MEK1 activation rescues Jurkat T cells from Fas-induced apoptosis.

Although the protease cascade initiated by Fas (CD95, Apo-1) is well characterized, there remains little known about how kinase pathways may impact on Fas-mediated apoptosis. We recently observed that in T lymphocytes Fas strongly induced activation of JNK (c-Jun N-terminal kinase) but not of second messengers leading to activation of ERK (extracellular regulated kinase). Additionally, Fas-mediated apoptosis was significantly inhibited with PMA, a potent activator of the ERK signaling pathway. This suggested a model whereby activation of the ERK pathway might attenuate Fas-mediated apoptosis. This was confirmed in the current study by showing that activation of MEK1, the upstream regulator of ERK, reduces Fas-mediated apoptosis, whereas inhibition of MEK1 augments apoptosis by Fas. Furthermore, Fas-mediated apoptosis of Jurkat T cells is not affected by constitutively active or dominant negative variants that modulate the JNK pathway. These results demonstrate that Fas-induced JNK activation is not required for apoptosis by Jurkat T cells, but rather is more likely secondary to cell stress during the early phases of apoptosis. This is supported by the ability of the caspase blocker zVAD to inhibit both apoptosis and JNK activation by Fas.     

Morphological changes in the nucleus and actin cytoskeleton in the process of Fas-induced apoptosis in Jurkat T cells

To investigate the early event of apoptosis, we monitored the morphological changes in the early stage of Fas-induced apoptosis in the human T-cell lymphoma cell line Jurkat, using confocal microscopy. Morphological changes in the nuclei were observed from 30min after stimulation, and preceded the changes in the cytoskeleton. This kind of change was enhanced in the presence of EGTA but decreased in the presence of dihydrocytochalasin B, without any changes in caspase-3 activation. During the changes in shape of the cells, the actin cytoskeleton collapsed and shrank in the center. Even though nuclei also changed their shapes in apoptotic cells, they were partially TUNEL-negative, suggesting that they were not yet damaged at the DNA level. Our results suggest that, in the process of apoptosis in Jurkat cells, cell nuclei and cytoskeleton are changed first, then membrane blebbing and caspase-3 activation occur, and fragmentation of chromosomal DNA is last.     

Functional characterization of Jurkat T cells rescued from CD95/Fas-induced apoptosis through the inhibition of caspases

The caspases are known to play a pivotal role in the triggering and execution of apoptosis in virtually all cell types. Because inappropriate apoptosis is a prominent feature of many human diseases, the caspases are attractive targets for therapeutic intervention. In the present study we investigated whether Jurkat T lymphocytes rescued from Fas-induced cell death through the inhibition of caspases are functional. Here we show that the pan-caspase, tripeptide inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Ome) fluoromethylketone (z-VAD-FMK), inhibited the activation of caspase-2, -3, -7, and -8, and subsequently apoptosis in Jurkat T lymphocytes induced by agonistic anti-Fas. The apoptotic signals induced by the cross-linking of the Fas antigen have a relatively long half-life, as z-VAD-FMK had to be continuously present in the culture medium for 72 h after Fas stimulation in order to maintain cell survival. After 72 h, the z-VAD-FMK-rescued cells proliferate normally and responded to activation induced cell death after phytohaemaglutinin treatment, and readily undergo apoptosis when restimulated with agonistic Fas antibodies. Taken together, our results demonstrate that Jurkat T cells rescued from Fas-mediated cell death through the inhibition of caspases are functional.     

Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13II sensitizes Jurkat T cells to Ras-mediated apoptosis

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13(II). We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13(II) localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13(II), using both transient and stable expression vectors. Our data indicate that p13(II)-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13(II)-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13(II)'s influence on lymphocyte survival. Our data are the first to demonstrate that p13(II) alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.     

Fas-induced apoptosis in B cells

Engagement of the cell surface receptor Fas/APO-1 (CD95) initiates a sequence of intracellular events that leads to apoptotic cell death, and this outcome occurs in B cells as it does in other cell types. Fas signaling for B cell death is of particular interest because the expression and function of Fas is altered by engagement of additional cell surface receptors, leading to marked receptor-specific variation in susceptibility to Fas-induced apoptosis. Evidence suggests that the sensitivity of B cells to Fas-mediated apoptosis is intimately connected to homeostasis in the serological arm of the immune system and plays a role in the dysregulation that occurs in certain autoimmune and malignant dyscrasias.     

Radiation-induced apoptosis and cell cycle progression in Jurkat T cells.

The purpose of this study was to explore the connection between radiation-induced apoptosis and progression of cells through the phases of the cell cycle. Cells of the human T-cell line Jurkat were separated by centrifugal elutriation into populations enriched in G(1)-, S- and G(2)/M-phase cells before irradiation. After a dose of 20 Gy, the onset of massive apoptosis occurred at about 6 h in all populations regardless of the phase of the cell cycle in which they were irradiated. In contrast, after 2 Gy, cells died at various times after a pronounced G(2)/M-phase arrest. These results indicate that radiation-induced apoptosis can occur independently of cell cycle arrest and that the time for onset of apoptosis may be dependent on the radiation dose.     

Dipropylcyclopentylxanthine triggers apoptosis in Jurkat T cells by a receptor-independent mechanism

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a xanthine analog used as selective antagonist of adenosine receptors, caused apoptosis in a human leukemia T cell line. Jurkat cells treated with DPCPX underwent apoptosis as demonstrated by flow cytometry, by DNA fragmentation and by accumulation of histones, H2A, H2B, H3 and H4, in the nucleoplasm of cells. Cell cycle and cell sorting analyses indicated an arrest of cells in G(2)/M followed by the appearance of apoptotic cells in G(1) and G(2)/M phases. The mechanism of programmed cell death does not seem to be mediated by signal transduction events at the plasma membrane since it did not involve activation of cell membrane receptors and modification of the intracellular levels of Ca(2+) or cAMP. Apoptosis by incorporation into DNA of a derivative of DPCPX is suggested in basis of the presence of radioactivity label in the DNA obtained from cells preincubated with [(3)H]DPCPX.     

Increase of Fas-induced apoptosis by inhibition of extracellular phosphorylation of Fas receptor in Jurkat cell line

Apoptosis signalling through the Fas pathway requires several steps of aggregation of the Fas receptor in the membrane, including aggregation that may occur in the absence of Fas ligand. Association of Fas domains is determinant to signal transmission following Fas ligand binding to a specific domain. The domains involved in Fas aggregation are located in its extracellular region and contain three potential protein kinase C-binding motifs. We therefore studied the possibility that phosphorylation of the extracellular region of Fas might be implicated in the regulation of Fas-mediated apoptosis. Inhibition experiments of extracellular phosphorylation were performed in human Jurkat T leukemia cells with K252b, an impermeant protein-kinase inhibitor. Extracellular phosphorylation of Fas receptor was related to ecto-kinase, as assessed by the [γ-³²P] ATP labelling of Fas-116 kDa aggregates, suppressed by K252b inhibitor which significantly increased the sensitivity to Fas-mediated apoptosis. Ecto-PKC involvement was demonstrated by bisindolylmaleimide VIII, a selective inhibitor of protein kinase C which significantly increased both Fas aggregation in the membrane and Fas-mediated apoptosis and by the addition of the PKC pseudo-substrate 19–36 which inhibited the phosphorylation of 116 kDa Fas aggregates. These data support a role for Fas phosphorylation in the decreased sensitivity to apoptosis in the Jurkat T leukemia cell line. ^*There was an equal contribution from these two authors.     

Combined action of ERK and NF kappa B mediates the protective effect of phorbol ester on Fas-induced apoptosis in Jurkat cells

The mechanisms whereby phorbol esters antagonize Fas-induced apoptosis in Jurkat T cells are poorly defined. In the present study, we report that protection from Fas-induced apoptosis by 12-O-tetradecanoylphorbol 13-acetate (TPA) is dependent on both ERK and NF kappa B activation. First, we showed that two specific mitogen-activated protein kinase/ERK kinase-inhibitors, PD98059 and U0126, both counteracted TPA-mediated suppression of Fas-induced apoptosis. Moreover, the dose-dependence of U0126-mediated inhibition of ERK phosphorylation correlated with that of reversion of the anti-apoptotic effect of TPA. Second, we observed an excellent correlation between repression of TPA-induced NF kappa B activation by an irreversible inhibitor of I kappa B alpha phosphorylation, BAY11-7082, and its ability to abrogate TPA-induced suppression of Fas-mediated apoptosis. Furthermore, we located the anti-apoptotic effect of both ERK and NF kappa B to lie upstream of the mitochondrial membrane potential depolarization event. Finally, although each inhibitor at optimal, non-toxic concentration by itself only partly reversed TPA-mediated repression of apoptosis, the combination of U0126 and BAY11-7082 completely abolished the anti-apoptotic effect of TPA. Together these findings suggest that TPA-induced activation of ERK and NF kappa B are parallel events that are both required for maximal inhibition of Fas-induced apoptosis in Jurkat T cells.     

Cleavage of nonmuscle myosin heavy chain-A during apoptosis in human Jurkat T cells

We have previously reported that calpastatin, an endogenous inhibitory protein of calpain, is cleaved by a caspase-3-like protease during apoptosis in human Jurkat T cells [Kato, M. et al. (2000) J. Biochem. 127, 297-305]. In this study, we found that nonmuscle myosin heavy chain-A (NMHC-A) is cleaved during apoptosis in Jurkat cells by using a cleavage-site-directed antibody for calpastatin. The cleavage-site-directed antibody was raised against the amino-terminal fragment of calpastatin, and this antibody detected the in vitro cleaved calpastatin fragment. Although cleaved calpastatin was not detected, a 95-kDa polypeptide (p95) was detected in apoptotic cells by this antibody. This p95 was identified as the carboxyl-terminal fragment of NMHC-A based on the results of peptide mass spectrometry fingerprinting and amino-terminal sequencing. Furthermore, two cleavage sites on NMHC-A, Asp-1153 and Asp-1948, were determined, and three cleaved fragments of NMHC-A, one cleaved at Asp-1153 and the other two cleaved at Asp-1948, were detected by cleavage-site-directed antibodies against each cleavage site. The results of confocal immunofluorescence microscopic analysis show that the cleavage at Asp-1948 occurs faster than that at Asp-1153 during apoptosis. In addition, the Asp-1153 cleaved fragment was distributed diffusely in the cytoplasm of apoptotic cells, whereas the Asp-1948 cleaved fragments were detected as condensed dots. In conclusion, our findings can be summarized as follows: (i) NMHC-A is cleaved at two sites during apoptosis, (ii) the timing of cleavage is different between these two cleavage sites, and (iii) the distribution of cleaved fragments is different in apoptotic cells.     

Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.     

Cleavage at the carboxyl-terminus of Ku80 during apoptosis in human Jurkat T cells

We have previously reported that the amount of Apg-2, an Hsp110 family protein, decreases during apoptosis in Jurkat T cells. Since we hypothesized that Apg-2 would be cleaved by caspase-3 during apoptosis, a cleavage-site-directed antibody was raised against the carboxyl-terminus of the Apg-2 fragment that appears after the cleavage by caspase-3. Although this antibody could not detect the Apg-2 fragment in apoptotic cells, three additional fragments were unexpectedly detected. Based on the results of microsequencing, one of these fragments was identified as Ku80. Ku80 is a nuclear protein and a component of DNA-dependent protein kinase (DNA-PK). In this study, we observed that Ku80 is cleaved at Asp-730 residue during apoptosis, and this cleavage occurs in the nucleus in the early apoptotic phase. Furthermore, Ku80 is distributed in the cytoplasm of nuclear fragmented apoptotic cells, although the cleaved fragment contains the nuclear-localization signal (NLS). Our study clearly shows that Ku80 is cleaved in the nucleus, and distributes in the cytoplasm during apoptosis.     

Effect of 4-hydroxynonenal on antioxidant capacity and apoptosis induction in Jurkat T cells

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation and may have either physiological or pathological significance regulating cell proliferation. We studied some biochemical effects of HNE, at various concentrations (0.1-100 μM), on Jurkat T cells incubated thereafter for 24, 48 and 72 h. HNE at low concentrations significantly enhanced the proliferation index, whereas at higher concentrations progressively blocked cell proliferation. Caspase 3 activity increased significantly at HNE concentrations between 1 and 10 μM and decreased at higher concentrations. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) increased progressively with HNE concentrations, particularly GSH-Px. Glucose-6-phosphate dehydrogenase (G6PDH) showed a different pattern, increasing at low HNE (1-5 μM) concentrations and rapidly declined thereafter. These results show that HNE may induce growth inhibition of Jurkat T cells and regulate the activity of typical antioxidant enzymes. Furthermore, the protective effect of doubling the foetal calf serum still points out the risk that cultured cells undergo oxidative stress during incubation.     

Protection against CD95-mediated apoptosis by inorganic mercury in Jurkat T cells.

Dysregulation of CD95/Fas-mediated apoptosis has been implicated as a contributing factor in autoimmune disorders. Animal studies clearly have established a connection between mercury exposure and autoimmune disease in rodents, while case reports have suggested a link between accidental mercury contamination and autoimmune disease in humans. The mechanism(s) for these associations are poorly understood. Using the Jurkat cell model, we have found that low levels (相似文献   

Phosphatidylserine exposure in Fas type I cells is mitochondria-dependent

Previous studies have demonstrated that Fas-triggered activation of effector caspases and subsequent nuclear apoptosis either is mitochondria-independent (type I cells) or relies on mitochondrial amplification of the initial stimulus (type II cells). We show herein that Bcl-2 overexpression in a prototypic type I cell line (SKW6.4) promotes mitochondrial generation of ATP and blocks Fas-triggered plasma membrane externalization of phosphatidylserine (PS). Moreover, overexpression of Bcl-2 attenuates macrophage engulfment of Fas-triggered cells. Fas-mediated DNA fragmentation, on the other hand, remains unaffected in SKW6.4-bcl-2 cells. These studies thus demonstrate that PS externalization and clearance of cell corpses are mitochondria-dependent events, and show that these events can be dissociated from other features of the apoptotic program, in Fas type I cells.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.