title: Constitutive nuclear localization and initial cytoplasmic apoptotic activation of endogenous caspase-3 evidenced by confocal microscopy.
abstract: The localization of caspases and their substrates in different cellular compartments may be one way to regulate apoptosis. Caspase-3-dependent proteolysis of inhibitor caspase-activated deoxyribonuclease (ICAD) activates caspase-activated deoxyribonuclease (CAD), which induces apoptotic internucleosomal DNA degradation. The nuclear localization of ICAD, pro- and active-caspase-3 molecules remains a controversial issue. Using a combination of immunodetection of endogenous molecules and confocal microscopy, we analysed the kinetics of the procaspase-3 and CAD activation induced by FAS triggering in Jurkat cells. Through a semi-quantitative image analysis, we showed a constitutive nuclear localization of pro-caspase 3 and ICAD in non-apoptotic cells. FAS stimulation induced 7A6 apoptotic antigen expression, which could be related to three different sequential patterns of nuclear chromatin organization. Active-caspase-3 first appeared in the cytoplasm and was next observed in the nucleus. Simultaneously, the amount of ICAD located in the nucleus decreased, whereas the amount of ICAD located in the cytoplasm remained unchanged. Thus, our experiments using in situ immunodetection of endogenous molecules show that the ICAD cleavage by the active-caspase-3 probably takes place in the nucleus. These results provide new perspectives about the subcellular compartmentation and traffic of caspases during the apoptotic process.
authors.affiliation: INSERM U119, Service d'Anatomie et Cytologie Pathologiques, Laboratoire d'Immunologie des Tumeurs, Institut Paoli-Calmettes, IFR 57 and Université de la Méditerranée, Marseille, France. anapath@marseille.fnclcc.fr
authors.forename: Olivier
authors.initials: O
authors.lastname: Ramuz
authors.forename: Daniel
authors.initials: D
authors.lastname: Isnardon
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Devilard
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Charafe-Jauffret
authors.forename: Jacques
authors.initials: J
authors.lastname: Hassoun
authors.forename: FranCoise
authors.initials: F
authors.lastname: Birg
authors.forename: Luc
authors.initials: L
authors.lastname: Xerri
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D011506:Proteins / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D004792:Enzyme Precursors; D011506:Proteins; C109664:caspase-activated DNase inhibitor; D019014:fas Receptor; D003851:Deoxyribonucleases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases