti-Fas mAb as specific suppressor for activated immune system on autoimmune disease and other diseases.

Animals↗

PubMed1998

The development of autoimmune inflammatory arthropathy in mice transgenic for the human T cell leukemia virus type-1 env-pX region is not dependent on H-2 haplotypes and modified by the expression levels of Fas antigen.

Previously, we reported that human T cell leukemia virus type-1 env-pX region-introduced transgenic (pX-Tg) mice develop an inflammatory polyarthropathy. Although autoimmune pathogenesis was suggested, the detailed mechanisms remain to be elucidated. In this report, we examined effects of the MHC and fas genes on the development of the disease. When pX-Tg mice were backcrossed with different inbred strains, the incidence of arthritis differed among strains; 64% and 72% in BALB/cAn (H-2d), 25% and 46% in C3H/HeN (H-2k), and 0% and 2% in C57BL/6J (H-2b) background at 3 and 6 months of age, respectively. Rheumatoid factor levels in the serum correlated with the susceptibility to the disease, whereas IL-1beta and MHC gene expression were similarly elevated in all of these strains, suggesting involvement of immune regulatory genes in this strain difference. However, introduction of the H-2d locus into C57BL/6J pX-Tg mice did not increase the incidence of arthritis, and substitution of the BALB/cAn H-2 locus with the H-2b did not decrease it. The results indicate that the H-2 locus is not the major determinant of the disease. Then, since previous study indicated a defect in Fas-mediated apoptosis of transgenic T cells, the effects of fas gene modification on the disease were examined. The incidence increased when these pX-Tg mice were crossed with lpr/lpr mice, while it decreased when crossed with fas-transgenic mice. These observations suggest that aberration of Fas-mediated apoptosis of peripheral lymphocytes, rather than negative selection in the thymus, is involved in the development of autoimmune arthropathy in pX-Tg mice.

Animals↗

PubMed1998

Redox regulation of caspase-3(-like) protease activity: regulatory roles of thioredoxin and cytochrome c.

Oxidative stress induces a variety of cellular responses, including apoptosis, and caspase family proteases are known to be involved in apoptosis. Caspase-3(-like) protease activity was examined in Jurkat T cells to investigate the mechanism of apoptosis induced by a thioloxidant, diamide. Caspase-3 was activated when cells were cultured with 200 microM diamide that induced apoptosis, whereas no caspase-3 activation was detected with 500 microM diamide that induced necrosis. When apoptosis was induced in cells with exposure to 200 microM diamide, the intracellular thioredoxin (TRX) levels were maintained and the intracellular generation of reactive oxygen intermediates was marginal. The cytosolic fractions of cytochrome c were increased earlier than the activation of caspase-3. In contrast, when cells were exposed to 500 microM diamide, intracellular reactive oxygen intermediate generation was increased and processing of caspase-3 was not detected despite cytochrome c release, resulting in necrosis. Caspase-3 activity in cell lysate precultured with anti-Fas Ab was suppressed dose dependently by diamide and restored by thiol-reducing agents, DTT or TRX. When cells were precultured with 5 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, intracellular TRX levels were maintained, and as low as 20 microM diamide could induce apoptosis associated with the increase of cytosolic cytochrome c and the activation of caspase-3. These results indicate that the activation of caspase-3 in diamide-induced apoptosis is mediated, at least partly, by cytochrome c release from mitochondria, and the cellular reducing environment maintained by TRX, as well as glutathione, is required for caspase-3 activity to induce apoptosis.

Antibodies, Monoclonal↗