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Titolo:
Activation of caspases measured in situ by binding of fluorochrome-labeledinhibitors of caspases (FLICA): Correlation with DNA fragmentation
Autore:
Bedner, E; Smolewski, P; Amstad, P; Darzynkiewicz, Z;
Indirizzi:
New York Med Coll, Brander Canc Res Inst, Valhalla, NY 10595 USA New York Med Coll Valhalla NY USA 10595 Res Inst, Valhalla, NY 10595 USA Pomeranian Sch Med, Dept Pathol, Szczecin, Poland Pomeranian Sch Med Szczecin Poland h Med, Dept Pathol, Szczecin, Poland Intergen Discovery Prod, Gaithersburg, MD 20877 USA Intergen Discovery Prod Gaithersburg MD USA 20877 thersburg, MD 20877 USA
Titolo Testata:
EXPERIMENTAL CELL RESEARCH
fascicolo: 1, volume: 259, anno: 2000,
pagine: 308 - 313
SICI:
0014-4827(20000825)259:1<308:AOCMIS>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
LASER-SCANNING CYTOMETRY; STRAND BREAKS; APOPTOSIS; ASSAYS; INDUCTION; CELLS;
Keywords:
camptothecin; laser scanning cytometry; DNA strand breaks; TUNEL;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
21
Recensione:
Indirizzi per estratti:
Indirizzo: Darzynkiewicz, Z NYMC, Brander Canc Res Inst, 19 Bradhurst Ave,Suite 2400,Hawthorne, NY 10532 USA NYMC 19 Bradhurst Ave,Suite 2400 Hawthorne NY USA 10532 A
Citazione:
E. Bedner et al., "Activation of caspases measured in situ by binding of fluorochrome-labeledinhibitors of caspases (FLICA): Correlation with DNA fragmentation", EXP CELL RE, 259(1), 2000, pp. 308-313

Abstract

Activation of caspases is the key event of apoptosis and new methods are needed to assay this event, particularly in situ, in individual cells. To measure in situ caspases activation in the present study we employed fam-VAD-fmk and fam-VEID-fmk, the fluorochrome (fam)-labeled inhibitors of caspases(FLICA), which through the fluoromethylketone (fmk) moiety bind to active center of the activated enzymes. The peptide moiety of these inhibitors defines their specificity; VAD is generic to most caspases and VEID is caspase-6 specific. The frequencies of cells showing caspases activation were compared with those showing DNA fragmentation (detected by the TUNEL assay) in the same cultures. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT) or tumor necrosis factor-a combined with cycloheximide (TNF-alpha + CHX). The cells that bound FLICA had morphological changes typical of apoptosis. The intensity of their fluorescence was measured by laser scanning cytometry. Maximal rate of activation of the caspases, measured by the increase in frequency of the cells that bound fam-VAD-fmk, occurred between 30 and 90 min after the administration of TNF-alpha + CHX and between 2 and 4 h after the administration of CPT. In the CPT-treated cultures about 30% fewer cells bound fam VEID-fmk than fam-VAD-fmk whichsuggests that the activation of caspase-6 was delayed or was not induced in some cells. A strong overall correlation between the cytometric assays ofthe apoptotic index based on the detection of caspases activation by the FLICA and the TUNEL assay was observed. The data indicate that FLICA offers a rapid and convenient method of assessing caspase's activation in individual cells and can also be used to estimate the frequency of apoptosis. (C) 2000 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 04/12/20 alle ore 00:57:25