Nuclear proto-oncogene products transactivate the human papillomavirus type 16 promoter.

Human papillomavirus (HPV) type 16 and 18 viral genomes are frequently detected in cervical and penile cancer biopsies. Although this strongly suggests a prominent role for HPV infection in the development of genital cancer, other genetic or environmental factors are also involved. Genital cancer is postulated to result from loss of cellular control functions, which leads to an unregulated expression of HPV oncogenic proteins. In our study, we determined the trans-activating properties of nuclear proto-oncogene proteins c-Fos, c-Jun and c-Myc on P97 enhancer/promoter activity of HPV16. Using a CAT-reporter construct containing the HPV16 enhancer/promoter element, we investigated the trans-activating effects of c-Fos, c-Jun, c-Myc, and E2 in cervical HT-3 cells. c-Fos and c-Jun overexpression resulted in a 3.3- and 3.1-fold up-regulation of CAT activity. Only 2-fold induction was determined by co-transfection with c-myc and the viral transcription factor E2. Based on these findings, we investigated the expression of HPV DNA (16 and 18) as well as nuclear proto-oncogenes (c-fos, c-jun and c-myc) in nine cervical cancers by in situ hybridisation. In six out of nine carcinomas, HPV16 and/or HPV18 DNA was detectable. All tumours showed an intense and homogeneous expression of c-fos and c-jun mRNA, while the signal for c-myc was detectable only in four specimens. These data suggest that deregulation of nuclear proto-oncogene expression may contribute to an overexpression of HPV-derived oncogenic proteins (E6 and E7), which is generally hypothesised to be an important step in the malignant transformation of HPV-associated tumours

Optimization of Naked DNA Delivery for Interferon Subtype Immunotherapy in Cytomegalovirus Infection

Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus (CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal efficacy was found with bupivacaine treatment prior to DNA inoculation of 200mg IFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule. Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMV gB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases

Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events

RAF Kinase Activity Regulates Neuroepithelial Cell Proliferation and Neuronal Progenitor Cell Differentiation during Early Inner Ear Development

Background: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. Methodology/Principal Findings: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. Conclusions/Significance: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG

Lysophosphatidic Acid Induces MDA-MB-231 Breast Cancer Cells Migration through Activation of PI3K/PAK1/ERK Signaling

Enhanced motility of cancer cells is a critical step in promoting tumor metastasis. Lysophosphatidic acid (LPA), representing the major mitogenic activity in serum, stimulates migration in various types of cancer cells. However, the underlying signaling mechanisms for LPA-induced motility of cancer cells remain to be elucidated.In this study, we found that LPA dose-dependently stimulated migration of MDA-MB-231 breast cancer cells, with 10 µM being the most effective. LPA also increased ERK activity and the MEK inhibitor U0126 could block LPA-induced ERK activity and cell migration. In addition, LPA induced PAK1 activation while ERK activation and cell migration were inhibited by ectopic expression of an inactive mutant form of PAK1 in MDA-MB-231 cells. Furthermore, LPA increased PI3K activity, and the PI3K inhibitor LY294002 inhibited both LPA-induced PAK1/ERK activation and cell migration. Moreover, in the breast cancer cell, LPA treatment resulted in remarkable production of reactive oxygen species (ROS), while LPA-induced ROS generation, PI3K/PAK1/ERK activation and cell migration could be inhibited by N-acetyl-L-Cysteine, a scavenger of ROS.Taken together, this study identifies a PI3K/PAK1/ERK signaling pathway for LPA-stimulated breast cancer cell migration. These data also suggest that ROS generation plays an essential role in the activation of LPA-stimulated PI3K/PAK1/ERK signaling and breast cancer cell migration. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

BACKGROUND: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. CONCLUSIONS/SIGNIFICANCE: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking