Purchase this article with an account.
Marko Pastak, Bernhard B. Singer, Anna Kovtun, Marta Czugala, Berthold Seitz, Matthias Epple, Klaus-Peter Steuhl, Süleyman Ergün, Thomas A. Fuchsluger; Kinetics of Intracellular Pro-Apoptotic Bax Protein Inducing Cell Death in Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5996.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
In the intrinsic apoptotic pathway, Bcl-2 family member protein Bax triggers cytochrome C release from mitochondria. Thus, it leads to an activation of the downstream caspase cascade ultimately resulting in apoptosis. The aim of the study was to investigate kinetics and up-regulation of this central pro-apoptotic factor in human corneal endothelial cells.
Human corneal endothelial cells (HCECs, 100% confluence) were incubated with the intrinsic apoptotic inducer Etoposide (a topoisomerase II inhibitor) in increasing concentrations (1.25, 2.5, 5.0 µg/ml). Bax protein level was determined within twelve hours using cell-based ELISA. These results were confirmed by Western blot. The rate of apoptotic HCECs was measured 6h, 12h, 24h and 48h after incubation with Etoposide by flow cytometric assay using Annexin V and Propidium Iodide.
We detected a positive correlation between increasing Bax protein level and apoptotic HCECs. Specifically, HCECs showed an early increase of Bax within six hours of incubation (Etoposide 2.5, 5.0 µg/ml). Interestingly, significant ratios of dying HCECs were subsequently detectable with a six hours delay, twelve hours after onset of Etoposide. In addition, after treatment with Etoposide, level of Bax was similar and independent of the respective inducer concentration. Once a certain Bax threshold was obtained, death of HCECs was unavoidable (up to 60% apoptotic cells).
In-depth knowledge of kinetics and level of this pro-apoptotic protein is highly relevant to understand intracellular mechanisms causing cell death in this peculiar cell type with very low proliferative capacity. Our results set the stage for further investigations of down-regulating Bax, thus preventing HCECs from undergoing apoptosis.
This PDF is available to Subscribers Only