Assay for detection of early stage apoptosis Abstract Detection of the early stages of apoptosis is of critical importance in many fields of biomedical research enterprise and especially in the development of the new drugs and therapeutic interventions. The long-term objective of this proposal is to introduce a new assay for the selective detection of the initial stages of apoptosis in fixed tissue sections.
Specific Aims of this project are: 1) To develop a new assay for specific detection of early stage apoptosis in fixed tissue sections. 2) To comprehensively test the newly developed assay using multiple experimental models of apoptotic death. The assay will use the unique enzymatic properties of vaccinia topoisomerase I to label characteristic DNA cuts produced by type II cellular topoisomerases in the initial stages of apoptosis. These double-strand DNA cuts contain 4 base-long overhangs with terminal 5'OH groups and cannot be selectively visualized by present techniques. The new assay will fill the gap in techniques for labeling early apoptosis in the tissue section format. It will have a single cell sensitivity limit and will be tested using multiple different models of apoptosis induction including corticosteroid-, temperature-, cancer-, and ischemia- induced apoptosis, as well as non-apoptotic DNA cleavage. The assay will be a convenient and practical tool in the apoptosis detection arsenal. It will have broad applicability in biomedicine and in the development of new drugs where detection of early apoptosis is important. The proposed project will result in the development of a new assay for the needs of medical diagnostics and pathology. The technology will simplify and make it cheaper to evaluate the effects of therapy in diseases where cell death and DNA damage have prognostic value, such as Alzheimer's disease, and various cancers. ? ? ?
