Inhibition of IGF-I receptor signaling in combination with rapamycin or temsirolimus increases MYC-N phosphorylation.

BACKGROUND: It has been previously shown that blockade of the type 1 insulin-like growth factor receptor (IGF1R) signaling combined with mTOR inhibition decreased neuroblastoma proliferation in vitro. MYC-N inactivation occurs through phosphorylation by downstream elements of the IGF1R signaling pathway. It was hypothesized that inhibition of IGF1R signaling would increase the inactivation of MYC-N. MATERIALS AND METHODS: BE-2(c) and IMR-32 neuroblastoma cell lines were treated with varying concentrations of alphaIR3, rapamycin and temsirolimus either alone or in combination and the expression of MYC-N and phosphorylated MYC-N proteins were evaluated by Western blotting. The number of apoptotic cells was evaluated through cleaved caspase-3 expression. RESULTS: IGF1R signaling blockade in combination with mTOR inhibition decreased MYC-N protein expression, increased MYC-N phosphorylation and significantly increased cleaved caspase-3 expression in treated cells. CONCLUSION: The combination of rapamycin or temsirolimus with alphaIR3 decreases MYC-N expression, increases MYC-N phosphorylation and induces apoptosis in vitro which may have clinical relevance to children with neuroblastoma.

Acute ethanol administration rapidly increases phosphorylation of conventional protein kinase C in specific mammalian brain regions in vivo.

BACKGROUND: Protein kinase C (PKC) is a family of isoenzymes that regulate a variety of functions in the central nervous system including neurotransmitter release, ion channel activity, and cell differentiation. Growing evidence suggests that specific isoforms of PKC influence a variety of behavioral, biochemical, and physiological effects of ethanol in mammals. The purpose of this study was to determine whether acute ethanol exposure alters phosphorylation of conventional PKC isoforms at a threonine 674 (p-cPKC) site in the hydrophobic domain of the kinase, which is required for its catalytic activity. METHODS: Male rats were administered a dose range of ethanol (0, 0.5, 1, or 2 g/kg, intragastric) and brain tissue was removed 10 minutes later for evaluation of changes in p-cPKC expression using immunohistochemistry and Western blot methods. RESULTS: Immunohistochemical data show that the highest dose of ethanol (2 g/kg) rapidly increases p-cPKC immunoreactivity specifically in the nucleus accumbens (core and shell), lateral septum, and hippocampus (CA3 and dentate gyrus). Western blot analysis further showed that ethanol (2 g/kg) increased p-cPKC expression in the P2 membrane fraction of tissue from the nucleus accumbens and hippocampus. Although p-cPKC was expressed in numerous other brain regions, including the caudate nucleus, amygdala, and cortex, no changes were observed in response to acute ethanol. Total PKCgamma immunoreactivity was surveyed throughout the brain and showed no change following acute ethanol injection. CONCLUSIONS: These results suggest that ethanol rapidly promotes phosphorylation of cPKC in limbic brain regions, which may underlie effects of acute ethanol on the nervous system and behavior.