Cell Density-dependent Apoptosis in HL-60 Cells, Which Is Mediated by an Unknown Soluble Factor, Is Inhibited By Transforming Growth Factor β1 and Overexpression of Bcl-2*

We report a novel mode of apoptosis induction observed in human leukemic HL-60 cells. These cells spontaneously underwent apoptosis in the course of proliferation when the cell density became higher than 1 × 106/ml. This occurred under ordinary in vitro culture conditions, with or without fetal calf serum. Even the low density cells were committed to undergo apoptosis if they were cultured under artificially concentrated conditions. Replacement of the culture supernatant of the low density cells by that of the high density ones resulted in apoptosis induction in the former cells. This apoptosis-inducing activity of the high density cell culture supernatant was completely eliminated by the action of trypsin but was fully restored following ultrafiltration by 3-kDa pore-sized membrane. A strong apoptosis-inducing activity was recovered from the culture supernatant of the high density HL-60 cells at a specific fraction in reverse-phase column chromatography. Neither an interleukin-β converting enzyme inhibitor nor CPP-32 inhibitor blocked the induction of cell density-dependent apoptosis in HL-60 cells, although overexpression of Bcl-2 protein markedly attenuated the induction of this mode. Surprisingly, transforming growth factor-β1 and activin A did not induce but, rather, inhibited the induction of cell density-dependent apoptosis. These data suggest that HL-60 cells release an unknown low molecular weight peptide-containing factor in response to an increase in cell density to induce apoptosis in an autocrine manner and that the interleukin-β converting enzyme-independent intracellular machinery for this mode of apoptosis is strongly affected by signaling events through the transforming growth factor-β1 receptor and by the action of Bcl-2 oncoprotein.