Role of interferon and 2‘,5‘-oligoadenylate synthetase in erythroid differentiation of Friend leukemia cells. Studies with interferon-sensitive and -resistant variants.

N Mechti,E Affabris,G Romeo,B Lebleu,G B Rossi

doi:10.1016/s0021-9258(17)43289-3

Abstract

It has been suggested that the interferon (IFN)-induced 2',5'-oligoadenylate (2-5A) synthetase, which polymerizes ATP into a series of 2',5'-linked oligomers with the general formula pppA(2'p5'A)n, plays a general role in cell growth and terminal differentiation. For instance, an increase in 2-5A synthetase activity has been described during dimethyl sulfoxide (Me2SO)-induced erythroid differentiation of Friend leukemia cells (FLC). 2-5A synthetase has been measured in two Friend leukemia cell sublines by various techniques including a radioimmunoassay of its products which would detect 10(-16) mol of 2-5A cores. Although cells of both sublines fully differentiate (as measured by benzidine staining), only in one subline was there an increase in 2-5A synthetase activity upon treatment with Me2SO. Hexamethylenebisacetamide, another potent agent of differentiation in this system, did not increase 2-5A synthetase activity in either of these two sublines. An IFN-resistant FLC variant differentiated normally upon treatment with Me2SO or hexamethylenebisacetamide while it was noninducible for 2-5A synthetase activity by exogenous IFN or by the inducers themselves. A similar situation has been observed with regard to the level of phosphorylation of the IFN-induced Mr = 67,000 protein band. In addition, treatment of IFN-sensitive and resistant FLC sublines with mouse alpha beta IFN antiserum did not affect differentiation. Even though we have duplicated previous findings on the increase of 2-5A synthetase activity in Me2SO-induced FLC, the lack of any such increase with other inducers or other sublines indicates that there is no causal relationship between the enzyme activation and FLC differentiation.

Highlights

I t has been suggested that the interferon (1FN)-in- independent 1FN’-induced enzymaticsystems,namely the duced 2’,5’-oligoadenylate(2-5A) synthetase, which protein kinase and2-5A synthetase pathways, both activated polymerizes ATPinto a seriesof 2‘,5‘-linked oligomers by double-stranded RNAs (for a review, see Lebleu and Conwith the general formulapppA(2’pB’A), plays a gen- tent, 1982)
2-5A synthetase has been measured in two Friend leukemia cell sublinebsy various techniques including a radioimmunoassay of itsproducts whichwould detect 10”‘ mol of2-5A cores
The terminal differentiationof one Friend leukemia cells (FLC) subline is accompanied by an increase of the IFN-induced2-5A synthetase, as originallydescribed by Kimchi(1981)

Summary

RESULTS

2-5A Synthetase Activity in Several Growing and Differentiating FLC Sublines Treated withMe2SO-Cells of two FLC sublines ( 7 4 5 A ~and 745AH) and of one IFN-resistant variant (745AR 1Cll) were seeded at lo cells/ml in medium supplemented with Me2S0 anwdere grown for 6 days under stationary conditions. Under these conditions, erythroid differentiation, as measured by hemoglobin-containing (benzidine-positive) cells, attained nearly 100% in all three lines (Fig. 1B). No modulation of 2-5A synthetase activit,y was detectable by this sensitive assay procedure in extractsprepared from Me2SO-treatedcells, even in the conditions described by Friedman-Einat et al (1982). BC values were determined as described under “Experimental Procedures.” 2-5A synthetase activities, measured with the conventional assay of Minks et al (1979), were expressed in nanomoles of ATP transformed/h/mg of protein and those measured with the radioimmunologicalassay were expressed in nanomoles of ATP transformed/106 cells (values in parentheses), as described in the text

FLC subline

Protein Kinase Activity in Growing and Differentiating

DISCUSSION

Findings

There are examples of excretion of minute amounts of IFN