SUMMARY:A membrane invasion culture system was used to study the ability of EBV to enhance invasion and migration of nasopharyngeal carcinoma (NPC) cells. Semi-reverse transcriptase-PCR analysis of matrix proteinases and angiogenic factors from EBV-infected, or EBV-positive (EBVϩ), cells demonstrated different degrees of elevated gene expression. In our animal model, EBVϩ tumors grew faster and larger than EBV-free, or EBV-negative (EBVϪ), tumors and also had clonal EBV terminal repeat sequences. Double-localization of EBV and certain host proteins in EBVϩ tumors and biopsy specimens demonstrated that EBV up-regulates host genes only in cells that express those genes but not in cells that do not express them. Double-localization of EBV and host genes in NPC biopsy specimens all showed EBVϪ tumor cells expressing those host genes. Our data strongly suggest that EBV infection enhances progression of NPC tumor growth. They do not rule out a role for EBV infection in the induction and early promotion of NPC development. Unidentified factors may also enhance NPC tumor growth independent of the effects of EBV. (Lab Invest 2003, 83:797-812).

Several lines of evidence suggest that the aggregation and deposition of amyloid-β peptide (Aβ) initiate the pathology of Alzheimer's disease (AD). Recently, a genomewide association study demonstrated that a single-nucleotide polymorphism proximal to the EPHA4 gene, which encodes a receptor tyrosine kinase, is associated with AD risk. However, the molecular mechanism of EphA4 in the pathogenesis of AD, particularly in Aβ production, remains unknown. Here, we performed several pharmacological and biological experiments both in vitro and in vivo and demonstrated that EphA4 is responsible for the regulation of Aβ production. Pharmacological inhibition of EphA4 signaling and knockdown of Epha4 led to increased Aβ levels accompanied by increased expression of β-site APP cleaving enzyme 1 (BACE1), which is an enzyme responsible for Aβ production. Moreover, EPHA4 overexpression and activation of EphA4 signaling via ephrin ligands decreased Aβ levels. In particular, the sterile-alpha motif domain of EphA4 was necessary for the regulation of Aβ production. Finally, EPHA4 mRNA levels were significantly reduced in the brains of AD patients, and negatively correlated with BACE1 mRNA levels. Our results indicate a novel mechanism of Aβ regulation by EphA4, which is involved in AD pathogenesis.

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Background: Subnanomolar ouabain binding to the Na,K-ATPase triggers intracellular signaling, prevents an overload of neurons with Ca2+, and their apoptosis caused by glutamate receptor agonists. Elevated plasma homocysteine (HCY), known as hyperhomocysteinemia, represents a risk factor for stroke and can exacerbate many neuronal disorders. HCY acts as a persistent N-methyl-D-aspartate receptor (NMDAR) agonist, which, in contrast to glutamate, desensitizes NMDARs containing GluN2B subunits. Mechanisms of HCY neurotoxicity remain not clearly understood since GluN2B-containing NMDARs provide a major contribution to excitotoxicity among glutamate receptors.Methods: Using fluorescent tools combined with the confocal microscopy, we compared 0.1 - 1 nM ouabain effects on the intracellular Ca2+ signaling, on the mitochondrial inner membrane voltage and the cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. We also studied an apoptosis-related protein expression and the involvement of some kinases in ouabain mediated effects.Results: In short insults HCY was less potent than glutamate as a neurotoxic agent. This amino acid induced the voltage loss (∆φmit) of 0.2 of the total mitochondrial inner membrane voltage (φmit) instead of ∆φmit = 0.7 for glutamate. We have found that subnanomolar ouabain exhibited rapid and postponed neuroprotective effects on neurons and (1) rapidly reduced the Ca2+ overload of neurons and the voltage loss of inner mitochondrial membranes evoked by glutamate and HCY, and (2) prevented neuronal apoptosis during 24 h treatments with glutamate or HCY. Using a set of specific kinase inhibitors such as PKA inhibitor, chelerythrine, and KN93, we demonstrated the role of multi-kinase signaling pathways involving PKC and PKA in neuronal survival caused by ouabain in hyperhomocysteinemia. Conclusions: Subnanomolar ouabain prevents neurodegeneration caused by glutamate and HCY. For both amino acids, ouabain evokes an acceleration of Ca2+ export by sodium-calcium exchangers from neurons preventing the voltage loss by mitochondrial inner membranes that rescue neurons in short insults. In prolonged insults, ouabain triggers intracellular neuroprotective cascades, including activation of PKA and PKC for HCY, but not for glutamate. This suggests that different appropriate pharmacology for hyperhomocysteinemia and glutamate excitotoxicity could be applied for clinical treatments.