Reduced proliferation of CD34(+) cells from patients with acute myeloid leukemia after gene transfer of INPP5D.

Acute myeloid leukemia (AML) is a malignant disease characterized by deregulated proliferation of immature myeloid cells. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently detected in approximately 50-70% of AML patients. The gene INPP5D encodes the SH2-containing inositol 5-phosphatase 1 (SHIP1), which is a negative regulator of PI3K/AKT signaling. After lentiviral-mediated gene transfer of INPP5D into CD34(+) cells derived from AML patients (n=12) the granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent proliferation was reduced in all samples analyzed (average 86%; range 72-93%). An enzymatically inactive form of SHIP1 (D672A) had no effect. In addition, SHIP1 reduced the autonomous proliferation of CD34(+) cells from a patient with a secondary AML who had a very high peripheral blast count (300 x 10(9) l(-1)). These data show that SHIP1 can effectively block GM-CSF-dependent and autonomous proliferation of AML cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP(+)) is an essential regulator of T-lymphocyte Ca(2+)-signaling.

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP(+)) dose-dependently stimulated intracellular Ca(2+)-signaling. At a concentration of 10 nM NAADP(+) evoked repetitive and long-lasting Ca(2+)-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca(2+)-peak followed by trains of smaller Ca(2+)-oscillations was observed. Higher concentrations of NAADP(+) (1 and 10 microM) gradually reduced the initial Ca(2+)-peak, and a complete self-inactivation of Ca(2+)-signals was seen at 100 microM. The effect of NAADP(+) was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4, 5-trisphosphate- and cyclic adenosine diphosphoribose-mediated Ca(2+)-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP(+). Most importantly, microinjection of a self-inactivating concentration of NAADP(+) completely abolished subsequent stimulation of Ca(2+)-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP(+) Ca(2+)-release system is essential for T-lymphocyte Ca(2+)-signaling.

Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate.

Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling.

Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230.

By constructing DNA probes we have identified and cloned a human PtdIns 4-kinase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a protein of 2044 amino acids. The C-terminal part of ca. 260 amino acids represents the catalytic domain which is highly conserved in all recently cloned PtdIns 4-kinases. N-terminal motifs indicate multiple heterologous protein interactions. Human PtdIns 4-kinase PI4K230 expressed in vitro exhibits a specific activity of 58 micromol mg-1min-1. The enzyme expressed in Sf9 cells is essentially not inhibited by adenosine, it shows a high Km for ATP of about 300 microM and it is half-maximally inactivated by approximately 200 nM wortmannin. These data classify this enzyme as type 3 PtdIns 4-kinase. Antibodies raised against the N-terminal part moderately activate and those raised against the C-terminal catalytic domain inhibit the enzymatic activity. The coexistence of two different type 3 PtdIns 4-kinases, PI4K92 and PI4K230, in several human tissues, including brain, suggests that these enzymes are involved in distinct basic cellular functions.

Beta1-integrins mediate Ca2+-signalling and T cell spreading via divergent pathways.

Interaction of Jurkat T-lymphocytes with two extracellular matrix (ECM) proteins of the basement membrane, laminin or collagen type IV, combined with poly-L-lysine resulted in a strong adhesion, a highly increased intracellular Ca2+-concentration ([Ca2]i), as compared to cells on laminin or collagen type IV alone and in spreading of the cells. The strong adhesion was independent of an increase in [Ca2+]i, was not mediated by a beta1-integrin, and was due to charge interaction between the positively charged polyaminoacid and the negatively charged cell surface. The latter was confirmed by substitution of poly-L-lysine by other positively charged polyaminoacids. In contrast, Ca+-signalling and spreading of the cells adhering to laminin or collagen type IV combined with poly-L-lysine was completely blocked by anti-beta1 mAb. However, spreading of the cells was independent of an increase in [Ca2+]i suggesting divergent signal transduction pathways leading to Ca2+-signalling and spreading of the cells. We elucidated these signal transduction pathways by inhibition of key enzymes involved. The tyrosine kinase inhibitor genistein blocked Ca2+-signalling as well as spreading, whereas inhibitors of PKC (calphostin C, GF109203x), PLCgamma (U73122) and PLA2 (bromophenacyl-bromide (BPB), 3-[4-octadecyl)benzoyl]acrylic acid (OBAA)) selectively blocked spreading of the cells.

Mechanisms involved in alpha6beta1-integrin-mediated Ca(2+) signalling.

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting alpha6beta1-integrin-mediated Ca(2+) signalling. Both Ca(2+) release from thapsigargin-sensitive Ca(2+) stores and capacitative Ca(2+) entry via Ca(2+) channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of alpha6beta1-integrin-mediated Ca(2+) signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca(2+)-releasing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cADPR.

Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

Unique properties of the capacitative Ca(2+)-entry antagonist LU 52396: its inhibitory activity depends on the activation state of the cells.

The pharmacological properties of the recently described antagonist for capacitative Ca2+ entry LU 52396 were investigated and compared to known Ca2+ antagonists in Jurkat T-lymphocytes. In the first set of experiments, cells were stimulated with the anti-CD3 monoclonal antibody OKT3 and, subsequently, Ca2+ antagonists were added. Under such conditions SK-F 96365, econazole, nitrendipine and ZnCl2 dose-dependently antagonized Ca2+ signaling, whereas LU 52396 in concentrations up to 100 microM did not. In contrast, when LU 52396 was added a few minutes before OKT3, a dose-dependent inhibition of the OKT3-stimulated Ca2+ signals by LU 52396 was observed. Likewise, by prior addition of LU 52396 to thapsigargin-stimulated Jurkat T cells, a dose-dependent inhibition of Ca2+ signals was achieved. The IC50 value of LU 52396 for both agonists was about 5 microM. LU 52396 also inhibited Jurkat T cell proliferation, but showed cytotoxic effects at concentrations > 50 microM. Our data indicate that, in contrast to the other Ca2+ antagonists SK-F 96365, econazole, nitrendipine and ZnCl2, LU 52396 recognized the channel for capacitative Ca2+ entry only when intracellular Ca2+ was low and the channel was in its closed state.

Suppression of thyrotropin receptor-G protein-phospholipase C coupling by activation of protein kinase C in thyroid carcinoma cells.

In human thyroid follicular cells TSH exerts its action on growth and function at least via two distinct pathways, the adenylate cyclase cascade and the phospholipase Cbeta (PLCbeta)-mediated inositol phosphate generation. We investigated the effect of TSH on activation of phosphoinositide hydrolysis and inositol phosphate generation by PLCbeta in HTh74 thyroid carcinoma cells that express functional TSH receptors and in HTC-TSHr thyroid carcinoma cells that are devoid of endogenous TSH receptors but express recombinant human TSH receptors. In both cell lines, TSH up to concentrations of 300 mU/ml failed to stimulate myo-inositol 1,4,5-trisphosphate and myo-inositol-tetrakisphosphate generation, but led to a decrease in these compounds within 1 min of stimulation. However, ATP and bradykinin increased concentrations of inositol phosphates in both thyroid carcinoma cell lines. In contrast, in differentiated FRTL5 thyroid cell line and CHO-TSHr cell line expressing recombinant human TSH receptors, TSH elicited a significant increase in myo-inositol 1,4,5-trisphosphate and its metabolic derivatives. However, when HTC-TSHr cells were pretreated with calphostin C or staurosporine, inhibitors of protein kinase C, a TSH concentration of 20 mU/ml enhanced generation of inositol phosphates in these cells. From our data we conclude that in HTC-TSHr and HTh74 thyroid carcinoma cells, the coupling within the TSH receptor-Gq protein-PLCbeta signaling pathway is impaired compared to that in nontransformed cells. It is conceivable that this is at least in part dependent on the level of protein kinase C activation in these cells.

A novel A-isoform-like inositol 1,4,5-trisphosphate 3-kinase from chicken erythrocytes exhibits alternative splicing and conservation of intron positions between vertebrates and invertebrates.

Based on the partial peptide sequence of inositol 1,4, 5-trisphosphate 3-kinase purified with 135 000-fold enrichment from chicken erythrocytes, cDNA-fragments were cloned by RT-PCR using degenerate oligonucleotides. Subsequent hybridization screening of an embryonic chicken cDNA library and 5'-RACE yielded a cDNA-contig of 2418 bp, encoding a 452 amino acid protein. The amino acid sequence shows the highest degree of homology with A-isoforms of inositol 1,4,5-trisphosphate 3-kinase (65% identities), whereas homology towards B and C isoforms was lower (57% and 52% amino acid identities respectively). These findings reveal a new tissue-specific pattern of A-isoform expression, a form which so far has only been found in brain and testes. Two overlapping lambda-genomic clones for chicken inositol 1,4,5-trisphosphate 3-kinase, isolated by hybridization screening, covered 18 499 bp of genomic sequence. This contig included four exons: three of them were present in all cDNA clones, whereas one was only represented in a single cDNA clone. In addition, the sequence of the latter differed from the other cDNAs by an in-frame deletion of 72 bp within the coding region for the catalytic domain of the enzyme. This divergent cDNA suggests the existence of alternative splice products, at least in embryonic tissue.A comparison of the position of introns, with the respective introns known from the corresponding gene from Caenorhabditis elegans, revealed a high degree of conservation of intron positions between vertebrates and invertebrates. Functional data for the enzyme suggests that the conserved exons represent defined functional protein modules.

The only inositol tetrakisphosphate detectable in avian erythrocytes is the isomer lacking phosphate at position 3: a NMR study.

Avian red blood cells contain a millimolar amount of inositol polyphosphate which plays a role as an allosteric effector of hemoglobin. We confirmed the structure of this substance by NMR techniques as purely myo-inositol 1,3,4,5,6-pentakisphosphate. Based on present knowledge this effector is synthesized from inositol trisphosphate by successive phosphorylation. In a search for biosynthetic and degradative intermediates of inositol pentakisphosphate we found only one inositol tetrakisphosphate isomer. Its structure was unambiguously assigned by proton, 13C- and 31P-NMR to myo-inositol 1,4,5,6-tetrakisphosphate. As in mammalian cells the major inositol tetrakisphosphate isomer is myo-inositol 1,3,4,5-tetrakisphosphate, there seem to be differences between avian red blood cells and mammalian cells in the routes of inositol polyphosphate formation and/or degradation.

Phosphofructokinase is a calmodulin binding protein.

A trial to purify myosin light chain kinase from crude myosin led to the isolation of a Mr 85 000 calmodulin binding protein different from this enzyme. Because it showed inherent phosphofructokinase activity we investigated its relation to this enzyme. We demonstrated identity to phosphofructokinase by a close to identical amino acid composition, by antigenic identity and a set of completely identical peptide maps. The calmodulin binding property was also shown for a fraction of the enzyme prepared by standard methods. First experiments show that Ca2+--calmodulin is a potent regulator of phosphofructokinase polymerization.

Skeletal muscle myosin light chain kinase. A refined structural model.

A hydrodynamic, enzymatic and CD spectroscopic study of skeletal muscle myosin light chain kinase, three proteolytic fragments and corresponding complexes with calmodulin was performed. A refined shape model was built for the enzyme. It was shown that a head-and-tail structure is formed from two major fragments which are aligned end-to-end. The one fragment (Mr 36000) is compact, of high alpha-helix content and contains the catalytic center with the light chain and the calmodulin binding domains. The other fragment (Mr 33000) with unknown function is asymmetric (a/b greater than 10), of low alpha-helix and of unusually high proline content.

Ectocellular CD38-catalyzed synthesis and intracellular Ca2+-signalling activity of cyclic ADP-ribose in T-lymphocytes are not functionally related.

Cyclic ADP-ribose (cADPR) is a natural metabolite of beta-NAD+ with a potent Ca2+-mobilizing activity in different cell types, including T-lymphocytes. We investigated (i) whether stimulation of T-lymphocytes with different agonists affects the intracellular concentration of cADPR, and (ii) whether the lymphocyte antigen CD38, through its ectocellular ADP-ribosyl cyclase and cADPR-hydrolase enzymatic activities, can account for the regulation of the intracellular levels of cADPR and the Ca2+-mobilizing effects of this nucleotide in Jurkat and HPB.ALL T-lymphocytes. The anti-CD3 antibody OKT3, the sphingolipid sphingosine and lysophosphatidic acid induced an increase in intracellular cADPR with concomitant increases in the intracellular Ca2+ concentration ([Ca2+]i). In contrast, activation of an ectocellular ADP-ribosyl cyclase by preincubation of cells with beta-NAD+ led to a dose-dependent increase in cADPR, but no changes in [Ca2+]i were observed. However, extensive washing of the cells following preincubation with NAD+ demonstrated that the increases in cADPR were not intracellular but due to cell surface-associated nucleotide. Accordingly, measurements of ADP-ribosyl cyclase activity in intact T-cells showed ectocellular synthesis of cADPR, but no evidence was obtained for a shift of this activity into the cells which could account for intracellular accumulation of cADPR. Taken together, the results indicate no direct involvement of the ADP-ribosyl cyclase activity of CD38 on the regulation of the cADPR-mediated intracellular Ca2+-signalling in T-lymphocytes.

Pharmacological activation of the ryanodine receptor in Jurkat T-lymphocytes.

1 Recently, we provided evidence for cyclic adenosine 5'-diphosphate-ribose, cADP-ribose, as a second messenger in Jurkat T-lymphocytes upon stimulation of the T-cell receptor/CD3- complex (Guse et al., 1999). cADP-ribose mobilizes Ca2+ from an intracellular Ca2+ store which is sensitive to caffeine and gated by the ryanodine receptor/Ca2+ release channel. In the present study we investigated the ability of the trypanocidal drug, suramin, to activate the ryanodine receptor of T-cells. Since suramin cannot permeate the plasma membrane, it was necessary to microinject the drug into Fura-2 loaded T-lymphocytes. 2 In a dose dependent manner suramin increased the intracellular Ca2+ concentration. The dose-response curve is very steep and calculates for an EC50 of 7. 6+/-2.9 mM suramin in the injection pipette. 3 Co-injection of the selective ryanodine receptor inhibitor ruthenium red completely abolished the suramin induced Ca2+ transient. This finding allows for the conclusion that the IP3-receptor sensitive Ca2+ pool is not the primary target of the suramin induced Ca2+ transient. 4 Furthermore, Ins(1,4,6)PS3, an antagonist of the InsP3-receptor could not suppress the suramin-induced Ca2+ signal. The suramin induced Ca2+ transients declined very slowly; however, in the presence of Ins(1,4,6)PS3 this decay was accelerated. In addition, suramin did not interact with the cADP-ribose binding site of the ryanodine receptor of T-cells. 5 In conclusion, suramin is found to be an agonist for the T-cell ryanodine receptor as previously found for the cardiac and skeletal muscle isoform. Therefore, suramin can be designated a universal ryanodine receptor agonist.

Ca(2+)-signalling in human T-lymphocytes. Potential roles for cyclic ADP-ribose and 2'-phospho-cyclic ADP-ribose.

Intracellular Ca(2+)-signals belong to the major events transducing extracellular signals into living cells. The discovery of (i) a caffeine-sensitive intracellular Ca(2+)-pool in Jurkat T-lymphocytes [1] and (ii) cyclic adenosine diphosphoribose (cADPR) as an agent that mobilizes Ca2+ from a caffeine- and ryanodine sensitive Ca(2+)-store in sea urchin egg homogenates [2] prompted us to investigate the potential role of this compound in T-lymphocyte Ca(2+)-signalling. cADPR, as well as its 2'-phosphorylated derivative, 2'-phospho-cADPR (2'-cADPR), released Ca2+ in a dose-dependent, specific manner from intracellular, non-endoplasmic reticular stores of permeabilized Jurkat and HPB. ALL T cells. In addition, attempts were made to prove the presence of endogenous cADPR and 2'-P-cADPR by HPLC. Several HPLC protocols, including microbore-HPLC were tested resulting in the detection of endogenous cADPR by sequential separation on strong-anion exchange HPLC and reverse-phase ion-pair HPLC.

Gene transfer of SHIP-1 inhibits proliferation of juvenile myelomonocytic leukemia cells carrying KRAS2 or PTPN11 mutations.

Juvenile myelomonocytic leukemia (JMML) is a malignant disease of early childhood characterized by a hypersensitivity to granulocyte/macrophage colony-stimulating factor (GM-CSF). Mutations in RAS or PTPN11 are frequently detected in JMML patients. The SH2-containing inositol 5-phosphatase 1 (SHIP-1) is a negative regulator of GM-CSF signaling, and inactivation of SHIP-1 in mice results in a myeloproliferative disease. Here, we report the effects of SHIP-1 expression on GM-CSF-dependent proliferation and colony formation of human hematopoietic cells. After retroviral-mediated transduction of SHIP-1 into CD34+ cells from cord blood of healthy newborns or peripheral blood of JMML patients carrying mutations in KRAS2 or PTPN11, we observed a reduction in GM-CSF-dependent proliferation and colony formation. An enzymatically inactive form of SHIP-1 (D672A) had no effect. These data indicate that SHIP-1 can effectively block GM-CSF hypersensitivity in JMML progenitor cells with mutations in KRAS2 or PTPN11 and may be a useful approach for the treatment of JMML patients.

Inositol 1,4-bisphosphate is an allosteric activator of muscle-type 6-phosphofructo-1-kinase.

The allosteric effects of various inositol biphosphate (InsP2) isomers and other inositol phosphates, of glycerophosphoinositol phosphates (GroPInsPx) and of phosphoinositides (PtdInsPx) on muscle-type 6-phosphofructo-1-kinase (PFK) were investigated. The binding of these substances to PFK was indirectly estimated by their ability to stabilize the tetrameric enzyme. At near-physiological concentrations of other allosteric effectors, muscle PFK was activated AMP-dependently by Ins(1,4)P2 (Ka = 43 microM), Ins(2,4)P2 (Ka = 70 microM) and GroPIns4P (Ka = 20 microM). These compounds activated PFK by a mechanism similar to that established for activating hexose bisphosphates. Indirect binding experiments indicated minimal Kd,app. values of about 5 microM for the binding of Ins(1,4)P2 in the presence of 0.1 mM-AMP at pH 7.4. This apparent affinity was comparable with that of fructose 1,6-bisphosphate and glucose 1,6-bisphosphate at identical conditions. The enzyme was also found to interact specifically with PtdIns4P (Kd,app. = 37 microM), the inositol phospholipid carrying Ins(1,4)P2 as its head group. The regulatory behaviour of muscle-type PFK in vitro and the concentrations of Ins(1,4)P2 in vivo (between 4 and greater than 50 nmol/g wet wt. of tissue) are consistent with the hypothesis that there is a functional interaction in vivo. Furthermore, a role of PtdIns4P in membrane compartmentation of PFK is suggested. Comparative experiments with liver PFK indicate that these regulatory properties may be relatively specific for the muscle isoform. Unlike muscle PFK, the liver isoform was slightly activated by sub-micromolar concentrations of Ins(1,4,5)P3.

A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called 'metal-dye detection' has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.