Abstract 2154: Simultaneous analysis of cell death mechanisms and oxidative stress using live cell fluorescence microscopy

Reactive oxygen species (ROS) can directly damage DNA, proteins and lipids, while increased oxidative stress has been reported to be associated with apoptotic and autophagic cell death. Importantly, increased oxidative stress and dysregulation of cell death mechanisms have been linked with cancer development. To better understand the connection between increased oxidative stress and cell death pathways, it is important to be able to monitor both of these events simultaneously in the same cell and amongst mixed populations of cells. The goal of this study was to utilize multi-parametric fluorescence microscopy to examine the temporal progression of oxidative stress relative to cell death. By using a fluorogenic caspase substrate in combination with a near infrared fluorescent probe for ROS, we observed that increased oxidative stress was followed by activation of caspase-3/7 after induction by several agonists. We examined the effect of various agonists in both primary cells as well as cancer cell lines. Loss of mitochondrial membrane potential, nuclear condensation, and cell proliferation were also measured to establish a more comprehensive assessment of the cellular response to stress in these models. By simultaneously examining multiple parameters over time, we were able to define the temporal progression of apoptosis relative to the onset of oxidative stress. Furthermore, we were able to characterize the mechanism of cell death by discriminating between cells which were apoptotic (active caspase-3/7), autophagic (LC3B-positive autophagosomes), or both. This multi-parametric approach provided detailed spatial information at the cellular level so that correlations and temporal resolution could be determined between oxidative stress and cell death mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2154. doi:10.1158/1538-7445.AM2011-2154