APOPTOSIS DURING HL-60 CELL-DIFFERENTIATION IS CLOSELY-RELATED TO A G0/G1 CELL-CYCLE ARREST

The cell cycle-associated differences in the susceptibility to apoptosis were examined in HL-60 cells before and after differentiation with phorbol 12, 13-dibutyrate (PDBu). HL-60 cells in various phases of the cell cycle were separated by the counterflow centrifugal elutriation and the susceptibility to apoptosis was measured by the morphological examination and by DNA fragmentation assay. Undifferentiated HL-60 cells in S phase showed a significantly higher susceptibility to apoptosis than those in G0/G1 or G2/M phase either in the absence or presence of apoptosis-inducing reagents such as A23187, actinomycin D (Act D), and cycloheximide (CHX). In contrast, PDBu-treated HL-60 cells preferentially underwent apoptosis in G0/G1 phase. When untreated HL-60 cells enriched for G0/G1 phase were recultured in a complete medium, the percentage of apoptotic cells increased after 6-12 h in correlation with the increase in S-phase cells. When the same experiment was performed with PBDu-treated cells, spontaneous increase of apoptotic cells was observed while almost all cells remained in G0/G1 phase. Northern blot analysis revealed that undifferentiated cells expressed the same amounts of bcl-2 mRNA in each cell cycle phase, whereas G0/G1-predominant reduction of bcl-2 mRNA was noted in PDBu-treated cells. There was no difference in the amounts of CD11b mRNA between G0/G1 fraction and S + G2/M fraction of differentiated HL-60 cells. BCL-2 overexpression could almost completely abrogate the G0/G1-predominant induction of apoptosis in differentiated HL-60 cells. These results suggest that G0/G1 cell cycle arrest and down-regulation of bcl-2 mRNA in G0/G1 phase might be associated with apoptosis in differentiated HL-60 cells whereas the weakness of chromatin structure in S phase might be related to apoptosis in undifferentiated HL-60 cells. (C) 1995 Wiley-Liss, Inc.