Salzberg S, Hyman T, Turm H, Kinar Y, Schwartz Y, Nir U, Lejbkowicz F, Huberman E
Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Cancer Res. 1997 Jul 1;57(13):2732-40.
Two variants of the human myeloid leukemia cell line HL-60 were used to study the possible involvement of the IFN-induced protein 2-5A synthetase in cell growth arrest and differentiation. The two variants, HL-205 and HL-525, are equally susceptible to differentiation to the granulocyte lineage by exposure to DMSO, but only HL-205 cells acquire the macrophage phenotype following exposure to phorbol esters. The kinetics of 2-5A synthetase activity was established in both variants exposed to either DMSO or phorbol 12-myristate 13-acetate. With DMSO treatment, 2-5A synthetase activity was markedly induced in both variants, although with slightly different kinetics. With phorbol 12-myristate 13-acetate treatment, 2-5A enzymatic activity increased only in HL-205; no activity was detected up to 96 h after treatment in HL-525. The induction of 2-5A synthetase activity is apparently alpha/beta-IFN dependent, because only antibodies directed against a mixture of alpha- and beta-IFN completely abolished the increase in activity detected during differentiation of HL-205 cells. To directly establish the role of 2-5A synthetase in differentiation, HL-205 cells were transfected with an expression vector harboring the cDNA for the 43-kDa isoform of murine 2-5A synthetase fused to the inducible metallothionein promoter. Two clones, clone 6, which yielded a low level of 2-5A synthetase activity in response to ZnCl2 (which activates the promoter), and clone 7, which was a high responder, were further analyzed and compared with the control clone, neo. Reductions in the rates of cell growth and thymidine incorporation were observed with both clone 6 and clone 7 cells exposed to ZnCl2; clone 7 was more responsive. In addition, the level of c-myc-specific RNA transcript was greatly reduced in ZnCl2 or beta-IFN-treated clone 7 cells, whereas the neo cells responded similarly only after beta-IFN treatment. Treatment of clone-neo cells with beta-IFN resulted in conversion of pRb protein from the phosphorylated to the underphosphorylated form within 24 h; ZnCl2 had no effect, even after 72 h. In contrast, the accumulation of the underphosphorylated form of pRb was observed in clone 7 cells treated either with beta-IFN or ZnCl2. Finally, a significant increase in nitro blue tetrazolium-positive cells, an indication of differentiation, was evident with ZnCl2-treated clone 6 and clone 7 cells; no such increase was observed with clone-neo cells under similar conditions. We conclude that ectopic expression of 2-5A synthetase in HL-205 cells results in cell growth arrest and facilitates the appearance of a myeloid differentiation marker.
利用人类髓系白血病细胞系HL - 60的两个变体来研究干扰素诱导蛋白2 - 5A合成酶在细胞生长停滞和分化中可能发挥的作用。这两个变体HL - 205和HL - 525，通过暴露于二甲基亚砜（DMSO），同样容易分化为粒细胞系，但只有HL - 205细胞在暴露于佛波酯后获得巨噬细胞表型。在暴露于DMSO或佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯的两个变体中确定了2 - 5A合成酶活性的动力学。用DMSO处理时，两个变体中2 - 5A合成酶活性均显著诱导，尽管动力学略有不同。用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯处理时，2 - 5A酶活性仅在HL - 205中增加；在HL - 525中，处理后96小时内未检测到活性。2 - 5A合成酶活性的诱导显然依赖于α/β干扰素，因为只有针对α - 和β - 干扰素混合物的抗体才能完全消除在HL - 205细胞分化过程中检测到的活性增加。为了直接确定2 - 5A合成酶在分化中的作用，用一个表达载体转染HL - 205细胞，该表达载体含有与可诱导的金属硫蛋白启动子融合的鼠2 - 5A合成酶43 kDa同工型的cDNA。进一步分析了两个克隆，克隆6在响应氯化锌（激活启动子）时产生低水平的2 - 5A合成酶活性，克隆7是高反应者，并与对照克隆neo进行了比较。暴露于氯化锌的克隆6和克隆7细胞均观察到细胞生长速率和胸苷掺入率降低；克隆7反应更明显。此外，在氯化锌或β - 干扰素处理的克隆7细胞中，c - myc特异性RNA转录水平大大降低，而neo细胞仅在β - 干扰素处理后有类似反应。用β - 干扰素处理克隆neo细胞导致pRb蛋白在24小时内从磷酸化形式转变为低磷酸化形式；即使72小时后，氯化锌也没有作用。相反，在β - 干扰素或氯化锌处理的克隆7细胞中观察到pRb低磷酸化形式的积累。最后，氯化锌处理的克隆6和克隆7细胞中，硝基蓝四氮唑阳性细胞显著增加，这表明分化，在类似条件下，克隆neo细胞未观察到这种增加。我们得出结论，HL - 205细胞中2 - 5A合成酶的异位表达导致细胞生长停滞并促进髓系分化标志物的出现。
