Clinical comparison of an enhanced-sensitivity branched-DNA assay and reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 RNA in plasma

We investigated whether inhibition of the BCR-ABL tyrosine kinase by the CGP57418B compound would render chronic myeloid leukaemia (CML) cells susceptible to Fas (CD95, Apo-1)-mediated cell death. Only two (AR230 and SD1) out of 10 BCR-ABL positive cell lines were found to express the CD95 protein. No change in Fas expression was observed in any of the 10 cell lines after 48 h exposure to CGP57418B. AR230 cells were resistant and SD1 cells were partially resistant to Fas-mediated apoptosis induced by ligation of the Fas receptor to an anti-Fas IgM antibody. Pre-incubation with 1 microM CGP57418B did not change the susceptibility of these cell lines to Fas-mediated cell death. Similar results were observed in experiments with CD34+ cells from CML patients and from normal individuals. The data suggest that, in contrast to some cytotoxic drugs, the CGP57148B tyrosine kinase inhibitor utilizes a pathway other than the CD95 system in order to induce apoptosis in CML cells.     

Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism

A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time- and concentration-dependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin- independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.     

Detection of platelet adhesion/aggregation to immobilized ligands on microbeads by an aggregometer

Platelet aggregation is believed to follow platelet adhesion to vascular injury sites. We have developed a turbidimetric assay for platelet aggregation following platelet adhesion to immobilized ligands using an aggregometer. The addition of polystyrene beads coated with von Willebrand factor (vWF) or fibrinogen (Fg) to platelet suspensions caused prompt aggregation of beads and platelets, which was detected as an increase in light transmission. Electron microscopic analysis revealed that platelets adhered to the bead surfaces and that additional platelets adhered to already adhering platelets, leading to the formation of platelet aggregates. vWF-coated beads induced larger aggregates than Fg-coated beads. The interaction of vWF-coated beads with platelets was abolished by both GPIb and GPIIb-IIIa blockers, while that of Fg-coated beads was abolished by GPIIb-IIIa blockers. vWF-coated beads induced modest secretion of granules from platelets but no thromboxane B2 synthesis. Fg-coated beads induced neither reaction. However, pleckstrin phosphorylation and protein tyrosine phosphorylation was induced by both types of bead. Platelet aggregation following platelet adhesion to both types of bead was inhibited by ADP scavengers, a protein kinase C inhibitor and a tyrosine kinase inhibitor, but not by aspirin. These findings suggest that vWF- and Fg-coated beads can induce platelet aggregation following platelet adhesion through specific ligand-receptor interactions and intracellular signaling. Our simple assay using these beads may represent a useful test for immobilized ligand-induced platelet adhesion and aggregation.     

The tetramerization domain-independent Ras activation by BCR-ABL oncoprotein in hematopoietic cells

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. We have previously shown that BCR-ABL activates Ras signaling pathways required for transformation. To elucidate the mechanisms whereby BCR-ABL induced transformation in hematopoietic cells, we examined the biological effects of expression of a series of BCR-ABL mutants. We found that the Grb2 binding site-deleted BCR-ABL and the SH2 domain-deleted BCR-ABL, as well as the tetramerization domain-deleted BCR-ABL do not diminish the transforming properties of BCR-ABL in hematopoietic cells, although these mutations were previously shown to drastically reduce the transforming activity of BCR-ABL in fibroblasts. The tetramerization domain-deleted BCR-ABL did not induce tyrosine phosphorylation of CrkL, SHP-2, Vav and the interactions of BCR-ABL and Shc. However, Ras is activated, Shc is tyrosine phosphorylated and binds to Grb2 in the tetramerization domain-deleted BCR-ABL expressing hematopoietic cells. These results suggest that the tetramerization domain-independent Ras activation is mediated by Shc proteins and induces the transformation of hematopoietic cells.     

Spurious platelet counts in acute leukaemia with DIC due to cell fragmentation

Automated platelet counts in a patient with newly diagnosed AML M5 with extreme leukocytosis were reported as 129, 166 and 121 x 10(9)/1. Routine blood films showed a corresponding number of platelet-sized particles, judged to be platelets. The patient was treated for DIC with low-dose heparin infusion. Platelet transfusions were not given initially. The patient died 14 h after admission from intracerebral haematoma. The origin of the platelet-sized particles seen in routine stained blood films was examined by cytochemical and immunological staining for peroxidase, non-specific esterase, CD 13 and CD 33. About 1/3 of the fragments had the same staining characteristics as the leukaemia cells, indicating leukaemia cell origin. Staining for platelet-specific antigen GpIIIa was positive only in 4% of the platelet-sized fragments, with a calculated true platelet count of 4 x 10(9)/1. The presence of cell fragments masquerading as platelets should be suspected in leukaemia patients with bleeding symptoms and normal or near normal platelet counts.     

p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin

To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-cbl in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-cbl was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120c-cbl was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-cbl may be involved in signal transduction following ligand binding to c-Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.     

Tyrphostin AG957, a tyrosine kinase inhibitor with anti-BCR/ABL tyrosine kinase activity restores beta1 integrin-mediated adhesion and inhibitory signaling in chronic myelogenous leukemia hematopoietic progenitors

Abnormal beta1 integrin receptor function may contribute to the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors in chronic myelogenous leukemia (CML). Previous studies suggest that abnormal integrin function in CML progenitors is related to the presence of the BCR/ABL oncogene. BCR/ABL may alter integrin function in CML by phosphorylating cytoskeletal and/or signaling proteins important for normal integrin function. We evaluated the effect of Tyrphostin AG957, a protein tyrosine kinase (PTK) inhibitor which has activity against the p210BCR/ABL kinase, on beta1 integrin function in CML progenitors. Incubation of CML marrow CD34+HLA-DR+ cells with Tyrphostin AG957 at concentrations that did not affect colony-forming cells (CFC) viability, but which partly inhibited p210BCR/ABL kinase activity, significantly increased CML CFC adhesion to stroma and alpha4beta1 and alpha5beta1 integrin binding fragments of fibronectin (FN). CML CFC proliferation, unlike that of normal CFC, is not inhibited following integrin receptor engagement with FN or anti-integrin antibodies. AG957 did not alter CML CFC proliferation by itself, but resulted in significant inhibition of CML CFC proliferation following integrin engagement. Another PTK inhibitor, Tyrphostin AG555, which does not have anti-p210BCR/ABL kinase activity, did not affect CML CFC adhesion or proliferation. Neither AG957 nor AG555 affected normal CFC adhesion or proliferation. In BCR/ABL expressing cells, AG957 partially inhibited phosphorylation of several proteins that are BCR/ABL PTK substrates and are involved in normal integrin signaling. These studies suggest that abnormal tyrosine phosphorylation may play an important role in defective integrin function in CML progenitors.     

Priming of platelet alphaIIbbeta3 by oxidants is associated with tyrosine phosphorylation of beta3

Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H2O2, a major cellular oxidant. Exposure to H2O2 triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced99032416 required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO4) and was dependent on the participation of integrin alphaIIbbeta3 (glycoprotein IIb-IIIa). A specific inhibitor of alphaIIbbeta3 blocked platelet aggregation induced by H2O2 and NaVO4, thus confirming that aggregation requires this receptor. In the presence of H2O2 and NaVO4, multiple platelet substrates were phosphorylated on tyrosine. Such tyrosine kinase response was necessary but not sufficient to activate alphaIIbbeta3, as detected by binding of soluble fibrinogen to platelets. Stirring of the platelets exposed to H2O2 and NaVO4 was also needed to allow for binding of fibrinogen to alphaIIbbeta3. The tyrosine kinase inhibitor genistein was able to block platelet aggregation induced by H2O2 and NaVO4, thus confirming that tyrosine kinase activity was needed to trigger alphaIIbbeta3 activation on stirring. N-Acetyl-L-cysteine, a cell-permeant antioxidant, blocked the tyrosine phosphorylation of platelet substrates and also the platelet aggregation induced by H2O2 and NaVO4. We found that beta3 was phosphorylated on tyrosine in platelets exposed to H2O2 and NaVO4, even in the absence of aggregation. Hence, tyrosine phosphorylation of beta3 might contribute to the "priming" of alphaIIbbeta3 induced by H2O2 and NaVO4, whereby the receptor can become activated on stirring of the platelets.     

Anti-Ca2+, Mg2+-dependent endonuclease antibody detects specifically a class of chromatin-bound endonuclease

The effect of N-beta-alanyl-5-S-glutathionyl-dopa (5-S-GAD), a compound originally isolated from Sarcophaga peregrina (a flesh fly) as an antibacterial substance, on protein phosphorylation was examined using v-src-transformed NIH3T3 cell lysates. 5-S-GAD was found to inhibit tyrosine phosphorylation of protein tyrosine kinase v-src, but not serine/threonine phosphorylation of protein kinase C. The potency of this compound was comparable to that of herbimycin A. Our results suggested that a substitution at position 5 of the catechol in 5-S-GAD with the sulfur of cysteine is essential for 5-S-GAD to inhibit protein tyrosine kinase v-src.     

The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells

The Philadelphia chromosome found in virtually all cases of chronic myeloid leukemia (CML) and in about one third of the cases of adult acute lymphoblastic leukemia is formed by a reciprocal translocation between chromosomes 9 and 22 that results in the fusion of BCR and ABL genetic sequences. This BCR-ABL hybrid gene codes for a fusion protein with deregulated tyrosine kinase activity that can apparently cause malignant transformation. CGP57148B, a 2-phenylaminopyrimidine derivative, has been shown to selectively inhibit the tyrosine kinase of ABL and BCR-ABL. We report here that this compound selectively suppresses the growth of colony-forming unit-granulocyte/macrophage (CFU-GM) and burst-forming unit-erythroid derived from CML over a 2-logarithmic dose range with a maximal differential effect at 1.0 micromol/L. However, almost all CML colonies that grow in the presence of 1.0 micromol/L CGP57148B are BCR-ABL-positive, which may reflect the fact that residual normal clonogenic myeloid precursors are infrequent in most patients with CML. We also studied the effects of CGP57148B on hematopoietic cell lines. Proliferation was suppressed in most of the BCR-ABL-positive lines; all five BCR-ABL-negative lines were unaffected. We conclude that this new agent may have significant therapeutic applications.     

Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated beta2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P approximately 110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P approximately 110. The inhibition of P approximately 110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin-transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin-IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin-IgG-triggered PMN/PMN aggregation as well as P approximately 110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered beta2-integrin-dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase-dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the beta2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P approximately 110.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

Bacterial community dynamics during start-up of a trickle-bed bioreactor degrading aromatic compounds

Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn(2+)-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn(2+)-dependent protein kinase activity of PI3K alpha immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3K alpha showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85 alpha or p110 alpha derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85 alpha and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3K alpha can autophosphorylate on serine and threonine, and both p85 alpha and p110 alpha are substrates for a constitutively-associated protein tyrosine kinase in platelets.     

Inhibition of BCR-ABL expression with antisense oligodeoxynucleotides restores beta1 integrin-mediated adhesion and proliferation inhibition in chronic myelogenous leukemia hematopoietic progenitors

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to beta1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210(BCR-ABL), interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210(BCR-ABL) in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34(+)HLA-DR+ (DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated beta1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210(BCR-ABL) levels in CML CD34(+) cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors.     

BCR-ABL activates pathways mediating cytokine independence and protection against apoptosis in murine hematopoietic cells in a dose-dependent manner

The hallmark of chronic myeloid leukemia (CML) is the chimeric tyrosine kinase oncogene bcr-abl. Since expression of bcr-abl mRNA frequently increases with disease progression and a duplication of the Philadelphia chromosome (harbouring the bcr-abl hybrid locus) represents the most frequent karyotypic abnormality in acute phase CML, we hypothesized that the level of BCR-ABL protein may affect the disease phenotype. Therefore, the biological effects of high and low levels of BCR-ABL expression were compared in growth factor-dependent and -independent myeloid and lymphoid cell lines. Our results demonstrated that low levels of BCR - ABL were sufficient to render these cell lines growth factor independent and tumorigenic, but higher levels were mandatory for additional protection against apoptotic stimuli. The provision of growth factor or an activated ras oncogene did not afford the same degree of protection as high levels of BCR-ABL and there were qualitative differences between the survival signals mediated by BCR-ABL and Bcl-2. These results have enabled us to establish a dose-dependent hierarchy of BCR-ABL induced biological effects, thus distinguishing the activation of pathways mediating protection from cytokine withdrawal from those protecting against other apoptotic stimuli.