Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival

Extent: 14 p.The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.Daniel Thomas, Jason A. Powell, Benjamin D. Green, Emma F. Barry, Yuefang Ma, Joanna Woodcock, Stephen Fitter, Andrew C.W. Zannettino, Stuart M. Pitson, Timothy P. Hughes, Angel F. Lopez, Peter R. Shepherd, Andrew H. Wei, Paul G. Ekert and Mark A. Guthridg

Gut Microbiota Is a Key Modulator of Insulin Resistance in TLR 2 Knockout Mice

A genetic and pharmacological approach reveals novel insights into how changes in gut microbiota can subvert genetically predetermined phenotypes from lean to obese

2-Aminophenoxazine-3-one and 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one cause cellular apoptosis by reducing higher intracellular pH in cancer cells

We examined intracellular pH (pHi) of ten cancer cell lines derived from different organs and two normal cell lines including human embryonic lung fibroblast cells (HEL) and human umbilical vein endothelial cells (HUVEC) in vitro, and found that pHi of most of these cancer cells was evidently higher (pH 7.5 to 7.7) than that of normal cells (7.32 and 7.44 for HEL and HUVEC, respectively) and that of primary leukemic cells and erythrocytes hitherto reported (≤7.2). Higher pHi in these cancer cells could be related to the Warburg effect in cancer cells with enhanced glycolytic metabolism. Since reversal of the Warburg effect may perturb intracellular homeostasis in cancer cells, we looked for compounds that cause extensive reduction of pHi, a major regulator of the glycolytic pathway and its associated metabolic pathway. We found that phenoxazine compounds, 2-aminophenoxazine-3-one (Phx-3) and 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one (Phx-1) caused a rapid and drastic dose-dependent decrease of pHi in ten different cancer cells within 30 min, though the extent of the decrease of pHi was significantly larger for Phx-3 (ΔpHi = 0.6 pH units or more for 100 µM Phx-3) than for Phx-1 (ΔpHi = 0.1 pH units or more for 100 µM Phx-1). This rapid and drastic decrease of pHi in a variety of cancer cells caused by Phx-3 and Phx-1 possibly perturbed their intracellular homeostasis, and extensively affected the subsequent cell death, because these phenoxazines exerted dose-dependent proapoptotic and cytotoxic effects on these cells during 72 h incubation, confirming a causal relationship between ΔpHi and cytotoxic effects due to Phx-3 and Phx-1. Phx-3 and Phx-1 also reduced pHi of normal cells including HEL and HUVEC, although they exerted less proapoptotic and cytotoxic effects on these cells than on cancer cells. Drugs such as Phx-3 and Phx-1 that reduce pHi and thereby induce cellular apoptosis might serve as benevolent anticancer drugs

Disorders of Consciousness

Disorders of Consciousness are a big challenge for the entire process of rehabilitation: assessment, diagnosis, pharmacological, and rehabilitation programs, including conventional treatments and the use of new technologies