Helga Rübsamen-Waigmann

Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer

Our previous data indicate that the expression of the PLK gene which codes for a serine/threonine kinase is restricted to proliferating cells. In Northern blot experiments PLK mRNA expression was at the limit of detection in normal lung tissue but elevated in most samples of non-small cell lung cancer (NSCLC). A very low frequency of PLK transcripts was only found in bronchiolo-alveolar carcinomas. NSCLC patients whose tumors showed moderate PLK expression survived significantly longer (5 year survival rate=51.8%) than those with high levels of PLK transcripts (24.2%, P=0.001). No statistically significant correlation was found between PLK mRNA expression and age, sex, TNM status, histological type or degree of differentiation. Interestingly, the prognosis of patients in post-surgical stages I and II was correlated with PLK expression (5 year survival rates in stage I: 69.1% (moderate PLK)  –  43.5% (high PLK), P=0.03 or in stage II: 51.9% (moderate PLK)  –  9.9% (high PLK), P=0.006). These results suggest that PLK mRNA expression provides a new independent prognostic indicator for patients with NSCLC.

New helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease

The vast majority of the world population is infected with at least one member of the human herpesvirus family. Herpes simplex virus (HSV) infections are the cause of cold sores and genital herpes as well as life-threatening or sight-impairing disease mainly in immunocompromized patients, pregnant women and newborns. Since the milestone development in the late 1970s of acyclovir (Zovirax), a nucleosidic inhibitor of the herpes DNA polymerase, no new non-nucleosidic anti-herpes drugs have been introduced. Here we report new inhibitors of the HSV helicase-primase with potent in vitro anti-herpes activity, a novel mechanism of action, a low resistance rate and superior efficacy against HSV in animal models. BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide), a well-tolerated member of this class of compounds, significantly reduces time to healing, prevents rebound of disease after cessation of treatment and, most importantly, reduces frequency and severity of recurrent disease. Thus, this class of drugs has significant potential for the treatment of HSV disease in humans, including those resistant to current medications.

Human serum albumin–polyethylenimine nanoparticles for gene delivery

Nanoparticles consisting of DNA, human serum albumin (HSA) and polyethylenimine (PEI) were formed and tested for transfection efficiency in vitro with the aim of generating a nonviral gene delivery vehicle. HSA-PEI-DNA nanoparticles containing the pGL3 vector coding for luciferase as reporter gene were formed by charge neutralization. The particles were characterized by gel retardation assay, dynamic light scattering (size) and electrophoretic mobility measurements (charge). Stability was determined by spectrophotometric analysis and transfection efficiency was evaluated in cell culture using human embryonic epithelial kidney 293 cells. HSA-PEI-DNA nanoparticles were prepared by co-encapsulation of PEI as a lysosomotropic agent at varying nitrogen to phosphate (N/P) ratios. An optimum transfection efficiency was achieved when the particles were prepared at N/P ratios between 4.8 and 8.4. Furthermore, they displayed a low cytotoxicity when tested in cell culture. Our results show that HSA-PEI-DNA nanoparticles are a versatile carrier for DNA that may be suitable for i.v. administration.

Potent In Vivo Antiviral Activity of the Herpes Simplex Virus Primase-Helicase Inhibitor BAY 57-1293

ABSTRACT BAY 57-1293 belongs to a new class of antiviral compounds and inhibits replication of herpes simplex virus (HSV) type 1 and type 2 in the nanomolar range in vitro by abrogating the enzymatic activity of the viral primase-helicase complex. In various rodent models of HSV infection the antiviral activity of BAY 57-1293 in vivo was found to be superior compared to all compounds currently used to treat HSV infections. The compound shows profound antiviral activity in murine and rat lethal challenge models of disseminated herpes, in a murine zosteriform spread model of cutaneous disease, and in a murine ocular herpes model. It is active in parenteral, oral, and topical formulations. BAY 57-1293 continued to demonstrate efficacy when the onset of treatment was initiated after symptoms of herpetic disease were already apparent.

Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

ABSTRACT Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

Cell-Cell Adhesion Mediated by Binding of Membrane-anchored Ligand LERK-2 to the EPH-related Receptor Human Embryonal Kinase 2 Promotes Tyrosine Kinase Activity

Human embryonal kinase 2 (HEK2) is a protein-tyrosine kinase that is a member of the EPH family of receptors. Transcripts for HEK2 have a wide tissue distribution. Recently, a still growing family of ligands, which we have named LERKs, for igands of the ph-elated inases, has been isolated. In order to analyze functional effects between the LERKs and the HEK2 receptor, we expressed HEK2 cDNA in an interleukin-3-dependent progenitor cell line 32D that grows as single cells in culture. Within the group of LERKs, LERK-2 and −5 were shown to bind to HEK2. Membrane-bound and soluble forms of LERK-2 were demonstrated to signal through HEK2 as judged by receptor phosphorylation. Coincubation of HEK2 and LERK-2 expressing cells induced cell-cell adhesion and formation of cell aggregates. This interaction could be inhibited by preincubation of HEK2 expressing cells with soluble LERK-2. Coexpression of HEK2 and LERK-2 in 32D cells showed reduced kinase activity and autophosphorylation of HEK2 compared with the juxtacrine stimulation, which seems to be due to a reduced sensitivity of the receptor.

Suppression of Acute Viremia by Short-Term Postexposure Prophylaxis of Simian/Human Immunodeficiency Virus SHIV-RT-Infected Monkeys with a Novel Reverse Transcriptase Inhibitor (GW420867) Allows for Development of Potent Antiviral Immune Responses Resulting in Efficient Containment of Infection

ABSTRACT A nonnucleoside reverse transcriptase (RT) inhibitor, GW420867, was tested for postexposure prophylaxis (PEP) in rhesus macaques experimentally infected with 100 50% tissue culture infective doses of a chimeric simian/human immunodeficiency virus (SHIV) containing the RT gene of HIV-1 (SHIV-RT). Animals were either mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867. While such therapy led to undetectable plasma viremia in three of six monkeys, a transient plasma viremia was noted in the other three treated animals at 2 to 4 weeks following cessation of therapy. Following this transient viremia all drug-treated animals showed low or undetectable levels of plasma viremia up to the last sample examined at 90 weeks p.i. Despite low and/or undetectable viremia, virus-specific cytotoxic T lymphocyte and viral Env-specific proliferative responses were seen in the peripheral blood mononuclear cells of both mock- and drug-treated animals as early as 3 weeks p.i. Such virus-specific cellular responses, however, were better maintained in the drug-treated animals than the mock-treated animals. In contrast to the virus-specific cellular response, the magnitude and kinetics of virus specific humoral responses appeared to correlate with the detection of viremia. These data support the view that a short-term PEP with GW420867 permits the generation and maintenance of long-lasting virus-specific cell-mediated immune responses while markedly reducing viral loads to undetectable levels for a prolonged period of time (90 weeks) and leads to long-term disease protection. This model provides a unique means to define mechanisms and correlates of disease protection.

Tyrosine-614, the major autophosphorylation site of the receptor tyrosine kinase HEK2, functions as multi-docking site for SH2-domain mediated interactions

HEK2 belongs to the family of EPH-related receptor tyrosine kinases (RTK) which are involved in axonal pathfinding and the formation of the embryonic body plan. The knowledge about intracellular pathways of signal transduction mediated by EPH-related receptors is still limited. Many of the known key players of cellular signalling contain Src homology 2 (SH2) domains, which recognize phosphotyrosine motifs in RTKs. Thus, we examined the interactions of various SH2-containing molecules like PLC-γ1, rasGAP, p85 subunit of PI3-kinase, Src, Fyn, Crk, Nck, Grb2 and Shc with HEK2 using in vitro binding assays, immunoprecipitations and yeast Two-Hybrid assays. We found that rasGAP, Crk and Fyn bind in a SH2-dependent manner to autophosphorylated HEK2. rasGAP, which contains two SH2- and one SH3-domain, was shown to associate with its N-terminal SH2-domain to HEK2. Furthermore, we demonstrated that a single amino acid substitution (Y614F) clearly reduces the phosphotyrosine content of HEK2 and abrogates its ability to bind rasGAP, Crk and Fyn indicating that this residue functions as major phosphorylation and multi-docking site. The conservation of this predicted binding site among various EPH-related RTKs provides evidence that Fyn, Crk and rasGAP are key players in signal transduction of at least a subset of these receptors.

Secretory repertoire of HIV-infected human monocytes/macrophages

Apart from lymphocytes, mononuclear phagocytes play an essential role as target cells for human immunodeficiency virus (HIV). Circulating blood monocytes (MOs) and tissue macrophages (M phi) may harbor and distribute the virus throughout the body. In addition, proinflammatory monokines [interleukin-1 (IL-1), IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha)] may contribute to the pathogenesis of HIV-mediated diseases. We have established a culture system on hydrophobic Teflon membranes for blood-borne MOs/M phi. Both freshly isolated MOs as well as MO-derived M phi could be infected with a monocytotropic HIV-1 isolate (HIV-1D117III) derived from a perinatally infected child. The virus production monitored by assay for viral antigen in cell-free supernatant is continuous for several weeks. We analyzed the stimulus response and the secretory repertoire of MOs/M phi early after infection with HIV as well as in long-term cultured, virus-replicating cells. Infected MOs/M phi respond to interferon-gamma more effectively than control cells as estimated from the release of neopterin. The response to lipopolysaccharide was regulated differently: whereas the proinflammatory cytokines IL-1, IL-6, IL-8 and TNF-alpha were up-regulated and even constitutively secreted upon infection, the production of the hematopoietin macrophage-colony-stimulating factor decreased. High levels of TNF-alpha and IL-1 might augment the infectibility of M phi by HIV in an autocrine manner. Our results may provide some explanation for the immunologic dysfunction, the hematopoietic failure and the chronic inflammatory disease occurring in HIV-infected patients.

Infection of monocytes/macrophages by HIV in vitro

Because of the very important role of the mononuclear phagocyte system in the immunopathogenesis of HIV infection, a culture system for in vitro studies of infection of monocytes/macrophages with HIV was developed. A method is described for the infection of human monocytes/macrophages cultured on hydrophobic membranes (Teflon) with different strains of HIV. The HIV isolates can be characterized according to their replication potential on monocytes/macrophages cultures. The biological properties of some HIV1 and HIV2 isolates are compared in lymphocyte and monocyte/macrophage cultures.