Rapamycin inhibits didemnin B-induced apoptosis in human HL-60 cells: evidence for the possible involvement of FK506-binding protein 25. (65/2714)

In the present paper we show that the immunosuppressant rapamycin inhibits the induction of apoptosis by didemnin B in human promyeloid HL-60 cells. The mechanism of this inhibition is investigated using FK506, which competes with rapamycin for binding to their common target FK506-binding protein (FKBP)12. The lack of competition for rapamycin-mediated inhibition of didemnin B-induced apoptosis by FK506 suggests that rapamycin inhibits apoptosis through some mechanism other than inhibition of p70 S6 kinase activation. The lack of inhibition of didemnin B-induced apoptosis by inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein (MAP) kinase kinase further supports the conclusion that rapamycin does not inhibit didemnin B-induced apoptosis through inhibition of the MAP kinase pathway. Furthermore, didemnin B-induced apoptosis is not inhibited by the inhibitors of cyclin-dependent kinase, roscovitine and olomoucine. This indicates that rapamycin does not act through inhibition of cyclin-dependent kinases. Together with the lack of competition for the effect of rapamycin by FK506, our data suggest the possible involvement of the FK506-binding protein, FKBP25, which is localized in the nucleus. This interpretation of our data gains support from the fact that didemnin B does not induce apoptosis in enucleated HL-60 cells, which supports the possible involvement of FKBP25 in the inhibition of apoptosis by rapamycin.  (+info)

Competitive and noncompetitive inhibition of the DNA-dependent protein kinase. (66/2714)

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase that is involved in mammalian DNA double-strand break repair. The catalytic subunit of DNA-PK (DNA-PKcs) shares sequence homology in its kinase domain with phosphatidylinositol (PI) 3-kinase. Here, we provide a detailed kinetic analysis of DNA-PK inhibition by the PI 3-kinase inhibitor wortmannin and demonstrate this inhibition to be of a noncompetitive nature, with a Ki of 120 nM. Another inhibitor of PI 3-kinase. LY294002, its parent compound, quercetin, and other derivatives have also been studied. These chemicals are competitive inhibitors of DNA-PK, with LY294002 having a Ki of 6.0 microM. Using an antibody to wortmannin, we found that this compound binds covalently to the kinase domain of DNA-PKcs both in vitro and in vivo. Binding of wortmannin to the active site of DNA-PKcs is inhibited by ATP but not by a peptide substrate. Furthermore, wortmannin is able to bind to DNA-PKcs independently of Ku, and it is not stimulated by the presence of DNA. This suggests that the ATP binding site of DNA-PKcs is open constitutively and that DNA activation of the kinase is mediated via another mechanism.  (+info)

Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis. (67/2714)

Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking. Hrs has a FYVE double zinc finger domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic. To understand the physiological role of Hrs, we generated mice carrying a null mutation of the gene. Hrs homozygous mutant embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes (cardia bifida), lacked a foregut, and died around embryonic day 11 (E11). These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0. Significant apoptosis was detected in the ventral region of mutant embryos within the definitive endoderm, suggesting an important role of this germ layer in ventral folding morphogenesis. Abnormally enlarged early endosomes were detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype. The vesicular localization of Hrs was disrupted in cells treated with wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking.  (+info)

Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation. (68/2714)

Chemotherapeutic drugs that damage DNA kill tumor cells, in part, by inducing the expression of a death receptor such as Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B (Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of EGF-stimulated extracellular signal-regulated kinase (ERK) activity did not affect EGF protection from Fas-mediated apoptosis. The findings indicate that EGF receptor stimulation of epithelial cells has a significant survival function against death receptor-induced apoptosis mediated by Akt.  (+info)

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism. (69/2714)

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.  (+info)

Phosphoinositide 3-kinase-dependent and -independent activation of the small GTPase Rac2 in human neutrophils. (70/2714)

The small GTPase Rac participates in various cellular events such as cytoskeletal reorganization. It has remained, however, largely unknown about intracellular signaling pathways for Rac activation because of the lack of a simple and reliable assay to estimate the activation. Here we describe a novel method to detect the GTP-bound, active Rac in cells by pulling it down with the Rac-binding domain of the protein kinase PAK. Experiments using this method reveal that stimulation of human neutrophils with the Gi-coupled receptor agonists N-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4) leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate (PMA) causes a slow but more sustained activation of Rac2. Pretreatment of cells with pertussis toxin results in defective activation of Rac2 in response to fMLP and LTB4, indicating that coupling of the receptors to Gi plays a crucial role in the activation. Furthermore, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 block Rac2 activation elicited by the receptor agonists, but not that by PMA. Thus the Gi-coupled receptors likely mediate Rac2 activation via PI3K, whereas PMA activates Rac2 in a PI3K-independent manner.  (+info)

Oncogenic Ras-induced germinal vesicle breakdown is independent of phosphatidylinositol 3-kinase in Xenopus oocytes. (71/2714)

A number of reports have identified phosphatidylinositol 3-kinase as a downstream effector of Ras in various cellular settings, in contrast to others supporting the notion that phosphatidylinositol 3-kinase acts upstream of Ras. Here, we used Xenopus oocytes, a model of Ras-mediated cell cycle progression (G2/M transition) to analyze the contribution of phosphatidylinositol 3-kinase to insulin/Ras-dependent signaling pathways leading to germinal vesicle breakdown and to ascertain whether phosphatidylinositol 3-kinase acts upstream or downstream of Ras in those signaling pathways. We analyzed the process of meiotic maturation induced by progesterone, insulin or micro-injected oncogenic Ras (Lys12) proteins in the presence and absence of specific inhibitors of phosphatidylinositol 3-kinase activity. As expected, the progesterone-induced maturation was independent of phosphatidylinositol 3-kinase since similar rates of germinal vesicle breakdown were produced by the hormone in the presence and absence of wortmannin and LY294002. In contrast, insulin-induced germinal vesicle breakdown was completely blocked by pre-incubation with the inhibitors prior to insulin treatment. Interestingly, similar rates of germinal vesicle breakdown were obtained in Ras (Lys12)-injected oocytes, independently of whether or not they had been pre-treated with phosphatidylinositol 3-kinase inhibitors. The effect of wortmannin or LY294002 on MAPK and Akt activation by progesterone, insulin or Ras was also analyzed. Whereas insulin activated those kinases in a phosphatidylinositol 3-kinase-dependent manner, progesterone and Ras were able to activate those kinases in the absence of phosphatidylinositol 3-kinase activity. Since Ras is a necessary and sufficient downstream component of insulin signaling pathways leading to germinal vesicle breakdown, these observations demonstrate that phosphatidylinositol 3-kinase is not a downstream effector of Ras in insulin/Ras-dependent signaling pathways leading to entry into the M phase in Xenopus oocytes.  (+info)

Persistent nocturnal cough: randomised controlled trial of high dose inhaled corticosteroid. (72/2714)

OBJECTIVE: To investigate the effect of a short course of inhaled corticosteroid in the treatment of isolated and persistent nocturnal cough in children. DESIGN: Randomised double blind placebo controlled study. SETTING: Subjects' homes in east London, England. SUBJECTS: Consecutively referred children, 1-10 years old, with persistent nocturnal cough. INTERVENTIONS: Placebo or fluticasone propionate 1 mg twice daily for three nights and 500 microg twice daily for 11 nights. Videotaping of children at night: two nights' baseline, nights 3 and 4 after three days of inhaled corticosteroid, and nights 15 and 16. MAIN OUTCOME MEASURE: A fall in 75% of coughs from baseline. RESULTS: 50 subjects were recruited. The median number of coughs in the baseline period for the inhaled corticosteroid group and placebo group were 92 and 71, respectively (p = 0.43) and, on nights 15 and 16, 8 and 36, respectively (p < 0. 01). Compared to baseline, both groups of subjects improved significantly by nights 15 and 16 (p < 0.01; p < 0.01). Comparing the inhaled corticosteroid and placebo groups, coughs fell to a median of 22% and 57% of baseline totals on nights 3 and 4, respectively (p = 0.38), and 8% and 35% on nights 15 and 16, respectively (p = 0.02). 17 of 24 subjects on inhaled corticosteroid who completed the study and 8 of 23 on placebo improved by 75% after two weeks (p = 0.03). CONCLUSIONS: Children with persistent nocturnal cough improve in two weeks after referral on placebo. There is a modest benefit from a two week course of high dose inhaled corticosteroid.  (+info)