Ryan Oyer

Epidermal Growth Factor Protects Epithelial Cells against Fas-induced Apoptosis REQUIREMENT FOR Akt ACTIVATION

Chemotherapeutic drugs that damage DNA kill tumor cells, in part, by inducing the expression of a death receptor such as Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B (Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of EGF-stimulated extracellular signal-regulated kinase (ERK) activity did not affect EGF protection from Fas-mediated apoptosis. The findings indicate that EGF receptor stimulation of epithelial cells has a significant survival function against death receptor-induced apoptosis mediated by Akt.

Increased Expression of Death Receptors 4 and 5 Synergizes the Apoptosis Response to Combined Treatment with Etoposide and TRAIL

ABSTRACT Chemotherapeutic genotoxins induce apoptosis in epithelial-cell-derived cancer cells. The death receptor ligand TRAIL also induces apoptosis in epithelial-cell-derived cancer cells but generally fails to induce apoptosis in nontransformed cells. We show here that the treatment of four different epithelial cell lines with the topoisomerase II inhibitor etoposide in combination with TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) induces a synergistic apoptotic response. The mechanism of the synergistic effect results from the etoposide-mediated increase in the expression of the death receptors 4 (DR4) and 5 (DR5). Inhibition of NF-κB activation by expression of kinase-inactive MEK kinase 1(MEKK1) or dominant-negative IκB (ΔIκB) blocked the increase in DR4 and DR5 expression following etoposide treatment. Addition of a soluble decoy DR4 fusion protein (DR4:Fc) to cell cultures reduced the amount of etoposide-induced apoptosis in a dose-dependent manner. The addition of a soluble TNF decoy receptor (TNFR:Fc) was without effect, demonstrating the specificity of DR4 binding ligands in the etoposide-induced apoptosis response. Thus, genotoxin treatment in combination with TRAIL is an effective inducer of epithelial-cell-derived tumor cell apoptosis relative to either treatment alone.

West Nile virus growth is independent of autophagy activation

West Nile virus (WNV) is an arthropod-borne virus with a worldwide distribution that causes neurologic disease and death. Autophagy is a cellular homeostatic mechanism involved in antiviral responses but can be subverted to support viral growth as well. We show that autophagy is induced by WNV infection in cell culture and in primary neuron cultures. Following WNV infection, lysosomes co-localize with autophagosomes resulting in LC3B-II turnover and autolysosomal acidification. However, activation or inhibition of autophagy has no significant effect on WNV growth but pharmacologic inhibition of PI3 kinases associated with autophagy reduce WNV growth. Basal levels of p62/sequestosome1(SQSTM1) do not significantly change following WNV-induced autophagy activation, but p62 is turned over or degraded by autophagy activation implying that p62 expression is increased following WNV-infection. These data show that WNV-induces autophagy but viral growth is independent of autophagy activation suggesting that WNV-specific interactions with autophagy have diverged from other flaviviruses.

West Nile and St. Louis encephalitis viruses

Abstract West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are arthropod-borne flaviviruses that belong to the Japanese encephalitis virus antigenic complex. SLEV transmission is limited to North and South America, whereas WNV infection occurs on six continents. WNV is now the most common cause of epidemic viral meningoencephalitis in the United States and greater than 30 000 human cases have been reported since its emergence in New York City in 1999. Both viruses are maintained in the natural environment in a cycle between mosquitoes and birds. Human infection is an incidental, non-amplifying, dead-end occurrence in the lifecycle of these enzoonitic viruses and neither WNV nor SLEV is naturally transmitted from person to person. The majority of infections are asymptomatic (80%) and most clinical illness manifests as a self-limited, febrile, flu-like syndrome. However, a small percentage of individuals (

In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain.

MEKK2 and MEKK3 are mitogen-activated protein kinase kinase kinases (MAP3 kinases) of 70 and 71 kDa respectively that are markedly homologous (94%) in their kinase domains. Both MEKK2 and MEKK3 are able to activate the Jun kinase pathway in vivo. However, following routine immunoprecipitation in Triton X-100, MEKK2 but not MEKK3 is able to effectively phosphorylate both SEK-1 and MEK-1 and to undergo autophosphorylation. Unexpectedly, both MEKK2 and MEKK3 are functional in an in vitro kinase assay when cells are solubilized with the closely related detergent, NP-40. Given the high homology between these kinases, we set out to relate this differential sensitivity to Triton X-100 to differences in primary structure. A set of chimeric molecules were generated and the loss of activity in Triton X-100 mapped to kinase domain II/III and specifically to serine 390 of MEKK3 and valine 384 of MEKK2, residues immediately N-terminal to the active site lysine. Mutation of serine 390 of MEKK3 to a valine (as is found in MEKK2) conferred catalytic activity to MEKK3 in Triton X-100 whereas the reciprocal alteration of valine 384 of MEKK2 to a serine conferred lack of activity in Triton X-100 to MEKK2. Search of the protein database identified only three kinases, MEKK3, Pbs2p and Dd-PKI, with a serine or threonine at this site. The presence of a serine or threonine adjacent to the active site lysine in protein kinases is rare and, in MEKK3, results in detergent instability.

Elevated CSF Cytokines in the Jarisch-Herxheimer Reaction of GeneralParesis

IMPORTANCE The Jarisch-Herxheimer reaction (JHR) is a well-recognized transient worsening of signs and symptoms occurring soon after the first dose of an appropriate antibiotic for several spirochetal infections. The pathogenesis of this reaction is poorly understood. In this case study of cerebrospinal fluid (CSF) cytokines, we aimed to improve understanding of the pathogenesis of JHR in patients with neurosyphilis who develop transient neurologic signs. OBSERVATIONS Four hours after receiving penicillin for general paresis, a 55-year-old man developed a severe JHR characterized by fever, tachycardia, hypertension, obtundation, seizures, and a neutrophilia lasting 18 hours. Cerebrospinal fluid obtained at the peak of the JHR demonstrated a switch from a mild lymphophilia to a moderate neutrophilia. He had markedly elevated CSF interleukin (IL) 8 and likely elevated IL-1β, IL-10, and IL-15 levels, which returned to normal in follow-up CSF examination results. CONCLUSIONS AND RELEVANCE To our knowledge, this is the first report of elevated CSF cytokines in a patient with a JHR, which possibly contributed to the neurologic signs of JHR. Further studies on the innate inflammatory response during episodes of acute infection and inflammation are needed to develop targeted therapies to modulate this system, which could, in turn, improve future outcomes and modify the JHR.