Yen A, Freeman L, Powers V, Van Sant R, Fishbaugh J
Exp Cell Res. 1986 Jul;165(1):139-51. doi: 10.1016/0014-4827(86)90539-2.
The putative role of Ca2+ and calmodulin in regulating cell proliferation and differentiation was tested in HL-60 human promyelocytic leukemia cells. The dependence of retinoic acid (RA)-induced terminal myeloid differentiation of HL-60 promyelocytic leukemia cells on calmodulin levels and calcium ion flux was ascertained. RA-treated and untreated control cells were stained for cellular DNA with a Hoechst dye. Populations of G1/0, S and G2 + M phase cells were isolated by fluorescence activated cell sorting (FACS). Cytosolic calmodulin levels were then measured as a function of cell cycle phase for RA-treated and untreated cells using a radioimmunoassay. RA-treated cells were measured at early times, corresponding to the pre-commitment state, and late times, when significant cell differentiation had occurred. Cellular calmodulin levels increased with progression through the cell cycle. In contrast, no difference in calmodulin levels was observed between RA-untreated or -treated cells in the same cell cycle phases at early or late times. RA-induced HL-60 terminal myeloid differentiation was thus apparently not regulated by cellular cytosolic calmodulin levels. These conclusions were supported by the effects of calmodulin antagonists and calcium flux inhibitors. The calmodulin antagonists trifluoperazine and compound 48/80 both retarded cell growth in a concentration-dependent manner. But at concentrations where cellular effect was evidenced by slight growth inhibition, neither antagonist inhibited RA-induced cell differentiation or G1/0 growth arrest. The same was true of the gated calcium channel inhibitors, verapamil and nitrendipene, and the passive calcium flux inhibitor, CoCl2. RA-induced HL-60 cell differentiation and arrest in G0 was thus apparently not strongly dependent on cellular calmodulin levels or calcium flux. This is in strong contrast to murine erythroleukemia cells. The results argue against a central regulatory role for calmodulin or calcium flux in control of HL-60 growth arrest or differentiation.
在HL-60人早幼粒细胞白血病细胞中测试了Ca2+和钙调蛋白在调节细胞增殖和分化中的假定作用。确定了维甲酸（RA）诱导的HL-60早幼粒细胞白血病细胞终末髓系分化对钙调蛋白水平和钙离子通量的依赖性。用Hoechst染料对经RA处理和未处理的对照细胞进行细胞DNA染色。通过荧光激活细胞分选（FACS）分离G1/0、S和G2+M期细胞群体。然后使用放射免疫测定法测量经RA处理和未处理细胞的细胞溶质钙调蛋白水平，作为细胞周期阶段的函数。在对应于预承诺状态的早期和发生显著细胞分化的晚期测量经RA处理的细胞。细胞钙调蛋白水平随着细胞周期的进展而增加。相比之下，在早期或晚期的相同细胞周期阶段，未处理或经RA处理的细胞之间未观察到钙调蛋白水平的差异。因此，RA诱导的HL-60终末髓系分化显然不受细胞溶质钙调蛋白水平的调节。这些结论得到了钙调蛋白拮抗剂和钙通量抑制剂作用的支持。钙调蛋白拮抗剂三氟拉嗪和化合物48/80均以浓度依赖性方式抑制细胞生长。但在通过轻微生长抑制证明有细胞效应的浓度下，两种拮抗剂均未抑制RA诱导的细胞分化或G1/0生长停滞。门控钙通道抑制剂维拉帕米和尼群地平以及被动钙通量抑制剂CoCl2也是如此。因此，RA诱导的HL-60细胞分化和G0期停滞显然不强烈依赖于细胞钙调蛋白水平或钙通量。这与小鼠红白血病细胞形成强烈对比。结果反对钙调蛋白或钙通量在控制HL-60生长停滞或分化中起核心调节作用。
