Flow cytometry detection of caspase 3 activation in preapoptotic leukemic cells.
BACKGROUND
Procaspase 3 is a constitutive proenzyme that is activated by cleavage during apoptosis. The resulting enzyme is able to cleave several target proteins after the second aspartate of a DEVD sequence common to all the substrates of caspases 3 and 7 (DEVDase). Because active caspase 3 is a common effector in several apoptotic pathways, it may be a good marker to detect (pre-)apoptotic cells by flow cytometry (FCM). Materials and Methods Apoptosis was induced in U937 or bone marrow mononuclear cells by daunorubicin (DNR), idarubicin (IDA), or camptothecin (CAM). Viable and apoptotic cells were sorted by FCM on the basis of either fluorescein isothiocyante (FITC)-annexin V binding or DiOC6(3) accumulation. DEVDase activity was measured in sorted populations by spectrofluorometry. Cleaved caspase 3 was labeled in situ with phycoerythrin (PE)-conjugated anti-activated caspase 3 antibodies and analyzed by FCM.


RESULTS
DEVDase activity was detected in sorted viable CAM- and DNR-treated U937 cells, demonstrating that a partial caspase activation occurred earlier than phosphatidyl-serine exposure and mitochondrial membrane potential dissipation. The presence of a low amount of active caspase 3 in the treated viable cells was confirmed in situ with PE-conjugated anti-active caspase 3 antibodies. The same antibody was used in combination with FITC-annexin V and CD45-PC5 to study caspase 3 activation in acute leukemia blast cells after in vitro DNR and IDA treatment. Both anthracyclines induced a caspase 3-dependent apoptosis that was more efficient in blast cells than in contaminating lymphocytes.


CONCLUSIONS
These results demonstrate that anti-active caspase 3 labeling can be an alternative to fluorogenic substrates to efficiently detect early apoptosis by FCM in heterogeneous samples. They also confirm that anthracyclines induce blast cell apoptosis by a caspase 3-dependent pathway.
Belloc
F
F
Laboratoire d'Hématologie, Hôpital Haut Lévêque, Pessac, France. francis.belloc@wanadoo.fr
Belaud-Rotureau
M A
MA
Lavignolle
V
V
Bascans
E
E
Braz-Pereira
E
E
Durrieu
F
F
Lacombe
F
F
D000328:Adult; D017304:Annexin A5 / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015415:Biomarkers; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology* / Q000201:enzymology; D002166:Camptothecin / Q000494:pharmacology; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D003630:Daunorubicin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D015255:Idarubicin / Q000494:pharmacology; D007938:Leukemia / Q000175:diagnosis / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D020298:U937 Cells
D017304:Annexin A5; D000903:Antibiotics, Antineoplastic; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010447:Peptide Hydrolases; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; C108711:DEVDase; D016650:Fluorescein-5-isothiocyanate; D002166:Camptothecin; D015255:Idarubicin; D003630:Daunorubicin