Pretreatment Of Jurkat Cells Research Articles

Acyclovir induces cell cycle perturbation and apoptosis in Jurkat leukemia cells, and enhances chemotherapeutic drug cytotoxicity

Aims and methodsMany antiviral agents have been reported to present direct cytotoxic activity in cancer, showing antiproliferative and proapoptotic effects through different mechanisms. In the present study, we took into account the cytotoxic action of the antiviral drug acyclovir (ACV) on leukemia cells, by investigating cell cycle perturbations and apoptosis induction upon drug administration to three still unexplored cell lines, namely Jurkat, U937, and K562. At the same time, the cytotoxicity of cisplatin (CDDP) and 5‑fluorouracil (5‑FU) in combination with ACV was assessed, thus to evaluate if the antiviral agent could enhance cancer cell sensitivity to these chemotherapeutic drugs. Findings and significanceOur results showed that ACV cytotoxic action was maximum in Jurkat cells (acute T cell leukemia), which showed a dose- and time-dependent reduction of cell viability after drug exposure. The flow cytometric analysis of cell cycle revealed a delay/block in S phase and an increase of the sub-G1 peak upon ACV administration, thereby indicating apoptotic cell death. The activation of caspase-3 and the presence of nuclear DNA fragmentation confirmed the induction of apoptosis in ACV-treated cells. Interestingly, the pre-treatment of Jurkat cells with ACV for 72 h or 7 days increased CDDP and 5-FU cytotoxicity, suggesting enhanced leukemia cell sensitivity to these anticancer drugs.

Post‐transcriptional regulation of interleukin‐3: roles of AREs, HuR and p38 (749.5)

Interleukin‐3 (IL‐3) is a pro‐inflammatory cytokine that is secreted by T‐cells upon stimulation, although its mRNA is constitutively expressed, suggesting that is regulated at this level. Our group has shown that the IL‐3 3’UTR is responsible for repressing its expression, being the Adenosine/Uridine‐Rich Elements (ARE) region mainly responsible for this repression. Chimeric luciferase constructs harboring either wild‐type IL‐3 3’ UTR or different mutations on its ARE region were transfected on Jurkat cells and the reporter activity was measured upon T‐cell stimulation. We observed that when the nonamer ARE is interrupted, the 3’UTR is not able to elicit the same response as the wild type 3’UTR. We carried out EMSAs using protein extracts from Jurkat cells at 6, 12 and 24hrs after activation and identified HuR as an ARE‐Binding Protein able to bind the IL‐3 3’UTR AREs. HeLa cells were co‐transfected with plasmids over‐expressing HuR and the luciferase construct which harbors the IL‐3 3’UTR. Luciferase activity and mRNA levels decreased when HuR was over‐expressed. Since p38 has been shown to increase pro‐inflammatory cytokine secretion and is known to phosphorylate HuR, we assessed its possible role in the regulation of IL‐3. After pretreatment of Jurkat cells with the p38 inhibitor SB202190, IL‐3 mRNA levels were significantly decreased upon T‐cell stimulation suggesting that p38 is indeed involved in the post‐transcriptional regulation of IL‐3.Grant Funding Source: Supported by U54 CA96297; NIGMS MBRS RISE R25GM061838

Down-Regulation of 11β-Hydroxysteroid Dehydrogenase Type 2 by Bortezomib Sensitizes Jurkat Leukemia T Cells against Glucocorticoid-Induced Apoptosis

11β-hydroxysteroid dehydrogenases type 2 (11β-HSD2), a key regulator for pre-receptor metabolism of glucocorticoids (GCs) by converting active GC, cortisol, to inactive cortisone, has been shown to be present in a variety of tumors. But its expression and roles have rarely been discussed in hematological malignancies. Proteasome inhibitor bortezomib has been shown to not only possess antitumor effects but also potentiate the activity of other chemotherapeutics. In this study, we demonstrated that 11β-HSD2 was highly expressed in two GC-resistant T-cell leukemic cell lines Jurkat and Molt4. In contrast, no 11β-HSD2 expression was found in two GC-sensitive non-hodgkin lymphoma cell lines Daudi and Raji as well as normal peripheral blood T cells. Inhibition of 11β-HSD2 by 11β-HSD inhibitor 18β-glycyrrhetinic acid or 11β-HSD2 shRNA significantly increased cortisol-induced apoptosis in Jurkat cells. Additionally, pretreatment of Jurkat cells with low-dose bortezomib resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis, more cells arrested at G1 stage and up-regulation of GC-induced leucine zipper which is an important mediator of GC action. Furthermore, we clarified that bortezomib could dose-dependently inhibit 11β-HSD2 messenger RNA and protein levels as well as activity (cortisol-cortisone conversion) through p38 mitogen-activated protein kinase signaling pathway. Therefore, we suggest 11β-HSD2 is, at least partially if not all, responsible for impaired GC suppression in Jurkat cells and also indicate a novel mechanism by which proteasome inhibitor bortezomib may influence GC action.

Bioactive Properties of Wood Knot Extracts on Cultured Human Cells

Not all felled wood is converted to timber or pulp, with the remaining material being a rich source of relatively unexplored and unexploited potentially novel bioactive compounds. Therefore the potential bioactive effects of two softwood knot (the part of the branch encased in the tree stem) extracts--namely, Pinus banksiana Lamb. (Jack pine) and Picea sitchensis (Bong.) Carr. (Sitka spruce)--were investigated by (1) determining their effects on the viability and antioxidant status of human Jurkat T cells, (2) investigating potential cytoprotective and genoprotective effects against oxidative stress in cultured cells, and (3) assessing their effects on concanavalin A (ConA)-induced interleukin-2 (IL-2) production. Initially, both Jack pine knot and Sitka spruce knot extracts were shown to possess strong antioxidant activity as determined by the ferric reducing antioxidant power assay. When added to Jurkat cells, Jack pine knot extract was more toxic compared with Sitka spruce knot extract, with concentrations that resulted in 50% cell death of 153.0 microg/mL and 376.1 microg/mL, respectively. Supplementation of Jurkat cells with wood knot extracts did not affect their glutathione content or catalase activity. Pretreatment of Jurkat cells with Sitka spruce or Jack pine knot extracts protected against H(2)O(2)-induced cell injury. However, none of the extracts protected against H(2)O(2)-induced DNA damage. Jack pine knots, at a concentration of 30 microg/mL, significantly suppressed ConA-induced IL-2 production. Although total phenol content did not differ between the two extracts, gas chromatography analysis did show variation in the types of constituents present. Further research is warranted to elucidate the selective bioactive properties of these softwood knot extracts.

Luteolin inhibits myelin basic protein‐induced human mast cell activation and mast cell‐dependent stimulation of Jurkat T cells

Allergic inflammation and autoimmune diseases, such as atopic dermatitis, psoriasis and multiple sclerosis (MS), involve both mast cell and T-cell activation. However, possible interactions between the two and the mechanism of such activations are largely unknown. Human umbilical cord blood-derived cultured mast cells (hCBMCs) and Jurkat T cells were incubated separately or together, following activation with myelin basic protein (MBP), as well as with or without pretreatment with the flavonoid luteolin for 15 min. The supernatant fluid was assayed for inflammatory mediators released from mast cells and interleukin (IL)-2 release from Jurkat cells. MBP (10 microM) stimulates hCBMCs to release IL-6, IL-8, transforming growth factor (TGF)-beta1, tumour necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF), histamine and tryptase (n=6, P<0.05). Addition of mast cells to Jurkat cells activated by anti-CD3/anti-CD28 increases IL-2 release by 30-fold (n=3, P<0.05). MBP-stimulated mast cells and their supernatant fluid further increase Jurkat cell IL-2 release (n=3, P<0.05). Separation of mast cells and activated Jurkat cells by a Transwell permeable membrane inhibits Jurkat cell stimulation by 60%. Pretreatment of Jurkat cells with a TNF-neutralizing antibody reduces IL-2 release by another 40%. Luteolin pretreatment inhibits mast cell activation (n=3-6, P<0.05), Jurkat cell activation and mast cell-dependent Jurkat cell stimulation (n=3, P<0.05). Mast cells can stimulate activated Jurkat cells. This interaction is inhibited by luteolin, suggesting that this flavonoid may be useful in the treatment of autoimmune diseases.

Calpain-dependent calpastatin cleavage regulates caspase-3 activation during apoptosis of Jurkat T cells induced by Entamoeba histolytica

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.

The Gα13-Rho Signaling Axis Is Required for SDF-1-induced Migration through CXCR4

The CXC chemokine stromal cell-derived factor-1alpha (SDF-1) binds to CXCR4, a seven-transmembrane G protein-coupled receptor that plays a critical role in many physiological processes that involve cell migration and cell fate decisions, ranging from stem cell homing, angiogenesis, and neuronal development to immune cell trafficking. CXCR4 is also implicated in various pathological conditions, including metastatic spread and human immunodeficiency virus infection. Although SDF-1-induced cell migration in CXCR4-expressing cells is sensitive to pertussis toxin treatment, hence involving heterotrimeric G proteins of the G(i) family, whether other G proteins participate in the chemotactic response to SDF-1 is still unknown. In this study, we took advantage of the potent chemotactic activity of SDF-1 in Jurkat T-cells to examine the nature of the heterotrimeric G protein subunits contributing to CXCR4-mediated cell migration. We observed that whereas G(i) and Gbetagamma subunits are involved in SDF-1-induced Rac activation and cell migration, CXCR4 can also stimulate Rho potently leading to the phosphorylation of myosin light chain through the Rho effector, Rho kinase, but independently of G(i). Furthermore, we found that Galpha(13) mediates the activation of Rho by CXCR4 and that the functional activity of both Galpha(13) and Rho is required for directional cell migration in response to SDF-1. Collectively, our data indicate that signaling by CXCR4 to Rho through Galpha(13) contributes to cell migration when stimulated by SDF-1, thus identifying the Galpha(13)-Rho signaling axis as a potential pharmacological target in many human diseases that involve the aberrant function of CXCR4.

Oxidative modification of IκB by monochloramine inhibits tumor necrosis factor α-induced NF-κB activation

We have previously reported that monochloramine (NH 2Cl), a neutrophil-derived oxidant, inhibited tumor necrosis factor α (TNFα)-induced expression of cell adhesion molecules and nuclear factor-κB (NF-κB) activation (Free Radical Research 36 (2002) 845–852). Here, we studied the mechanism how NH 2Cl inhibited TNFα-induced NF-κB activation, and compared the effects with taurine chloramine (Tau–NHCl). Pretreatment of Jurkat cells with NH 2Cl at 70 μM resulted in suppression of TNFα-induced IκB phosphorylation and degradation, and inhibited NF-κB activation. In addition, a slow-moving IκB band appeared on SDS-PAGE. By contrast, Tau–NHCl for up to 200 μM had no effects. Interestingly, NH 2Cl did not inhibit IκB kinase activation by TNFα. Protein phosphatase activity did not show apparent change. When recombinant IκB was oxidized by NH 2Cl in vitro and phosphorylated by TNFα-stimulated Jurkat cell lysate, its phosphorylation occurred less effectively than non-oxidized IκB. In addition, when NF-κB–IκB complex was immunoprecipitated from NH 2Cl-treated cells and phosphorylated in vitro by recombinant active IκB kinase, native IκB but not oxidized IκB was phosphorylated. Amino acid analysis of the in vitro oxidized IκB showed methionine oxidation to methionine sulfoxide. Although Tau–NHCl alone had little effects on TNFα-induced NF-κB activation, simultaneous presence of Tau–NHCl and ammonium ion significantly inhibited the NF-κB activation, probably through the conversion of Tau–NHCl to NH 2Cl. These results indicated that NH 2Cl inhibited TNFα-induced NF-κB activation through the oxidation of IκB, and that NH 2Cl is physiologically more relevant than Tau–NHCl in modifying NF-κB-mediated cellular responses.

The phosphorylation of phospholipase C-gamma1, Raf-1, MEK, and ERK1/2 induced by a conserved retroviral peptide

A synthetic 17-amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins has been found to exhibit suppressive properties for numerous immune functions. It has been shown that CKS-17 causes an imbalance of human types 1 and 2 cytokines and inhibition of the immune responses of lymphocytes, monocytes, and macrophages. CKS-17 induced increased intracellular levels of cAMP, which plays an important role in regulation of cytokine biosynthesis. In this study, using a Jurkat T-cell line and Western blot analysis, CKS-17 induced phosphorylation of PLC-gamma1, Raf-1, MEK and ERK1/2. Using a PLC selective inhibitor U73122 or PLC-gamma1-deficient Jurkat cell line, phosphorylation induced by CKS-17 of ERK1/2, PLC-gamma1, or Raf-1, respectively, were undetectable or significantly reduced. Reintroduction of PLC-gamma1 into the PLC-gamma1-deficient Jurkat cells restored the phosphorylation of ERK1/2 and PLC-gamma1 induced by CKS-17. Further, pretreatment of Jurkat cells with PKC inhibitors blocks the phosphorylation of Raf-1, MEK, and ERK1/2 induced by CKS-17. These results indicate that CKS-17 induces the PLC-gamma1-PKC-Raf-1-MEK-ERK1/2 signaling pathway.

Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro.

DNA is a nuclear macromolecule that circulates in the blood where its levels can reflect the activity of inflammatory and malignant diseases. While dead and dying cells have usually been considered the source of blood DNA, the mechanisms for its release during apoptosis and necrosis are not well defined. To elucidate DNA release, an in vitro model system was used, assessing DNA in the media of living, apoptotic or necrotic Jurkat and U937 cells. Apoptosis was induced by etoposide, camptothecin or staurosporine, while necrosis was induced by heating at 56 degrees C. DNA release was measured by fluorometry with the dye PicoGreen while the extent of death was measured by fluorescence-activated cell sorter analysis with propidium iodide and annexin. Apoptotic Jurkat cells released significantly more DNA in the media than untreated cells while necrotic cells did not show significant DNA release. U937 cells showed similar findings. Pretreatment of Jurkat cells with z-VAD-fmk, a caspase inhibitor, reduced both apoptosis and DNA release. By gel electrophoresis, extracellular DNA from apoptotic cells showed laddering with low molecular weight fragments. These studies suggest that extracellular release of DNA is a consequence of apoptosis and may account for some of the DNA in the blood.

Caspase-2 and caspase-7 are involved in cytolethal distending toxin-induced apoptosis in Jurkat and MOLT-4 T-cell lines.

Cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is a G2/M cell-cycle-specific growth-inhibitory toxin that leads to target cell distension followed by cell death. To determine the mechanisms by which A. actinomycetemcomitans CDT acts as an immunosuppressive factor, we examined the effects of highly purified CDT holotoxin on human T lymphocytes. Purified CDT was cytolethal toward normal peripheral T lymphocytes that were activated by in vitro stimulation with phytohemagglutinin. In addition, purified CDT showed cytolethal activity against Jurkat and MOLT-4 cells, which are known to be sensitive and resistant, respectively, to Fas-mediated apoptosis. Death in these cell lines was accompanied by the biochemical features of apoptosis, including membrane conformational changes, intranucleosomal DNA cleavage, and an increase in caspase activity in the cells. Pretreatment of Jurkat cells with the general caspase inhibitor z-VAD-fmk mostly suppressed CDT-induced apoptosis. Furthermore, specific inhibitors of caspase-2 and -7 showed significant inhibitory effects on CDT-induced apoptosis in Jurkat cells, and these inhibitory effects were fully associated with reduced activity of caspase-2 or -7 in the CDT-treated Jurkat cells. These results strongly suggest that CDT possesses the ability to induce human T-cell apoptosis through activation of caspase-2 and -7.

Signaling through P2X7 Receptor in Human T Cells Involves p56 lck , MAP Kinases, and Transcription Factors AP-1 and NF-κB

ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate multiple downstream signaling events in a human T-lymphoblastoid cell line.

Entamoeba histolytica-induced dephosphorylation in host cells.

Activation of host cell protein tyrosine phosphatases (PTPases) and protein dephosphorylation is an important mechanism used by various microorganisms to deactivate or kill host defense cells. To determine whether protein tyrosine dephosphorylation played a role in signaling pathways affecting Entamoeba histolytica-mediated host cell killing, we investigated the involvement of PTPases during the attachment of E. histolytica to target cells. We observed a rapid decrease in cellular protein tyrosine levels in Jurkat cells, as measured with an antiphosphotyrosine monoclonal antibody, following adherence to E. histolytica. Ameba-induced protein dephosphorylation was contact dependent and required intact parasite, since blocking amebic adherence with galactose inhibited tyrosine dephosphorylation and amebic lysates had no effect on phosphotyrosine levels. Moreover, disruption of amebic adherence with galactose promoted recovery of phosphorylation in Jurkat cells, indicating that dephosphorylation precedes target cell death. The evidence suggests that ameba-induced dephosphorylation is mediated by host cell phosphatases. Prior treatment of Jurkat cells with phenylarsine oxide, a PTPase inhibitor, inhibited ameba-induced dephosphorylation. We also found proteolytic cleavage of the PTPase 1B (PTP1B) in Jurkat cells after contact with amebae. The calcium-dependent protease calpain is responsible for PTP1B cleavage and enzymatic activation. Pretreatment of Jurkat cells with calpeptin, a calpain inhibitor, blocked PTP1B cleavage and inhibited ameba-induced dephosphorylation. In addition, inhibition of Jurkat cell PTPases with phenylarsine oxide blocked Jurkat cell apoptosis induced by E. histolytica. These results suggest that E. histolytica-mediated host cell death occurs by a mechanism that involves PTPase activation.

FAS Activation Induces Dephosphorylation of SR Proteins: DEPENDENCE ON THE DE NOVO GENERATION OF CERAMIDE AND ACTIVATION OF PROTEIN PHOSPHATASE 1

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.

Stromal cell-derived factor 1 alpha-induced chemotaxis in T cells is mediated by nitric oxide signaling pathways.

Stromal cell-derived factor 1 alpha (SDF1 alpha) and its cognate chemokine receptor CXCR4 act as potent chemoattractants and regulate trafficking and homing of hematopoietic progenitor cells and lymphocytes. However, the molecular mechanisms regulating SDF1 alpha-driven cell migration are not well defined. In this study, we have explored the roles of the second messenger NO and the transcription factor NF-kappa B in SDF1 alpha-induced T cell migration. SDF1 alpha treatment of Jurkat T cells increased the activity of NO synthase, which catalyzes the generation of NO. We observed that pretreatment of Jurkat cells or activated PBLs with several NO donors significantly enhanced the SDF1 alpha-induced migration, whereas various inhibitors of NO synthase markedly abrogated the chemotactic response in a concentration-dependent manner. Furthermore, we observed that inhibitors of the transcription factor NF-kappa B, which is linked to NO signaling pathways, also significantly blocked the SDF1 alpha-induced chemotactic response. However, these compounds did not have a significant effect on SDF1 alpha-induced mitogen-activated protein kinase activity. In addition, the MAP/Erk kinase kinase inhibitor PD98059 did not abrogate SDF1 alpha-induced chemotaxis. AKT, which has been shown to mediate NO production, was also phosphorylated upon SDF1 alpha stimulation. These studies suggest that NO-related signaling pathways may mediate SDF1 alpha-induced chemotaxis, but not mitogen-activated protein kinase activation.

Inositol 1,4,5-Trisphosphate Receptor Type 1 Is a Substrate for Caspase-3 and Is Cleaved during Apoptosis in a Caspase-3-dependent Manner

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.

Differential Activation of c-Jun NH2-Terminal Kinase and p38 Pathways During FTY720-Induced Apoptosis of T Lymphocytes That Is Suppressed by the Extracellular Signal-Regulated Kinase Pathway

FTY720 is a novel immunosuppressive drug derived from a metabolite from Isaria sinclairii that is known to induce apoptosis of rat splenic T cells. In this study, we examined the intracellular signaling pathway triggered by FTY720. Treatment of human Jurkat T lymphocytes with FTY720-induced apoptosis characterized by DNA fragmentation. The same treatment induced activation of protein kinases such as c-Jun NH2-terminal kinase (JNK), p38/CSBP (CSAID-binding protein), and a novel 36-kDa myelin basic protein (MBP) kinase, but not extracellular signal-regulated kinase (ERK). Pretreatment of Jurkat cells with DEVD-CHO blocked FTY720-induced DNA fragmentation as well as the activation of p38/CSBP. However, DEVD-CHO treatment failed to inhibit FTY720-induced activation of JNK and the 36-kDa MBP kinase. We have also demonstrated that activation of the ERK signaling pathway completely suppressed the FTY720-induced apoptotic process including activation of caspase 3 and activation of JNK and the 36-kDa MBP kinase. Furthermore, transient expression of constitutively active mitogen-activated protein kinase/ERK kinase (MEK) protected the cells from FTY720-induced cell death. The effect of MEK was canceled by coexpression of a mitogen-activated protein kinase phosphatase, CL100. These results indicate that JNK and p38 pathways are differentially regulated during FTY720-induced apoptosis and that activation of ERK pathway alone is sufficient to cancel the FTY720-induced death signal.

CD45 and Src-Related Protein Tyrosine Kinases Regulate the T Cell Response to Phorbol Esters

Protein kinase C (PKC)-dependent activation of the Ras signal transduction cascade is essential for induction of the IL-2 promoter during stimulation of T lymphocytes via the T cell receptor (TCR). In this study, the effects of PKC-activating phorbol myristate acetate (PMA) on Ras-dependent activation of transcription from the ets/AP-1 Ras-responsive promoter element were examined in human T cells. Pretreatment of Jurkat cells with the Src-family PTK inhibitor herbimycin A resulted in a 50% inhibition of transactivation of the reporter following incubation with PMA. Evidence was also obtained to suggest the participation of the leukocyte-specific protein tyrosine phosphatase CD45, a regulator of Src-like PTKs, in the PMA-induced activation of the Ras/Raf pathway. First, PMA-induced transactivation of ets/AP-1 is diminished 75% in CD45-negative variants, compared with CD45-positive cells. Second, engagement of CD45 by monoclonal antibodies suppresses the PMA response from the reporter construct. Taken together, these data suggest that Src-related proteins mediate PKC-dependent activation of the Ras/Raf pathway and implicate CD45 in the TCR-independent activation of T lymphocytes induced by agents such as PMA.

Evidence that a kinase distinct from protein kinase C and phosphatidylinositol 3-kinase mediates ligation-dependent serine/threonine phosphorylation of the T-lymphocyte co-stimulatory molecule CD28.

The CD28 cytoplasmic tail contains several potential phosphorylation sites for the serine/threonine kinase protein kinase C (PKC) and/or proline-directed serine/threonine kinases, such as extracellular signal-regulated kinases. We demonstrate that ligation of CD28 by B7.1 results in strong serine/threonine phosphorylation of CD28. It is unlikely that ligation-stimulated phosphorylation of CD28 is mediated via activation of PKC, since it was not prevented by pre-treatment of Jurkat cells with inhibitors of PKC, and it was not mimicked by treatment with PKC activators such as PMA. Nevertheless, despite for lack of detectable effects of PMA treatment on CD28 phosphorylation, PMA did partially inhibit the association of CD28 with the putative signalling molecule phosphatidylinositol 3-kinase (PI 3-kinase) and the subsequent accumulation of PtdIns(3,4,5)P3. PI 3-kinase exhibits dual specificity as both a lipid kinase and a protein serine kinase, and site-specific mutagenesis of the Tyr173 residue in the CD28 cytoplasmic tail, which abolishes CD28 coupling to PI 3-kinase [Pages, Ragueneau, Rottapel, Truneh, Nunes, Imbert and Olive (1994) Nature (London) 369, 327-329], also prevents ligation-stimulated phosphorylation of CD28. However, the two PI 3-kinase inhibitors wortmannin and LY294002 had no effect on phosphorylation of CD28 after ligation by B7.1. This study therefore demonstrates that (1) a CD28-activated serine/threonine kinase distinct from both PKC and PI 3-kinase mediates ligation-stimulated CD28 phosphorylation, and (2) the PMA-stimulated down-regulation of the coupling of CD28 to PI 3-kinase is not due to PMA-stimulated phosphorylation of CD28.

Activation of Src-family tyrosine kinases during Fas-induced apoptosis.

Stimulation of several human and murine hematopoietically derived cell lines with anti-Fas antibodies induced increased tyrosine phosphorylation of a panel of proteins observed in whole-cell lysates. In the human T cell line Jurkat, the activity of a 56-kDa tyrosine kinase was likewise activated by anti-Fas antibodies. Immunoprecipitation studies of anti-Fas-stimulated human Jurkat and murine 2B4.11 T cells revealed activation of the Src-family tyrosine kinases Lck and Fyn. Fas receptor-induced tyrosine phosphorylation of p120 c-cbl proto-oncogene product was observed in Jurkat T cells. Pharmacological experiments demonstrated that pretreatment of Jurkat cells with tyrphostins inhibited Fas-induced apoptosis; likewise, Lck activity was inhibited by tyrphostins in a dose-dependent fashion. Finally, Lck derived from unstimulated Jurkat T cells formed stable complexes with the intracellular domain of the Fas receptor. These data are consistent with the notion that expression and activation of members of the Src-family kinases is required for Fas-induced cell death in T lymphocytes and consistent with recent findings demonstrating decreased Fas-mediated thymocytic death in Fyn-knockout mice.