Monitoring Caspase-3 Activation with a Multimodality Imaging Sensor in Living Subjects

• Published in: Clinical cancer research Vol. 14; no. 18; pp. 5801 - 5809
• Main Authors: Ray, Pritha, De, Abhijit, Patel, Manishkumar, Gambhir, Sanjiv Sam
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 15.09.2008
• Subjects: Animals; Antineoplastic agents; Apoptosis; Biological and medical sciences; Bioluminescence; Caspase 3 - genetics; Caspase-3; Cell Line; Cell Line, Tumor; Diagnostic Imaging - methods; Enzyme Activation; Genetic Vectors; Green Fluorescent Proteins; Humans; Luciferases, Firefly; Luminescent Proteins - analysis; Medical sciences; Melanoma; Mice; micro PET; Models, Biological; Pharmacology. Drug treatments; Recombinant Fusion Proteins - metabolism; Thymidine Kinase; Triple fusion reporter; Human; Peptidases; Regulation(control); Surveillance; Enzyme; Cysteine endopeptidases; Imaging; Hydrolases; Medical imagery; Activation; Monitoring
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1078-0432.CCR-07-5244

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 in TK ( P = not significant), and a 2-fold increase in mRFP1 ( 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.