Multiple pathways leading to apoptosis of cancer cells have been discovered. To understand the mechanisms controlling cell elimination in each specific situation, it is necessary to have probes which can distinguish between different apoptotic routes. One of the most recently identified apoptotic programs is the elastase inhibitor-driven apoptosis. However, this particular pathway still lacks specific tools for its visualizationin the tissue section format, which is most relevant for pathology studies. Monocyte neutrophil elastase inhibitor (MNEI) is the main member of this pathway responsible for its activation. It acts by self-converting into an active nuclease and can rapidly dispose of tumor cells. The conditions promoting such a unique transition in brain tumors are not fully understood at this time. To a large extent this is because of the absence of specific tools for selective labeling of MNEI-driven apoptosis in tissue sections and in live cell cultures. In the absence of specific in situ methods for this apoptotic pathway, it is currently studied by using the bulk biochemical approaches which have limited value in heterogeneous pathology samples. In this project we will overcome this obstacle and will develop new enabling imaging technologies. We will apply the new probes to visualize MNEI-driven apoptosis in glioblastoma (GBM).
Specific aims : 1. To develop the first methodology for selective detection of the elastase inhibitor- driven apoptotic pathway in tissue sections. The approach will label the key event of this pathway and visualize the characteristic DNA cleavage activity of monocyte neutrophil elastase inhibitor (MNEI) after its transformation into an active DNase. To test this newly developed imaging tool in glioblastoma sections. 2. To develop the new methodology for selective labeling of the elastase inhibitor-driven apoptotic pathway in live cell cultures. The new fluorescent sensors specific for MNEI pathway will produce fluorescence only after they detect a specific marker of this apoptotic route. The project will introduce enabling technologies for apoptosis research in general, and for cancer studies in particular. Their application to glioblastoma will provide information useful for future clinical and research investigations of these tumors, and for the development of effective therapeutic interventions.
The proposed project will result in the development of a new assay for the needs of medical diagnostics and pathology. The technology will allow precise evaluation of the effects of therapy in diseases where cell death and DNA damage have prognostic value, such as various cancers, including glioblastoma, as well as stroke and Alzheimer's disease.