Menachem Ailenberg

Trichostatin A-histone deacetylase inhibitor with clinical therapeutic potential-is also a selective and potent inhibitor of gelatinase A expression.

Modulation of histone acetylation is currently being explored as a therapeutic strategy in treatment of cancer. Specifically, inhibition of histone deacetylase by trichostatin A (TSA) has been shown to prevent tumorigenesis and metastasis. In the present paper we demonstrate that increased histone acetylation by TSA-treated 3T3 cells decreases mRNA as well as zymographic activity of gelatinase A, a matrix metalloproteinase, which is itself, implicated in tumorigenesis and metastasis. Furthermore, TSA inhibits cytochalasin D-induced activation of gelatinase A, but TSA does not affect other members of the gelatinase A activation complex, MT1-MMP and TIMP-2. Thus, TSA is a selective and potent inhibitor of expression and activation of gelatinase A. This finding not only strengthens the rationale for continuing to investigate the therapeutic utility of TSA in cancer, but also, provides evidence that TSA inhibition of gelatinase A expression and activation can be used as a biological marker to monitor and determine end-points of clinical trials involving TSA.

Cytochalasin D disruption of actin filaments in 3T3 cells produces an anti-apoptotic response by activating gelatinase A extracellularly and initiating intracellular survival signals

Disruption of actin filaments affects multiple cell functions including motility, signal transduction and cell division, ultimately culminating in cell death. Although this is the usual sequence of events, we have made the interesting observation that disruption of actin filaments by the potent toxin cytochalasin D (Cyto D) causes one cell type, mouse mesangial cells (MMC), to undergo apoptosis, while in another cell type (NIH 3T3), it has the opposite effect, resulting in production of survival signals. The purpose of this study was to investigate the molecular basis for these observed differences. In the present communication, we demonstrate that exposure to Cyto D induces the pro-apoptotic pathways, p38 and stress-activated protein kinase (SAPK)/jun amino-terminal kinase (JNK), in both cell types. However, in 3T3, but not MMC, the extracellular signal regulated kinase (ERK) 1/2 pathway is protected from inhibition following treatment with Cyto D-leading to phosphorylation of Bclxi/Bcl 2-associated death promoter (BAD). Inhibition of Cyto D-induced secretion and activation of gelatinase A in 3T3 cells reverses the production of survival signals by Cyto-D. To investigate this effect further we employed CS-1 cells, a well-characterized melanoma cell line that lacks integrin beta3, and also does not secrete gelatinase A. Co-transfection of CS-1 cells with integrin beta3 and a gelatinase A transgene, which enables the cells to secrete constituitively active gelatinase A, enhances CS-1 cell survival signals. Together, our findings suggest that extracellularly activated gelatinase A, through interaction with integrin alphaVbeta3, elicits survival signals mediated through ERK 1/2 that override activation of p38 and SAPK/JNK stress pathways.

Differential effects of trichostatin A on gelatinase A expression in 3T3 fibroblasts and HT-1080 fibrosarcoma cells: implications for use of TSA in cancer therapy

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor with potential in cancer therapeutics. In a recent communication, we demonstrated that TSA is a selective, potent inhibitor of gelatinase A in 3T3 fibroblasts. In the present study, we extend these observations and examine the effects of TSA in 3T3 fibroblasts compared to HT-1080 fibrosarcoma cells with respect to gelatinase A expression, cell viability, and apoptosis. We find that while expression of gelatinase A in 3T3 fibroblasts is exquisitely sensitive to inhibition by TSA, expression of this enzyme in HT-1080 cells is minimally affected by this compound. Moreover, we show that TSA is pro-apoptotic in HT-1080 cells, but is anti-apoptotic in 3T3 cells. We propose a two-pronged model for the therapeutic action of TSA. On the one hand TSA selectively decreases cancer cell viability, while enhancing the viability of stromal cells. On the other hand, by selectively decreasing gelatinase A expression in stromal but not cancer cells, TSA acts to control metastatic potential by reducing the ability of metastatic cells to recruit stromal cells to secrete gelatinase A.

An improved Huffman coding method for archiving text, images, and music characters in DNA.

An improved Huffman coding method for information storage in DNA is described. The method entails the utilization of modified unambiguous base assignment that enables efficient coding of characters. A plasmid-based library with efficient and reliable information retrieval and assembly with uniquely designed primers is described. We illustrate our approach by synthesis of DNA that encodes text, images, and music, which could easily be retrieved by DNA sequencing using the specific primers. The method is simple and lends itself to automated information retrieval.

Oxidative Stress Increases Surface Toll-Like Receptor 4 Expression in Murine Macrophages Via Ceramide Generation.

ABSTRACT Multiorgan failure is a major cause of late mortality following trauma. Oxidative stress generated during shock/resuscitation contributes to tissue injury by priming the immune system for an exaggerated response to subsequent inflammatory stimuli, such as lipopolysaccharide (LPS). We recently reported that oxidative stress causes rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, thus increasing LPS responsiveness and cellular priming. We hypothesized that activation of Src family kinases by oxidants might contribute to these events. We utilized microscopy, flow cytometry, Western blotting, and thin-layer chromatography methods. Using hydrogen peroxide in vitro and hemorrhagic shock/resuscitation in vivo, oxidant-induced TLR4 translocation in macrophages occurred in an Src-dependent manner. Approaches supporting this conclusion included pharmacologic inhibition of the Src family kinases by PP2, Src inhibition by a molecular approach of cell transfection with Csk, and genetic inhibition of all Src kinases relevant to the monocyte/macrophage lineage in hck−/−fgr−/−lyn−/− triple knockout mice. To evaluate the upstream molecules involved in Src activation, we evaluated the ability of oxidative stress to activate the bioactive lipid molecule ceramide. Oxidants induced ceramide generation in macrophages both in vitro and in vivo, an effect that appears to be due to activation of the acid sphingomyelinase. Using pharmacological approaches, ceramide was shown to be both necessary and sufficient to mediate TLR4 translocation to the plasma membrane in an Src-dependent manner. This study identifies a hierarchy of signaling molecules following oxidative stress that might represent novel targets for therapy in critical illness and organ injury.

ROS stimulate reorganization of mesangial cell-collagen gels by tyrosine kinase signaling

Reactive oxygen species (ROS) initiate multiple pathological and physiological cellular responses, including tyrosine phosphorylation of proteins. In this study, we investigated the effects of ROS on cell-extracellular matrix interactions utilizing the floating three-dimensional collagen gel assay. Exposure of mesangial cells grown in three-dimensional culture to H2O2, 3-amino-1,2,4-triazole (a catalase inhibitor), or puromycin is associated with gel reorganization accompanied by tyrosine phosphorylation of multiple proteins, including focal adhesion kinase (FAK). Neutrophils cocultured with mesangial cells in three-dimensional culture also induce mesangial cell-collagen gel reorganization and initiate tyrosine phosphorylation of a similar set of proteins. Collectively, these results show that ROS of either endogenous or exogenous origin can modulate mesangial cell-extracellular matrix interactions through initiation of a phosphotyrosine kinase signaling cascade. Consequently, ROS may play a role as signaling molecules that regulate mesangial cell-extracellular matrix interactions in both physiological and pathological conditions.Reactive oxygen species (ROS) initiate multiple pathological and physiological cellular responses, including tyrosine phosphorylation of proteins. In this study, we investigated the effects of ROS on cell-extracellular matrix interactions utilizing the floating three-dimensional collagen gel assay. Exposure of mesangial cells grown in three-dimensional culture to H2O2, 3-amino-1,2,4-triazole (a catalase inhibitor), or puromycin is associated with gel reorganization accompanied by tyrosine phosphorylation of multiple proteins, including focal adhesion kinase (FAK). Neutrophils cocultured with mesangial cells in three-dimensional culture also induce mesangial cell-collagen gel reorganization and initiate tyrosine phosphorylation of a similar set of proteins. Collectively, these results show that ROS of either endogenous or exogenous origin can modulate mesangial cell-extracellular matrix interactions through initiation of a phosphotyrosine kinase signaling cascade. Consequently, ROS may play a role as signaling molecules that regulate mesangial cell-extracellular matrix interactions in both physiological and pathological conditions.

Description of a one step staggered reannealing method for directional cloning of PCR‐generated dna using sticky‐end ligation without employing restriction enzymes

A novel method is described for ligation of PCR products into vectors. It utilizes formation of sticky end sequences compatible with those generated on the plasmid. Four primers are used: two primers are designed to contain the desired sequence of the sticky end plus the sequence of the gene and the other two primers contain the same sequence as the first two primers without the additional bases. The primers are paired to create two PCR products containing the additional bases in a staggered 5′ position. Following melting and reannealing, the newly formed products contain the additional sequences as sticky overhangs compatible with the sticky ends of the plasmid.

Effect of a matrix metalloproteinase sequestering biomaterial on Caco-2 epithelial cell barrier integrity in vitro

A novel matrix metalloproteinase sequestering biomaterial (MI Theramer beads) restored the epithelial barrier in a double chamber in vitro test system after disruption by Cytochalasin D and the secretion of the metalloproteinase MMP-2. MI beads are chemically modified (hydroxamated) poly(methacrylic acid-co-methyl methacrylate). We are exploring the utility of this material in inflammatory bowel disease (IBD), in which one manifestation is a compromised intestinal epithelial barrier. In a first step towards this goal we incubated MI beads (or polymethyl methacrylate control beads) with Caco-2 epithelial cells and mesenchymal 3T3 fibroblasts on two sides of a Matrigel((R))-coated filter and used Cytochalasin D (Cyto D) to activate MMP-2 (secreted by the 3T3 cells), disrupt actin filaments of Caco-2 cells and render the epithelial barrier leaky, as measured by dextran fluorescein equilibration. Addition of MI beads to Cyto D-treated cells inhibited active MMP-2 and prevented equilibration of dextran fluorescein. This study is the first step in showing a potential benefit to local (as opposed to systemic) inhibition of metalloproteinases in IBD or other intestinal inflammatory diseases.

Dynasore enhances the formation of mitochondrial antiviral signalling aggregates and endocytosis-independent NF-κB activation.

Dynasore has been used extensively as an inhibitor of clathrin‐mediated endocytosis. While studying the role of endocytosis in LPS‐induced signalling events, we discovered that dynasore itself induced activation of NF‐κB, independently of its effects on endocytosis and without involving the Toll‐like receptor 4 signalling pathways. The purpose of this study was to characterize this novel effect and to explore the underlying mechanism of action.