A STUDY OF THE INTRACELLULAR ROUTING OF CYTOTOXIC RIBONUCLEASES

Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pa ncreatic ribonuclease A superfamily can be toxic when applied to the o utside of cells, they become thousands of times more toxic when artifi cially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents th at disrupt the Gels apparatus by distinct mechanisms, retinoic acid, b refeldin A, and monensin, to probe the intracellular pathways ribonucl eases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and h uman ribonuclease A 100 times or more. Retinoic acid-mediated potentia tion of ribonucleases is completely blocked by brefeldin A. Ribonuclea ses appear to route more efficiently into the cytosol through the Golg i apparatus disrupted by monensin or retinoic acid. Intracellular RNA degradation by BS-RNase increased more than 100 times in the presence of retinoic acid confirming that the RNase reaches the cytosol and ind icating that degradation of RNA is the intracellular lesion causing to xicity. As retinoic acid alone and Onconase are in clinical trials for cancer therapy, combinations of RNases and retinoic acid in vivo may offer new clinical utility.