sq-23377 and herbimycin

Endothelium-derived hyperpolarizing factor (EDHF) mediates NO/prostacyclin-independent relaxation in the coronary circulation. Because hemodynamic stimuli modulate endothelial gene expression and because coronary arteries are subjected to pronounced variations in vessel distension, we determined the effects of cyclic stretch on the expression and activity of the coronary EDHF synthase/cytochrome P450 (CYP) 2C8/9. In cultured porcine coronary and human umbilical vein endothelial cells, acute application of cyclic stretch (6%, 1 Hz, 10 minutes) elicited the generation of 8,9-epoxyeicosatrienoic acid (EET), 11,12-EET, and 14,15-EET. Prolonged stretch (4 to 36 hours) increased the expression of CYP 2C mRNA and protein 5- to 10-fold and was accompanied by a 4- to 8-fold increase in EET generation. A corresponding increase in CYP 2C mRNA and protein was also observed in pressurized segments of porcine coronary artery perfused under pulsatile conditions (8%, 1 Hz) for 6 hours. Although in cultured endothelial cells, cyclic stretch elicited the rapid activation of tyrosine kinases as well as Akt and the p38 mitogen-activated protein kinase, the mechanism by which cyclic stretch induces the expression of CYP 2C could not be elucidated, because inhibitors of these pathways induced CYP 2C expression in cells maintained under static conditions. These results have identified coronary EDHF synthase/CYP 2C as a novel mechanosensitive gene product in native and cultured endothelial cells. Because this enzyme generates both EETs and superoxide anions, this finding has wide-reaching implications for vascular homeostasis in conditions of manifest endothelial dysfunction.

Topics: 8,11,14-Eicosatrienoic Acid; Androstadienes; Animals; Benzoquinones; Cells, Cultured; Coronary Vessels; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Endothelium, Vascular; Enzyme Inhibitors; Humans; Ionomycin; Lactams, Macrocyclic; Oxygenases; Patch-Clamp Techniques; Periodicity; Pulsatile Flow; Quinones; Rifabutin; Sirolimus; Stress, Mechanical; Swine; Umbilical Veins; Vasodilation; Wortmannin

Previous studies have demonstrated that a mAb that recognizes the leukocyte surface Ag V7 inhibits TCR/CD3-dependent T cell activation. In the current study, we demonstrate that in addition to inhibiting T cell proliferation and IL-2 production, anti-V7 blocks tyrosine phosphorylation of TCR/CD3-associated substrates. PMA overcomes this effect, and both PMA and exogenous IL-2 overcome anti-V7-mediated inhibition of T cell proliferation and IL-2 production. T cells stimulated with anti-CD3 in the absence of CD28 or V7 ligation become unresponsive (anergic) to restimulation with anti-CD3; T cells primed in the presence of either anti-V7 or anti-CD28 retain their ability to respond to restimulation with anti-CD3. When T cells are primed in the presence of optimal concentrations of anti-V7 and anti-CD28 Abs, they proliferate normally, indicating that the costimulatory signals generated through CD28 dominate the inhibitory signals generated through V7. However, as the anti-CD28 stimulus is diluted, the V7 effect becomes dominant and proliferation is inhibited. Thus, although both anti-V7 and anti-CD28 Abs prevent anergy, they induce distinct, competing intracellular signals. Wortmannin, which blocks phosphoinositol 3-kinase-dependent signaling, has little effect on V7-mediated inhibition, while herbimycin, an inhibitor of tyrosine kinase, synergizes with anti-V7 to inhibit T cell activation. On the basis of these findings, V7-mediated signals appear to inhibit TCR-dependent tyrosine kinases that are required for IL-2 production and cellular proliferation.

Topics: Androstadienes; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Benzoquinones; CD28 Antigens; Cell Survival; Clonal Anergy; Humans; Interleukin-2; Ionomycin; Lactams, Macrocyclic; Ligands; Lymphocyte Activation; Membrane Glycoproteins; Mycotoxins; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Receptor-CD3 Complex, Antigen, T-Cell; Rifabutin; Signal Transduction; Substrate Specificity; T-Lymphocytes; Tetradecanoylphorbol Acetate; Wortmannin

Leukemia with megakaryocytic involvement has a poor prognosis. MX2 is a new morpholino anthracycline that is effective against various leukemic cell lines. This study examined the antitumor activity of MX2 against human megakaryocytic cell lines, including CMK, CMK11-5, MEG-01, and UT-7, and investigated the role of apoptosis in the cytotoxicity of this drug. To quantify the extent of apoptosis induced by MX2, we used the in situ terminal deoxynucleotide transferase assay and the histone-associated DNA fragmentation assay. The cytotoxic effect of MX2 on CMK cells was reduced by various inhibitors of apoptosis. To our knowledge, this is the first report showing that apoptosis is involved in the killing of megakaryocytic cell lines by an antileukemic agent. We suggest that MX2 may be useful for the treatment of megakaryocytic leukemia.

Topics: Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Carubicin; DNA Fragmentation; Enzyme Inhibitors; Humans; Ionomycin; Lactams, Macrocyclic; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Microscopy, Electron; Protein-Tyrosine Kinases; Quinones; Rifabutin; Staurosporine; Tumor Cells, Cultured

To investigate the novel interaction between endothelin-1 (ET-1) and cellular protein tyrosine kinases (PTK), we asked whether Ca2+ influx links ET-1 receptors to PTK activation. In glomerular mesangial cells, ET-1 stimulated a biphasic increase in PTK activity in anti-phosphotyrosine immunoprecipitates that temporally correlated with increased tyrosine phosphorylation of cellular proteins. ET-1 increased tyrosine phosphorylation of proteins in the cytosol and in a puncture distribution consistent with focal adhesions. Addition of ionomycin to increase Ca2+ influx stimulated PTK activity, and inhibition of extracellular Ca2+ influx blocked PTK activation by ET-1. ET-1 increased autophosphorylation of pp60c-src, which was mimicked by addition of ionomycin and inhibited by chelation of extracellular Ca2+. In addition, a selective PTK inhibitor blocked induction of c-fos mRNA by ionomycin, suggesting that Ca(2+)-stimulated PTKs contribute to a signaling pathway regulating immediate early gene expression. Taken together, these results demonstrate that ET-1 stimulates nonreceptor PTK activity, including pp60c-src, by activating Ca2+ channels and subsequent influx of extracellular Ca2+.

Topics: Animals; Benzoquinones; Calcium; Calcium Channels; Endothelin-1; Enzyme Activation; Ionomycin; Lactams, Macrocyclic; Male; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fos; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; RNA, Messenger; src-Family Kinases

Interactions of Fas (CD95) and its ligand (Fas-L) has been shown to play a pivotal role in T cells receptor (TCR)/CD3 activation-induced cell death via apoptosis. Although several lines of evidence suggest involvement of protein tyrosine kinase (PTK) activity in this process, the role of src family PTK p561ck (lck) is not known. We report here that, contrary to wild type Jurkat, the lck-deficient mutant JCaM is resistant to anti-CD3-induced apoptosis and fails to express Fas-L mRNA upon anti-CD3 treatment. However, both Jurkat and JCaM were found to constitutively express Fas and were equally sensitive to anti-Fas-mediated apoptosis. If stimulated with PMA plus ionomycin, JCaM expressed Fas-L mRNA and underwent apoptosis. These findings indicate that p56lck is required for TCR/CD3-mediated Fas-L induction but not for the transduction of Fas receptor-mediated death signal.

Topics: Antigens, Surface; Apoptosis; Base Sequence; Benzoquinones; CD3 Complex; Fas Ligand Protein; fas Receptor; Humans; Ionomycin; Lactams, Macrocyclic; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Membrane Glycoproteins; Molecular Sequence Data; Oligodeoxyribonucleotides; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Several studies have demonstrated that addition of soluble anti-CD6 mAbs to 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated naive T cells can induce cell proliferation. We showed in the present study that cell proliferation in TPA-treated T cell cultures can be enhanced several fold when the anti-CD6 mAbs are either immobilized or crosslinked with rabbit anti-mouse immunoglobulins (RAM Ig). Using a src family protein tyrosine kinase (PTK) inhibitor, herbimycin A, the cell proliferation induced by the anti-CD6 mAb, IOR-T1, in TPA-treated T cells were effectively abolished. Analysis of the cellular proteins in these cells after crosslinking the CD6 receptor with IOR-T1 (followed by RAM Ig) in the presence of TPA resulted in an increased level of tyrosine phosphorylation. Pretreatment of native T cells with herbimycin A (0.5 and 1 microgram/ml) for 18 hr completely inhibited the tyrosine phosphorylation on cellular substrates in T cell cultures stimulated with IOR-T1/RAM Ig and TPA. Similar concentrations of herbimycin A also inhibited the increase in IL-2 mRNA expression and cell proliferation in T cell cultures after IOR-T1/RAM Ig and TPA treatment. Furthermore, the increase in cytosolic free Ca2+ concentration in naive T cells after crosslinking of the CD6 receptor with IOR-T1/RAM Ig was also inhibited by herbimycin A. Taken together, our results suggest that CD6-mediated T cell proliferation is IL-2 dependent, and involves tyrosine kinase activity which is strictly dependent on protein kinase C activation.

Topics: Adult; Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Base Sequence; Benzoquinones; Calcium; Cross-Linking Reagents; Humans; Interleukin-2; Ionomycin; Ionophores; Lactams, Macrocyclic; Lymphocyte Activation; Molecular Sequence Data; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rabbits; Rifabutin; T-Lymphocytes; Tetradecanoylphorbol Acetate

Mast cells, mastocytoma cells and basophil leukaemia cells are well-established producers of leukotrienes when grown and stimulated appropriately. I report that the cells' ability to produce leukotrienes is dependent on the cells' proliferative status or their provision with growth factors. Proliferating MC/9 and subconfluent RBL2H3 cells respond maximally to stimulation by 1 microM ionomycin with the production of 56 and 32 pmol of cysteinyl-leukotrienes/10(6) cells respectively. In contrast, confluent RBL2H3 or growth-arrested MC/9 cells lose their ability to generate leukotrienes in response to ionomycin treatment. This rapid down-regulation of leukotriene synthesis is also observed when proliferating RBL2H3 cells are transferred to growth-factor-free medium, wherein cellular leukotriene-synthesis capacity has an apparent half-lifetime of 60 min. Transfer back into growth medium results in the regeneration of leukotriene synthesis capacity within 6 h. In growth-arrested MC/9 cells, leukotriene production ability can at least partially be restored by priming the cells with interleukin 3, but not with interleukin 4. In RBL2H3 cells, pretreatment with protein tyrosine kinase inhibitors such as genistein (5 min, 37 microM), herbimycin A (6 h, 3 microM) or tyrphostin 25 (16 h, 100 microM) completely inhibits leukotriene generation, whereas okadaic acid (15 min, 0.5 microM) has no effect. Under these conditions, both genistein and herbimycin A strongly impair ionomycin-induced protein tyrosine phosphorylation. Our study indicates that leukotriene generation in these tumour cells is tightly regulated by their proliferation status and supply with growth factors, and cell stimulation towards leukotriene synthesis appears to involve protein tyrosine kinase activity.

Topics: Animals; Benzoquinones; Catechols; Cell Division; Cell Line; Genistein; Interleukin-3; Interleukin-4; Ionomycin; Isoflavones; Kinetics; Lactams, Macrocyclic; Leukemia, Basophilic, Acute; Leukotrienes; Mast-Cell Sarcoma; Mice; Nitriles; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tumor Cells, Cultured; Tyrosine; Tyrphostins

Fas/APO-1 is a member of the tumor necrosis factor receptor family of proteins that induces apoptosis when cross-linked with monoclonal antibody (mAb) or with its physiological ligand. Recently, both a perforin-based and a Fas-based mechanism have been proposed to account for T cell-mediated cytotoxicity. In the present study we used a murine CD8+ cytotoxic T lymphocyte (CTL) clone (KB5 C20) specific for H-2Kb and a T cell receptor (TcR)-negative variant of the same clone (2005-D4) to test (i) whether the same cell can exert both cytotoxic effector mechanisms and (ii) the role of TcR engagement in the induction of Fas-based cytotoxicity. We demonstrate that both the TcR+ and TcR- clones were able to express the Fas ligand after stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and that TcR engagement of the KB5.C20 clone by means of antigen-bearing cells or of its anticlonotypic mAb (Désiré-1), which leads to Ca(2+)-dependent, presumably perforin-based, cytotoxicity, was also able to induce Fas-based cytotoxicity. In addition, using inhibitors we investigated the signal transduction pathway(s) involved in the induction of Fas-based cytotoxicity and expression of the Fas ligand mRNA in the CTL clones. The involvement of src-like protein tyrosine kinases (PTK) in Fas ligand induction through TcR engagement, was strongly suggested by inhibition with the src-like PTK inhibitor herbimycin A. Inhibition of Fas ligand induction by genistein, a more general TPK inhibitor, even upon stimulation by PMA plus ionomycin, suggested the possible involvement of PTK activities downstream of protein kinase C (PKC) in Fas ligand induction in CTL. Finally, the implication of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in Fas ligand induction was demonstrated by the partial inhibition of Fas ligand induction with cyclosporin A. Thus, in CTL clones, Fas ligand expression is inducible by TcR engagement through a pathway similar to that involved in expression of some lymphokine genes.

Topics: Animals; Antigens, Surface; Benzoquinones; CD3 Complex; Cell Survival; Clone Cells; Cyclosporine; fas Receptor; Gene Expression; Genistein; In Vitro Techniques; Ionomycin; Isoflavones; Lactams, Macrocyclic; Mice; Protein-Tyrosine Kinases; Quinones; Receptors, Antigen, T-Cell; Rifabutin; RNA, Messenger; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate

The T cell surface molecule CD28 binds to ligands on accessory cells and APCs, playing an important costimulatory role in the response of T cells to Ags. Our knowledge of the intracellular signaling pathways coupled to this receptor is incomplete. In addition to activation of phospholipase C gamma 1, ligation of this receptor also seems to activate a calcium-independent, CD28-specific pathway. In this paper, we report that cross-linking of CD28 (but not CD2, CD5, LFA-1, or CD7) leads to an elevation of c-jun mRNA, with only minimal activation of c-fos expression. CD28-dependent induction of c-jun expression requires protein tyrosine kinase activity, but does not depend on activation of a phorbol ester-responsive protein kinase C or elevation of cytosolic calcium. Furthermore, CD28-dependent elevation of c-jun mRNA does not appear to be mediated at the level of mRNA stability. A mechanism is suggested whereby expression of c-jun and junB, in the absence of members of the fos family, can prevent inappropriate activation of T cells caused by ligation of CD28 in the absence of a specific antigenic stimulus.

Topics: Base Sequence; Benzoquinones; CD28 Antigens; Cells, Cultured; Dactinomycin; Egtazic Acid; Gene Expression Regulation; Humans; Ionomycin; Lactams, Macrocyclic; Lymphocyte Activation; Molecular Sequence Data; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Quinones; Rifabutin; Signal Transduction; T-Lymphocytes; Tetradecanoylphorbol Acetate

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein ki

Topics: Benzoquinones; Bone Marrow; Bone Marrow Cells; Carrier Proteins; Cell Division; Cell Line; Ethers, Cyclic; Genistein; Glucose; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Ionomycin; Isoflavones; Kinetics; Lactams, Macrocyclic; Naphthalenes; Okadaic Acid; Phosphoprotein Phosphatases; Polycyclic Compounds; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate