This project aims to assess the role of caspases in T cell death generally. In order to better study their functional role, we have developed a new fluorogenic caspase substrate termed PhiPhiLux which can detect caspase activation in intact apoptotic cells. This reagent is a peptide derived from the caspase-3 cleavage site of poly-ADP- ribose polymerase containing two rhodamine dyes substituted at the ends of the peptide so that they form a self-quenched cyclic compound which is disrupted by proteolytic cleavage. Extracts from apoptotic but not normal thymocytes cleave this substrate after DEVD, and the fluorescence increase is selectively blocked by caspase- 3 inhibitors. PhiPhiLux can be loaded into intact thymocytes in vitro, and in response to apoptotic stimuli thymocytes undergo a time-dependent about 10x increase in rhodamine fluorescence by flow cytometry. This staining increase is specifically blocked by preincubation with the caspase inhibitor ZVAD-FMK. Two-color staining with FITC- annexin-V shows that PhiPhiLux+ cells are apoptotic as seen by their exposed phosphatidyl serine. Confocal microscopy shows that the PhiPhiLux stain is cytoplasmic and in T hybridomas treated with staurosporine appears kinetically after the loss of mitochondrial psi. In assessing the general importance of caspases in T cell death, we have found that virtually all T cell death which is apoptotic by traditional criteria can be specifically inhibited by peptide-FMK caspase inhibitors. This includes the effect of a variety of chemotherapeutic agents on T cell tumors. However, since tumor cell death assessed in vitro may not be relevant to in vivo tumor eradication, we have tested the ability of caspase inhibitors to block the ability of various agents to decrease the formation of colonies in soft agar. Using both chemotherapeutic agents and anti-Fas, we have been unable to observe a blocking effect in this assay by either peptide-FMK or baculovirus p35 caspase inhibitors, even though they efficiently block traditional cell death readouts in the same experiment. We are currently attempting to ascertain whether these differences are due to the different kinetics of the two types of assay, or whether inhibiting colony formation occurs by a caspase-independent pathway even when triggered by Fas- crosslinking.
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