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.
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.
Bressenot
Aude
A
Service d'Anatomie et de Cytologie Pathologiques, Hôpital de Brabois, Centre Hospitalier Régional et Universitaire Nancy, Vandoeuvre-lès-Nancy, France.
Marchal
Sophie
S
Bezdetnaya
Lina
L
Garrier
Julie
J
Guillemin
François
F
Plénat
François
F
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D053179:Caspase 7 / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D008652:Mesoporphyrins / Q000494:pharmacology; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D017239:Paclitaxel / Q000494:pharmacology; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000378:metabolism*; D014183:Transplantation, Heterologous; D014226:Triazenes / Q000494:pharmacology
D000970:Antineoplastic Agents; D008652:Mesoporphyrins; D014226:Triazenes; C008848:1-phenyl-3,3-dimethyltriazene; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053179:Caspase 7; C072269:temoporfin; D017239:Paclitaxel