Assessment of Apoptosis by Immunohistochemistry to Active Caspase-3, Active Caspase-7, or Cleaved PARP in Monolayer Cells and Spheroid and Subcutaneous Xenografts of Human Carcinoma
Assessment of Apoptosis by Immunohistochemistry to Active Caspase-3, Active Caspase-7, or Cleaved PARP in Monolayer Cells and Spheroid and Subcutaneous Xenografts of Human Carcinoma
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DOI：
10.1369/jhc.2008.952044
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发表时间：
2009-04-01
影响因子：
3.2
通讯作者：
Plenat, Francois
中科院分区：
文献类型：
--
作者：
Bressenot, Aude;Marchal, Sophie;Plenat, Francois
最近推荐用于凋亡检测的活性半胱天冬酶-3的免疫组织化学不适合检测涉及半胱天冬酶-7的凋亡。多聚腺苷三磷酸核糖聚合酶1（PARP-1），两种半胱天冬酶的主要底物的切割是细胞凋亡的有价值的标志物。无论使用何种抗体，紫杉醇或Foscan光动力学处理（PDT）在HT 29或KB单层细胞和HT 29球状体中诱导的体外细胞凋亡评价均产生了接近百分比的标记细胞，而在对照标本中，裂解PARP（c-PARP）免疫染色未能像活性caspase-3或-7免疫染色那样有效地检测细胞凋亡。在MDA-MB 231单层细胞和HT 29异种移植物中进行或未进行Foscan-PDT的研究导致活性caspase-3标记细胞的数量显著增加，尽管免疫荧光分析显示c-PARP和活性caspase-3在肿瘤中完美共定位。在对照肿瘤的caspase-3表达细胞的大部分中，c-PARP的限制性表达是明显的，而光敏化肿瘤显示出更高数量的表达来自活性caspase-3和c-PARP的大荧光点的细胞。这些结果支持c-PARP表达依赖于处理诱导的细胞凋亡的假设。在经历Foscan-PDT的一些表达半胱天冬酶-3的细胞中不存在半胱天冬酶-7活化，这显示了使用可以区分半胱天冬酶依赖性凋亡途径的抗体的相关性。（J Histochem Cytochem 57：289- 300，2009）
Immunohistochemistry to active caspase-3, recently recommended for apoptosis detection, is inappropriate to detect apoptosis involving caspase-7. Cleavage of poly-ADPribose polymerase 1 (PARP-1), a major substrate of both caspases, is a valuable marker of apoptosis. Apoptosis evaluation induced in vitro either by paclitaxel or by photodynamic treatment (PDT) with Foscan in HT29 or KB monolayer cells and HT29 spheroids yielded a close percentage of labeled cells whatever the antibody used, whereas in control specimens, cleaved PARP (c-PARP) immunostaining failed to detect apoptosis as efficiently as active caspase-3 or -7 immunostaining. Studies in MDA-MB231 monolayer cells and HT29 xenografts either subjected or not subjected to Foscan-PDT resulted in a significant higher number of active caspase-3-labeled cells, although immunofluorescence analysis showed c-PARP and active caspase-3 perfectly colocalized in tumors. A restricted expression of c-PARP was obvious in the greater part of caspase-3 expressing cells from control tumor, whereas photosensitized tumors showed a higher number of cells expressing large fluorescent spots from both active caspase-3 and c-PARP. These results support the assumption that c-PARP expression was dependent on treatment-induced apoptosis. The absence of caspase-7 activation in some caspase-3-expressing cells undergoing Foscan-PDT shows the relevance of using antibodies that can discriminate caspase-dependent apoptotic pathways. (J Histochem Cytochem 57:289-300,2009)