Differential Activity of the 30-kD and the 100-kD Forms of 2′-5′A n Synthetase Induced by Recombinant Human Interferon-α and Interferon-γ

Abstract

Two molecular weight variants of the interferon (IFN)-induced intracellular enzyme 2′,5′-oligoadenylate (2-5A) synthetase have been described recently; a 100-kD (cytoplasmic) and a 30-kD (intranuclear) form of the enzyme. The 30-kD form has been located primarily in the nucleus, while the 100-kD enzyme was found mainly in the cytoplasm. We examined 2-5A synthetase activity in extracts of human melanoma (Hs294t) cells treated with either recombinant (r) IFN-αA or rIFN-γ to determine the differential regulation of these enzyme subtypes by treatment with the two types of IFN. Cells were treated (continuous exposure) with doses of rIFN-αA (1,000 U/ml) or rIFN-γ (5,000 U/ml), each of which reduced the number of viable cells to 50% of control values (ED₅₀) by day 3 of treatment. At equieffective doses, the maximal increase in 2-5A synthetase occurred at 48 h for both rIFN-αA and rIFN-γ continuous exposure. The maximal 2-5A intracellular activities at 48 h were 800 ± 40 and 160 ± 15 nmoles/mg protein for rIFN-αA and rIFN-γ treatment, respectively. High-performance gel permeation chromatography of cell lysates resolved the 2-5A activity into both 100-kD and 30-kD fractions. At 48 h after treatment with rIFN-αA, the activity of the 30-kD synthetase was approximately twofold greater than that of the 100-kD enzyme. In contrast, the activity of both 30- and 100-kD enzymes were equivalent 48 h after treatment with rIFN-γ. The 100-kD enzyme activity did not vary substantially in the first 24 h after exposure to IFN-γ. However, the change in total 2-5A synthetase activity 24 h after IFN-γ treatment appeared to be due primarily to changes in the 30-kD enzyme. These studies show that the activities of the two molecular weight species of 2-5A synthetase which may exist in different subcellular locations are regulated differently at various times after exposure to IFNs. In addition, the importance of intracellular 2-5A activity to the antiproliferative activity of the IFNs may depend upon the location and activity of the various enzymatic forms within the cell.