Secretion of ribonucleases by normal and immortalized cells grown in serum-free culture conditions

Abstract

The requirement of serum in cell culture is a major limitation for studies on secreted ribonucleases (RNases) because serum contains a high amount of ribonucleolytic activity. Defined culture condition is thus of interest to improve our knowledge of the RNase biology. We report here that cells from three different types and origins, Chinese hamster lung fibroblasts, bovine smooth muscle cells, and human endothelium-derived EA.hy926 cells, proliferate consistently in the presence of a basal medium supplemented with bovine serum albumin, high-density lipoproteins, basic fibroblast growth factor, insulin, and transferrin. Using a new quantitative radio-RNase inhibitor assay, two distinct ribonucleolytic assays, and a radioimmunoassay against angiogenin, it is shown that RNases became apparent in media conditioned by cell monolayers. Both the hamster lung fibroblast and the EA.hy926 cell lines secreted larger amounts of RNase inhibitor-interacting factors and RNase activity than normal smooth muscle cells. The serum-free medium represents an alternative way to grow these cells and allows investigation of biosynthesis and functions of RNases in culture. It should be useful to identify and quantitate unambiguously specific members of the RNase family secreted by normal versus tumor cells in culture.

Internalization and Processing of Human Angiogenin by Cultured Aortic Smooth Muscle Cells

“…AALTR 16-2 cells were used between 20 and 26 subcultures. Bovine SMC were cultured in serum-free medium in the conditions described in (18). Cells were grown at 37°C in a humidified atmosphere of 5% CO 2 , 95% air.…”

Extracellular RNA Liberates Tumor Necrosis Factor-α to Promote Tumor Cell Trafficking and Progression

“…The natural counterpart of eRNA in the vascular system is circulating RNase1, which is predominantly expressed and released by endothelial cells (12)(13)(14). Immediate release of RNase1 from endothelial cell storage granules (designated Weibel-Palade bodies) is promoted by proinflammatory and prothrombotic agents including eRNA itself.…”

Basic homopolyamino acids, histones and protamines are potent antagonists of angiogenin binding to ribonuclease inhibitor

“…The specific radioactivity of the labelled protein was between 220 000 and 450 000 cpm/ng (1.5–3 Ci/μmol). RRIA was performed at 20°C essentially as described [14]. 125 I‐Angiogenin (7000–15 000 cpm) and the test samples were diluted in RRIA buffer (MBS, 1 mg/ml BSA, 5 mM EDTA, 0.02% NaN 3 ; final volume 460 μl).…”

“…Then, 0.015 U RI in 20 μl MBS, 5 mM DTT, pH 7.2 was added and incubation was continued for 10 min. 125 I‐Angiogenin⋅RI complexes were precipitated in 50% ammonium sulfate in the presence of the globulin fraction of fetal calf serum [14]. Free 125 I‐angiogenin was measured by counting aliquots of the supernatants.…”

“…On the basis of the angiogenin interaction with RI, an in vitro binding assay has been developed to detect members of the RNase superfamily whatever their catalytic activities [14]. Using this assay, we now report that basic homopolyamino acids as well as the lysine‐ and/or arginine‐rich proteins histones and protamines, although structurally unrelated to RNases, are potent competitors of angiogenin binding to RI.…”