Susan M. Fennewald

Inhibition of high affinity basic fibroblast growth factor binding by oligonucleotides.

Oligonucleotides can be used to inhibit the binding of basic fibroblast growth factor to cells. Though standard phosphodiester oligonucleotides show a slight inhibition of binding, the oligonucleotides with phosphorothioate internucleoside linkages have inhibition levels equivalent to that of the polyanion heparin. Variations in sequence of the oligonucleotides does lead to differences in the inhibitory action of the oligonucleotides. This inhibition of basic fibroblast growth factor by phosphorothioate oligonucleotides may account for much of the published data on inhibition of various genes by proposed antisense oligonucleotides and needs to be taken into account when considering the mechanism of action of oligonucleotides in biological systems.

Identification of Differentially Activated Cell-Signaling Networks Associated with Pichinde Virus Pathogenesis by Using Systems Kinomics

ABSTRACT Phosphorylation plays a key role in regulating many signaling pathways. Although studies investigating the phosphorylated forms of signaling pathways are now commonplace, global analysis of protein phosphorylation and kinase activity has lagged behind genomics and proteomics. We have used a kinomics approach to study the effect of virus infection on host cell signaling in infected guinea pigs. Delineating the host responses which lead to clearance of a pathogen requires the use of a matched, comparative model system. We have used two passage variants of the arenavirus Pichinde, used as a biosafety level 2 model of Lassa fever virus as it produces similar pathologies in guinea pigs and humans, to compare the host cell responses between infections which lead to either a mild, self-limiting infection or lethal disease. Using this model, we can begin to understand the differences in signaling events which give rise to these markedly different outcomes. By contextualizing these data using pathway analysis, we have identified key differences in cellular signaling matrices. By comparing these differentially involved networks, we have identified a number of key signaling “nodes” which show differential phosphorylations between mild and lethal infections. We believe that these nodes provide potential targets for the development of antiviral therapies by acting at the level of the host response rather than by directly targeting viral proteins.

Alterations in NF-κB and RBP-Jκ by Arenavirus Infection of Macrophages In Vitro and In Vivo

ABSTRACT Pichinde virus is an arenavirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-κB and RBP-Jκ, we compared the activities of NF-κB and RBP-Jκ in P388D1 cells following infection with Pichinde virus. The attenuated P2 virus inhibited NF-κB activation and caused a shift in the size of the RBP-Jκ complex. The virulent P18 virus showed less inhibition of NF-κB and failed to alter the size of the RBP-Jκ complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-κB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jκ. By contrast, P18 induced large amounts of the NF-κB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jκ size. Taken together, these changes suggest that the attenuated viral strain induces an “activation” of macrophages, while the virulent form of the virus does not.

Inhibition of herpes simplex virus in culture by oligonucleotides composed entirely of deoxyguanosine and thymidine

Oligodeoxynucleotides (ODNs) composed entirely of deoxyguanosine and thymidine, but not specifically designed to act as antisense agents, were able to significantly inhibit herpes simplex virus growth in acute infection assay systems. The guanosine/thymidine (GT) ODNs which demonstrated this antiviral activity contained either natural phosphodiester (PO) or phosphorothioate (PS) modified internucleoside linkages. In all experiments, the antiviral activity of the ODNs was enhanced when the backbone was modified to contain the PS linkages. When present during the time of virus addition, the ODNs were able to block the adsorption of virus to Vero cells. In this assay the PS-containing ODNs had ID50 values of approximately 0.020 microM for HSV-2 and of 0.3 microM for HSV-1. When these same PS-containing ODNs were used against HSV-2 in single-cycle viral yield assays, designed to minimize the effects due to external blockage of virus, the ID50 values rose to 0.2 microM. Analysis of viral DNA obtained 14 h post-HSV-2 infections in the single-cycle assay, revealed a decrease in replicated viral DNA in cells treated with PS-ODNs. Analysis of viral mRNA obtained 4 h post-HSV-2 infection revealed, in cells treated with the PS-ODNs, a decrease in measurable HSV-2 alpha- and beta-mRNAs. Although the mechanism of action of the antiviral activity (beyond adsorption blocking) is not fully understood, the toxicity of these compounds was low, giving high therapeutic indices for the GT-rich PS-ODNs. The good therapeutic index of GT-ODNs make this a class of compounds which warrant investigation as therapeutic agents to be used against herpes viruses.

Differential Signaling Networks Induced by Mild and Lethal Hemorrhagic Fever Virus Infections

ABSTRACT The family Arenaviridae includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants of Pichinde virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified key intermediates which may be targets for therapeutic intervention.

L-selectin-mediated Lymphocyte-Cancer Cell Interactions under Low Fluid Shear Conditions

Cell migration in blood flow is mediated by engagement of specialized adhesion molecules that function under hemodynamic shear conditions, and many of the effectors of these adhesive interactions, such as the selectins and their ligands, are well defined. However, in contrast, our knowledge of the adhesion molecules operant under lymphatic flow conditions is incomplete. Among human malignancies, head and neck squamous cell cancer displays a marked predilection for locoregional lymph node metastasis. Based on this distinct tropism, we hypothesized that these cells express adhesion molecules that promote their binding to lymphoid tissue under lymphatic fluid shear stress. Accordingly, we investigated adhesive interactions between these and other cancer cells and the principal resident cells of lymphoid organs, lymphocytes. Parallel plate flow chamber studies under defined shear conditions, together with biochemical analyses, showed that human head and neck squamous cell cancer cells express heretofore unrecognized L-selectin ligand(s) that mediate binding to lymphocyte L-selectin at conspicuously low shear stress levels of 0.07–0.08 dynes/cm2, consistent with lymphatic flow. The binding of head and neck squamous cancer cells to L-selectin displays canonical biochemical features, such as requirements for sialylation, sulfation, and N-glycosylation, but displays a novel operational shear threshold differing from all other L-selectin ligands, including those expressed on colon cancer and leukemic cells (e.g. HCELL). These data define a novel class of L-selectin ligands and expand the scope of function for L-selectin within circulatory systems to now include a novel activity within shear stresses characteristic of lymphatic flow.

Solution structure and design of dithiophosphate backbone aptamers targeting transcription factor NF-κB☆

Abstract A variety of monothio- and dithiosubstituted duplex aptamers targeting NF-κB have been synthesized and designed. The specificity and affinity of the dithioate aptamers of p50 and RelA(p65) NF-κB homodimers was determined by gel shift experiments. The NMR solution structures for several unmodified and dithioate backbone modified 14-base paired duplex aptamers have been determined by a hybrid, complete matrix (MORASS)/restrained molecular dynamics method. Structural perturbations of the dithioate substitutions support our hypothesis that the dithioate binds cations less tightly than phosphoryl groups. This increases the electrostatic repulsion across the B-form narrow minor groove and enlarges the minor groove, similar to that found in A-form duplexes. Structural analysis of modeled aptamer complexes with NF-κB homo- and heterodimers suggests that the dithioate backbone substitution can increase the aptamer’s relative affinity to basic groups in proteins such as NF-κB by helping to “strip” the cations from the aptamer backbone.

Attenuated and lethal variants of pichindé virus induce differential patterns of NF-κB activation suggesting a potential target for novel therapeutics

Lassa virus pathogenesis is believed to involve dysregulation of cytokines. We have previously shown nuclear factor-kappaB (NF-kappaB) inhibition using a BSL-2 model for Lassa fever. Here we further define the potential mechanism for NF-kappaB inhibition as involving increased levels of repressive p50/p50 homodimers, and suggest a novel therapeutic strategy that acts via modulation of host signaling.

Silencing Met receptor tyrosine kinase signaling decreased oral tumor growth and increased survival of nude mice

OBJECTIVES The hepatocyte growth factor receptor (Met) is frequently overexpressed in Head and Neck Squamous Cell Carcinoma (HNSCC), correlating positively with high-grade tumors and shortened patient survival. As such, Met may represent an important therapeutic target. The purpose of this study was to explore the role of Met signaling for HNSCC growth and locoregional dissemination. MATERIALS AND METHODS Using a lentiviral system for RNA interference, we knocked down Met in established HNSCC cell lines that express high levels of the endogenous receptor. The effect of Met silencing on in vitro proliferation, cell survival and migration was examined using western analysis, immunohistochemistry and live cell imaging. In vivo tumor growth, dissemination and mouse survival was assessed using an orthotopic tongue mouse model for HNSCC. RESULTS We show that Met knockdown (1) impaired activation of downstream MAPK signaling; (2) reduced cell viability and anchorage independent growth; (3) abrogated HGF-induced cell motility on laminin; (4) reduced in vivo tumor growth by increased cell apoptosis; (5) caused reduced incidence of tumor dissemination to regional lymph nodes and (6) increased the survival of nude mice with orthotopic xenografts. CONCLUSION Met signaling is important for HNSCC growth and locoregional dissemination in vivo and that targeting Met may be an important strategy for therapy.

Laminin interactions with head and neck cancer cells under low fluid shear conditions lead to integrin activation and binding.

Background: Clinically relevant tumor cell/lymph node interactions are likely initiated in the context of lymphodynamic flow. Results: β1 integrins mediate interaction with laminins under lymphodynamic shear stress, resulting in a discrete calcium signal. Conclusion: β1 integrins mediate tumor cell/lymph node interactions active under lymphodynamic flow. Significance: These novel interactions may drive growth and immunomodulation in this niche. Lymphatic metastasis of cancer cells involves movement from the primary tumor site to the lymph node, where the cells must be able to productively lodge and grow. It is there that tumor cells encounter cellular and non-cellular constituent elements that make up the lymph node parenchyma. Our work shows that head and neck squamous cell carcinoma (HNSCC) cell lines are able to bind to laminin, fibronectin, vitronectin, and hyaluronic acid, which are extracellular matrix elements within the lymph node parenchyma. HNSCC cell lines bound to laminin under lymphodynamic low shear stress (0.07 dynes/cm2), consistent with lymph flow via β1 integrins, including α2β1, α3β1, and α6β1. Binding occurred in the presence of shear stress and not in the absence of flow. Additionally, tumor cell binding to laminin under flow did result in calcium signaling. Our data indicate a novel role for β1 integrin-mediated binding of HNSCC cells to laminin under conditions of lymphodynamic flow that results in intracellular calcium signaling within the cancer cell.