A Previously Unknown Unique Challenge for Inhibitors of SYK ATP-Binding Site: Role of SYK as A Cell Cycle Checkpoint Regulator

The identification of SYK as a molecular target in B-lineage leukemia/lymphoma cells prompted the development of SYK inhibitors as a new class of anti-cancer drug candidates. Here we report that induction of the SYK gene expression in human cells causes a significant down-regulation of evolutionarily conserved genes associated with mitosis and cell cycle progression providing unprecedented evidence that SYK is a master regulator of cell cycle regulatory checkpoint genes in human cells. We further show that SYK regulates the G2 checkpoint by physically associating with and inhibiting the dual-specificity phosphatase CDC25C via phosphorylation of its S216 residue. SYK depletion by RNA interference or treatment with the chemical SYK inhibitor prevented nocodazole-treated human cell lines from activating the G2 checkpoint via CDC25C S216-phosphorylation and resulted in polyploidy. Our study provides genetic and biochemical evidence that spleen tyrosine kinase (SYK) has a unique role in the activation of the G2 checkpoint in both non-lymphohematopoietic and B-lineage lymphoid cells. This previously unknown role of SYK as a cell cycle checkpoint regulator represents an unforeseen and significant challenge for inhibitors of SYK ATP binding site

Structure-Based Design and Engineering of a Nontoxic Recombinant Pokeweed Antiviral Protein with Potent Anti-Human Immunodeficiency Virus Activity

A molecular model of pokeweed antiviral protein (PAP)-RNA interactions was used to rationally engineer FLP-102((151)AA(152)) and FLP-105((191)AA(192)) as nontoxic PAPs with potent anti-human immunodeficiency virus (anti-HIV) activities. FLP-102 and FLP-105 have been produced in Escherichia coli and tested both in vitro and in vivo. These proteins depurinate HIV type 1 (HIV-1) RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP. They are substantially less toxic than native PAP in BALB/c mice and exhibit potent in vivo activities against genotypically and phenotypically nucleoside reverse transcriptase inhibitor-resistant HIV-1 in a surrogate human peripheral blood lymphocyte (Hu-PBL) SCID mouse model of human AIDS. Rationally engineered nontoxic recombinant PAPs such as FLP-102 and FLP-105 may provide the basis for effective salvage therapies for patients harboring highly drug-resistant strains of HIV-1. The documented in vitro potencies of FLP-102 and FLP-105, their in vivo antiretroviral activities in the HIV-infected Hu-PBL SCID mouse model, and their favorable toxicity profiles in BALB/c mice warrant the further development of these promising new biotherapeutic agents

One-Step, Rapid, F-18-F-19 Isotopic Exchange Radiolabeling of Difluoro-dioxaborinins: Substituent Effect on Stability and In Vivo Applications

The beta-diketone moiety is commonly present in many anticancer drugs, antibiotics, and natural products. We describe a general method for radiolabeling beta-diketone-bearing molecules with fluoride-18. Radiolabeling was carried out via F-18-F-19 isotopic exchange on nonradioactive difluoro-dioxaborinins, which were generated by minimally modifying the beta-diketone as a difluoroborate. Radiochemistry was one-step, rapid (80%) and proceeded at room temperature to accommodate the half-life of F-18 (t(1/2) = 110 min). High molar activities (7.4 Ci/mu mol) were achieved with relatively low starting activities (16.4 mCi). It was found that substituents affected both the solvolytic stability and fluorescence properties of difluoro-dioxaborinins. An F-18 radiolabeled difluoro-dioxaborinin probe that was simultaneously fluorescent showed sufficient stability for in vivo positron emission tomography (PET)/fluorescence imaging in mice, rabbits, and patients. These findings will guide the design of probes with specific PET/fluorescence properties; the development of new PET/fluorescence dual-modality reporters; and accurate in vivo tracking of beta-diketone molecules

Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

<div><p>Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros.</p></div

MMP-2 and MMP-9 Alteration in Response to Collaring in Rabbits: The Effects of Endothelin Receptor Antagonism

WOS: 000271727400005PubMed ID: 19734325Matrix metalloproteinases (MMPs), and, in particular, gelatinases (MMP-2 and MMP-9), have been implicated in vascular cell proliferation and/or migration, contributing to intimal thickening, an essential stage in the development of atherosclerosis and restenosis following balloon angioplasty. Endothelin, a strong chemoatractant and mitogen, has been shown to promote smooth muscle cell proliferation and migration by activating MMPs via endothelin-A (ETA) receptors. The positioning of a soft silicon collar around the left carotid artery in rabbits results in intimal thickening. In this study, we investigate the possible role of gelatinases and the effect of a nonselective ETA/ETB receptor antagonist, TAK-044 (5 mg/kg body weight/day, subcutaneously [sc]), on these enzymes. Our results demonstrated that both MMP-2 and MMP-9 activities increased in response to collaring in placebo group, while treatment with TAK-044 significantly suppressed both gelatinase activities and proMMP-2 levels, and inhibited intimal thickening in collared arteries. These results suggest that either enhanced MMP expression or endothelin receptor antagonism may be involved in the formation of intimal thickening in this model.Novartis Pharmaceutical Company Pharmacological Research Grant; Ege University Science and Technology Centre (EBILTEM) Research Project AdministrationEge University [2004 BIL 002, ZK]Authors would like to thank TAKEDA Chemical Industries, Ltd., Japan, for kindly providing us with a sample of TAK-044. This work was substantially supported by a grant of Novartis Pharmaceutical Company Pharmacological Research Grant for 2003, Istanbul, Turkey (ZK) and also by Ege University Science and Technology Centre (EBILTEM) Research Project Administration (2004 BIL 002, ZK)

BTK Expression Levels Control Nuclear Localization of Ikaros.

<p>[<b>A</b>] Cells were stained with mouse monoclonal antibody (mAb) against human BTK and an anti-IK mouse mAb, which was raised against murine IK and which cross-reacts with human IK. TOTO-3 was used for nuclear staining. Depicted are the confocal two-color (green/blue: BTK/TOTO-3 and IK/TOTO-3) merge images of abundantly BTK⁺ (high BTK) leukemic cells from a pediatric B-precursor ALL case vs. leukemic cells with low BTK expression level from an infant B-precursor ALL case. Nuclear staining for IK was observed in the confocal fluorescence images of primary leukemic B-cell precursors with high BTK expression level. By contrast, leukemic cells with low BTK expression levels showed an aberrant, predominantly cytoplasmic localization of IK (System Magnification: 500×). [<b>B</b>] The DT-40 cell line and its subclones were stained with the rabbit polyclonal, H-100 (sc-13039) antibody against the N-terminus of IK. Depicted are the confocal two-color (red/blue) IK/DAPI merge images of wildtype (WT) DT40 cells, BTK-deficient DT40 cells, and BTK-deficient DT40 cells reconstituted with wildtype <i>btk</i>. The contour of the DAPI-stained nuclei (blue) was marked with a dotted line and shows a significant amount of IK protein (red) outside the nucleus of the BTK⁻ DT40 cells. [<b>C1</b>] Upper panel: Depicted are the confocal single-color and two-color merge images of control Hek293T cells stained with the secondary goat anti-mouse (GAM) antibody and DAPI. No false positive green fluorescence was detected (System Magnification: 630×). Lower panel: Depicted are the confocal single-color and two-color (green/blue: BTK/DAPI) merge images of test Hek293T cells stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI. BTK (green fluorescence) was localized primarily in the cytoplasm of Hek293T cells around the DAPI-stained blue nucleus. [<b>C2</b>] Left panel: Merge confocal images of untreated control Hek293T cells two-color stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI. Right panel: Merge confocal images of BTK-siRNA transfected Hek293T cells two-color stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI 72 h post-transfection. (System Magnification: 630×). BTK depletion was confirmed by Western blot analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071302#pone-0071302-g003" target="_blank">Figure 3</a>) [<b>D</b>] Confocal images of Hek293T cells expressing the HA-tagged mutant IK proteins D₆ or A₁₂ stained with the mouse monoclonal anti-HA antibody (HA-probe F-7)(primary Ab)/Alexa Fluor 488 goat anti-mouse IgG (secondary Ab) (green) antibody and blue fluorescent DNA dye 4′, 6-diamidino-2-phenylindole (DAPI) following treatment with BTK siRNA (50 nM) vs. PBS (CON). (System Magnification: 630×).</p