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A study of the intracellular routing of cytotoxic ribonucleases
1Biochemistry Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pancreatic ribonuclease A superfamily can be toxic when applied to the outside of cells, they become thousands of times more toxic when artificially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents that disrupt the Golgi apparatus by distinct mechanisms, retinoic acid, brefeldin A, and monensin, to probe the intracellular pathways ribonucleases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times or more. Retinoic acid-mediated potentiation of ribonucleases is completely blocked by brefeldin A. Ribonucleases appear to route more efficiently into the cytosol through the Golgi 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 indicating that degradation of RNA is the intracellular lesion causing toxicity. 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.
Insights
Ribonucleases (RNases) are more toxic inside cells. Disrupting the Golgi apparatus with retinoic acid or monensin enhances RNase entry into the cytosol, increasing their cancer-fighting potential.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Ribonucleases (RNases) are enzymes involved in host defense and programmed cell death.
- Certain RNases exhibit significant cytotoxicity, but their efficacy is limited by cellular internalization.
Purpose of the Study:
- To investigate the intracellular pathways of RNase internalization and cytotoxicity.
- To explore the role of the Golgi apparatus in RNase entry into the cytosol.
- To evaluate the potential of agents disrupting the Golgi apparatus to enhance RNase-mediated cytotoxicity.
Main Methods:
- Utilized retinoic acid, brefeldin A, and monensin to disrupt the Golgi apparatus via distinct mechanisms.
- Assessed the potentiation of cytotoxicity for bovine seminal RNase (BS-RNase), Onconase, angiogenin, and human ribonuclease A.
- Quantified intracellular RNA degradation as a measure of RNase activity within the cytosol.
Main Results:
- Retinoic acid and monensin potentiated RNase cytotoxicity by over 100-fold.
- Retinoic acid-mediated potentiation was completely inhibited by brefeldin A, suggesting Golgi-dependent pathways.
- Intracellular RNA degradation by BS-RNase increased >100-fold with retinoic acid, confirming cytosolic entry and RNA degradation as the toxic mechanism.
Conclusions:
- The Golgi apparatus plays a crucial role in the efficient cytosolic delivery of ribonucleases.
- Disruption of the Golgi apparatus by agents like retinoic acid can significantly enhance RNase cytotoxicity.
- Combinations of RNases and retinoic acid may offer novel therapeutic strategies for cancer treatment.