Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells
Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells
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DOI：
10.1074/jbc.m108419200
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发表时间：
2002-01-04
影响因子：
4.8
通讯作者：
Kaufmann, SH
中科院分区：
文献类型：
--
作者：
Kottke, TJ;Blajeski, AL;Kaufmann, SH
MCF-7人乳腺癌细胞被广泛用于研究凋亡过程。最近的研究表明，这些细胞缺乏procaspase-3。在本研究中，半胱天冬酶的激活和活性进行了检查，在这个细胞系处理后的微管毒药紫杉醇。当用100 nm紫杉醇处理24小时后72小时收获细胞时，37 +/- 5%的细胞为非粘附性的，并显示凋亡形态学变化。虽然线粒体细胞色素c释放和半胱天冬酶-9裂解可通过免疫印迹检测，但从凋亡细胞制备的细胞质和细胞核的测定未能证明存在裂解合成半胱天冬酶底物LEHD-7-氨基-4-三氟甲基香豆素（LEHD-AFC）、DEVD-AFC和VEID-AFC的活性。同样，紫杉醇处理的MCF-7细胞不能切割多种半胱天冬酶底物，包括核纤层蛋白A、β-连环蛋白、凝溶胶蛋白、蛋白激酶C δ、拓扑异构酶1和半胱天冬酶原-6、-8和-10。用野生型半胱天冬酶原-3转染MCF-7细胞部分恢复了这些多肽的切割，但没有导致可检测到的可切割合成半胱天冬酶底物的活性。免疫印迹显示，caspase-9和-3，这是蛋白水解裂解在紫杉醇处理的MCF-7/caspase-3细胞，被隔离在耐盐的可沉降部分，而不是释放到胞质溶胶。免疫荧光显示，在这些凋亡细胞中含有裂解的caspase-3的大细胞质聚集体。这些观察结果表明，在某些模型系统中可能发生半胱天冬酶的螯合，导致基于四肽的活性测定低估了原位发生的半胱天冬酶活化的量。
MCF-7 human breast cancer cells are widely utilized to study apoptotic processes. Recent studies demonstrated that these cells lack procaspase-3. In the present study, caspase activation and activity were examined in this cell line after treatment with the microtubule poison paclitaxel. When cells were harvested 72 h after the start of a 24-h treatment with 100 nm paclitaxel, 37 +/- 5% of the cells were nonadherent and displayed apoptotic morphological changes. Although mitochondrial cytochrome c release and caspase-9 cleavage were detectable by immunoblotting, assays of cytosol and nuclei prepared from the apoptotic cells failed to demonstrate the presence of activity that cleaved the synthetic caspase substrates LEHD-7-amino-4-trifluoromethylcoumarin (LEHD-AFC), DEVD-AFC, and VEID-AFC. Likewise, the paclitaxel-treated MCF-7 cells failed to cleave a variety of caspase substrates, including lamin A, beta-catenin, gelsolin, protein kinase Cdelta, topoisomerase 1, and procaspases-6, -8, and -10. Transfection of MCF-7 cells with wild type procaspase-3 partially restored cleavage of these polypeptides but did not result in detectable activities that could cleave the synthetic caspase substrates. Immunoblotting revealed that caspase-9, and -3, which were proteolytically cleaved in paclitaxel-treated MCF-7/caspase-3 cells, were sequestered in a salt-resistant sedimentable fraction rather than released to the cytosol. Immunofluorescence indicated large cytoplasmic aggregates containing cleaved caspase-3 in these apoptotic cells. These observations suggest that sequestration of caspases can occur in some model systems, causing tetrapeptide-based activity assays to underestimate the amount of caspase activation that has occurred in situ.