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Microbial ribonucleases possess a broad spectrum of biological activities, which demonstrate stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. Mechanisms of their penetration into the cells still remain unclear. In this study penetration of Bacillus intermedius RNase (binase) in alveolar lung epithelial cells, type II (ATII) pneumocytes, has been investigated. Using immunofluorescence analysis we have shown for the first time internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in MLE-12 (Murine Lung Epithelial-12 cells). However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. These results clearly indicate higher sensitivity of tumor cells to binase compared to normal cells; they also demonstrate that penetration of the enzyme into alveolar epithelial cells is not directly associated with their death. © 2012 Pleiades Publishing, Ltd.

Some RNases including ones of microbial origin possess antitumor activity, which mechanisms remains unclear. Here we investigated the first step of RNase action towards eukaryotic cells which is connected with increase of cell permeability for ions and macromolecules. Using radiological analysis of 45Ca2+uptake by Candida yeast and fluorescence imaging of human embryo kidney cells HEK stained by Ca2+-specific Fura-2/AM day the level of intracellular Ca2+ under treatment with the RNase of Bacillus intermedius (binase) was studied. Viability of lung carcinoma epithelial cells A549 treated by binase was measured by WST proliferation kit, stability of erythrocytes was tested by lysis assay. We have shown that binase induces the permeability increase of lower and higher eukaryotic cells for Ca2+as well as the increase of protein permeability of A549 cells. Binase treatment protects erythrocytes from osmotic shock. The protective or cytotoxic binase effect followed by increase of cellular permeability is realized depending on the dell type, where the expression of Kca channels and of certain oncogens, particularly of ras family, is crucial. The obtained data supports the significance of the cell permeability increase as a primary step in the mechanisms of binase-induced biological effects.