COFACTORS IN IN-VITRO INDUCTION OF APOPTOSIS IN HL60 CELLS BY ALL-TRANS-RETINOIC ACID (ATRA)

The aim of this study was to determine the culture conditions that cou ld modulate the induction of apoptosis by all-trans retinoic acid (ATR A). Cell viability was evaluated by trypan blue test, differentiation by nitro blue tetrazolium test, and apoptosis by morphological analysi s. ATRA induced apoptosis in HL60 cells only when more than 100,000 ce lls/ml were seeded, while differentiation was induced regardless of th e seeded cell concentration. Reduction in the concentration of foetal calf serum or glutamine in the medium led to a weak increase in apopto sis. In contrast, a dramatic enhancement of apoptosis occurred when th e culture medium was supplemented with glucose or when the culture pH was decreased. These characteristics were independent of the mechanism of action of ATRA, but the action of glucose could be of significance in diabetic patients. An exchange of supernatants after 3 days of cul ture showed that supernatants from control cultures seeded at high cel l density were better apoptosis inducers than supernatants from cultur es treated with ATRA, but seeded at low cell density. Factor(s) in thi s supernatant which induced apoptosis was (were) removed by ultrafiltr ation. In conclusion, our results showed that ATRA alone cannot induce apoptosis, but can do so in conjunction with cofactors. The depletion of some components of the medium and the appearance of secreted macro molecule(s) could be cofactor(s) in the induction of apoptosis. (C) 19 98 Elsevier Science Inc.