Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects.
Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects.
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DOI：
10.1158/1078-0432.ccr-07-5244
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发表时间：
2008-09-15
期刊：
影响因子：
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通讯作者：
Gambhir SS
中科院分区：
文献类型：
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作者：
Ray P;De A;Patel M;Gambhir SS
Capsase-3 在许多癌症化疗诱导的细胞凋亡中发挥着重要作用。在此，我们应用多模态报告载体来间接监测正在经历细胞凋亡的活体动物的活细胞和肿瘤中 caspase-3 的激活。通过组合三种不同的报告蛋白、红色荧光蛋白 (mRFP1)、萤火虫荧光素酶 (FL) 和 HSV1-sr39 截短胸苷激酶 (TK)，通过 caspase-3 可识别的多肽接头连接，构建了融合蛋白 (MTF)。 caspase-3 裂解后，通过荧光激活细胞分选和基于酶的测定观察到 mRFP1、FL 和 TK 活性显着增加。生成稳定表达 mtf（用于测量 caspase-3 激活）和 hrl-IRES-gfp（用于确定活细胞数量减少）载体的黑色素瘤细胞系 (B16F10-mtf-hrl)，以测量治疗后的两个独立分子事件。用 8 μmol/L 星形孢菌素诱导后，融合蛋白在 293T 细胞中显示 FL 增加 2.8 倍（P = 0.03），TK 增加 1.5 倍（P = 不显着），mRFP1 活性增加 2 倍（P = 0.05）。当用 RL 活性标准化时，凋亡 B16F10-mtf-hrl 肿瘤的生物发光和微正电子发射断层扫描成像显示，与对照肿瘤相比，FL 活性高 2 倍（897 比 416），TK 活性高 2 倍（10.3 比 3.87）。使用类似的标准化方法，在活体小鼠中无创监测两种蛋白激酶 C 抑制剂激活 caspase-3 的时间动力学。这种多模态 caspase 传感器载体可以有效、无创地监测从单个活细胞到多细胞肿瘤环境的 caspase-3 激活，因此，将成为临床前模型中药物筛选和未来基于患者细胞的治疗的有价值的工具。
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. 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. 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. 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.