Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects

Abstract

Purpose: Capsase-3 plays an important role in chemotherapy-induced apoptosis in many cancers. Herein, we applied a multimodality reporter vector to monitor caspase-3 activation indirectly in live cells and tumors of living animals undergoing apoptosis. Experimental Design: A fusion protein (MTF) was constructed by combining three different reporter proteins, red fluorescent protein (mRFP1), firefly luciferase (FL), and HSV1-sr39 truncated thymidine kinase (TK), linked through a caspase-3 recognizable polypeptide linker. After cleavage by caspase-3, a significant gain in mRFP1, FL, and TK activity are observed by fluorescence-activated cell sorting and enzyme-based assays. A melanoma cell line (B16F10-mtf-hrl) stably expressing mtf (to measure caspase-3 activation) and hrl-IRES-gfp (to determine the decrease in a number of viable cells) vectors was generated to measure two independent molecular events upon treatment. Results: Upon induction with 8 μmol/L staurosporine, the fusion protein showed a 2.8-fold increase in FL (P = 0.03), a 1.5-fold increase inTK (P = not significant), and a 2-fold increase in mRFPI (P = 0.05) activity in 293T cells. Bioluminescence and micropositron emission tomography imaging of the apoptotic B16F10-mtf-hrl tumors showed a 2-fold higher FL activity (897 versus 416) and a 2-fold higher TK activity (10.3 versus 3.87) than control tumors when normalized with RL activity. Using a similar normalization approach, the time kinetics of caspase-3 activation by two protein kinase-C inhibitors was noninvasively monitored in living mice. Conclusion: This multimodality caspase sensor vector could effectively and noninvasively monitor caspase-3 activation from single live cells to a multicellular tumor environment and, thus, would be a valuable tool for drug screening in preclinical models and future patient cell based therapy. © 2008 American Association for Cancer Research.