Laboratory of Cancer Cell Biology, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China.
Nat Commun. 2013;4:2157. doi: 10.1038/ncomms3157.
Cytosolic caspase-3-like proteases, such as caspase-3 and caspase-7, have a central role in mediating the progress of apoptosis. Here to conveniently monitor caspase-3-like activity in the multicellular environment, we have developed genetically encoded switch-on fluorescence-base indicators that are cyclized chimeras containing a caspase-3 cleavage site as a switch. When cleaved by caspase-3-like proteases, the non-fluorescent indicator rapidly becomes fluorescent, and thus detects in real-time the activation of such caspases. We generate cultured cells constitutively expressing these chimeras, and all the healthy cells are non-fluorescent. When these cells are exposed to apoptotic stimuli, dead cells show strong fluorescence depending on caspase activation. With these tools, we monitor in real-time caspase-3-like activity in each cell under various conditions, and show for the first time that the environment of cancer cells affects their sensitivity to chemotherapeutic drugs in a modified soft agar assay. These biosensors should enable better understanding of the biological relevance of caspase-3-like proteases.
细胞质中的胱天蛋白酶-3 样蛋白酶，如 caspase-3 和 caspase-7，在介导细胞凋亡的进程中起着核心作用。为了方便在多细胞环境中监测胱天蛋白酶-3 样活性，我们开发了遗传编码的开关型荧光基指示剂，这些是包含半胱天冬酶切割位点的环化嵌合体作为开关。当被胱天蛋白酶-3 样蛋白酶切割时，非荧光指示剂迅速变为荧光，从而实时检测此类胱天蛋白酶的激活。我们生成了持续表达这些嵌合体的培养细胞，所有健康的细胞都没有荧光。当这些细胞暴露于凋亡刺激时，根据胱天蛋白酶的激活，死亡细胞显示出强烈的荧光。有了这些工具，我们可以实时监测各种条件下每个细胞中的胱天蛋白酶-3 样活性，并首次表明癌细胞的环境会影响它们在改良软琼脂测定法中对化疗药物的敏感性。这些生物传感器应该能够更好地理解胱天蛋白酶-3 样蛋白酶的生物学相关性。
