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

J Histochem Cytochem. 2009 Apr;57(4):289-300. doi: 10.1369/jhc.2008.952044. Epub 2008 Nov 24.


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.

MeSH terms

  • Adenocarcinoma / metabolism
  • Adenocarcinoma / pathology*
  • Animals
  • Antineoplastic Agents / pharmacology
  • Apoptosis*
  • Caspase 3 / metabolism*
  • Caspase 7 / metabolism*
  • Colonic Neoplasms / metabolism
  • Colonic Neoplasms / pathology*
  • Humans
  • Immunohistochemistry
  • Mesoporphyrins / pharmacology
  • Mice
  • Mice, Nude
  • Neoplasm Transplantation
  • Paclitaxel / pharmacology
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Spheroids, Cellular / metabolism*
  • Transplantation, Heterologous
  • Triazenes / pharmacology


  • Antineoplastic Agents
  • Mesoporphyrins
  • Triazenes
  • 1-phenyl-3,3-dimethyltriazene
  • PARP1 protein, human
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases
  • Caspase 3
  • Caspase 7
  • temoporfin
  • Paclitaxel