Overexpression of CREB protein protects from tunicamycin-induced apoptosis in various rat cell types.

Endoplasmic reticulum (ER) stress plays an essential role in unfolded protein response induced apoptosis contributing to several pathological conditions. Glycogen synthase kinase-3ß (GSK-3ß) plays a central role in several apoptotic signaling, including ER stress, as the active form of GSK-3ß induces apoptosis. The phosphorylation of cAMP responsive element (CRE) binding protein (CREB) Ser-133 (S133) residue is the end-point of various signaling pathways, like growth factor signaling, while the Ser-129 (S129) residue is phosphorylated by GSK-3ß. The significance of the ubiquitously expressed transcription factor CREB is demonstrated in prolonged, tunicamycin (TM)-induced ER stress in this study. In the experiments wild-type (wt) CREB, S129Ala, S133Ala or S129Ala-S133Ala mutant CREB expressing PC12 rat pheochromocytoma cell lines showed increased survival under TM-evoked prolonged ER stress compared to wtPC12 cells. After TM treatment ER stress was activated in all PC12 cell types. Lithium and SB-216763, the selective, well-known inhibitors of GSK-3ß, decreased TM-induced apoptosis and promoted cell survival. The proapoptotic BH3-only Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim) level was decreased in the different CREB overexpressing PC12 cells as a result of TM treatment. CREB overexpression also inhibited the sequestration of Bim protein from tubulin molecules, as it was demonstrated in wtPC12 cells. Transient expression of wtCREB diminished TM-induced apoptosis in wtPC12, Rat-1 and primary rat vascular smooth muscle cells. These findings demonstrate a novel role of CREB in different cell types as a potent protector against ER stress.

A simple fluorescent labeling technique to study virus adsorption in Newcastle disease virus infected cells.

The present study demonstrates that the fluorescent general membrane dyes PKH67 and PKH26 are suitable to label Newcastle disease virus, an enveloped virus belonging to the family of paramyxoviridae. Adsorption of the labeled virus particles was tracked, visualized and quantitated using confocal laser scanning microscopy. The specificity of PKH-labeling was determined by colocalization analysis of the PKH signal with NDV-specific immunolabeling, and by using mock-infected controls and infection with detergent-pretreated labeled virus particles. The infectivity of the NDV particles was not affected by the labeling procedure as indicated by the results of a cytotoxicity ATP assay, an apoptosis assay and detection of virus-specific RNA and protein by qPCR and Western blotting, respectively, in cells infected with PKH-labeled and unlabeled virus particles. This technique can be used as an inexpensive, sensitive and rapid alternative method in the analysis of adsorption and internalization of enveloped viruses by the infected cells.