CASPASE ACTIVATION IS AN EARLY EVENT IN ANTHRACYCLINE-INDUCED APOPTOSIS AND ALLOWS DETECTION OF APOPTOTIC CELLS BEFORE THEY ARE INGESTED BYPHAGOCYTES

An increasing number of methods are being described to detect apoptoti c cells. However, attempts to detect apoptotic cells in clinical sampl es are rarely successful, A hypothesis is that apoptotic cells are cle ared from the circulation by phagocytosis before they become detectabl e by conventional morphological or cytometric methods. Using LR73 adhe ring cells as phagocytes in a model of in vitro phagocytosis, we found that phagocytosis of daunorubicin (DNR)treated U937, HL60, or K562 le ukemia cell lines occurred prior to phosphatidylserine externalization , DNA hydrolysis, chromatin condensation, nuclear fragmentation, or mi tochondrial potential alteration. Moreover DNR-treated K562 cells were eliminated by phagocytes while apoptosis was never observed by any of the above methods. By contrast, using a fluorometric batch analysis a ssay to detect caspase activity in ceramide-or DNR-treated cells (fluo rogenic substrate for caspase), we found that caspase activity increas ed in apoptosis-committed cells before they were detected by flow cyto metry or recognized by phagocytes, Similarly a caspase activity increa se was detected in circulating mononuclear cells of leukemic patients 15 h after the beginning of anthracyclin treatment. We suggest that re cent findings on enzymatic events (caspase activation) occurring in th e early events of apoptosis must now allow the development of new mark ers for apoptosis, irrespective of the morphological features or inter nucleosomal fragmentation which are late events in apoptosis. (C) 1998 Academic Press.