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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

Aude Bressenot, Sophie Marchal, Lina Bezdetnaya, Julie Garrier, François Guillemin and François Plénat

Service d'Anatomie et de Cytologie Pathologiques, Hôpital de Brabois, Centre Hospitalier Régional et Universitaire Nancy, Vandoeuvre-lès-Nancy, France (AB,FP), and Centre de Recherche en Automatique de Nancy-Centre National de la Recherche Scientifique Unité Mixte de Recherche 7039 Nancy Université, Centre Alexis Vautrin, Vandoeuvre-lès-Nancy, France (SM,LB,JG,FG)

Correspondence to: F. Plénat, Service d'Anatomie et de Cytologie Pathologiques, CHU de Brabois, Allée du Morvan, 54511 Vandoeuvre-lès-Nancy, France. E-mail: f.plenat{at}chu-nancy.fr

Immunohistochemistry to active caspase-3, recently recommended for apoptosis detection, is inappropriate to detect apoptosis involving caspase-7. Cleavage of poly-ADP-ribose 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)

Key Words: apoptosis • caspase-3 • caspase-7 • poly-ADP-ribose polymerase

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