Release of angiopoietin-1 by primary human acute myelogenous leukemia cells is associated with mutations of nucleophosmin, increased by bone marrow stromal cells and possibly antagonized by high systemic angiopoietin-2 levels.

The balance between proangiogenic Angiopoietin-1 (Ang-1) and the antagonistic Ang-2 is important both for leukemogenesis and chemosensitivity in human acute myelogenous leukemia (AML). We examined the release of Ang-1 and Ang-2 by AML cells cultured alone and in cocultures with stromal cells. Detectable Ang-1 release from AML cells was observed for most patients (62/91), whereas Ang-2 release was detected only for a minority (23/91). Coculture of AML and stromal cells led to increased Ang-1 levels. Furthermore, the role of the angiopoietin system was investigated by characterizing whether the differences in angiopoietin expression in AML patients can be related to nucleophosmin (NPM1) mutations. We compared the gene expression profiles of AML cells derived from 19 patients with FLT3 mutations and normal cytogenetics with and without NPM1 mutations and observed increased expression of Ang-1 in patients with NPM1 mutations. Finally, we found significantly higher Ang-2 levels in serum of AML patients compared with healthy controls. Our results suggest that AML cells are a major source of Ang-1 in leukemic bone marrow, especially in patients with NPM1 mutations, but the local levels are also influenced by stromal cells. Local Ang-2 release from AML cells is less common, but high systemic levels of Ang-2 may affect bone marrow angioregulation.

Histone deacetylase inhibitors in cancer treatment: a review of the clinical toxicity and the modulation of gene expression in cancer cell.

Characterization of epigenetic events in carcinogenesis has led to the discovery of a new class of oncogenes and thereby a new class of therapeutic targets. Among the new therapeutic approaches are modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs). HDACs deacetylate histones as well as transcription factors and can modulate gene expression through both these mechanisms in normal and malignant cells. Furthermore, acetylation is an important posttranslational modulation of several proteins involved in the regulation of cell proliferation, differentiation and apoptosis in normal as well as cancer cells. Even though several HDAC inhibitors have been characterized in vitro, only a limited number of these agents are in clinical trials. Various HDAC inhibitors differ in their toxicity profile when comparing the side effects described in the available clinical studies of HDAC inhibition in the treatment of cancer. These drugs may also affect normal hematopoiesis; hematologic toxicity is common to many drugs but stimulation of hematopoiesis seems to occur for others. HDAC inhibitors usually affect <10% of the genes in cancer cells. Divergent effects of HDAC inhibition on the global gene expression profiles have been described when testing various cancer cells, and this is further complicated by altered HDAC expression induced by HDAC inhibitors. However, increased p21 expression seems to be a common characteristic for most studies, suggesting an important role of this molecule during HDAC inhibitory treatment. Even though the initial studies are encouraging, additional in vitro and in vivo pharmacological characterization is definitely needed.

Induction of apoptosis in human cells by RNAi-mediated knockdown of hARD1 and NATH, components of the protein N-alpha-acetyltransferase complex.

Protein N-epsilon-acetylation is recognized as an important modification influencing many biological processes, and protein deacetylase inhibitors leading to N-epsilon-hyperacetylation of histones are being clinically tested for their potential as anticancer drugs. In contrast to N-epsilon-acetyltransferases, the N-alpha-acetyltransferases transferring acetyl groups to the alpha-amino groups of protein N-termini have only been briefly described in mammalians. Human arrest defective 1 (hARD1), the only described human enzyme in this class, complexes with N-acetyltransferase human (NATH) and cotranslationally transfers acetyl groups to the N-termini of nascent polypeptides. Here, we demonstrate that knockdown of NATH and/or hARD1 triggers apoptosis in human cell lines. Knockdown of hARD1 also sensitized cells to daunorubicin-induced apoptosis, potentially pointing at the NATH-hARD1 acetyltransferase complex as a novel target for chemotherapy. Our results argue for an essential role of the NATH-hARD1 complex in cell survival and underscore the importance of protein N-alpha-acetylation in mammalian cells.

Role of autocrine stimulation on the effects of cyclic AMP on protein and lipid phosphorylation in collagen-activated and thrombin-activated platelets.

We compared several responses in thrombin-stimulated and collagen (type I)-stimulated platelets with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP-removing system of creatine phosphate/creatine phosphokinase, Arg-Gly-Asp-Ser peptide to prevent fibrinogen/fibronectin binding to GPIIb/IIIa, SQ 29.548 as a thromboxane A2 receptor antagonist, cyproheptadine as a serotonin receptor antagonist, BN 52021 as a platelet-activating factor receptor antagonist). The pattern of tyrosine-phosphorylated proteins, the phosphorylation of lipids in the polyphosphoinositide cycle and phosphorylation of pleckstrin (P47) were studied as markers for signal-transducing responses, exposure of CD62 (P-selectin) and CD63 (Glycoprotein 53), as well as secretion of ADP + ATP and beta-N-acetyl-glycosaminidase were studied as final activation responses. Clear differences between thrombin-stimulated and collagen-stimulated platelets were observed. First, practically all protein-tyrosine phosphorylation induced by thrombin was inhibited by IAS, while a partial inhibition was observed for collagen; the phosphorylation due to collagen alone was apparently stimulated by elevation of cAMP. Secondly, the other responses to thrombin were inhibited by increased levels of cAMP, independent of autocrine stimulation. In contrast, only the autocrine part of the collagen-induced responses was inhibited by elevation of cAMP. Thus, the inhibition by elevated cAMP seen in collagen-stimulated platelets seems to be due to removal of the G-protein-mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure collagen effect which is not affected by elevated levels of cAMP.

Role of autocrine stimulation for the effects of cyclic AMP on protein and lipid phosphorylation in platelets activated by particles.

We have compared responses in platelets stimulated with the particulate materials, Intralipid (liposome-suspension) and a potential contrast medium IEEC (1'-(ethyloxycarbonyloxy)-ethyl-5-acetyl-amino-3-(N-methyl-acetyla mino)-2,4,6-triiodo-benzenecarboxylate coated with human serum albumin), with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP-removing system of creatine phosphate/creatine phosphokinase; RGDS to prevent fibrinogen/fibronectin binding to GPIIb/IIIa; SQ 29.548 as a TXA2 receptor antagonist; cyproheptadine as a serotonin receptor antagonist; BN 52021 as a platelet-activating factor receptor antagonist). The pattern of tyrosine-phosphorylated proteins, phosphorylation of initial lipids and phosphorylation of pleckstrin (P47) were used as markers for early signal transducing responses, while secretion of ADP+ATP and beta-N-acetyl-glycosaminidase were used as final responses. Intralipid showed no platelet activation except for some weak tyrosine protein phosphorylation that was inhibited by elevated cAMP. IEEC induced strong platelet activation that was partly inhibited by increased levels of cAMP and IAS. The inhibition of elevated cAMP seemed to be due to removal of the G protein-mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure effect from IEEC which is not affected by elevated cAMP.

Elevation of cyclic AMP decreases phosphoinositide turnover and inhibits thrombin-induced secretion in human platelets.

Elevation of cyclic AMP (cAMP) in platelets inhibits agonist-induced, G protein-mediated responses and activation of polyphosphoinositide-specific phospholipase C (PLC) by ill-defined mechanism(s). Signal transduction steps downstream of PLC are inhibited by elevated cAMP, suggesting an inhibitory effect of cAMP, via protein kinase A, on PLC. In [32P]i-prelabeled platelets, forskolin increased intracellular cAMP (104 nmol/1011 cells at 10-5 M forskolin) and [32P]phosphatidylinositol 4-phosphate (Delta[32P]PIP) (30% at 10-7-10-6 M forskolin). The thrombin-induced (0.1 U/ml) increase in production of [32P]PA, 'overshoots' in [32P]PIP and [32P]PIP2 ([32P]phosphatidylinositol 4,5-bisphosphate), and the increase in [32P]PI and secretion of ADP+ATP were abolished by forskolin (10-7 M). Forskolin stimulated total [32P]Pi uptake in resting platelets (48%), increased 32P incorporation into PIP (110%), and inhibited 32P incorporation into PI (50%). The latter inhibition was most likely considerably greater due to the forskolin-induced stimulation of [32P]Pi uptake. The changes in radioactive PA, PIP and PIP2 are regarded as being proportional with their masses in the prelabeled platelets, while the increase in PI (phosphatidylinositol) is regarded as a change in specific radioactivity, and hence in its synthesis. The results suggest that cAMP elevation inhibits the flux in the polyphosphoinositide cycle through both inhibition of PIP 5-kinase and PI synthesis. The inverse relation between forskolin-produced DeltaPIP and [32P]PA production suggests that the PLC reaction is inhibited by elevated cAMP through reduction of substrate (PIP2) resynthesis, and not by inhibition of the PLC enzyme.

Thrombin per se does not induce tyrosine protein phosphorylation in human platelets as judged by western blotting, while collagen does: the significance of synergistic, autocrine stimulation.

Thrombin elicits responses in platelets such as shape change, aggregation, arachidonate liberation and secretion of the contents of three storage granules, processes that coincide with serine/threonine and tyrosine phosphorylation of numerous proteins, hydrolysis of polyphosphoinositides and mobilisation of Ca2+ within the cell. However, the significance of these parallel signal transduction processes has not been clearly elucidated in the light of the prevalent autocrine stimulation in platelets: a great amplification of the thrombin signal through secreted ADP, by production of thromboxane A2 from the liberated arachidonic acid, by the close cell contact produced by aggregation caused by exposure of integrin receptors that become ligated by fibrinogen and other platelet-produced factors. In the present communication five pathways of autocrine stimulation have been prevented by appropriate inhibitors. Under these conditions thrombin stimulated platelet secretion with little tyrosine phosphorylation, except for a 125-130 kDa protein that was tyrosine-phosphorylated in response to one of the inhibitors, the peptide Arg-Gly-Asp-Ser (RGDS) used to block aggregation. In sharp contrast, collagen elicits massive tyrosine phosphorylation and platelet secretion in the absence of autocrine stimulation. When the thrombin-induced tyrosine phosphorylations was corrected for RGDS-induced phosphorylation, the presence of inhibitors of autocrine stimulation reduced the thrombin-induced phosphorylation by 97%. Our results strongly suggests that tyrosine phosphorylation is not part of the signal transduction pathway initiated by thrombin per se, but it represents an integral part of signal transduction initiated by collagen.

Differential secretion of blood platelet storage granules.

Platelets contain three types of secretory granules, dense granules, α-granules and lysosomes, which are characterized by their different contents. Dense granule and α-granule secretion appear to be similar in responsiveness to dose and types of agonists, whereas lysosomal secretion is observed only with higher doses of strong agonists such as thrombin. Recently, with the advent of flow cytometry, surface expression of membrane granule proteins, which are claimed to be specific for granule type, has come into use as a monitor for secretion. Expression of CD62 (PADGEM) in particular has become synonymous with α-granule secretion, based on comparisons with measurements of ß-thromboglobulin release by a method in which secretion is not stopped by fixation. We have now developed an immunoassay for fibrinogen that tolerates fixation stopping and have compared the release of dense and α-granule markers in the same platelet supernatants with the expression of CD62 and CD63 in gel-filtered platelets. At thrombin concentrations less than 0.04 U/ml, secretion of α-granule fibrinogen was both more rapid and quantitatively greater than that of dense granule serotonin, ATP and ADP. Comparison of the secretion of granule markers (contents) with the expression of granule membrane markers on the platelet surface showed that surface expression of CD62 (P-selectin, PADGEM) corresponded to fibrinogen secretion, and CD63 correlated reasonably well with the release of dense granule contents. Pretreatment of platelets with acetylsalicylic acid (ASA) before gel-filtration moderately inhibited thrombin-induced dense and α-granule release in GFP at a concentration range of 0.01-0.03 U/ml. The agonist effect of a thrombin receptor agonist peptide (TRAP) was comparable to that of thrombin with respect to all measured markers except for ß-hexosaminidase release, which was significantly less with TRAP.