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PubMed1991

Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production.

CD45, a hematopoietic cell-specific surface antigen, has recently been shown to be a protein tyrosine phosphatase. Expression of CD45 is essential for the T-cell antigen receptor to couple with the phosphatidylinositol second messenger pathway and for antigen-mediated proliferation of T lymphocytes. In this report we describe a CD45-deficient mutant of the human T-cell leukemia line Jurkat. CD45 expression is required for the activation of a T-cell receptor-associated tyrosine kinase as well as the phosphatidylinositol pathway. Additionally, stimulation of T lymphocytes by way of the accessory molecule CD2 requires the expression of CD45. The mutation in the CD45-deficient cell specifically impairs signal transduction by the T-cell receptor and CD2 because activation events by way of another accessory molecule, CD28, are unimpaired.

Antibodies, Monoclonal↗
PubMed1994

Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.

Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipitation of JAK1 with the G-CSF receptor suggested a physical association which existed prior to G-CSF stimulation.

Animals↗
PubMed1995

Alternatively spliced juxtamembrane domain of a tyrosine kinase receptor is a multifunctional regulatory site. Deletion alters cellular tyrosine phosphorylation pattern and facilitates binding of phosphatidylinositol-3-OH kinase to the hepatocyte growth factor receptor.

The hepatocyte growth factor (HGF) receptor is a tyrosine kinase receptor that mediates signal transduction upon ligand stimulation. This receptor is present in mouse tissues as two major isoforms differing by a 47-amino acid segment in the juxtamembrane domain, an alternatively spliced cytoplasmic region adjacent to the transmembrane domain of the receptor. We report here that the juxtamembrane domain of the receptor is involved in the regulation of downstream signal transduction. The two receptor isoforms were transiently expressed in COS-7 cells. Both exogenous receptors underwent autophosphorylation and subsequently stimulated a set of protein tyrosine phosphorylations that were not present in control cells. Comparisons of phosphotyrosine profiles of transfected cell lysates induced by receptor isoforms demonstrated that at least three phosphorylated proteins of approximately 62, approximately 35, and approximately 30 kDa were differentially induced by the receptor isoforms, suggesting that the juxtamembrane domain of a kinase receptor can play a role in selective signal transduction. Furthermore, the p85 subunit of phosphatidylinositol-3-OH kinase (PI3 kinase) co-precipitated with the small isoform of the HGF receptor, and this association was dramatically inhibited by treatment with 12-O-tetradecanoylphorbol-13-acetate. Since removal of the juxtamembrane domain facilitates the binding of p85 to the receptor, it is likely that the juxtamembrane region plays a role in negative regulation of the binding of PI3 kinase to the HGF receptor. Our study establishes novel molecular sequelae of alternative splicing of an intracellular domain of the HGF receptor.

Alternative Splicing↗
PubMed1995

An SH3-binding site conserved in Bruton's tyrosine kinase and related tyrosine kinases mediates specific protein interactions in vitro and in vivo.

Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

Protein-tyrosine kinases activate while protein-tyrosine phosphatases inhibit L-type calcium channel activity in pituitary GH3 cells.

The aim of this study was to evaluate the effect of protein-tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) inhibitors on Ca2+ channels in GH3 cells. The activity of Ca2+ channels was monitored either by single-cell microfluorometry or by the whole-cell configuration of the patch-clamp technique. Genistein (20-200 micron) and herbimycin A (1-15 micron) inhibited [Ca2+]i rise induced either by 55 mM K+ or 10 micron Bay K 8644. In addition, genistein and lavendustin A inhibited whole-cell Ba2+ currents. By contrast, daidzein, a genistein analogue devoid of PTK inhibitory properties, did not modify Ca2+ channel activity. The inhibitory action of genistein on the [Ca2+]i increase was completely counteracted by the PTP inhibitor vanadate (100 micron). Furthermore, vanadate alone potentiated -Ca2+-i response to both 55 mM K+ and 10 micron Bay K 8644. The possibility that genistein could decrease the [Ca2+]i elevation by enhancing Ca2+ removal from the cytosol seems unlikely since genistein also reduced the increase in fura-2 fluorescence ratio induced by Ba2+, a cation that enters into the cells through Ca2+ channels but cannot be pumped out by Ca2+ extrusion mechanisms. Finally, in unstimulated GH3 cells, genistein caused a decline of [Ca2+]i and the disappearance of [Ca2+]i oscillations, whereas vanadate induced an increase of [Ca2+]i and the appearance of [Ca2+]i oscillations in otherwise non-oscillating cells. The present results suggest that in GH3 cells PTK activation causes an increase of L-type Ca2+ channel function, whereas PTPs exert an inhibitory role.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Syk tyrosine kinase is required for immunoreceptor tyrosine activation motif-dependent actin assembly.

Clustering of several multisubunit receptors on hematopoetic cells results in a signaling cascade involving the phosphorylation of immunoreceptor tyrosine activation motifs, or "ITAMs," and actin polymerization. Recent experiments indicate that direct clustering of the ITAM-binding protein, p72(syk) (Syk), is capable of transmitting a phagocytic signal in COS cells (Greenberg, S., Chang, P., Wang, D., Xavier, R., and Seed, B.(1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1103-1107). However, the possibility of redundant signaling pathways makes it difficult to test the requirement for Syk in ITAM-dependent actin polymerization in hematopoetic cells. We developed a model system to study ITAM-dependent actin assembly. DT40 lymphocytes were transfected with fusion proteins encoding the transmembrane and cytosolic domains of the ITAM-containing gamma subunit of Fc receptors. Clustering the gamma-containing fusion proteins with IgG-coated erythrocytes triggered submembranous actin assembly. This response depended on an intact ITAM, was absent in cell lines that had been engineered to lack Syk, and was augmented in cell lines that stably overexpressed Syk. These experiments demonstrate an absolute requirement for Syk tyrosine kinase in ITAM-dependent actin assembly in transfected lymphocytes.

Actins↗
PubMed2006

Role of tyrosine kinase Csk in G protein-coupled receptor- and receptor tyrosine kinase-induced fibroblast cell migration.

Tyrosine kinase Csk is essential for mouse embryonic development. Csk knock-out mice died at early stages of embryogenesis (around embryonic day 10). The molecular mechanism for this defect is not completely understood. Here we report that Csk deficiency in mouse embryonic fibroblast cells blocked cell migration induced by lysophosphatidic acid through G protein-coupled receptors, by platelet-derived growth factor and epidermal growth factor through receptor tyrosine kinases, and by serum. Re-expression of Csk in these Csk-deficient cells rescued the migratory phenotype. Furthermore, deletion of Csk did not interfere with Rac activation and lamellipodia formation, but impaired the focal adhesions. Our data demonstrate a critical role for Csk in cell migration.

Actins↗
PubMed1999

Protein tyrosine phosphatase alpha (PTPalpha) and contactin form a novel neuronal receptor complex linked to the intracellular tyrosine kinase fyn.

Glycosyl phosphatidylinositol (GPI)-linked receptors and receptor protein tyrosine phosphatases (RPTPs), both play key roles in nervous system development, although the molecular mechanisms are largely unknown. Despite lacking a transmembrane domain, GPI receptors can recruit intracellular src family tyrosine kinases to receptor complexes. Few ligands for the extracellular regions of RPTPs are known, relegating most to the status of orphan receptors. We demonstrate that PTPalpha, an RPTP that dephosphorylates and activates src family kinases, forms a novel membrane-spanning complex with the neuronal GPI-anchored receptor contactin. PTPalpha and contactin associate in a lateral (cis) complex mediated through the extracellular region of PTPalpha. This complex is stable to isolation from brain lysates or transfected cells through immunoprecipitation and to antibody-induced coclustering of PTPalpha and contactin within cells. This is the first demonstration of a receptor PTP in a cis configuration with another cell surface receptor, suggesting an additional mode for regulation of a PTP. The transmembrane and catalytic nature of PTPalpha indicate that it likely forms the transducing element of the complex, and we postulate that the role of contactin is to assemble a phosphorylation-competent system at the cell surface, conferring a dynamic signal transduction capability to the recognition element.

Animals↗
PubMed1995

Impaired development of V gamma 3 dendritic epidermal T cells in p56lck protein tyrosine kinase-deficient and CD45 protein tyrosine phosphatase-deficient mice.

To determine whether p56lck protein tyrosine kinase and CD45 protein tyrosine phosphatase are involved in the signal transduction during intrathymic differentiation of gamma/delta T cells, we have examined the development of T cells expressing V gamma 3 T cell receptor (TCR) in mice deficient for either protein. The skin from both mice contained significantly reduced numbers of dendritic epidermal T cells expressing decreased levels of V gamma 3 TCR at the cell surface. Analysis of the fetal thymus from these mice suggested that maturation of V gamma 3 thymocytes was blocked at the immature stage that was characterized by the low level of V gamma 3 TCR and the high level of heat stable antigen. These results imply that both p56lck and CD45 are involved in the signal transduction during maturation of V gamma 3 T cells in the fetal thymus.

Animals↗
PubMed1996

Genetic analysis reveals cell type-specific regulation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase SHP1.

Receptor protein tyrosine kinases (RTKs) transmit downstream signals via interactions with secondary signaling molecules containing SH2 domains. Although many SH2-phosphotyrosyl interactions have been defined in vitro, little is known about the physiological significance of specific RTK/SH2 interactions in vivo. Also, little is known about the mechanisms by which specific RTKs interact with and/or are regulated by specific protein tyrosine phosphatases (PTPs). To address such issue, we carried out a genetic analysis of the previously reported biochemical interaction between the RTK c-Kit, encoded at the W locus, and the SH2-containing non-transmembrane PTP SHP1, encoded at the motheaten (me) locus (1). Mice carrying a kinase-defective allele of c-Kit (Wv/+) were crossed with me/+ mice, which carry one effectively null allele of SHP1, and then backcrossed to generate all possible allelic combinations. Our results indicate strong intergenic complementation between these loci in hematopoietic progenitor cells. Compared to progenitors purified from normal mice, bone marrow progenitor cells (lin-) from me/me mice markedly hyper-proliferated in response to Kit ligand (KL). stimulation. Superimposition of the me/me genotype increased the number of one marrow-derived CFU-E from Wv/+ mice. Conversely, the presence of one or two copies of Wv decreased the number of macrophages and granulocytes in me/me lung, skin, peripheral blood and bone marrow, thereby decreasing the severity of the me/me phenotype. The decrease in dermal mast cells in Wv/Wv mice was rescued to levels found in Wv/+mice by superimposition of the me/me genotype. Surprisingly, however, the presence or absence of SHP1 had no effect on the proliferative response of bone marrow-derived cultured mast cells to KL or IL3 ex vivo. Nevertheless, the immediate-early response to KL stimulation, as measured by KL-induced tyrosyl phosphorylation, was substantially increased in mast cells from Wv/+:me/me compared to Wv/ +:+/+ mice, strongly suggesting that SHP1 directly dephosphorylates and regulates c-Kit. Taken together, our results establish that SHP1 negatively regulates signaling from c-Kit in vivo, but in a cell type-specific manner.

Animals↗
PubMed2006

RNA interference targeting protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta suppresses glioblastoma growth in vitro and in vivo.

The protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta (PTPzeta/RPTPbeta) and its ligand pleiotrophin (PTN) are overexpressed in human glioblastomas. Both molecules are involved in neuronal cell migration during CNS development. In addition, PTN can induce glioma cell migration which is at least in part mediated through binding to PTPzeta/RPTPbeta. To study the relevance of this ligand-receptor pair for glioma growth in vitro and in vivo, we transfected the human glioblastoma cell line U251-MG with small interfering RNA (siRNA) directed against PTPzeta/RPTPbeta. Stable siRNA transfection resulted in strong down-regulation of PTPzeta/RPTPbeta expression. When injected subcutaneously into nude mice, clones that expressed normal levels of PTPzeta/RPTPbeta (PTPzeta + clones) formed exponentially growing tumours, whereas tumour growth was almost completely abrogated for clones that expressed reduced PTPzeta/RPTPbeta levels (PTPzeta - clones). Similar results were obtained using an orthotopic intracerebral model. Proliferation of PTPzeta - cells in vitro was significantly reduced compared with that of control clones. Matrix-immobilized PTN stimulated the proliferation of PTPzeta + cells but not of PTPzeta - cells. Haptotactic migration induced by PTN was reduced for PTPzeta - clones compared with control clones. Our findings suggest that antagonization of PTPzeta/RPTPbeta expression can inhibit glioma growth in vivo and may thus represent a potentially promising treatment strategy.

Animals↗
PubMed2002

Modulation of Kv1.5 currents by protein kinase A, tyrosine kinase, and protein tyrosine phosphatase requires an intact cytoskeleton.

The regulation of cardiac delayed rectifier potassium (Kv) currents by cAMP-dependent protein kinase (PKA) contributes to the control of blood pressure and heart rate. We investigated the modulation by PKA and protein phosphatases of cloned Kv1.5 channels expressed in Xenopus laevis oocytes. Exposure of oocytes to activators of PKA (100 nM forskolin, 1 mM 8-bromo-cAMP, or 1 mM 3-isobutyl-1-methylxanthine) had no effect on the amplitude of Kv1.5 currents. Inhibition of PKA by injection of protein kinase A inhibitor peptide or exposure to myristoylated protein kinase A inhibitor peptide (M-PKI; 100 nM) reduced currents mediated by Kv1.5. M-PKI also reduced the amplitude of currents mediated by mutated Kv1.5 channels in which the COOH terminal PKA phosphorylation sites and PSD-95, Disc-large, and ZO-1-binding domain were removed. The reduction of Kv1.5 currents by M-PKI was attenuated by inhibition of actin polymerization by 1 microM cytochalasins B and D, but was not affected by 10 microM phalloidin (stabilizes actin filaments) or 50 microM colchicine (disrupts microtubules). Treatment of oocytes with antisense oligonucleotides against alpha-actinin-2 abolished the reduction in Kv1.5 current by M-PKI. These observations suggest that Kv1.5 currents are activated by endogenous PKA in "resting" oocytes and that inhibition of PKA activity reveals the action of endogenous phosphatases. Indeed, injection of alkaline phosphatase reduced currents mediated by Kv1.5. Further preincubation of oocytes with 1 mM sodium orthovanadate (a protein tyrosine phosphatase inhibitor) abolished the reduction in Kv1.5 currents by M-PKI. We conclude that currents encoded by Kv1.5 are regulated by PKA and protein tyrosine phosphatase and that this regulation requires an intact actin cytoskeleton and alpha-actinin-2.

Actinin↗
PubMed1998

Protein tyrosine phosphatase 1B antagonizes signalling by oncoprotein tyrosine kinase p210 bcr-abl in vivo.

The p210 bcr-abl protein tyrosine kinase (PTK) appears to be directly responsible for the initial manifestations of chronic myelogenous leukemia (CML). In contrast to the extensive characterization of the PTK and its effects on cell function, relatively little is known about the nature of the protein tyrosine phosphatases (PTPs) that may modulate p210 bcr-abl-induced signalling. In this study, we have demonstrated that expression of PTP1B is enhanced specifically in various cells expressing p210 bcr-abl, including a cell line derived from a patient with CML. This effect on expression of PTP1B required the kinase activity of p210 bcr-abl and occurred rapidly, concomitant with maximal activation of a temperature-sensitive mutant of the PTK. The effect is apparently specific for PTP1B since, among several PTPs tested, we detected no change in the levels of TCPTP, the closest relative of PTP1B. We have developed a strategy for identification of physiological substrates of individual PTPs which utilizes substrate-trapping mutant forms of the enzymes that retain the ability to bind to substrate but fail to catalyze efficient dephosphorylation. We have observed association between a substrate-trapping mutant of PTP1B (PTP1B-D181A) and p210 bcr-abl, but not v-Abl, in a cellular context. Consistent with the trapping data, we observed dephosphorylation of p210 bcr-abl, but not v-Abl, by PTP1B in vivo. We have demonstrated that PTP1B inhibited binding of the adapter protein Grb2 to p210 bcr-abl and suppressed p210 bcr-abl-induced transcriptional activation that is dependent on Ras. These results illustrate selectivity in the effects of PTPs in a cellular context and suggest that PTP1B may function as a specific, negative regulator of p210 bcr-abl signalling in vivo.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Molecular cloning of a tyrosine kinase gene from the marine sponge Geodia cydonium: a new member belonging to the receptor tyrosine kinase class II family.

We have isolated and characterized a cDNA from the marine sponge Geodia cydonium coding for a new member of the tyrosine protein kinase (TK) family. The cDNA encodes a protein of M(r) = 68,710, termed GCTK, which is homologous to class II receptor tyrosine kinases (RTKs). GCTK contains conserved amino acids (aa) characteristic of all protein kinases, and the sequences DLATRN and PIRWMATE which are highly specific for TKs. Furthermore, the sequence N-L-Y-x(3)-Y-Y-R is highly homologous to the sequence D-[LIV]-Y-x(3)-Y-Y-R found only in class II RTKs. The sponge TK, when compared with mammalian class II RTKs, shows maximum 31% homology in the TK domain indicating that this the oldest member of class II RTK started to diverge from the common ancestral protein kinase approximately 650 million years ago. Using GCTK as a probe we identified three mRNA signals ranging from 2.6 to 0.6 kb. Kinase activity was localized only in the cell membranes from G. cydonium (M(r) = 65,000), and was not detected in the cytosol of this organism. Antibodies raised against a synthetic peptide, corresponding to the aa residues within the catalytic domain of the sponge TK, recognized strongly two proteins of M(r) = 65,000; these proteins, present in membrane fractions, also bound to the antiphosphotyrosine antibody. These data suggest that the TK cloned from the sponge is a membrane-associated 65 kDa protein. Moreover these results demonstrate that RTKs are present from the lowest group of multicellular eukaryotes, sponges, to mammals, and may suggest that RTKs are involved in a signal transduction pathway.

Amino Acid Sequence↗
PubMed2002

Expression of the tyrosine phosphatase SRC homology 2 domain-containing protein tyrosine phosphatase 1 determines T cell activation threshold and severity of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a CD4 Th1-mediated inflammatory demyelinating disorder of the CNS and a well-established animal model for multiple sclerosis. Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) is a cytosolic tyrosine phosphatase that is involved in regulating the T cell activation cascade from signals initiated through the TCR. To study the role of SHP-1 in EAE pathogenesis, we immunized B10.PL mice heterozygous for deletion of the SHP-1 gene (me(v+/-)) and B10.PL wild-type mice with the immunodominant epitope of myelin basic protein (MBP Ac1-11). T cell proliferation and IFN-gamma production were significantly increased in me(v+/-) mice after immunization with MBP Ac1-11. The frequency of MBP Ac1-11-specific CD4 T cells, analyzed by staining with fluorescently labeled tetramers (MBP1-11[4Y]: I-A(u) complexes), was increased in the draining lymph node cells of me(v+/-) mice compared with wild-type mice. In addition, me(v+/-) mice developed a more severe course of EAE with epitope spreading to proteolipid protein peptide 43-64. Finally, expansion of MBP Ac1-11-specific T cells in response to Ag was enhanced in me(v+/-) T cells, particularly at lower Ag concentrations. These data demonstrate that the level of SHP-1 plays an important role in regulating the activation threshold of autoreactive T cells.

Animals↗
PubMed2004

Macrophage-stimulating protein, the ligand for the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase, inhibits IL-12 production by primary peritoneal macrophages stimulated with IFN-gamma and lipopolysaccharide.

IL-12, produced by APCs during the initial stages of an immune response, plays a pivotal role in the induction of IFN-gamma by NK and gammadeltaT cells and in driving the differentiation of Th1 cells, thus providing a critical link between innate and acquired immunity. Due to the unique position occupied by IL-12 in the regulation of immunity, many mechanisms have evolved to modulate IL-12 production. We have shown previously that macrophage-stimulating protein (MSP), the ligand for the stem cell-derived tyrosine kinase/recepteur d'origine nantais (RON) receptor, inhibits NO production by macrophages in response to IFN-gamma and enhances the expression of arginase. Mice lacking RON exhibit increased inflammation in a delayed-type hypersensitivity reaction and increased susceptibility to endotoxic shock. In this study we demonstrate that pretreatment of macrophages with MSP before IFN-gamma and LPS results in the complete inhibition of IL-12 production due to suppression of p40 expression. This response is mediated by the RON receptor, and splenocytes from RON(-/-) animals produce increased levels of IFN-gamma. MSP pretreatment of macrophages resulted in decreased tyrosine phosphorylation of Stat-1 and decreased expression of IFN consensus sequence binding protein in response to inflammatory cytokines. In addition to IL-12, the expression of IL-15 and IL-18, cytokines that are also dependent on IFN consensus sequence binding protein activation, is inhibited by pretreatment with MSP before IFN-gamma and LPS. We also show that the ability of MSP to inhibit IL-12 production is independent of IL-10. Taken together, these results suggest that MSP may actively suppress cell-mediated immune responses through its ability to down-regulate IL-12 production and thus inhibit classical activation of macrophages.

Animals↗
PubMed1976

Enzymatic microassay of pyridoxal-5'-phosphate using L-tyrosine apodecarboxylase and L-(1-14C) tyrosine.

A microassay for pyridoxal-5'-phosphate (PLP) is described, which incorporates both the enzymatic and radioisotopic techniques. The method is based upon the stoichiometric decarboxylation of L-(1-14C) tyrosine by L-tyrosine apodecarboxylase and PLP. This method is an extensive modification of the ones reported by us previously, the main features of this present method being the ease of preparation of the enzyme extract and the substrate, elimination of the preincubation period and shortening of the 14CO2-trapping step. The assay as described here is rapid, highly sensitive and specific.

Apoenzymes↗
PubMed2004

[Effects of protein tyrosine kinase, protein tyrosine phosphatase and protein kinase C on the apoptosis of arsenic trioxide treated NB4 cells and human cortex neurons].

OBJECTIVE: To investigate the effects of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP) and protein kinase C (PKC) on apoptosis and observe the changes of cytosolic calcium ([Ca(2+)]i) of arsenic trioxide (As2O3) treated human leukemia cells NB4 and cortex neurons. METHODS: [Ca(2+)]i of NB4 cells and cortex neurons was probed with Fluo-3/AM, its changes were assayed with laser confocal microscopy in real-time after As2O3 treatment at different concentrations, the effects of PTK and PTP and the activation of PKC on these changes with confocal microscopy and phosphorus radioisotope assay. DNA ladders of NB4 cells and cortex neurons after exposed to As2O3 were observed. RESULTS: As2O3 at 1 micromol/L could remarkably increase the [Ca(2+)]i of NB4 cells but had no effects on neurons. Vanadate, a kind of PTP inhibitor, could promote the increase of [Ca(2+)]i treated by 2, 5, 10 micromol/L As2O3 in a dose-dependent manner. The mean total increase rates at 240 seconds after exposed to As2O3 at different concentrations were (6.5 +/- 2.3)%, (21.7 +/- 2.1)%, (49.2 +/- 2.5)% for NB4 cells, and (6.7 +/- 2.1)%, (19.4 +/- 2.5)%, (52.3 +/- 2.7)% for cortex neurons, respectively. Genistein, a kind of PTK inhibitor, could decrease the increase of [Ca(2+)]i treated by 2, 5, 10 micromol/L As2O3 in a dose-dependent manner. The mean total inhibited rates at 240 seconds after As2O3 treatment at different concentrations were (6.7 +/- 2.9)%, (25.6 +/- 2.5)%, (52.2 +/- 3.5)% for NB4 cells, and (7.8 +/- 3.1)%, (18.1 +/- 2.8)%, (51.3 +/- 3.3)% for cortex neurons, respectively. The activation of PKC began to increase as exposed to As2O3 at 1 micromol/L for 3 h, and kept rising continuously in NB4 cells and at 24 h DNA ladders emerged. However, none of the above results was found in human cortex neurons, but when exposed to 2 micromol/L As2O3, the activation of PKC and DNA ladders did emerge in neurons. CONCLUSIONS: The phosphorylation and dephosphorylation of PTK and PTP participated in nonspecific apoptosis signal transduction pathway related to As2O3, and accompanied with PKC activation. The [Ca(2+)]i elevation was closely related to increased PKC activation. There existed difference in dose tolerances to As2O3 between NB4 cell and cortex neurons.

Adult↗