Ectopic expression of 2-5A synthetase in myeloid cells induces growth arrest and facilitates the appearance of a myeloid differentiation marker

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 α/β-IFN dependent, because only antibodies directed against a mixture of α- and β- 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 ZnCI₂ (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 tn ZnCI₂; clone 7 was more responsive. In addition, the level of c-myc-specific RNA transcript was greatly reduced in ZnCI₂ or β-IFN-treated clone 7 cells, whereas the neo cells responded similarly only after β-IFN treatment. Treatment of clone- neo cells with β-IFN resulted in conversion of pRb protein from the phosphorylated to the underphosphorylated form within 24 h; ZnCI₂ 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 β-IFN or ZnCI₂. Finally, a significant increase in nitro blue tetrazolium-Positive cells, an indication of differentiation, was evident with ZnCI₂-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.