Anti-elF-2α (Phospho S51) monoclonal antibody

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 - a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors: (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of elF2 alpha and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions. (C) 2011 Elsevier Inc. All rights reserved.

In response to different cellular stresses, a family of protein kinases phosphorylates eIF2 alpha (alpha subunit of eukaryotic initiation factor-2), contributing to regulation of both general and gene-specific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2 alpha(P) (phosphorylated eIF2 alpha) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 19 1 194]. We show in the present study that one eIF2 alpha kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2 alpha(P) in these areas. In PKR-deficient animals subjected to SE, eIF2 alpha phosphorylation was clearly evident coincident with activation of a secondary eIF2 alpha kinase, PEK/PERK (pancreatic eIF2 alpha kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2 alpha phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2 alpha(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2 alpha contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2 alpha phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.