ABSTRACT
BACKGROUND: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. METHODS: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. RESULTS: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21(Waf1/Cip1) and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. CONCLUSION: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies.
Subject(s)
Histones/metabolism , Neuroblastoma/metabolism , Ouabain/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Comet Assay , Female , Flow Cytometry , Humans , Mice , Mice, Nude , Ouabain/pharmacology , Real-Time Polymerase Chain Reaction , Transplantation, HeterologousABSTRACT
Glial cell line-derived neurotrophic factor family ligands act through the receptor tyrosine kinase Ret, which plays important roles during embryonic development for cell differentiation, survival, and migration. Ret signaling is markedly affected by compartmentalization of receptor complexes into membrane subdomains. Ret can propagate biochemical signaling from within concentrates in cholesterol-rich membrane microdomains or lipid rafts, or outside such regions, but the mechanisms for, and consequences of, Ret translocation between these membrane compartments remain largely unclear. Here we investigate the interaction of Shc and Frs2 phosphotyrosine-binding domain-containing adaptor molecules with Ret and their function in redistributing Ret to specialized membrane compartments. We found that engagement of Ret with the Frs2 adaptor results in an enrichment of Ret in lipid rafts and that signal transduction pathways and chemotaxis responses depend on the integrity of such rafts. The competing Shc adaptor did not promote Ret translocation to equivalent domains, and Shc-mediated effects were less affected by disruption of lipid rafts. However, by expressing a chimeric Shc protein that localizes to lipid rafts, we showed that biochemical signaling downstream of Ret resembled that of Ret signaling via Frs2. We have identified a previously unknown mechanism in which phosphotyrosine-binding domain-containing adaptors, by means of relocating Ret receptor complexes to lipid rafts, segregate diverse signaling and cellular functions mediated by Ret. These results reveal the existence of a novel mechanism that could, by subcellular relocation of Ret, work to amplify ligand gradients during chemotaxis.
