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


METHODS
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 mumol/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.


CONCLUSIONS
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.
Ray
Pritha
P
Molecular Imaging Program at Stanford, Departments of Radiology and Bioengineering, Bio-X Program, School of Medicine, Stanford University, California 94305-5427, USA.
De
Abhijit
A
Patel
Manishkumar
M
Gambhir
Sanjiv Sam
SS
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D003952:Diagnostic Imaging / Q000379:methods*; D004789:Enzyme Activation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D049409:Luciferases, Firefly; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013937:Thymidine Kinase
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly; D013937:Thymidine Kinase; D053148:Caspase 3
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