Ability of Inhibitors of Glycosylation and Protein Synthesis to Sensitize Cells to Abrin, Ricin, Shigella Toxin, and Pseudomonas Toxin
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[CANCER RESEARCH 46, 6418-6422, December 1986] Ability of Inhibitors of Glycosylation and Protein Synthesis to Sensitize Cells to Abrin, Ricin, Shigella Toxin, and Pseudomonas Toxin Kirsten Sandvig, Tor Inge T0nnessen, and Sjur Olsnes Norsk Hydro's Institute for Cancer Research and The Norwegian Cancer Society, Montebello, Oslo, Norway ABSTRACT MATERIALS AND METHODS A number of compounds that interfere with glycoprotein synthesis and Toxins. Abrin, ricin, and Shigella toxin were purified as earlier transport have been tested for their ability to sensitize cells to cancero- described (10-12). Purified Pseudomonas aeruginosa exotoxin A was a static protein toxins. Tunicamycin, swainsonine, cycloheximide, and pur- generous gift from Dr. Stephen H. Leppla (SAMRIID, Frederick, MD). omycin sensitized Vero cells and HeLa cells to abrin and ricin, as we Ricin was coupled covalently to horseradish peroxidase with SPDP1 have found previously with monensin (K. Sandvig and S. Olsnes, J. Biol. essentially as described by Carlsson et al. (13). Ricin was treated with Chem., 257: 7504-7513, 1982). Cycloheximide, but not swainsonine, a 10-fold molar excess of SPDP, and horseradish peroxidase was treated sensitized Vero cells to Pseudomonas exotoxin A and Shigella toxin. The with a 20-fold molar excess. Unreacted SPDP was removed by gel ability of ricin to intoxicate cells was much lower at 19°Cthan at 37°C filtration on Sephadex G-25. Derivatized HRP was reduced with 50 and there was almost no sensitizing effect of cycloheximide and monensin mM dithiothreitol for 20 min at room temperature, run through a at 19°C.Studies by electron microscopy showed that ricin conjugated to Sephadex G-25 column, and then reacted with derivatized ricin over horseradish peroxidase appeared in trans Golgi elements in Vero cells. night at room temperature. Polyacrylamide gel electrophoresis revealed Possibly, transport of ricin into the cytosol requires passage through the that the conjugates formed contained ricin and horseradish peroxidase Golgi apparatus. The possibility that the sensitizing agents here described (1:1) as well as higher molecular weight conjugates. In order to obtain may be valuable in enhancing the action of immunotoxins is discussed. monovalent conjugates (i.e., ricin:HRP, 1:1), ricin-HRP was run through a Sephacryl S-200 column equilibrated with 0.14 M NaCl, 20 mM sodium phosphate (pH 7.4), and 0. l M lactose. Conjugates were INTRODUCTION well separated according to their molecular weight. Monovalent con jugates as shown by sodium dodecyl sulfate-polyacrylamide gel electro In recent years there has been an increasing interest in protein phoresis were used in the experiments. toxins with intracellular sites of action. A main reason for this Materials. Monensin and puromycin were obtained from Calbi- interest is the cancerostatic properties of the plant toxins abrin ochem-Behring Corp., La Jolla, CA; swainsonine and tunicamycin were and ricin (1-3) as well as the use of these toxins in the construc from Boehringer Mannheim GmbH, Mannheim, West Germany; cy tion of target-specific cytotoxic conjugates. Toxins that act on cloheximide and horseradish peroxidase type VI were from Sigma intracellular targets include the plant toxins abrin, ricin, mo- Chemical Co., St. Louis, MO; and SPDP was from Pharmacia, Upps deccin, and viscumin and the bacterial toxins diphtheria toxin, ala, Sweden. Cell Culture. HeLa cells, Vero cells (from African green monkey Shigella toxin, and Pseudomonas toxin. The mechanism of kidney), fetal hamster kidney cells, and mouse L-cells were propagated action of these toxins involves binding to cell surface receptors in monolayer cultures in minimum essential medium (Gibco, Europe, and subsequent endocytosis of the bound toxin. An enzymatic- Ltd., Paisley, Scotland) with 10% fetal calf serum. ally active part of the toxins then crosses the limiting membrane Measurement of Protein Synthesis. After incubation with toxin in a of the vesicle and enters the cytosol where it inhibits protein Ar-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid containing me synthesis (for review, see Ref. 4). Due to the enzymatic effect dium with and without the indicated compounds as described in the of the toxins, few molecules are sufficient to kill cells. Whole legends to the figures, the cells were transferred to medium without toxins or enzymatically active fragments of toxins in this group inhibitors and the ability of the cells to incorporate [3H]leucine during 15 min was measured as earlier described (8). Incorporation of [3HJ- have therefore been conjugated to proteins, e.g., antibodies, that bind to defined cell surface structures (immunotoxins). In those leucine into cells that had previously been exposed to monensin, swain cases where intact toxins are used the native binding sites are sonine, tunicamycin, cycloheximide, and puromycin but not to toxin was essentially the same as in cells that had not been treated with these commonly blocked by chemical modification or by addition of compounds. carbohydrates to which the toxins bind (5). Electron Microscopy. The cells were fixed with 1.5% glutaraldehyde The mechanism of toxin entry into the cytosol is only par in 0. l M cacodylate buffer at pH 7.4 for 20 min. Cytochemical dem tially understood. In spite of the structural similarity of these onstration of ricin-HRP was performed as described by Graham and toxins they have different requirements for entry and they seem Karnovsky (14). Cells were postfixed in 1% OsO4 in 0.1 M cacodylate to enter the cytosol from different intracellular compartments. buffer, pH 7.4. They were dehydrated in 50-96% ethanol and embedded Compounds that enhance the entry of the enzymatically active in Luft Epon through propylene oxide. Specimens were stained with part of the toxins into the cytosol may therefore give valuable lead citrate for 10 min. Ultrathin sections were examined with a Jeol clues to the nature of the entry mechanism. Such compounds 1200 electron microscope. may also improve the action of immunotoxins. Thus, NH4C1 and monensin, which sensitize cells to abrin and ricin (6-8), RESULTS AND DISCUSSION are now widely used to sensitize cells to immunotoxins. Since It has been shown earlier that low concentrations of monensin the low concentrations of monensin often used for this purpose sensitize cells to abrin and ricin as well as to a number of are known to affect the glycosylation of proteins (9) we decided immunotoxins (6-8, 15, 16). Although the low concentrations to investigate if other compounds that interfere with glycosyl of monensin commonly used for this purpose do not strongly ation or inhibit formation of glycoproteins have a similar sen affect the acidification of endosomes and lysosomes, they in sitizing effect. hibit the processing of glycoproteins by affecting transport Received 8/12/85; revised 2/28/86, 5/1/86, 7/22/86; accepted 8/18/86. within the Golgi apparatus (9, 17). We therefore tested the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in 1The abbreviations used are: SPDP, Ar-succinimidyl-3-(2-pyridylthio)pro- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. pionate; HRP, horseradish peroxidase. 6418 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. SENSITIZATION TO PROTEIN TOXINS effect on toxin entry of two other compounds known to interfere but an even larger fraction of the ricin-HRP was now found in with the assembly of glycoproteins. Tunicamycin inhibits the secondary lysosomes. In cells treated with 10~7 M monensin there was a marked addition of carbohydrate to asparagine by preventing the syn thesis of the lipid intermediate involved in this process (18). difference in morphological appearance. Cells were preincu Swainsonine, which inhibits the Golgi enzyme, mannosidase II, bated for 30 min with monensin and then ricin-HRP was added to the cells. After l h incubation at 4°Cthe cells were washed prevents the removal of carbohydrate from one branch of the high mannose intermediate formed during glycosylation (19), and then incubated at 37"C. After 15 min at 37°Cricin-HRP and it thus inhibits the subsequent additions of carbohydrates. was found in the same elements as in cells not treated with As shown in Fig. 1, swainsonine sensitized Vero cells to ricin monensin, but the Golgi stacks and trans-Go\gi elements had to the same extent as monensin. Also tunicamycin had a sen already started to swell. After 45 min numerous large vesicular sitizing effect, although smaller than that found with the other profiles with electron-dense vesicles around them were seen in two compounds. Tunicamycin, swainsonine, and monensin sen the Golgi region. These probably represent swollen Golgi ele sitized Vero cells to abrin to the same extent as to ricin (data ments, and they contained large amounts of ricin-HRP. After 2 h incubation at 37°Cthere were fewer typical second not shown). The sensitizing effect was observed also at a toxin concentration high enough to saturate all the cell surface- ary lysosomes than in cells not treated with monensin indicating binding sites (10 Mg/ml toxin). Furthermore, the effect was not that ricin-HRP had been trapped in the Golgi region (Fig. 2b). limited to Vero cells. Thus, tunicamycin, swainsonine, and In another experiment cells were treated with swainsonine (1 monensin also sensitized HeLa cells and mouse L-cells to ricin ¿ig/ml)in the same way as described for monensin. After 15 and abrin. min ricin-HRP was found in the same elements as in the control To study the effect of the inhibitors on the uptake and experiment, but there was less labeling of endosomes and intracellular routing of ricin, cells were treated with ricin con somewhat more labeling of tubular elements.