The goal of this Phase II project is to continue development of a novel molecular imaging reporter construct that can report induction of apoptosis in intact cells and animals. Under normal biological conditions a tight balance between proliferation and cell death (apoptosis) is essential. An imbalance in these two opposing processes has been implicated in a variety of diseases which includes neurdegenerative disorders (e.g. Alzheimer's disease), myelodysplastic syndromes, AIDS, ischemic injury, autoimmune disease and cancer. The ability to quantitatively detect (image) apoptosis is a significant advancement for its use in high-throughput screening and validation of novel therapeutic molecules for the above diseases. During the Phase I award period, we have developed and validated a novel molecular reporter cassette which when transfected into mammalian cells results in a polypeptide that has significantly attenuated levels of reporter activity. If the cefl undergoes apoptosis, a caspase (a protease activated during apoptosis) specific cleavage of the reporter protein occurs which activates the reporter enabling detectionhmaging of the apoptotic event. In this Phase II proposal, we will test the specifity of this approach to image three different proteases involved in apoptosis (Caspase-3, -8 and -9). Three different reporter molecules, each containing a different cleavage site specific for one of three Caspases will be constituatively expressed in D54 human glioma cells with either luciferase (for bioluminescence detection) or green fluorescent protein (for fluorescence detection) as the activatable reporter (Specific Aim 1). These cell lines will be used to evaluate the specificity and sensitivity of these reporter molecules to """"""""report"""""""" on specific Caspases activated during different anticancer therapies. Moreover, in Specific Aim 2 transgenic mice will be developed using these reporter molecules wherein the activation of apoptosis within the skin in response to UV radiation can be imaged. Overall, this reporter molecule will provide a unique opportunity to image specific apoptotic events non-invasively and in real time in intact cell and animals. This feature will provide exciting and important opportunities for both the pharmaceutical industry and scientists to study apoptosis using in vitro high throughput screening compounds with pro- and anti- apoptotic activity as well as for target validation in vivo in intact animals.
