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• W2045816749 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. 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. Ribonucleases serve as selective cytotoxic agents in host defense and physiological cell death pathways in bacteria, higher plants, and mammals (reviewed in (1Youle R.J. Newton D. Wu Y.N. Gadina M. Rybak S.M. C.R.C. Cryt. Rev. Ther. Drug Carrier Syst. 1993; 10: 1-28PubMed Google Scholar) and (2D'Alessio G. Trends Cell Biol. 1993; 3: 106-109Abstract Full Text PDF PubMed Scopus (97) Google Scholar)), and they have potential use as therapeutic agents for human disorders either alone (3Mikulski S.M. Grossman A.M. Carter P.W. Shogen K. Costanzi J.J. Int J. Oncol. 1993; 3: 57-64PubMed Google Scholar, 4Youle R.J. Wu Y.N. Mikulski S.M. Shogen K. Hamilton R.S. Newton D. D'Alessio G. Gravell M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6012-6016Crossref PubMed Scopus (63) Google Scholar) or after conjugation to targeting molecules(5Rybak S.M. Saxena S.K. Ackerman E.J. Youle R.J. J. Biol. Chem. 1991; 266: 21202-21207Abstract Full Text PDF PubMed Google Scholar, 6Newton D. Ilercil O. Laske D.W. Oldfield E. Rybak S.M. Youle R.J. J. Biol. Chem. 1992; 267: 19572-19578Abstract Full Text PDF PubMed Google Scholar). They appear to bind cell surface receptors and enter the cytosol where they degrade RNA to kill the target cell(1Youle R.J. Newton D. Wu Y.N. Gadina M. Rybak S.M. C.R.C. Cryt. Rev. Ther. Drug Carrier Syst. 1993; 10: 1-28PubMed Google Scholar). Bovine seminal ribonuclease, a homodimer purified from bull semen with 80% amino acid sequence homology to RNase A, is toxic to certain mammalian cells in culture and expresses anti-cancer activity in animal models(7Vescia S. Tramontano D. Augusti-Tocco G. D'Alessio G. Cancer Res. 1980; 40: 3740-3744PubMed Google Scholar, 8Lacceti P. Portella G. Mastronicola M.R. Russo A. Piccoli R. D'Alessio G. Cancer Res. 1992; 52: 4582-4586PubMed Google Scholar).1 1M. R. Mastronicola, R. Piccoli, and G. D'Alessio, submitted for publication. Another member of the RNase A superfamily, Onconase, isolated from frog eggs(10Ardelt W. Mikulski S.M. Shogen K. J. Biol. Chem. 1991; 266: 245-251Abstract Full Text PDF PubMed Google Scholar), also expresses anti-tumor activity in animals and is now in phase II clinical trials for cancer therapy(3Mikulski S.M. Grossman A.M. Carter P.W. Shogen K. Costanzi J.J. Int J. Oncol. 1993; 3: 57-64PubMed Google Scholar). Angiogenin, originally purified based upon angiogenesis activity(11Fett J.W. Strydom D.J. Lobb R.R. Alderman E.M. Bethune J.L. Riordan J.F. Vallee B.L. Biochemistry. 1985; 24: 5480-5486Crossref PubMed Scopus (846) Google Scholar), is also homologous to ribonuclease A and can be cytotoxic when fused with targeting molecules(12Rybak S.M. Hoogenboom H.R. Meade H.M. Raus J.C.M. Schwartz D. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3165-3169Crossref PubMed Scopus (76) Google Scholar). Although these RNases can be toxic when applied to the outside of cells, RNases become thousands of times more toxic when artificially introduced into the cytosol(13Saxena S.K. Rybak S.M. Winkler G. Meade H.M. McGray P. Youle R.J. Ackerman E.J. J. Biol. Chem. 1991; 266: 21208-21214Abstract Full Text PDF PubMed Google Scholar), indicating that internalization is the rate-limiting step. The pathways ribonucleases take to cross the membrane surrounding the cytosol remains unknown. Retinoic acid and monensin disrupt the Golgi apparatus by distinct mechanisms, yet both potentiate the cytotoxicity of ricin A chain immunotoxins(14Wu Y.N. Gadina M. Tao-Cheng J.H. Youle R.J. J. Cell Biol. 1994; 125: 743-753Crossref PubMed Scopus (48) Google Scholar). These agents appear to alter the intracellular trafficking of certain protein toxins and facilitate their transport into the cytosol. Here we report an investigation of the intracellular route ribonucleases take to the cytosol. All-trans-retinoic acid was purchased from Calbiochem; brefeldin A was from Sigma; bovine seminal ribonuclease was purified from bull semen or seminal vesicles as reported(15Tamburrini M. Piccoli R. De Prisco R. Di Donato A. D'Alessio G. Ital. J. Biochem. (Engl. Ed.). 1986; 35: 22-32PubMed Google Scholar); and Onconase was isolated from Rana pipiens eggs as described(10Ardelt W. Mikulski S.M. Shogen K. J. Biol. Chem. 1991; 266: 245-251Abstract Full Text PDF PubMed Google Scholar); alkylated Onconase was prepared as described (16Crestfield A.M. Stein W.H. Moore S. J. Biol. Chem. 1963; 238: 2413-2420Abstract Full Text PDF PubMed Google Scholar) with 98% of the ribonuclease activity being inactivated; the catalytically active monomeric derivatives of BS-RNase,2 2The abbreviations used are: BS-RNasebovine seminal ribonucleaseMSSRmonomeric Cys-31, 32-S-ethylamine BS-RNaseMCMmonomeric Cys-31, 32-carboxymethyl BS-RNaseBFAbrefeldin AERendoplasmic reticulumNBD12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). MSSR (monomeric Cys-31, 32-S-ethylamine-BS-RNase), and MCM (monomeric Cys-31, 32-S-carboxymethyl-BS-RNase) were prepared by stably blocking the sulfhydryls exposed after selective reduction with dithiothreitol of the intersubunit disulfides as described previously (17D'Alessio G. Malorni M.C. Parente A. Biochemistry. 1975; 14: 1116-1121Crossref PubMed Scopus (77) Google Scholar). bovine seminal ribonuclease monomeric Cys-31, 32-S-ethylamine BS-RNase monomeric Cys-31, 32-carboxymethyl BS-RNase brefeldin A endoplasmic reticulum 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). 9L (rat glioma) cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 10 εg/ml gentamicin. Protein synthesis inhibition by RNases was determined as described previously(14Wu Y.N. Gadina M. Tao-Cheng J.H. Youle R.J. J. Cell Biol. 1994; 125: 743-753Crossref PubMed Scopus (48) Google Scholar, 18Wu Y. Mikulski S.M. Ardelt W. Rybak S.M. Youle R.J. J. Biol. Chem. 1993; 268: 10686-10693Abstract Full Text PDF PubMed Google Scholar). Briefly, cells in 100 εl were plated at concentrations of 2 × 105 cells/ml in 96-well microtiter plates overnight in Dulbecco's modified Eagle's complete medium. Retinoic acid (15 mM in dimethyl sulfoxide), monensin (2 mM in ethanol), and brefeldin A (BFA, 10 mg/ml in ethanol) stock solutions were diluted into leucine-free RPMI 1640 medium without fetal calf serum to the appropriate concentrations. The same amounts of dimethyl sulfoxide and/or ethanol were added in the control solutions. After removing the complete Dulbecco's modified Eagle's medium, cells were incubated in the above leucine-free RPMI 1640 medium containing increasing concentrations of ribonucleases with or without retinoic acid or monensin and/or BFA for 16 h followed by a 1-h pulse with 0.1 εCi of [14C]leucine. Cells were harvested onto glass fiber filters using a PHD cell harvester, washed with water, dried with ethanol, and counted. The results were expressed as the percentage of [14C]leucine incorporation in mock-treated control cells. 9L cells cultured in 96-well plates were incubated with increasing concentrations of BS-RNase in leucine-free RPMI 1640 medium with or without 10 εM retinoic acid for either 6 h or 16 h. Cells were then trypsinized by adding trypsin in each well without removing the incubation medium, resuspended, mixed with trypan blue, and counted. Cell viability was calculated as the percentage of trypan blue excluding cells with respect to the total cell counts. 9L cells (1 × 106 cells/ml) cultured in 75-cm2 flasks were treated with increasing concentrations of BS-RNase in leucine-free RPMI 1640 medium with or without 10 εM retinoic acid. After 6 h, cells were trypsinized, washed, and processed for total RNA isolation using the RNAzol™ method supplied by TEL-TEST, Inc. Briefly, cells were homogenized in RNAzol™ (2 ml of RNAzol™ per 1 × 107 cells), RNA was then extracted with 0.1 volume of chloroform, precipitated with 1 volume of isopropyl alcohol, and finally washed with 75% ethanol. Total RNA was analyzed either on a 1.4% agarose gel or a polyacrylamide gel containing 7.5 M urea as described(19Saxena S.K. Ackerman E.J. J. Biol. Chem. 1991; 265: 17106-17109Abstract Full Text PDF Google Scholar). The human RNase A gene was synthesized using an Escherichia coli codon bias(20Grantham R. Gaufier C. Gouy M. Jacobzone M. Mercier R. Nucleic Acids Res. 1981; 9: r43Crossref PubMed Scopus (156) Google Scholar). Twelve oligonucleotides for assembling the synthetic human ribonuclease A gene were synthesized on a Cyclone™ Plus DNA Synthesizer. After being phosphorylated with T4 kinase, these oligonucleotides were ligated together with DNA ligase. The ligated product was used as the template for amplification with the polymerase chain reaction. The amplified polymerase chain reaction product was then cloned into the PET-11d plasmid using BamHI and XbaI restriction sites and sequenced. The final sequence (Fig. 1) was that desired to generate the human pancreatic RNase protein lacking the leader sequence with an additional Met-1 residue. Human RNase A was then expressed in BL21-DE3 E. coli cells with isopropyl-1-thio-β-D-galactopyranoside as the inducing agent. The fraction of inclusion bodies that contains the expressed protein was isolated and treated as described(21Schultz D. Baldwin R.L. Protein Sci. 1992; 1: 910-916Crossref PubMed Scopus (73) Google Scholar). The refolded ribonuclease was then purified by ion exchange chromatography on S-Sepharose followed by size exclusion chromatography on Sephacryl S-100. The S-Sepharose column was developed with a linear sodium chloride gradient (0.35-0.5 M) in 0.15 M sodium acetate buffer, pH 5.0. The main peak was collected, concentrated by ultrafiltration, and run on a Sephacryl column in 0.075 M ammonium bicarbonate. The pooled peak fractions were lyophilized. The resulting preparation was homogeneous in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Ribonuclease activity of the recombinant (Met-1) human RNase was about 90% relative to the specific activity of bovine pancreatic RNase A. The human angiogenin cDNA gene (22Saxena S.K. Rybak S.M. Davey Jr., R.T. Youle R.J. Ackerman E.J. J. Biol. Chem. 1992; 267: 21982-21986Abstract Full Text PDF PubMed Google Scholar) was cloned into the PET-11d plasmid and expressed in BL21-DE3 E. coli by induction with isopropyl-1-thio-β-D-galactopyranoside. The expressed protein was purified using the same method as described under the purification of human ribonuclease A and was homogeneous by Coomassie staining of SDS-polyacrylamide gels. When incubated with 9L glioma cells, BS-RNase inhibited cellular protein synthesis only 30% at 1 × 10−6M (Fig. 2A). In the presence of either retinoic acid or monensin, however, the inhibition of protein synthesis reached 98% at the same concentration of BS-RNase and reached 50% at about 1 × 10−8M. Thus, these drugs increase the cytotoxicity of BS-RNase over 100-fold. BS-RNase is a disulfide-linked dimer (17D'Alessio G. Malorni M.C. Parente A. Biochemistry. 1975; 14: 1116-1121Crossref PubMed Scopus (77) Google Scholar) which may be reduced and alkylated to generate single chain monomers(17D'Alessio G. Malorni M.C. Parente A. Biochemistry. 1975; 14: 1116-1121Crossref PubMed Scopus (77) Google Scholar). The dimer form is much more potent than the monomer in a variety of biologic activities of this protein (2D'Alessio G. Trends Cell Biol. 1993; 3: 106-109Abstract Full Text PDF PubMed Scopus (97) Google Scholar). Alkylation with ethyleneimine results in a Cys-31, Cys-32-S-ethylamine derivative (MSSR), whereas alkylation with iodoacetamide leads to a Cys-31, Cys-32-carboxymethyl monomer of BS-RNase (MCM). Neither MSSR nor MCM were toxic to 9L glioma cells within the employed concentration range (Fig. 2, B and C, respectively). However, they both were quite potent in the presence of either retinoic acid or monensin. The respective values of IC50 were 5 × 10−8M for MSSR and 1 × 10−7M for MCM. The potency of the two monomers was within one order of magnitude of the dimer in the presence of either retinoic acid or monensin. Dimerization is not essential for BS-RNase toxicity in the presence of Golgi disrupting drugs. Fig. 3 shows the cell viability compared to protein synthesis inhibition after treatment of cells with BS-RNase in the presence or absence of 10 εM retinoic acid. Although protein synthesis was inhibited by 60% after a 6-h incubation with BS-RNase in the presence of 10 εM retinoic acid, the cells remained intact as measured by trypan blue exclusion (Fig. 3A). However, incubation of the cells with BS-RNase in the presence of retinoic acid for 16 h resulted in the complete inhibition of protein synthesis and cell lysis as shown in Fig. 3B. After 16 h of treatment, no cells were lysed by BS-RNase alone at 10−6M, whereas, in the presence of retinoic acid, 90% of the cells were lysed. Thus, the potentiation of RNase inhibition of protein synthesis seen with retinoic acid effects a large decrease in cell viability. Although protein synthesis was inhibited up to 60%, cells remained intact for at least 6 h after treatment with BS-RNase in the presence of retinoic acid (Fig. 3A). We examined the status of RNA in the cells treated with BS-RNase with or without retinoic acid for 6 h (Fig. 4). There was no detectable RNA degradation in cells treated with BS-RNase up to a 100 nM concentration. At a concentration of 1.0 εM BS-RNase, 28 S and 18 S rRNA began to be degraded, whereas there was no detectable degradation of 5.8 S, 5 S, or tRNA (Fig. 4). Thus, 28 S and 18 S rRNA are the more susceptible substrates for BS-RNase than 5.8 S, 5 S rRNA, and tRNA. However, in the presence of retinoic acid, the 28 S, 18 S, 5.8 S, 5 S rRNA, and tRNA were all degraded to some extent, even at BS-RNase concentrations as low as 10 nM (Fig. 4). In retinoic acid-treated cells, RNA is degraded at concentrations of BS-RNase 100 times lower than that needed for RNA degradation in cells not exposed to retinoic acid. However, the 28 S and 18 S rRNA seem to be more susceptible to BS-RNase than 5.8 S, 5 S, and tRNA both in the presence and absence of BS-RNase. These results indicate that BS-RNase gets into cytosol more efficiently in the presence than in the absence of retinoic acid. As rRNA degradation correlates with cytotoxicity, both in the presence and absence of retinoic acid, these results substantiate the model that rRNA degradation after cytosolic entry of BS-RNase is the mechanism of BS-RNase protein synthesis inhibition. A number of proteins discovered based on a variety of biologic activities have recently been found to be homologous to RNase A and to express RNase activity (reviewed in Refs. 1, 2, and 23). We compared the cytotoxicity of different RNases with and without Golgi apparatus selective drugs. In addition to BS-RNase and its monomers, retinoic acid potentiated the cytotoxicity of Onconase, angiogenin, and human RNase A as shown in Fig. 5. Onconase is a cytotoxic ribonuclease isolated from R. pipiens eggs and early embryos based upon its anti-cancer activity both in vitro and in vivo(10Ardelt W. Mikulski S.M. Shogen K. J. Biol. Chem. 1991; 266: 245-251Abstract Full Text PDF PubMed Google Scholar, 18Wu Y. Mikulski S.M. Ardelt W. Rybak S.M. Youle R.J. J. Biol. Chem. 1993; 268: 10686-10693Abstract Full Text PDF PubMed Google Scholar, 24Darzynkiewicz Z. Carter S.P. Mikulski S.M. Ardelt W.J. Shogen K. Cell Tissue Kinet. 1988; 21: 169-182PubMed Google Scholar, 25Mikulski S.M. Ardelt W.J. Shogen K. J. Natl. Cancer Inst. 1990; 82: 151-153Crossref PubMed Scopus (93) Google Scholar, 26Mikulski S.M. Viera A. Ardelt W.J. Menduke H. Shogen K. Cell Tissue Kinet. 1990; 23: 237-246PubMed Google Scholar). Alone, Onconase has an IC50 of 10−6M and in the presence of retinoic acid cytotoxicity increased 100-fold to around 10−8M (Fig. 5A), quite close to that of BS-RNase. Angiogenin, a ribonuclease originally purified because of its angiogenesis activity(11Fett J.W. Strydom D.J. Lobb R.R. Alderman E.M. Bethune J.L. Riordan J.F. Vallee B.L. Biochemistry. 1985; 24: 5480-5486Crossref PubMed Scopus (846) Google Scholar), is not detectably toxic alone, but, in the presence of retinoic acid, has an IC50 of 3 × 10−6M (Fig. 5B). Recombinant human pancreatic RNase A was also more toxic with retinoic acid (Fig. 5C), yet the potency was relatively low compared to the other RNases. Although all four members of the RNase A superfamily examined were more toxic in the presence of retinoic acid, their potency varied 10,000-fold from 2 × 10−9 to 2 × 10−5M. What molecular features account for these large differences remain unknown. As shown in Table 1, Onconase ribonuclease activity is essential for its cytotoxicity. Inactivation of Onconase ribonuclease activity by 98% with iodoacetic acid abolished its cytotoxicity. Recombinant Onconase which only contains 2% of the native Onconase ribonuclease activity also lost its cytotoxicity, although the crystal structure showed that recombinant Onconase has been properly folded.3 3S. C. Mosimann and M. N. G. James, unpublished data. Tabled 1 Open table in a new tab It has been demonstrated that retinoic acid disrupts the Golgi apparatus and potentiates the cytotoxicity of ricin A chain immunotoxins(14Wu Y.N. Gadina M. Tao-Cheng J.H. Youle R.J. J. Cell Biol. 1994; 125: 743-753Crossref PubMed Scopus (48) Google Scholar). To examine whether or not retinoic acid affects the routing of RNases through the Golgi apparatus, the effect of brefeldin A (BFA) on the retinoic acid potentiation of BS-RNase and Onconase was examined (Fig. 6). BFA, by inhibiting vesicular transport from the endoplasmic reticulum (ER) to the Golgi, results in collapse of the cis-Golgi apparatus blocking the retrograde vesicular transport of vesicles from the Golgi to the ER (27Doms R.W. Russ G. Yewdell J.W. J. Cell Biol. 1989; 109: 61-72Crossref PubMed Scopus (429) Google Scholar, 28Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1315) Google Scholar). BFA was incubated with 9L cells in the presence of retinoic acid and BS-RNase or Onconase. Fig. 6 shows that BFA completely blocks the potentiation of toxicity of both BS-RNase and Onconase by retinoic acid. BFA, however, has no effect on the cytotoxicity caused by Onconase and BS-RNase themselves. This indicates that BS-RNase and Onconase route through the Golgi apparatus in the presence of retinoic acid. The results also indicate that Onconase routes to the cytosol through a BFA insensitive compartment in the absence of retinoic acid. Thus, Onconase likely routes to the cytosol normally without going through the Golgi apparatus, and addition of retinoic acid alters the intracellular routing through the Golgi where Onconase accesses the cytosol 100-fold more efficiently. BS-RNase is also routed more than 100 times more efficiently through the Golgi to the cytosol in the presence than in the absence of retinoic acid. To test whether the RNase affects cell death via some signaling pathway that can be enhanced by retinoic acid through transcriptional controls, cells were incubated with actinomycin D before exposure to retinoic acid and Onconase. Table 2 showed that pretreatment with actinomycin D did not prevent the potentiation of Onconase cytotoxicity by retinoic acid. Thus, the well established transcription activation activity of retinoic acid does not appear to be the mechanism by which retinoic acid increases cell sensitivity to RNases.Tabled 1 Open table in a new tab Ribonucleases have potential as therapeutic agents for a number of human disorders including cancer and HIV(1Youle R.J. Newton D. Wu Y.N. Gadina M. Rybak S.M. C.R.C. Cryt. Rev. Ther. Drug Carrier Syst. 1993; 10: 1-28PubMed Google Scholar, 2D'Alessio G. Trends Cell Biol. 1993; 3: 106-109Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 3Mikulski S.M. Grossman A.M. Carter P.W. Shogen K. Costanzi J.J. Int J. Oncol. 1993; 3: 57-64PubMed Google Scholar, 4Youle R.J. Wu Y.N. Mikulski S.M. Shogen K. Hamilton R.S. Newton D. D'Alessio G. Gravell M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6012-6016Crossref PubMed Scopus (63) Google Scholar, 5Rybak S.M. Saxena S.K. Ackerman E.J. Youle R.J. J. Biol. Chem. 1991; 266: 21202-21207Abstract Full Text PDF PubMed Google Scholar, 6Newton D. Ilercil O. Laske D.W. Oldfield E. Rybak S.M. Youle R.J. J. Biol. Chem. 1992; 267: 19572-19578Abstract Full Text PDF PubMed Google Scholar). Certain members of the ribonuclease family can be used as a toxin moiety to construct chimeric or fusion proteins for targeted therapy (5Rybak S.M. Saxena S.K. Ackerman E.J. Youle R.J. J. Biol. Chem. 1991; 266: 21202-21207Abstract Full Text PDF PubMed Google Scholar, 6Newton D. Ilercil O. Laske D.W. Oldfield E. Rybak S.M. Youle R.J. J. Biol. Chem. 1992; 267: 19572-19578Abstract Full Text PDF PubMed Google Scholar, 12Rybak S.M. Hoogenboom H.R. Meade H.M. Raus J.C.M. Schwartz D. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3165-3169Crossref PubMed Scopus (76) Google Scholar) and some members of the RNase family, such as Onconase, are cytotoxic by themselves(18Wu Y. Mikulski S.M. Ardelt W. Rybak S.M. Youle R.J. J. Biol. Chem. 1993; 268: 10686-10693Abstract Full Text PDF PubMed Google Scholar). How RNases kill cells is poorly understood. It has been proposed that the mechanism of cytotoxic RNases resembles that of plant and bacterial toxins to some degree whereby the RNases bind the mammalian cell surface, enter into the cell cytosol, and degrade RNA resulting in cell death(18Wu Y. Mikulski S.M. Ardelt W. Rybak S.M. Youle R.J. J. Biol. Chem. 1993; 268: 10686-10693Abstract Full Text PDF PubMed Google Scholar).1 How the RNases cross the membrane and enter the cytosol remains unclear. It has been demonstrated recently that retinoic acid specifically disrupts the Golgi apparatus(14Wu Y.N. Gadina M. Tao-Cheng J.H. Youle R.J. J. Cell Biol. 1994; 125: 743-753Crossref PubMed Scopus (48) Google Scholar). Golgi-specific staining with mannosidase II (28Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1315) Google Scholar) and NBD-ceramide (29Lipsky N.G. Pagano R.E. Science. 1985; 228: 745-747Crossref PubMed Scopus (241) Google Scholar, 30Misumi Y. Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11399-11403Abstract Full Text PDF Google Scholar) was reduced by treating cells with 10 εM retinoic acid; after removal of the retinoic acid, the Golgi apparatus staining rapidly reappeared as normal. By electron microscopy, the Golgi apparatus appeared swollen in the presence of retinoic acid and rapidly assumed normal morphology upon removal of retinoic acid. The morphology of the Golgi apparatus under electron microscopy in the presence of retinoic acid resembled that of monensin-treated cells in contrast to NBD-ceramide staining of cells where retinoic acid-treated cells were markedly different from monensin-treated cells. Retinoic acid and monensin increase the cytotoxicity of ricin A chain immunotoxins by increasing the intracellular routing of the toxins to the cytosol(14Wu Y.N. Gadina M. Tao-Cheng J.H. Youle R.J. J. Cell Biol. 1994; 125: 743-753Crossref PubMed Scopus (48) Google Scholar). A variety of data suggest that ricin immunotoxins route through the Golgi apparatus on the way to the cytosol. We examined the effect of retinoic acid and monensin on the cytotoxicity of a series of homologous ribonucleases. Retinoic acid and monensin increased the cytotoxicity of all four members of the ribonuclease family examined. This indicates that ribonucleases reach the cytosol after endocytosis and do not cross directly across the plasma membrane. These results also indicate that ribonucleases can reach the cytosol more efficiently when routed through a disrupted Golgi apparatus, and that, normally, transmembrane transport of the ribonucleases is rate-limiting for cytotoxicity. The sensitization of cells to ribonucleases by retinoic acid is completely blocked by BFA. BFA blocks the vesicular transport from the ER to the cis-Golgi apparatus causing a collapse of the cis-Golgi and a termination of the retrograde vesicular transport from the cis-Golgi back to the ER(27Doms R.W. Russ G. Yewdell J.W. J. Cell Biol. 1989; 109: 61-72Crossref PubMed Scopus (429) Google Scholar, 28Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1315) Google Scholar). The block of the retinoic acid potentiation by BFA suggests that retinoic acid stimulates transport of the ribonucleases through the cis-Golgi, possibly to the ER, en route to the cytosol. BFA has also been shown to affect endosomes, lysosomes, and the trans-Golgi network(9Lippincott-Schwartz J. Yuan L. Tipper C. Amherdt M. Orci L. Klausner R.D. Cell. 1991; 67: 601-616Abstract Full Text PDF PubMed Scopus (683) Google Scholar, 31Pelham H.R.B. Cell. 1991; 67: 449-451Abstract Full Text PDF PubMed Scopus (286) Google Scholar), and we cannot ascertain at this time which of the cellular effects of BFA cause the blockage of ribonuclease potentiation by retinoic acid. BFA, however, does not block the cytotoxicity caused by Onconase itself, indicating that the pathway through which Onconase goes to the cytosol in the absence of retinoic acid is qualitatively different from that of the retinoic acid-induced intracellular routing of ribonucleases. Previous experiments indicate that cytotoxic RNases can enter cells and cause RNA degradation to effect cell cytotoxicity and death (18Wu Y. Mikulski S.M. Ardelt W. Rybak S.M. Youle R.J. J. Biol. Chem. 1993; 268: 10686-10693Abstract Full Text PDF PubMed Google Scholar).1 The finding that retinoic acid potentiated BS-RNase toxicity orders of magnitude allowed a further test of this hypothesis. If intracellular RNA degradation was the lesion that caused cell death, then RNA degradation in retinoic acid-treated cells should occur at orders of magnitude lower RNase concentrations, correlating with protein synthesis inhibition. BS-RNase caused 28 S and 18 S rRNA degradation only at 1.0 εM in the absence of retinoic acid, without any detectable degradation of 5.8 S, 5 S rRNA, or tRNA. In retinoic acid-treated cells, all RNA species showed some degradation at a 10 nM concentration of BS-RNase, 100 times lower than the concentration of BS-RNase needed for cells not exposed to retinoic acid. This result indicates that BS-RNase gets into the cytosol more efficiently in the presence of retinoic acid. In both retinoic acid-treated and untreated cells, 28 S and 18 S rRNA are more readily degraded by BS-RNase than 5.8 S, 5 S rRNA, or tRNA. The correlation between the degradation of cytosolic RNA by BS-RNase and cytotoxicity indicates that retinoic acid increases delivery of BS-RNase to the cytosol. It has been shown that dimerization of the bovine seminal ribonuclease is necessary for its anti-cancer activity both in cell culture and in animal models(23D'Alessio G. Di Donato A. Parente A. Piccoli R. Trends Biochem. Sci. 1991; 16: 104-106Abstract Full Text PDF PubMed Scopus (135) Google Scholar). However, we found that two monomers of the bovine seminal ribonuclease showed similar cytotoxicity to 9L cells in the presence of retinoic acid compared with dimerized BS-RNase. This indicates that monomer forms of bovine seminal ribonuclease cannot efficiently get into the cytosol by themselves, but, once in the cytosol, they can be almost as toxic as the dimer. Perhaps the dimer form of BS-RNase functions to route the RNase through the Golgi to effect more efficient entry into the cytosol compared to the monomer. It is interesting that the human RNase A, another monomer form of RNase that is 73% identical in sequence with BS-RNase monomer, is 300 times less toxic in the presence of retinoic acid than are the BS-RNase monomers. The reason for the large biological difference resulting from the small sequence difference may be a key to further understanding the mechanism of cytotoxicity of ribonucleases. Onconase is in clinical trials for cancer therapy(3Mikulski S.M. Grossman A.M. Carter P.W. Shogen K. Costanzi J.J. Int J. Oncol. 1993; 3: 57-64PubMed Google Scholar), and BS-RNase has interesting anti-cancer activity in animal models(7Vescia S. Tramontano D. Augusti-Tocco G. D'Alessio G. Cancer Res. 1980; 40: 3740-3744PubMed Google Scholar, 8Lacceti P. Portella G. Mastronicola M.R. Russo A. Piccoli R. D'Alessio G. Cancer Res. 1992; 52: 4582-4586PubMed Google Scholar). Retinoic acid is also in several experimental trials for cancer therapy. Retinoic acid caused potentiation of BS-RNase cytotoxicity results in greater protein synthesis inhibition and cell death than in the absence of retinoic acid. Combination of RNases and retinoic acid in vivo may improve the clinical utility of ribonucleases. After completion of this manuscript, an interesting study of BS-RNase toxicity was published (32Kim J.S. Souček J. Matoušek J. Raines R.T. J. Biol. Chem. 1995; 270: 10525-10530Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We are grateful to Prof. Matteo Adinolfi for his supervision in chemical synthesis, to Dr. Renata Piccoli for discussions, and to Patricia Johnson for her technical assistance." @default.
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