Baffy G, Miyashita T, Williamson J R, Reed J C
Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia 19104.
J Biol Chem. 1993 Mar 25;268(9):6511-9.
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.
在白细胞介素-3（IL-3）依赖的造血细胞系32D中研究了细胞内Ca2+池的调节情况。当缺乏淋巴因子时，该细胞系会发生程序性细胞死亡（“凋亡”）。将其与稳定转染了bcl-2表达质粒的32D细胞进行比较，该质粒编码一种26 kDa的细胞内整合膜蛋白，已知其可消除因IL-3撤除导致的凋亡。从32D细胞或对照转染的32D-NEO细胞培养物中去除IL-3 1 - 2天会导致细胞周期停滞和寡核小体DNA片段化，并与较低的胞质游离Ca2+浓度（[Ca2+]i）相关，这是通过在含Ca(2+)的培养基中用Indo-1荧光测量活细胞来测定的。在表达bcl-2的32D-BCL2细胞中，IL-3撤除也导致增殖停止，但[Ca2+]i水平未降低，并且DNA片段化明显受到抑制。基于在无Ca(2+)条件下细胞暴露于毒胡萝卜素（内质网Ca(2+)-ATP酶的抑制剂）后Ca2+释放到胞质溶胶中的测量结果，IL-3缺乏的32D-NEO细胞中非线粒体Ca2+储存也显著减少，但32D-BCL2细胞中没有。相反，使用氧化磷酸化解偶联剂1799（2,6 - 二羟基 - 1,1,1,7,7,7 - 六氟 - 2,6 - 双（三氟甲基）庚 - 4 - 酮[双（六氟乙酰基）]丙酮）对线粒体Ca2+储存的估计表明，IL-3缺乏导致32D-NEO细胞中该细胞内Ca2+池增加，但32D-BCL2细胞中没有。将IL-3重新添加到缺乏因子的32D-NEO细胞中可逆转毒胡萝卜素和1799可释放Ca2+池的变化，并使许多细胞免于死亡。总细胞Ca2+的测量显示，IL-3撤除前后32D-NEO细胞没有差异，这表明观察到的线粒体和非线粒体Ca2+池的改变是由Ca2+的细胞内重新分配引起的。用Ca2+离子载体处理IL-3缺乏的32D-NEO细胞可阻断DNA片段化并延长细胞存活，而向IL-3刺激的32D细胞中添加Ca2+螯合剂会导致寡核小体DNA片段化和细胞死亡，这表明胞质溶胶、内质网或其他细胞内区室中Ca2+浓度的降低直接或间接调节这些淋巴因子依赖的造血细胞中的凋亡。
