Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production.

Abstract

The regulation of intracellular pools of Ca2+ was investigated in an interleukin-3 (IL-3)-dependent hematopoietic cell line 32D that undergoes programmed cell death ("apoptosis") when deprived of lymphokine. Comparisons were made with 32D cells that had been stably transfected with a bcl-2 expression plasmid that encodes a 26-kDa intracellular integral-membrane protein known to abrogate apoptosis resulting from IL-3 withdrawal. Removal of IL-3 from cultures of 32D cells or control-transfected 32D-NEO cells for 1-2 days led to cell cycle arrest and oligonucleosomal DNA fragmentation and was associated with lower cytosolic free Ca2+ concentrations ([Ca2+]i), as measured by Indo-1 fluorescence of viable cells in Ca(2+)-containing media. In bcl-2-expressing 32D-BCL2 cells, IL-3 withdrawal also resulted in cessation of proliferation, but [Ca2+]i levels were not decreased and DNA fragmentation was markedly suppressed. Nonmitochondrial stores of Ca2+ were also significantly diminished in IL-3-deprived 32D-NEO but not in 32D-BCL2 cells, based on measurements of Ca2+ release into the cytosol following exposure of cells to thapsigargin (an inhibitor of endoplasmic reticulum Ca(2+)-ATPases) under Ca(2+)-free conditions. In contrast, estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation 1799 (2,6-dihydroxy-1,1,1,7,7,7-he xafluoro-2,6-bis(trifluoromethyl)heptan-4-one[bis(he xafluoroacetonyl)]acetone) suggested that IL-3 deprivation leads to an increase in this intracellular pool of Ca2+ in 32D-NEO but not in 32D-BCL2 cells. Re-addition of IL-3 to factor-deprived 32D-NEO cells reversed the changes in thapsigargin- and 1799-releasable Ca2+ pools and rescued many of the cells from death. Measurements of total cellular Ca2+ revealed no difference in 32D-NEO cells before and after IL-3 withdrawal, suggesting that the observed alterations in mitochondrial and nonmitochondrial Ca2+ pools result from intracellular repartitioning of Ca2+. Treatment of IL-3-deprived 32D-NEO cells with Ca2+ ionophores blocked DNA fragmentation and prolonged cell survival, whereas addition of Ca2+ chelators to IL-3-stimulated 32D cells resulted in oligonucleosomal DNA fragmentation and cell death, suggesting that diminutions in the concentrations of Ca2+ in cytosol, endoplasmic reticulum, or other intracellular compartments either directly or indirectly regulate apoptosis in these lymphokine-dependent hematopoietic cells.