ABSTRACT
Ataxia telangiectasia (A-T) is a rare cancer-predisposing genetic disease, caused by the lack of functional ATM kinase, a major actor of the double strand brakes (DSB) DNA-damage response. A-T patients show a broad and diverse phenotype, which includes an increased rate of lymphoma and leukemia development. Fas-induced apoptosis plays a fundamental role in the homeostasis of the immune system and its defects have been associated with autoimmunity and lymphoma development. We therefore investigated the role of ATM kinase in Fas-induced apoptosis. Using A-T lymphoid cells, we could show that ATM deficiency causes resistance to Fas-induced apoptosis. A-T cells up-regulate FLIP protein levels, a well-known inhibitor of Fas-induced apoptosis. Reconstitution of ATM kinase activity was sufficient to decrease FLIP levels and to restore Fas sensitivity. Conversely, genetic and pharmacologic ATM kinase inactivation resulted in FLIP protein up-regulation and Fas resistance. Both ATM and FLIP are aberrantly regulated in Hodgkin lymphoma. Importantly, we found that reconstitution of ATM kinase activity decreases FLIP protein levels and restores Fas sensitivity in Hodgkin lymphoma-derived cells. Overall, these data identify a novel molecular mechanism through which ATM kinase may regulate the immune system homeostasis and impair lymphoma development.
Subject(s)
Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Hodgkin Disease/metabolism , Lymphocytes/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , fas Receptor/metabolism , Animals , Apoptosis/genetics , Ataxia Telangiectasia , Ataxia Telangiectasia Mutated Proteins , Autoimmunity/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Hodgkin Disease/genetics , Hodgkin Disease/pathology , Homeostasis/genetics , Humans , Leukemia/genetics , Leukemia/metabolism , Leukemia/pathology , Lymphocytes/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , fas Receptor/geneticsABSTRACT
We identified Caspase-8 as a new substrate for Src kinase. Phosphorylation occurs on Tyr380, situated in the linker region between the large and the small subunits of human Procaspase-8, and results in downregulation of Caspase-8 proapoptotic function. Src activation triggers Caspase-8 phosphorylation on Tyr380 and impairs Fas-induced apoptosis. Accordingly, Src failed to protect Caspase-8-defective human cells in which a Caspase-8-Y380F mutant is expressed from Fas-induced cell death. Remarkably, Src activation upon EGF-receptor stimulation triggers endogenous Caspase-8 phosphorylation and prevents Fas-induced apoptosis. Tyr380 is phosphorylated also in human colon cancers where Src is aberrantly activated. These data provide the first evidence for a direct role of tyrosine phosphorylation in the control of caspases and reveal a new mechanism through which tyrosine kinases inhibit apoptosis and participate in tumor progression.
Subject(s)
Apoptosis , Caspases/metabolism , Colonic Neoplasms/enzymology , Tyrosine/metabolism , src-Family Kinases/metabolism , Amino Acid Sequence , Caspase 8 , Caspases/genetics , Enzyme Activation , ErbB Receptors/agonists , Humans , Molecular Sequence Data , Mutation , Schizosaccharomyces/enzymology , Tumor Cells, Cultured , fas Receptor/pharmacologyABSTRACT
The morphogenetic role of the neurotransmitter acetylcholine was studied in cultures of dorsal root ganglia (DRG) neurons obtained from E12 and E18 chick embryos. With this model we have evaluated neurofilament expression and neurite outgrowth following cholinergic agonist and antagonist treatment. Morphometric analysis undertaken to evaluate fiber outgrowth has indicated that E12 DRG cultures treated with cholinergic agonists, such as muscarine and carbachol, when compared with untreated cultures, have longer fibers and a higher number of fibers per neuron. Concomitant treatment with agonists and the antagonists atropine or mecamylamine counteracts the increase in fiber outgrowth, suggesting that the cholinergic agonist effects were mediated by both muscarinic and nicotinic receptors. The expression of the three neurofilament proteins was also evaluated. Western blot analysis showed that, in E12 DRG cultures, both muscarine and carbachol induce a significant increase in neurofilament protein expression and that this effect is inhibited by cholinergic antagonist treatment. Moreover, Northern blot analysis has demonstrated that the increased expression of 68- and 145-kDa neurofilament proteins is dependent on cholinergic modulation of the neurofilament transcripts. Modulated expression of neurofilament proteins by cholinergic agonists was not evident in E18 DRG cultures, suggesting that, when sensory neurons have completed their differentiation, the cholinergic system might be involved in other functions. In conclusion, our data demonstrate that, during sensory neuron development, acetylcholine modulates neurite outgrowth controlling neurospecific marker expression.
