Accentuation of Production of Human Interferon by Metabolic Inhibitors 1

Abstract

Vilcek (1, 2) has shown that the production of interferon by rabbit kidney cells stimulated with poly (I):poly (C) can be enhanced by certain inhibitors of protein synthesis. Accentuated interferon production in the presence of protein inhibitors such as cycloheximide can occur in several different tissues (4) and paradoxically requires concomitant protein synthesis (5). Maximal enhancement is achieved if inhibitors are used sequentially to block the synthesis of a postulated regulatory protein (3). This procedure involves treatment of cells with a reversible inhibitor of protein synthesis during the synthesis of interferon mRNA. Prior to reversal of protein blockade, actinomycin D treatment is initiated to inhibit transcription of the regulatory protein. In this paper we show that human interferon production can also be enhanced by cycloheximide and actinomycin D.

Materials and Methods. Cell cultures. Human embryonic fibroblasts (HEF) were obtained from tissues of an approximately 4-week embryo (about 3 cm long) by treatment with 0.25% trypsin. Cultures were grown in medium consisting of Hanks' balanced salt solution (HBSS) supplemented with 0.5% lactalbumin hydrolyzate, 2 mM L-glutamine, Eagle's vitamin mixture, antibiotics, and 5% inactivated (56° for 30 min) newborn calf serum. Subcultures were prepared as needed by treatment of monolayers with 0.25% trypsin and 0.2% EDTA in phosphate-buffered saline (PBS). The cells were centrifuged, resuspended at 2 × 10⁵ cells/ml, and dispensed into suitable culture vessels. Stock cultures were carried in glass prescription bottles but experimental work was performed with cultures in 5-cm plastic dishes or 6-mm micro wells (Falcon Microtest II plates) incubated under 5% CO₂. Stock cultures could be stored at room temperature for several weeks.

Interferon inducers. Polyinosinic acid and polycytidylic acid (Mann or P-L) were dissolved in PBS (pH 7.2) at 1 mg/ml and complexed by mixing at 37° immediately prior to use (4).