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The process of controlled cellular death known as apoptosis has an important central role not only in normal homeostatic maintenance of tissues, but also in numerous diseases such as cancer, neurodegenerative, autoimmune, and cardiovascular diseases. As a result, new technologies with the capability to selectively detect apoptotic cells represent a central focus of research for the study of these conditions. We have developed a new biosensor for the detection of apoptotic cells, incorporating the targeted selectivity for apoptotic cells from Annexin V with the sensitivity of bioluminescence signal generation from a serum-stable mutant of Renilla luciferase (RLuc8). Our data presents a complete characterization of the structural and biochemical properties of this new Annexin-Renilla fusion protein (ArFP) construct, as well as a validation of its ability to detect apoptosis in vitro. Moreover, this work represents the first report of a bioluminescent Annexin V apoptosis sensor utilized in vivo. With this new construct, we examine apoptosis within disease-relevant animal models of surgery-induced ischemia/reperfusion, corneal injury, and retinal cell death as a model of age-related macular degeneration. In each of these experiments, we demonstrate successful application of the ArFP construct for detection and bioluminescence imaging of apoptosis within each disease or treatment model. ArFP represents an important new tool in the continuously growing kit of technologies for apoptosis detection, and our results from both in vitro and in vivo experiments suggest a diverse range of potential clinically relevant applications including cancer therapeutic screening and efficacy analysis, atherosclerosis and cardiovascular disease detection, and the monitoring of any number of other conditions in which apoptosis has a central role.

The ability to selectively induce apoptosis in tumor cells is the prime goal in cancer immunotherapy and aims at identifying potential molecular targets, regulating this process. Here we show that the sera from the animals which had spontaneously rejected the AK-5 tumor (a rat histiocytoma) had an effective and potent ability to counteract and kill tumor cells by inducing apoptosis, with a high degree of specificity. Apoptosis induced by the serum factor involved the activation of caspases and cytochrome c release to the cytosol. A reduction in mitochondrial transmembrane potential (Dc m ) occurred considerably later than cytochrome c translocation. The antiapoptotic protein Bcl-2 and the pancaspase inhibitor zVADfmk did not prevent cytochrome c release, but completely blocked the reduction in Dc m , DNA fragmentation and apoptosis. Cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition (MPT) pore had no effect on cytochrome c release and apoptosis mediated by serum factor in AK-5 cells, suggesting that apoptosis was independent of MPT. Taken together these results suggest that the serum factor in conjunction with the immune cells may be participating in the efficient rejection of the tumor in syngeneic hosts and Dc m disruption but not cytochrome c release, is a critical and decisive event to trigger apoptotic cell death induced by the serum factor in AK-5 tumor cells.

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