The Binding of Abrin and Ricin by Ehrlich Ascites Tumor Cells1

[CANCER RESEARCH 33, 2688 2691, November 1973) The Binding of Abrin and Ricin by Ehrlich Ascites Tumor Cells1

Jung-Yaw Lin, Shyr-Te Ju, Hua Lin Wu, and Ta-Cheng Tung

Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China

SUMMARY Louis, Mo. L-Leucine-4.5-3H with a specific activity of 5.7 mCi/mmole was purchased from New England Nu Abrin and ricin cause 50% agglutination of Ehrlich as- clear Corp., Boston, Mass. Yeast RNA was further puri cites tumor cells (5 x 106/ml) at a concentration of 8 and fied from crude yeast RNA extract (Sigma Chemical Co.) 4 Â¿ig/ml, respectively. Addition of D-galactose or its by phenol method (5). Na'3'I was obtained from National derivatives, lactose or raffmose, inhibits the agglutinating Chin-Hua University, Sin-Chu, Taiwan. activity of the toxic proteins. Binding of 131I-toxic pro Preparation of Abrin and Ricin. Abrin and ricin were teins on the tumor cells was shown to be specific, pH de isolated and crystallized as described previously (9, 10). pendent, and concentration dependent. Native ricin, abrin, lodination of Abrin and Ricin. Abrin and ricin were D-galactose, and its sterically related saccharides inhibit labeled with CP31I at the ratio of 1:4 in 2 ml of pH 9.0 this specific binding, while concanavalin A, bovine serum glycine :NaOH buffer as described previously (8). At these albumin, heat-denatured abrin or ricin, and other sac conditions, about 60% of toxicity was retained in iodinated charides do not. In the presence of D-galactose, the inhibi toxic proteins. tion of protein biosynthesis by ricin does not occur in vitro. Agglutination of Ehrlich Ascites Tumor Cells. Ehrlich ascites tumor cells were obtained by centrifuging the 10- day-old Ehrlich ascites fluid with an International refriger INTRODUCTION ated centrifuge at 600 x g and 4Â° for 5 min. The cell Con A2 isolated from Canavalia ensiformis is a well- pellet was then washed 3 times with 10 ml of ice-cold 0.01 M phosphate-buffered saline, pH 7.4. Agglutination ex known PHA (16). The interaction between Con A and its periments were performed by incubating the reaction mix receptors on cell membranes was thoroughly studied by ture at room temperature for 10 min containing 5 x IO6 Burger and Noonan (1-3), and it was found that virus- tumor cells and various amounts of protein in the pres transformed cells behave differently toward the agglutinat ence or absence of various saccharides. The results were ing activity of Con A. Similar approaches have been taken observed under a microscope as described by Burger and with a variety of plant agglutinins as well as hormones to Noonan (3). study the 1st step of action of these substances (4, 15). Binding of 131I-labeled Proteins on Ehrlich Ascites Ricin was shown to agglutinate all kinds of erythrocytes Tumor Cells. Five ml of Krebs-Ringer buffer solution con regardless of types and sources (13). D-Galactose and its taining various amounts of 131I-toxic proteins and 5 x derivatives have been found to inhibit the hemagglutinat- IO7 Ehrlich ascites tumor cells were incubated at 37Â°for ing activity of ricin (14). Since the phytotoxic proteins abrin 60 min. The reaction was terminated by centrifugation, and and ricin were shown to have a strong inhibitory effect on the packed cells were quickly washed 3 times, each with the growth of Ehrlich ascites tumors, it is the purpose of 5 ml of ice-cold Krebs-Ringer buffer solution. The radio these experiments to study the effect of abrin and ricin activity of each suspension was determined by paper disc on the agglutination of Ehrlich ascites tumor cells. method (12). Amino Acid Incorporation in Ehrlich Ascites Tumor MATERIALS AND METHODS Cells. Protein biosynthesis of Ehrlich ascites tumor cells was carried out as described before (6). After addition of 40 Con A, 3 times crystallized into a desalted lyophilized Hg of ricin into the 5 ml of incubation mixture containing powder, was obtained from Miles-Yeda, Ltd. (Stock 5 x 10' Ehrlich ascites tumor cells, 1 mmole of D-galac Court, Stock Poges, Slough, England). /J-D-GIucose, tose was added. The radioactivity of each sample was (8-D-fructose, /8-methyl-D-xyloside, a-D-glucuronic acid measured by paper disc method with a Nuclear-Chicago HC1, a-methyl-D-xyloside, a-L-(-)-fucose, D-arabinose, Mark I liquid scintillation counter. D-( + )-xylose, D-galactose, 1-erythritol, lactose, raffmose, and BSA were obtained from Sigma Chemical Co., St. RESULTS

'This work was supported by a grant from the National Council of Agglutination of Ehrlich Ascites Tumor Cells by Abrin Science, Republic of China. "The abbreviations used are: Con A, concanavalin A; PHA, phyto- and Ricin. The amounts of abrin and ricin that caused a hemagglutinin; BSA, bovine serum albumin. half maximal agglutination of Ehrlich ascites tumor cells Received March 7, 1973; accepted July 11, 1973. were 8 and 4 ng/ml, respectively. In the presence of 65

2688 CANCER RESEARCH VOL. 33

/ig of abrin per ml or 32 fig of ricin per ml, complete agglutination of tumor cells occurred while BSA and carboxymethylated abrin did not agglutinate Ehrlich ascites tumor cells under the same conditions. The toxic protein treated with heating at 100Â°for 30 min also lost the agglutinating activity. O Since agglutinating activity of plant agglutinins was in hibited by certain saccharides (14), various saccharides S were used to investigate their effects on the agglutination of D tumor cells caused by the phytotoxic proteins. D-Galac- tose and its derivatives, lactose and raffinose, were found O to be not only able to prevent the agglutination of tumor a cells but also able to dissociate the agglutinated tumor cells into homogeneously dispersed state. The concentrations of these saccharides required to inhibit the agglutination of tumor cells in the presence of 64 /ig of abrin per ml or 32 /Â¿gofricin per ml were 0.67 x 10"3 M of D-galactose, 0.34 x 10-3 Mof lactose, and 4.76 x IO'3 Mof raffinose. Binding of Ricin-131! on Tumor Cells. Binding of ricin- 13'1 on Ehrlich ascites tumor cells was a saturable process with respect to the concentration of ricin-131! (Chart 1), while the binding of BSA-131! on the tumor cells was quite ,H low compared to that of ricin-131!; this suggests that the binding of ricin-131! on Ehrlich ascites tumor cells was Chart 2. Effect of pH on the binding of ricin-131!. The experiments specific to the phytotoxic proteins. The binding reac were carried out by changing the pH in the reaction mixture. Acetate tion took place rapidly, so that 30 sec after the addition buffer was used instead of phosphate buffer at pH below 6. Glycine: NaOH buffer was used at pH above 8. After incubating at 37Â°for 60 min, cells were centrifuged and then the packed cells were washed 3 times with the same buffer. Finally, the packed cells were suspended with 5 ml of the same buffer and the radioactivity of each sample was counted as described in "Materials and Methods."

Table 1 Effect of saccharides on the binding of ricin-'"! The experiments were performed by incubating 100 Â¿/gof ricin-131! in 5 ml of Krebs-Ringer buffer solution in which the indicated amounts of materials were previously added. After incubating at 37Â°for 60 min. the reaction mixtures were centrifuged and the packed cells were washed 3 times with ice-cold Krebs-Ringer buffer solution. The radio activity of each sample was counted as described in "Materials and Methods." Each value represents the mean of 3 experimental results.

Material at 0.02 \i binding of concentrationNonea-n-Glucuronic-HCla-L-(-)-Fucosea-Methyl-D-mannosidea-Methyl-D-xylosideÃŸ-n-ricin-'31! (Mg/10'cells)6.6 Â±0.36.31 Â±0.607.03 Â±0.366.02 Â±0.606.61 Â±0.296.61 Fructose0-o-Glucose/3-Methyl-n-xylosideD-ArabinoseD-( Â±0.106.26 Â±0.606.66 Â±0.286.52 Â±0.1256.86 +)-Xylose1-Erythritoln-GalactoseLactoseRaffinoseSpecificÂ±0.446.66 Â±1.191.35 10 20 30 40 Â±0.140.85 Â±0.2251.65 CONC. OF131 I-PROTEIN Â±0.7

Chart I. Concentration (Co/if.)-depenaence of binding of ricin-'31!...... â€ž¿__ ...... The experiments were earned out as described in "Materials and Â°f"CUI-131! more than 80% of binding was completed. Methods." Each point represents the mean of 5 experimental results. â€¢¿ By varying NaCl concentration from 0.07 to 0.56 M, binding of ricin-131! on Ehrlich ascites tumor cells; O, binding of tne amounts of ricin-131! found on tumor cells were not BSA-131lonEhrlichascitestumorcells. affected significantly. Varying the concentrations of cal-

NOVEMBER 1973 2689

100 The concentrations of inhibitors required to produce 50% inhibition of binding were 1.5 mM for D-galactose, 0.6 mM for lactose, and 5 mM for raffinose. Native abrin also can inhibit the binding of ricin-131! on Ehrlich ascites tumor cells, but Carboxymethylated abrin, BSA, Con A, and yeast RNA did not show any inhibitory effect. The amount of abrin required to produce 50% inhibition of bindings was found to be 400 ^g (Table 2, Chart 4). Dissociation of Bound Ricin-131!. Ricin-131! and tumor cell complex were incubated with D-galactose, lactose, raffinose, and abrin separately; and the dissociation of ricin-131! from the complex was observed. Under the same conditions, Con A, BSA, RNA and other saccha rides had no effect on the dissociation (Table 3).

100

-i O COMC OF 3ACCHARIDE (mM) ce Chart 3. Effect of various concentrations (cone.) of D-galactose and its derivatives on the binding of ricin-'31!. The experiments were per o formed by incubating the reaction mixtures which contained 100 jig of u ricin-131! and various concentrations of D-galactose, lactose, and raffinose at 37Â°for 60 min. After incubation, the cells were centrifuged, o washed, and counted as described in "Materials and Methods." Sac- charides added: O, galactose: â€¢¿,lactose:â€¢¿raffmose. *

Table 2 Effect ofabrin on the binding of ricin-131/ The experiments were performed as described in Table 1, except that 40"0~ indicated materials were used instead of saccharides. Each value repre 600 800 sents the mean of 3 experimental results.

bindingof ABRIN ( jig/ 5ml ) ricin-131!(/ig/ MaterialNoneBSACon (mg)10.81O.I0.8SpecificIO7cells)6.6 Chart 4. Effect of the concentrations of abrin on the binding of ricin-131!. The experiments were performed as described in Chart 3 Â±0.36.55 except that abrin was used instead of D-galactose. Â±0.356.84 ACarboxymethylated Â±0.756.76 abrinRicinAbrinAmount Â±0.53.69 Table 3 Â±0.332.15 Dissociation of bound ricin-13'! Â±0.22 Cells bound with ricin-131! were further incubated with 5 ml of Krebs- Ringer buffer (KRB) solution that contained the indicated amounts of added materials except the control (*) in which the KRB solution was cium and magnesium ion from 1 x 10~3 to 1.6 x 10~2 added. After incubating at 37Â°for 60 min, the cells were centrifuged. washed, and counted as described in "Materials and Methods." Results Malso had no effect on the binding. were averages of 3 experimental values. The binding was pH dependent and the optimal pH of Ricin-131!dissociated binding was found in the range between 6.0 and 7.5 (Chart (Â¿ig/IO7cells) 2). Material Amount The binding properties of abrin-131! are similar to those Â«KRB0-o-GlucoseD-GalactoseLactoseRaffinoseBSARNARicinAbrin0.Â±0.030.70 of ricin-131!. 1mmole0. Â±0.145.19 Effect of Saccharides and Abrin on the Binding of 1mmole0. Â±0.605.39 Ricin-131!. Saccharides that prevented the agglutination of 1mmole0. Â±0.024.11 1mmole1 Â±0.400.73 Ehrlich ascites tumor cells are also able to inhibit the mg1 Â±0.210.60 binding of ricin-131! on the tumor cells. D-Galactose and mg0. Â±0.103.32 its derivatives inhibited the binding while other saccha 1mg0.2 Â±0.243.07 rides showed very little effect, if any (Table 1, Chart 3). mg0.57 Â±0.11

2690 CANCER RESEARCH VOL. 33

Table 4 Abrin and ricin resembled each other in many respects, Effect of D-galaclose on prolein biosynthesis of Ehrlich osciles especially in their biological functions (11). The fact that tumor cells in the presence of ricin abrin can dissociate the bound ricin-131! from a tumor The experiments were performed as described in "Materials and cell: ricin-131! complex suggests that abrin may bind to the Methods." Each value represents average of 2 experimental results. receptors that are very similar if not identical to the ricin Cell treatment cpm/2.5 x 10" cells receptors. No treatment 272 Â±9 n-Galactose (0.2 mmole/ml) 296 Â±4 REFERENCES Ricin (8 Mg/ml) 102 Â±2 n-Galactose (0.2 mmole/ml) 296 Â±5 Ricin (8 /ig/ml) 1. Burger, M. M. Isolation of a Receptor Complex for a Tumor-Specific Determinant on Neoplastic Cell Surface. Proc. Nati. Acad. Sci. U. S., 57: 359 366, 1967. 2. Burger, M. M. Difference in the Architecture of the Surface Mem Effect of D-Galactose on Protein Biosynthesis. In the brane of Normal and Virally Transformed Cells. Proc. Nati. Acad. presence of 0.2 Mof D-galactose, the saccharide completely Sei. U. S., 62: 994 1001, 1969. prevented the inhibition of protein biosynthesis of Ehrlich 3. Burger, M. M., and Noonan, K. K. Restoration of Normal Growth ascites tumor cells caused by ricin while D-galactose only, by Covering of Agglutinin Sites on Tumor Cell Surface. Nature, 228: 512-515, 1970. at the same conditions, had no effect on the protein biosyn 4. Cuatrecasas, P. Isolation of the Insulin Receptor of Liver and Fat- thesis (Table 4). Ricin itself at the concentration of 8 n%/m\ Cell Membranes. Proc. Nati. Acad. Sei. U. S., 69: 318-322, 1972. caused 70% of inhibition of protein biosynthesis. 5. Kirby, K. S. A New Method for the Isolation of Ribonucleic Acids from Mammalian Tissues. Biochem. J., 64: 405 408, 1956. 6. Lin, J. Y., Kao, W. Y., Tserng, K. Y., Chen, C. C., and Tung, T. C. DISCUSSION Effect of Crystalline Abrin on the Biosynthesis of Protein, RNA, and DNA in Experimental Tumors. Cancer Res., 30: 2431 2433, 1970. Ehrlich ascites tumor cells agglutinated by abrin and 7. Lin, J. Y., Pao, C. C., Ju, S. T., and Tung, T. C. Polyseme Disag- ricin were still viable as judged by trypan blue exclusion gregation in Rat Liver Following Administration of the Phytotoxic method. Similar results were also observed in the case of Proteins, Abrin and Ricin. Cancer Res., 32: 943-947, 1971. Ehrlich ascites carcinoma cells agglutinated by PHA. This 8. Lin, J. Y., Shaw, Y. S., and Tung, T. C. Distribution of I'"-Labeled phenomenon seems to indicate that agglutination of tumor Abrin in Vivo. Toxicon, 8: 197 201, 1970. cells by abrin and ricin cannot cause the cell destruction 9. Lin, J. Y., Shaw, Y. S., and Tung, T. C. Studies on the Active Prin but is rather a surface phenomenon that is followed by the ciple from Ahrus precatorius L. Leguminosae Seed Kernels Toxicon, 9:97-101, 1971. alternation of metabolic processes such as inhibition of pro 10. Lin, J. Y., Tserng, K. Y.. and Tung, T. C. Purification of Ricin from tein and DNA biosynthesis (7). Ricinus communi!. T'ai-Wan I Hsueh Hui Tsa Chih, 69:48-52, 1970. The present investigations demonstrated that D-galactose 11. Lin, J. Y., and Tung, T. C. Studies on Phytotoxic Proteins: Abrin and its derivatives, lactose and raffinose, inhibited the and Ricin. J. Chinese Biochem. Soc., /: 1-20, 1972. agglutinating activity of abrin and ricin. The dissociation 12. Mans, R. J., and Novelli, G. D. Measurement of Incorporation of of agglutinated tumor cells by D-galactose and its deriva Radioactive Amino Acids into Protein by Filter-Paper Disc Method. tives indicates that there is a stage of reversible binding of Arch. Biochem. Biophys., 94: 48-52, 1961. abrin and ricin on tumor cells, subsequently followed by an 13. Osawa, T., Phytohemagglutinin. Tampakushitsu Kakusan Koso, 16: irreversible stage of inhibition of protein and DNA bio 335-349, 1971. synthesis. 14. Sharon, N.. and Lis, H. Lectins: Cell-Agglutinating and Sugar- Specific Proteins. Science, 177: 949 959, 1972. As Steck and Wallach (15) showed that binding of PHA 15. Steck, T. L., and Wallach, D. F. The Binding of Kidney-Bean Phyto- to Ehrlich ascites carcinoma cells was a prerequisite step agglutinin by Ehrlich Ascites Carcinoma. Biochim. Biophys. Acta., for agglutination, we were able to demonstrate also the 97:510-522, 1965. binding of abrin or ricin on Ehrlich tumor cells. The binding 16. Summer, J. B., and Howell, S. F. The Identification of the Hemag- reaction was shown to be specific, concentration dependent, glutinin of the Jack Bean with Concanavalin A. J. Bacteriol., 32: 227- and pH dependent. 237, 1936.

NOVEMBER 1973 2691

Jung-Yaw Lin, Shyr-Te Ju, Hua-Lin Wu, et al.

Cancer Res 1973;33:2688-2691.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/33/11/2688

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/33/11/2688. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.