Background: Procaspase 3 is a constitutive proenzyme that is activated by c
leavage during apoptosis. The resulting enzyme is able to cleave several ta
rget proteins after the second aspartate of a DEVD sequence common to all t
he 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 d
etect (pre-)apoptotic cells by flow cytometry (FCM).
Materials and Methods: Apoptosis was induced in U937 or bone marrow mononuc
lear cells by daunorubicin (DNR), idarubicin (IDA), or camptothecin (CAM).
Viable and apoptotic cells were sorted by FCM on the basis of either fluore
scein isothiocyante (FITC)-annexin V binding or DiOC6(3) accumulation. DEVD
ase activity was measured in sorted populations by spectrofluorometry. Clea
ved caspase 3 was labeled in situ with phycoerythrin (PE)-conjugated anti-a
ctivated caspase 3 antibodies and analyzed by FCM.
Results: DEVDase activity was detected in sorted viable CAM- and DNR-treate
d U937 cells, demonstrating that a partial caspase activation occurred earl
ier than phosphatidyl-serine exposure and mitochondrial membrane potential
dissipation. The presence of a low amount of active caspase 3 in the treate
d 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 conta
minating 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 anthracy
clines induce blast cell apoptosis by a caspase 3-dependent pathway. Cytome
try 40:151-160, 2000. (C) 2000 Wiley-Liss. Inc.