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 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.
Salzberg
S
S
Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Hyman
T
T
Turm
H
H
Kinar
Y
Y
Schwartz
Y
Y
Nir
U
U
Lejbkowicz
F
F
Huberman
E
E
D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002712:Chlorides / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006098:Granulocytes / Q000201:enzymology; D018922:HL-60 Cells; D006801:Humans; D016899:Interferon-beta / Q000276:immunology / Q000502:physiology; D007951:Leukemia, Myeloid / Q000201:enzymology* / Q000473:pathology; D008264:Macrophages / Q000201:enzymology; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D016271:Proto-Oncogene Proteins c-myc; D016160:Retinoblastoma Protein; D017967:Zinc Compounds; D016899:Interferon-beta; C016837:zinc chloride; D015088:2',5'-Oligoadenylate Synthetase; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide