Cell surface and nuclear changes during TNF-α-induced apoptosis in WEHI 164 murine fibrosarcoma cells

Abstract

Tumour necrosis factor (TNF)-alpha-induced apoptosis is associated with several nuclear and cell surface alterations, in particular with the condensation of chromatin and the fragmentation of the cell nucleus, formation of blebs on the cell surface and breakdown of the plasma membrane. However, there is little information about the relationship between the cell surface alterations and the nuclear changes during apoptosis. To study this, cultured WEHI cells were exposed to TNF-alpha over different time periods. The cytological changes were studied using a correlative approach, which allowed observation of the same cell consecutively under light, scanning and transmission electron microscopy. The earliest sign of cell alteration was a reduction of the number of microvilli after 15 min of TNF-alpha exposure. This reaction was reversible (reappearance of microvilli) and took place during the first hour, in which neither nuclear alterations nor plasma membrane breakdown were observed. The changes in the nucleus began with condensation of chromatin after approximately 1 h of TNF-alpha-exposure. After 4-5 h the microvilli disappeared again, particularly in areas where the formation of blebs (blebbing) was observed. Strikingly, cell surface alterations (bleb formation) were detected only in those cells that presented with condensed chromatin, and not in cells with a normal chromatin pattern, proving at least a close correlation between nuclear and cell surface changes during the process of apoptosis.

Directed apoptosis in Cox-2-overexpressing cancer cells through expression-targeted gene delivery

“…This molecule was selected because it has been shown experimentally to be an initiator of apoptosis. 27 TNF-a is a type II membrane protein that acts as a ligand to TNF receptors to bring about the activation of pro-forms of initiator caspases, in much the same way that the Fas ligand interacts with Fas receptors to spark the apoptotic cascade. 6 We observed a lack of apoptotic response to TNF-a, which served to verify that the cells used were apoptosis resistant.…”

Up-regulation of Multidrug Resistance P-glycoprotein via Nuclear Factor-κB Activation Protects Kidney Proximal Tubule Cells from Cadmium- and Reactive Oxygen Species-induced Apoptosis

“…In the present study, we first wanted to characterize the concentration dependence and kinetics of cadmium-mediated apoptosis in the rat kidney PT cell line in more detail. By subsequent incubation with the vital bisbenzimidazole dye H-33342 and the DNA intercalating agent EtBr, apoptotic cells, whose plasma membrane integrity is still intact, could easily be distinguished from cells that underwent necrosis and therefore can be labeled by the plasma membrane-impermeable charged dye EtBr (20). Incubation of PT cells with 2.5-10 M CdCl 2 for 5 h induced typical features of apoptosis, such as chromatin condensation (arrows) or fragmentation (asterisk) (see Fig.…”

“…Cells were stained intravitally with the DNA dyes Hoechst 33342 (H-33342; 2 g/ml) for 20 min and ethidium bromide (EtBr; 5 g/ml) for 10 min, as described previously (20). Under ultraviolet (UV) epi-illumination ( ex , 330 -380 nm; em , Ͼ430 nm), necrotic cells fluoresce pink due to EtBr, whereas normal and apoptotic cells emit blue fluorescence due to H-33342.…”

Entrapment of Embryonic Stem Cells-Derived Cardiomyocytes in Macroporous Biodegradable Microspheres: Preparation and Characterization

“…Dehydration and drying of the suspension was carried out in increasing series of ethanol, while the last dehydrating step was performed in acetone using the Polaron high resolution sputter coater SC 7640 (Quorum Technologies Ltd.). The CultiSpher-S microcarriers entrapped with the cells were prepared for block-face scanning electron microscopy (SEM) as previously published [12]. Briefly the microspheres entrapped with the cells were embedded in epoxy resin, crosssectioned with ultra-microtome.…”

“…The suspension was washed with sodium cacodylate buffer, pH 7.2, and was embedded in 2% low melting agarose (Sigma-Aldrich, Taufkirchen, Germany). The post fixation was performed by the treatment of the microcarriers suspension in three steps using 2% osmium tetroxide, 1% tannic acid and 1% uranyl acetate in water as previously described [12]. Dehydration and drying of the suspension was carried out in increasing series of ethanol, while the last dehydrating step was performed in acetone using the Polaron high resolution sputter coater SC 7640 (Quorum Technologies Ltd.).…”