cytochrome-c-t and Carcinoma--Embryonal

Angiogenin is a 14 kDa protein originally identified as an angiogenic protein. Recent development has shown that angiogenin acts on both endothelial cells and neuronal cells. Loss-of-function mutations in the coding region of the ANG gene have recently been identified in patients with amyotrophic lateral sclerosis. Angiogenin has been shown to control motor neuron survival and protect neurons from apoptosis under various stress conditions. In this article, we characterize the anti-apoptotic activity of angiogenin in pluripotent P19 mouse embryonal carcinoma cells. Angiogenin prevents serum withdrawal-induced apoptosis. Angiogenin upregulates anti-apoptotic genes, including Bag1, Bcl-2, Hells, Nf-kappab and Ripk1, and downregulates pro-apoptotic genes, such as Bak1, Tnf, Tnfr, Traf1 and Trp63. Knockdown of Bcl-2 largely abolishes the anti-apoptotic activity of angiogenin, whereas the inhibition of Nf-kappab activity results in a partial, but significant, inhibition of the protective activity of angiogenin. Thus, angiogenin prevents stress-induced cell death through both the Bcl-2 and Nf-kappab pathways.

Topics: Animals; Apoptosis; Carcinoma, Embryonal; Caspases; Culture Media, Serum-Free; Cytochromes c; Gene Expression Regulation, Neoplastic; Mice; NF-kappa B; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease, Pancreatic; Tumor Cells, Cultured

Spinal muscular atrophy (SMA) is a common neurodegenerative disease that is caused by mutations in the survival of motor neuron gene (SMN), leading to reduced levels of the SMN protein in affected individuals. In SMA, motor neurons selectively degenerate, however, the mechanism of cell death and the precise role of SMN in this process are not completely understood. In this study, we apply RNA interference (RNAi) to knockdown Smn gene expression in the murine embryonal carcinoma stem cell line P19, which can be differentiated into neuronal cells. A direct effect of Smn loss on apoptotic cell death in differentiated P19 neuronal cells, and to a lesser extent in undifferentiated cells was observed. Apoptosis could be partly reversed by expression of an SMN rescue construct, was reversible by the addition of the caspase-inhibitor ZVAD-fmk and involved the cytochrome c pathway. This study shows for the first time that knockdown of SMN results in apoptosis in mammalian neuronal cells and has implications for understanding the cause of motor neuron-specific cell loss in SMA, and for identifying novel therapeutic targets for this disease.

Topics: Acridine Orange; Animals; Apoptosis; Carcinoma, Embryonal; Caspase Inhibitors; Cell Differentiation; Cell Line; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Cytochromes c; Enzyme Inhibitors; Fluorescent Dyes; Immunohistochemistry; Mice; Nerve Tissue Proteins; Neurons; Plasmids; RNA Interference; RNA-Binding Proteins; SMN Complex Proteins; Stem Cells