ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.

Identification of Zfp521/ZNF521 as a cooperative gene for E2A-HLF to develop acute B-lineage leukemia.

E2A-hepatic leukemia factor (HLF) is a chimeric protein found in B-lineage acute lymphoblastic leukemia (ALL) with t(17;19). To analyze the leukemogenic process and to create model mice for t(17;19)-positive leukemia, we generated inducible knock-in (iKI) mice for E2A-HLF. Despite the induced expression of E2A-HLF in the hematopoietic tissues, no disease was developed during the long observation period, indicating that additional gene alterations are required to develop leukemia. To elucidate this process, E2A-HLF iKI and control littermates were subjected to retroviral insertional mutagenesis. Virus infection induced acute leukemias in E2A-HLF iKI mice with higher morbidity and mortality than in control mice. Inverse PCR detected three common integration sites specific for E2A-HLF iKI leukemic mice, which induced overexpression of zinc-finger transcription factors: growth factor independent 1 (Gfi1), zinc-finger protein subfamily 1A1 isoform a (Zfp1a1, also known as Ikaros) and zinc-finger protein 521 (Zfp521). Interestingly, tumors with Zfp521 integration exclusively showed B-lineage ALL, which corresponds to the phenotype of human t(17;19)-positive leukemia. In addition, ZNF521 (human counterpart of Zfp521) was found to be overexpressed in human leukemic cell lines harboring t(17;19). Moreover, both iKI for E2A-HLF and transgenic for Zfp521 mice frequently developed B-lineage ALL. These results indicate that a set of transcription factors promote leukemic transformation of E2A-HLF-expressing hematopoietic progenitors and suggest that aberrant expression of Zfp521/ZNF521 may be clinically relevant to t(17;19)-positive B-lineage ALL.

Overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induced acute leukemia in p210BCR/ABL transgenic mice.

Chronic myelogenous leukemia (CML) is a hematopoietic disorder, which begins as indolent chronic phase but inevitably progresses to fatal blast crisis. p210BCR/ABL, a constitutively active tyrosine kinase, is responsible for disease initiation but molecular mechanism(s) underlying disease evolution remains largely unknown. To explore this process, we employed retroviral insertional mutagenesis to CML-exhibiting p210BCR/ABL transgenic mice (Tg). Virus infection induced acute lymphoblastic leukemia (ALL) in p210BCR/ABL Tg with a higher frequency and in a shorter latency than wild-type littermates, and inverse PCR detected two retrovirus common integration sites (CISs) in p210BCR/ABL Tg tumors. Interestingly, one CIS was the transgene itself, where retrovirus integrations induced upregulation of p210BCR/ABL and production of truncated BCR/ABL with an enhanced kinase activity. Another CIS was Notch1 gene, where retrovirus integrations resulted in overexpression of Notch1 and generation of Notch1 lacking the C-terminal region (Notch1DeltaC) associated with stable expression of its activated product, C-terminal-truncated Notch intracellular domain (NICD Delta C). In addition, generation of Tg for both p210BCR/ABL and Notch1DeltaC developed ALL in a shortened period with Stat5 activation, demonstrating the cooperative oncogenicity of Notch1DeltaC/NICD Delta C with p210BCR/ABL involving Stat5-mediated pathway. These results demonstrated that overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induces acute leukemia in a transgenic model for CML.

Thermodynamic properties in the approach to the quantum critical point of the spin-ladder material Na2Co2(C2O4)3(H2O)2.

Magnetic susceptibility and heat capacity measurements as a function of temperature on a single-crystal sample of a spin-ladder material, Na2Co2(C2O4)3(H2O)2, are reported. Principal susceptibilities, parallel and perpendicular to the ladder direction, respectively, show broad maxima around 22 and 17 K. Both susceptibilities decay exponentially down to about 5 K and thereafter they are essentially independent of temperature. These findings amount to a signature of a quantum phase transition from a spin-liquid to Néel ordered state previously predicted theoretically. No anomaly is found in the heat capacity around the transition temperature.

Zeeman levels with exotic field dependence in the high field phase of an S=1 Heisenberg antiferromagnetic chain.

We have performed electron spin resonance measurements over a wide frequency and magnetic field range on a single crystal of the S=1 quasi-one-dimensional Heisenberg antiferromagnet Ni(C5H14N2)2N3(PF6). We observed gapped excitation branches above the critical field H(c) where the Haldane gap closes. These branches are analyzed by a phenomenological field theory using the complex-field phi(4) model. A satisfactory agreement between experiment and theory is obtained.

Quasielastic neutron scattering in the high-field phase of a Haldane antiferromagnet.

Inelastic neutron scattering experiments on the Haldane-gap quantum antiferromagnet Ni(C5D14N2)2-N3(PF6) are performed in magnetic fields below and above the critical field H(c) at which the gap closes. Quasielastic neutron scattering is found for H>H(c), indicating topological excitations in the high-field phase.

Field-induced three- and two-dimensional freezing in a quantum spin liquid.

Field-induced commensurate transverse magnetic ordering is observed in the Haldane-gap compound Ni(C(5)D(14)N(2))2N(3)(PF(6)) by means of neutron diffraction. Depending on the direction of applied field, the high-field phase is shown to be either a three-dimensional ordered Néel state or a short-range ordered state with dominant two-dimensional spin correlations. The structure of the high-field phase is determined, and properties of the observed quantum phase transition are discussed.

Overexpression of the glycogen targeting (G(M)) subunit of protein phosphatase-1.

The G(M) glycogen-targeting subunit of protein phosphatase-1 (PP1) is believed to be involved in dephosphorylation of the enzymes of glycogen metabolism. To assess the roles of G(M) on glycogen metabolism, we created site-directed G(M) mutants and overexpressed them in Chinese hamster ovary (CHO) cells expressing human insulin receptor. Overexpressed G(M) recruited glycogen synthase as well as PP1 to the glycogen pellet, and upregulated basal glycogen synthase activity. Overexpressed G(M)-67A (Ser-67 replaced with alanine) exhibited decreased sensitivity to suppression of glycogen synthase activity by forskolin, while overexpression of G(M)-48A (Ser-48 replaced with alanine) preserved glycogen synthase activation in response to insulin. These observations indicate that in CHO cells overexpressing G(M); (1) G(M) translocates glycogen synthase to the glycogen pellet and affected basal glycogen synthase, (2) Ser-67 might be involved in the suppression of glycogen synthase activity by glycogenolytic agents, and (3) Ser-48 might not commit to activation of glycogen synthase by insulin.

Differential involvement of Src family kinases in Fc gamma receptor-mediated phagocytosis.

The tyrosine phosphorylation cascade originated from Fc gamma receptors (Fc gamma Rs) is essential for macrophage functions including phagocytosis. Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined. In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the Fc gamma R-mediated signaling that leads to phagocytosis in a kinase-dependent manner. We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed Fc gamma R-mediated Syk activation, filamentous actin rearrangement, and phagocytosis. In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling. Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but Fc gamma RIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck. These findings suggest that Src family kinases are differentially involved in Fc gamma R-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.

Sequential requirements of the N-terminal palmitoylation site and SH2 domain of Src family kinases in the initiation and progression of FcepsilonRI signaling.

Initial biochemical signaling originating from high-affinity immunoglobulin E receptor (FcepsilonRI) has been ascribed to Src family kinases. To understand the mechanisms by which individual kinases drive the signaling, we conducted reconstitution experiments: FcepsilonRI signaling in RBL2H3 cells was first suppressed by a membrane-anchored, gain-of-function C-terminal Src kinase and then reconstructed with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-myc epitope. Those constructs derived from Lyn and Fyn, which are associated with detergent-resistant membranes (DRMs), physically interacted with resting FcepsilonRI and reconstructed clustering-induced signaling that leads to calcium mobilization and ERK1 and -2 activation. c-Src-derived construct, which was excluded from DRMs, failed to interact with FcepsilonRI and to restore the signaling, whereas creation of palmitoylatable Cys3 enabled it to interact with DRMs and with FcepsilonRI and to restore the signaling. Deletion of Src homology 3 (SH3) domain from the Lyn-derived construct did not alter its ability to transduce the series of signaling. Deletion of SH2 domain did not affect its association with DRMs and with FcepsilonRI nor clustering-induced tyrosine phosphorylation of FcepsilonRI beta and gamma subunits, but it almost abrogated the next step of tyrosine phosphorylation of Syk and its recruitment to FcepsilonRI. These findings suggest that Lyn and Fyn could, but c-Src could not, drive FcepsilonRI signaling and that N-terminal palmitoylation and SH2 domain are required in sequence for the initial interaction with FcepsilonRI and for the signal progression to the molecular assembly.

Interferon-gamma upregulates the c-Met/hepatocyte growth factor receptor expression in alveolar epithelial cells.

In the repair process after lung injury, the regeneration of alveolar epithelial cells plays an important role by covering the damaged alveolar wall and preventing the activated fibroblasts from invading the intra- alveolar spaces. Hepatocyte growth factor (HGF) is a potent mitogen for alveolar epithelial cells and has been reported to be capable of repressing the fibrosing process by connecting to the c-Met/HGF receptor on alveolar epithelial cells. However, it has been reported that the c-Met expression was downregulated in an acute phase of lung injury, which may limit the effect of HGF for therapeutic use. In the present study we observed that interferon (IFN)-gamma upregulates the c-Met messenger RNA (mRNA) and protein expression in A549 alveolar epithelial cells. We analyzed the mechanism of this upregulation and found that IFN-gamma enhances the transcription of the c-met proto-oncogene, and that it does not prolong the stability of the c-Met mRNA. HGF is known to act as a motogen as well as a mitogen for epithelial cells. We also found that the migratory activity of A549 cells induced by HGF is strongly enhanced by preincubation with IFN-gamma. Finally, we administered recombinant IFN-gamma to C57BL/6 mice and confirmed that this upregulation is also observed in vivo. These results suggest that the combination of HGF and IFN-gamma could be a new therapeutic approach for fibrosing pulmonary diseases.

Essential roles of Lyn in fibronectin-mediated filamentous actin assembly and cell motility in mast cells.

Although the requirement for c-Src in extracellular matrix (ECM)-mediated fibroblast motility has been well established, the roles of hemopoietic Src family protein tyrosine kinases in leukocyte migration have not been fully elucidated. To address the issue, we analyzed fibronectin (Fn)-mediated adhesion signaling in rat basophilic leukemia (RBL) 2H3 cells overexpressing 1) Csk, 2) a membrane-anchored, gain-of-function Csk (mCsk), and 3) a kinase-defective mCsk (mCsk(-)). Parent RBL2H3 cells, expressing autoactivated c-kit, readily adhered to Fn-coated surface, developed typical leukocyte adhesion machinery (podosome), and migrated toward Fn without cytokine priming, thus provided a simple experimental system to analyze Fn-mediated outside-in signaling. While overexpression of Csk or the Csk mutants did not significantly affect cell adhesion to the Fn surface or alpha5 integrin recruitment to the attachment sites, Csk suppressed and mCsk almost abolished Fn-mediated tyrosine phosphorylation of paxillin, filamentous actin assembly to podosomes, and cell migration, but mCsk(-) did not. Coexpression of LynA devoid of C-terminal negative regulatory tyrosine in mCsk cells successfully restored Fn-mediated podosome formation and cell migration. Coexpression of c-Src lacking the C-terminal tyrosine reconstructed podosomes, but could not restore the cell migration regardless of its expression level. Collectively, these observations provide evidence that Src family protein tyrosine kinases are required, and that Lyn could transmit sufficient signal for Fn-mediated cytoskeletal changes leading to cell locomotion in RBL2H3 cells, and they suggest that Lyn and c-Src are differentially involved in cell motility.

Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas.

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Roles of C-terminal Src kinase in the initiation and the termination of the high affinity IgE receptor-mediated signaling.

As an attempt to analyze the roles of C-terminal Src kinase (Csk) in the high affinity IgE receptor (FcepsilonRI)-mediated signaling, we overexpressed Csk, a membrane-targeted form of Csk (mCsk), and a kinase-defective, membrane-targeted form of Csk (mCsk(-)) in rat basophil leukemia (RBL) 2H3 cells. Specific activity of Lyn at the basal state was decreased in Csk-expressing cells, and further decreased in mCsk-expressing cells. In mCsk(-)-expressing cells, basal specific activity of Lyn was increased, thereby indicating that mCsk(-) functioned as a dominant negative molecule. The onset of FcepsilonRI-mediated Lyn activation was delayed in Csk-expressing cells, and further delayed in mCsk-expressing cells. In mCsk(-)-expressing cells, Lyn activation was rapid and quite long lasting. These findings indicate (i) Csk negatively regulates rapid FcepsilonRI/Lyn coupling, and (ii) Csk activity is potentially required for its termination. The onsets of the series of events including tyrosyl phosphorylation of Syk, mitogen-activated protein (MAP) kinase activation, elevation of intracellular calcium concentration ([Ca2+]i), and histamine release were all stepwisely delayed in Csk-expressing cells and in mCsk-expressing cells. The durations of Syk phosphorylation and MAP kinase activation also closely correlated with those of Lyn activation, but [Ca2+]i elevation and histamine release followed different temporal patterns: the delayed responses in Csk-expressing cells and in mCsk-expressing cells led to sustained [Ca2+]i oscillation and histamine release, while the prompt responses in parent cells and mCsk(-)-expressing cells rapidly subsided. These findings provide further evidence that the initiations of the FcepsilonRI-mediated signals are upstreamly regulated by Src family protein tyrosine kinases and revealed that their terminations are regulated by Lyn-dependent (Syk and MAP kinase) and -independent ([Ca2+]i elevation and histamine release) mechanisms.

Mutant of insulin receptor substrate-1 incapable of activating phosphatidylinositol 3-kinase did not mediate insulin-stimulated maturation of Xenopus laevis oocytes.

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin.

Micro-trap phosphorylation assay of mitogen-activated protein (MAP) kinases to detect their activation by lipopolysaccharides.

We designed a microplate-based assay method for mitogen-activated protein (MAP) kinase. Using anion-exchanger resin, MAP kinases from murine macrophages were partially purified in 96-well plates. The activities of these purified enzymes correlated well with those detected in heretofore used assays. The micro-trap phosphorylation assay has advantages over conventional methods (immunoprecipitation, Western blotting for the detection of mobility shift, or kinase detection assay in myelin basic protein (MBP)-containing gel), in terms of sensitivity, economy and rapid execution for hundreds of samples. Using micro-trap phosphorylation assay, it was demonstrated that MAP kinase activities in macrophages were persistently increased by lipopolysaccharide (LPS) stimulation, and this activation was inhibited by polymyxin B or tyrosine kinase inhibitors. This method is expected to give a wide range of application, such as determining effects of drug inhibitors or antisense oligonucleotides on MAP kinases, or measuring the various protein kinases after specificity controls were done.

Amino-acid sequence and tissue distribution of guinea-pig leukotriene A4 hydrolase.

The guinea-pig leukotriene A4 hydrolase (LTA4H)-encoding cDNA was isolated from a guinea-pig lung cDNA library by cross-hybridization using a human probe. The deduced amino acid (aa) sequence consists of 611 aa (68 756 Da) and contains all twelve internal peptide and N-terminal sequences determined from the purified enzyme from guinea-pig intestine. The aa identity of the guinea-pig enzyme with its human, mouse and rat counterparts was 92.9, 90.5 and 90.4%, respectively. The previously characterized zinc-binding motif and a putative active site were highly conserved, supporting the aminopeptidase activity described for this enzyme. RNA blot analysis demonstrated ubiquitous expression of the LTA4H mRNA.

Prostaglandin F2 alpha stimulates formation of p21ras-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via Gq-protein-coupled pathway.

Prostaglandin (PG) F2 alpha activated mitogen-activated protein (MAP) kinase and MAP kinase kinase in NIH-3T3 cells by a mechanism that was completely inhibited by protein kinase inhibitors, staurosporine (20 nM) or H-7 (20 microM), but was insensitive to pretreatment with islet-activating protein (100 ng/ml; 24 h) or 12-O-tetradecanoylphorbol 13-acetate (2.5 microM; 24 h). PGF2 alpha stimulation also led to a significant increase in Ras.GTP complex. Transfection of a cDNA encoding a constitutively active mutant of Gq alpha-subunit (Q209L) mimicked PGF2 alpha-induced MAP kinase activation, increase in Ras.GTP complex, and DNA synthesis in these cells, suggesting that activation of Gq mediates the PGF2 alpha-activation of Ras-MAP kinase pathway and mitogenesis in NIH-3T3 cells. These data provide a new insight into regulatory mechanisms of Ras-MAP kinase pathway through heterotrimeric G-protein-mediated pathways.