Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2218-2221, May 1979 Inhibition of human neuroblastoma DNA polymerase activities by plant and (DNA nucleotidyltransferase/control of DNA replication/agarose-conca javalin A column) PRABIR BHATTACHARYA, IRA SIMET, AND SUBHASH BASU* Department of Chemistry, Biochemistry and Biophysics Program, University of Notre Dame, Notre Dame, Indiana 46556 Communicated by Edwin T. Mertz, February 23,1979

ABSTRACT The effects of and (RCAII, human neuroblastoma IMR-32 cells by the highly toxic plant Mr 65,000) on [3Hlthymidine incorporation into human neuro- ricin. Our studies with the cell-free system indicate that blastoma IMR-32 DNA showed reduction of total DNA synthesis to 50% and 70% of control, respectively. Two DNA polymerase ricin exerts its toxic effect on human cells by inhibiting DNA (DNA nucleotidyltransferase, EC 2.7.7.7) activities (a and P) polymerase a activity, in addition to the widely accepted theory involved in the biosynthesis in vitro of DNA were separated by of inhibition of protein synthesis by plant toxins (12-15). sucrose density gradient centrifugation from IMR-32 cell ho- mogenate. The DNA polymerase a activity was also purified MATERIALS AND METHODS by selective precipitation with polyethylene glycol (Mr 6000) followed by agarose-concanavalin A column chromatography. Materials. Unlabeled deoxynucleoside triphosphates were The activities of both DNA polymerases were examined at purchased from Sigma. [5-3H]dCTP (30 Ci/mmol) and various concentrations of mutagenic and nonmutagenic plant [methyl-3H]thymidine (40-60 Ci/mmol) were purchased from agglutinins and the ricin. Concanavalin A and ricin spe- New England Nuclear (1 Ci = 3.7 X 1010 becquerels). cifically inhibited DNA polymerase a activity (activity reduced RCAI, to 19% and 10%, respectively), whereas DNA polymerase P RCA11, peanut agglutinin (PNA), and soybean agglutinin (SBA) activity was inhibited (reduced to 16%) by red bean were purchased from E. Y. Laboratories (San Mateo, CA). agglutinin (PHA-P). (WGA) and Con A were purchased from Vector Laboratories and Sigma, respectively. PHA-P and Various plant lectinst are toxic to human and other animal cells PHA-M were purchased from Difco. Abrus precatorius grown in vivo (2, 3) or in vitro (3-5). The tumor-suppressive agglutinin (APA) was a gift sample from A. Sen of The Bose effects of two lectins, concanavalin A (Con A) (3-5) and phy- Institute (Calcutta, India). tohemagglutinin (PHA) (6-8), and differential toxic effects of Isolation of DNA Polymerases a and ft. Human neuro- a few plant toxins such as ricin (RCAII, a ; Mr blastoma clone IMR-32 cells were purchased from the Ameri- 65,000) and (a toxin isolated from Abrus precatorius; Mr can Type Culture Collection and maintained in our laboratory 63,000) (9-11) have been demonstrated on tumor and trans- as described (26-28). Confluent monolayers (5 to 7 X 106 cells formed cells. It has been proposed that ricin interferes in the per 250-ml Falcon plastic flask) were harvested with phos- peptide chain elongation step by interacting with 60S ribosomal phate-buffered saline [7.0 mM potassium phosphate/0.14 M subunits (12); furthermore, the inhibitory property increases NaCl buffer, pH 7.2 (P1/NaCl)l containing 0.1% EDTA for after treatment of the toxin with 2-mercaptoethanol (13). The enzymatic studies. A 25% (vol/vol) homogenate of cells in 0.32 cell surface-binding properties, but not the protein synthesis- M sucrose containing 10 mM Tris1HCI buffer (pH 7.8) was inhibitory properties, of ricin (14) and abrin (15) are inhibited obtained, and DNA polymerase a and 3 activities were sub- by methyl f-D-galactoside and galactose-containing carbo- sequently separated on a 5-20% continuous sucrose gradient hydrates, respectively. containing 10 mM Tris- HCl (pH 8.0), 1 mM 2-mercaptoetha- Previously, the stimulation of [3H]thymidine incorporation nol, and 100 mM KCl as described (27). The gradient (5.0 ml) has been observed during transformation by PHA was centrifuged for 16 hr in a Beckman SW 50.1 swinging- (16, 17). Increased DNA polymerase a (DNA nucleotidyl- bucket rotor at 149,000 X g. Fractions (0.25 ml) were collected transferase, EC 2.7.7.7) activity has been observed in PHA- from the bottom with a peristaltic pump. The activity of DNA transformed normal human (18), Con A-treated polymerase a appeared between fractions 7 and 9, and the rabbit cells (19), and Corynebacterium parvum vac- activity of DNA polymerase 3 between fractions 15 and 17 (Fig. cine-treated mouse spleen cells (20). Recently, cell culture 1). studies have established that PHA and Con A are inhibitors of The identification of DNA polymerase a activity in tubes [3H]thymidine incorporation into nuclear DNA of Limnaea 7-9 was confirmed by its sensitivity to N-ethylmaleimide (26). eggs (21) and Chinese hamster cells (22), respectively. Treat- The activity was reduced to 30% by 5-10 mM N-ethylmalei- ment of PHA with periodate destroys its mitogenic and inhib- mide. The selective precipitation (90%) of DNA polymerase itory effects on proliferation of mouse leukemic granulocytes (23). It also has been reported that PHA-P inhibits DNA syn- Abbreviations: Con A, ensiformis agglutinin or concana- thesis in cultured Gross ascites lymphoma cells at concentrations valin A; APA, Abrus precatorius agglutinin; RCAI, communis stimulatory for DNA synthesis in normal mouse spleen cells agglutinin; RCAII, Ricinus communis toxin or ricin; PNA, Arachis (24). However, effects of lectins or toxins on the cell-free DNA hypogaea or peanut agglutinin; SBA, Glycine max or soybean agglu- tinin; WGA, Triticum vulgaris or wheat germ agglutinin; PHA, replication complex have not been demonstrated until recently phytohemagglutinin; PHA-P, Phaseolus vulgaris or red kidney bean (25). We now report on the inhibition of DNA biosynthesis in agglutinin; PHA-M, Phaseolus vulgaris or red kidney bean ; P1/NaCl, phosphate-buffered saline. The publication costs of this article were defrayed in part by page * To whom correspondence should be addressed. charge payment. This article must therefore be hereby marked "ad- t "," as used in this paper, denotes -binding properties and vertisement" in accordance with 18 U. S. C. §1734 solely to indicate does not imply [in contrast to the suggestion of Boyd (1)1 hem- this fact. agglutinative capacity. 2218 Downloaded by guest on October 2, 2021 Biochemistry: Bhattacharya et al. Proc. Natl. Acad. Sci. USA 76 (1979) 2219 Whatman borosilicate filter discs (GF/A; porosity 1.0 ,im; di- ameter 2.4 cm) in a Millipore apparatus. The discs were dried c 0.6 at 80°C and radioactivity was measured in a Triton X-100 scintillation system as described (27). RESULTS 0 8.(N Effect of Con A and Ricin on [3HlThymidine Incorpora- tion into IMR-32 DNA. The effects of Con A and ricin on [3H]thymidine incorporation into IMR-32 DNA were tested 0 : 0.2 in the presence and absence of serum (Table 1). DNA synthesis was higher in IMR-32 cultures treated with 10% fetal bovine 0.0 serum. The number of cells stimulated to enter the S phase was a function of both cell density and the concentration of added 2 4 6 8 10 12 14 16 18 20 serum. Con A and ricin reduced total DNA synthesis to 50% and Fraction 27-70%, respectively. It became apparent that the inhibition FIG. 1. Sucrose density gradient analysis of IMR-32 DNA poly- of DNA synthesis in intact cells by Con A was reversible in the merases. The IMR-32 cells were grown in Eagle's minimal essential presence of fetal bovine serum, while the effects of reduced and medium as described for Table 1. The semiconfluent cells were har- nonreduced ricin were irreversible (Table 1). vested, homogenized in 0.32 M sucrose/10.0 mM Tris-HCl (pH 7.8)/1.0 Inhibition of IMR-32 DNA Polymerases. The activities of mM dithiothreitol, and sonicated for six 15-sec periods in a Heat- human neuroblastoma IMR-32 DNA polymerases a and were system Ultrasonicator with a microtip. The sample was kept at 4°C # between sonications and then centrifuged at 30,000 X g for 30 min. examined at various concentrations of Con A (Fig. 2). Under The supernatant was dialyzed against 10.0 mM Tris-HCl (pH 7.8)/1.0 the present assay conditions, we observed that Con A in the mM 2-mercaptoethanol. The dialyzed supernatant was applied to 4.5 absence of mercaptoethanol specifically inhibited DNA poly- ml of a 5-20% continuous sucrose gradient containing 10.0 mM merase a activity at concentrations from 2.5 to 25 ,g/ml. DNA Tris-HCl (pH 8.0)/1.0 mM 2-mercaptoethanol/0.1 M KCI and cen- polymerase was insensitive to Con A even at a 10-fold higher trifuged for 16 hr in a Beckman SW 50.1 swinging-bucket rotor at concentration. 40,000 rpm. Bovine gamma globulin (7S) was run simultaneously as a molecular weight marker. Fractions (0.25 ml) were collected from The inhibition by Con A of DNA polymerase a activity in the bottom with a peristaltic pump. Aliquots (10 Ml) of the indicated the IMR-32 cell-free system (25) led us to investigate this effect fractions were assayed as described in the text. The ratio of the in greater detail. We examined IMR-32 DNA polymerase a and [3H]dCMP incorporation in activated calf thymus DNA was pro- portional to 10-30 ml of sucrose density gradient fractions and re- mained constant with time of incubation up to 45 min at 37°C. Table 1. Incorporation of [3H]thymidine into DNA of lectin- or toxin-treated IMR-32 cells

a with polyethylene glycol (Mr 6000) at high ionic strength (2 Fetal [3H]Thymidine M NaCI) (28) was also used for further identification of DNA bovine incorporated, polymerase a. Lectin or toxin serum cpm/106 cells Affinity Chromatographic Purification of DNA Poly- None - 2054 ± 353 merase a. Attempts were made to purify DNA polymerase a + 3012±59 activity by on an agarose-Con A Con A (30 Ag/ml) - 934 ± 200 (Glycosylex A; Miles) column in the presence of 50 mM Tris. + 1608 ± 266 HCl (pH 7.4). The DNA pQlymerase a fraction (1.0 ml; 4 mg Ricin, nonreduced (0.3 Mg/ml) - 1359 ± 110 of protein) obtained after polyethylene glycol precipitation was + 1240 ± 237 applied to the agarose-Con A column (1 X 5 cm) equilibrated Ricin, reduced (0.3 jg/ml) - 878 ± 73 with 50 mM Tris-HCl (pH 7.4) buffer. The column was washed + 792+9 with 40 ml of the same buffer and bound DNA polymerase a IMR-32 cells were maintained on Eagle's minimal essential medium (92% of applied activity) was eluted with 30 ml of 0.3 M methyl (F-16, GIBCO) supplemented with L-glutamine, nonessential amino a-Dmannoside in 50mM Tris.HCI buffer (pH 7.4). The sample acids, and 10% fetal bovine serum. Cultures were grown in duplicate with peak activity was dialyzed and concentrated to 1.0 ml by in 75-cm2 T-flasks for 4 days with a single change of medium (10 ml). ultrafiltration. Purified DNA polymerase a was obtained, with Just before the experiment was started, the existing media were a specific activity 75 times that of whole cytosol, and this withdrawn and new media containing 1 ACi of [3H]thymidine per ml of cell culture medium and Con A or ricin (reduced or nonreduced) fraction was also used for lectin inhibition studies (Table 2). in the presence and absence of 10% fetal bovine serum were added as DNA Polymerase Assays. The complete incubation mixture indicated. Ricin was reduced with 1% 2-mercaptoethanol in 10 mM contained the following components in a total volume of 0.1 ml: Tris-HCl, pH 7.4, at 370C for 4 hr. The cells were maintained for 14 5 Mg of activated calf thymus DNA [treated with pancreatic hr at 370C in an atmosphere containing 95% air/5% CO2 at 85% rela- DNase for 15 min, followed by heat treatment at 80°C (26)]; tive humidity. Under these experimental conditions, the rate of into DNA IMR-32 0.1 M Tris*HCI buffer (pH 8.5 for DNA polymerase a activity, [3H]thymidine incorporation by cells was pro- to the cell number and remained constant with time of in- 8.0 DNA mM portional pH for polymerase activity); 10 mM MgCl2; 20 cubation. Both the control and the lectin- or toxin-treated cells were KCI; 0.2 mM dithiothreitol; 0.2 mM [3H]dCTP (specific activity washed with two 15-ml portions of Pi/NaCl after removal of the cul- 100 cpm/pmol); 0.2 mM each dNTP; and enzyme (sucrose ture fluid, and finally harvested with 5 ml of Pi/NaCl containing 0.1% density gradient fractions 7-9, 20 Ml for DNA polymerase a EDTA (pH 7.2). Viable cell counts were obtained in Pi/NaCl con- activity, and fractions 15-17, 30 Al for DNA polymerase ac- taining 0.025% EDTA and 0.2% trypan blue. A 1.0-ml aliquot was tivity). filtered through Whatman GF/A borosilicate discs. The discs were washed with cold 10% for 30 The mixtures were at trichloroacetic acid and dried at 80°C incubated for 30 min 37°C, and the min, and [3H]thymidine content was determined quantitatively in reaction was stopped by addition of I ml of 15% perchloric acid a Triton X-100/toluene scintillation system (27) with a Beckman containing 20 mM sodium pyrophosphate at 4°C. The mixtures scintillation counter (LS-3133 T). The values given are averages + were kept at 4°C for 20 min, and aliquots were filtered through SEM of four aliquots measured in duplicate experiments. Downloaded by guest on October 2, 2021 2220 Biochemistry: Bhattacharya et al. Proc. Natl. Acad. Sci. USA 76 (1979) respectively. APA, even at a 5-fold higher concentration, did not inhibit DNA polymerase activities in the cell-free system. 3 80 A However, an inhibitory effect of abrin toxin cannot be ruled out because further supplies were not available. E 60 - E\ x 40 DISCUSSION E Specific sugar-binding lectins are widely used in structural and 20 - functional studies of . Ricin and abrin originate in the of two unrelated plants, have different molecular weights, 0L . and are immunologically distinct (15). Both toxins are more 5 10 15 20 25 toxic to malignant and tumor cells than to normal cells. Olsnes Con A, Mg/ml et al. (12) have reported that toxins split into their constituent A and B chains FIG. 2. Effect of Con A concentration on IMR-32 DNA poly- by 2-mercaptoethanol inhibit protein biosyn- merases. The complete incubation mixtures contained the same thesis in a cell-free system much more strongly than the intact components as described for DNA polymerase assays except that Con toxins do. Under our assay conditions (30 min incubation in the A was used at the indicated concentrations. Aliquots (20 Ml) of sucrose presence of 0.2 mM dithiothreitol), ricin is most probably dis- density gradient fractions 8 and 16 (described in Fig. 1) were used as sociated into its A and B chains, one of which may inhibit the the sources of DNA polymerase a (@) and (3(A) activities, respec- cell-free DNA replication complex. In addition, both the intact tively. The mixtures were incubated at 370C for 30 min and aliquots toxin ( bridge-linked A and B chains) and the reduced were then assayed for incorporation of [3H]dCMP as described in the text. toxin have toxic effects on intact cells, with the inhibitory effect (27-43% of control activity) of reduced ricin being more pro- nounced (Table 1). Recently Saltvedt (29) has shown that, upon d activities in the presence of various lectins afld toxins (Table treatment with 2-mercaptoethanol, highly purified Ricinus 2). Under these experimental conditions, Con A and ricin communis agglutinin (RCAI) inhibits protein synthesis in a (RCA11) specifically reduced DNA polymerase a activity to 25% cell-free system, probably because of the presence of the A and 15%, respectively, whereas only PHA-P inhibited DNA chain, which is also present in ricin (RCA11). In contrast, we have polymerase d activity (reduction to 16%). Lectins were tested observed (Table 2) that the reduced RCA1 inhibits DNA poly- at various concentrations (10-130 /Ag/ml); the values in Table merase a and # activities very little (80-95% of control). These 2 were obtained at the concentrations at which maximal inhi- results suggest that the mode of inhibition by RCAn of the bition was observed. In a separate experiment (Table 2), Con cell-free DNA polymerase a complex is different from the A and ricin also inhibited purified (agarose-Con A column el- mechanism proposed for cell-free protein synthesis. uate) DNA polymerase a activity to 19% and 10% of control, Holley (30) has suggested that modification of the cell membrane may serve to increase or decrease the transport of serum nutrients or growth factors into the cell, leading even- Table 2. Inhibition of IMR-32 DNA polymerase activities by tually to reinitiation or blockage of the cell cycle. The inability lectins and toxins of serum to induce DNA synthesis in ricin-treated cells suggests DNA polymerase activity, either that ricin, by combining with cell surfaces (31), influences Agglutinin % of control the ability of the cell to respond to agents that initiate DNA or toxin a ( synthesis or that ricin inactivates directly some component of the DNA polymerase complex. Our results, obtained in a cell- None 100.0 100.0 free system, support the latter hypothesis. However, additional Con A 24.9 (18.8)* 79.3 inhibition of DNA biosynthesis in whole cells as a result of in- APA 98.0 82.1 hibition of protein synthesis by ricin cannot be ruled out. RCA1 77.5 97.8 Multiple forms of DNA polymerase (a have been reported RCA11 15.3 (10.4)* 101.4 our lectin the PNA 96.6 85.1 (32-36); present inhibition studies, and purifi- a SBA 81.1 106.3 cation of DNA polymerase activity from the agarose-Con A WGA 101.7 95.8 column, indicate that some of these proteins may be glyco- PHA-P 77.3 15.7 proteins. However, the exact mode of action of Con A and ricin PHA-M 76.1 78.5 on the active site of DNA polymerase a has yet to be deter- mined. The complete incubation mixtures contained the same components The inhibitory action of PHA-P on the growth of two human as described for DNA polymerase assays. The conditions used in the epithelioid cell lines, HeLa and L-32, has been observed by Caso assays for DNA polymerases a and ( had been optimized with respect to pH and concentrations of Mg2+ and KCJ. The lectins (or aggluti- (6). Growth of cell cultures, determined by Caso (6) as a func- nins) and a toxin were used in the indicated amounts: Con A, 25 Ag/ml; tion of total DNA content, is reduced to 50% in the presence of APA, 127 og/ml; RCAI, 25 ,ug/ml; RCA11, 10 ,g/ml; PNA, 25 Mg/ml; PHA-P after 4 days of incubation. This growth inhibition of SHA, 25 .tg/ml; and WGA, 25 Aig/ml. Lyophilized samples (5( mg) of human cells in culture can now be partly explained in view of PHA-P and PHA-M, purchased from Difco, were reconstituted with our observations in a cell-free system that DNA polymerase ( 5 ml of distilled water, and 10-A1 aliquots were used in the assay activity is reduced to 16% PHA-P. mixtures. The 100% value was 1200 cpm (12.0 pmol) for a 30-min in- by cubation period. * These incubation mixtures contained the same components as de- The generous cooperation of Miss Kathleen Presper in cell culture scribed above, except that the purified DNA polymerase a fraction work is acknowledged with gratitude. We thank Dr. A. Sen and Dr. (eluate from the agarose-Con A affinity column) was used as the J. Roy for their generous gift sample of pure APA. We also are grateful enzyme source (2.0 ,ug of protein per 100-jul incubation volume). The to Dr. Steven E. Brooks for providing an IMR-32 clone (passaged rate of reaction was proportional to enzyme concentration between through nude mice). This work was supported by Grants CA-14764 0.5 and '3.0 ,tg of protein per incubation volume of 100,l and re- and NS-09541 from the National Institutes of Health and a Grant- mained constant with time of incubation up to 45 min. in-Aid from Miles Laboratories (Elkhart, IN), to S.B. Downloaded by guest on October 2, 2021 Biochemistrv: Bhattacharva et al. Proc. Natl. Acad. Sci. USA 76 (1979) 2221

I. Bovd, W. C. (1970) Ann. N. Y. Acad. Sci. 169,168-190. 20. Maruyama, Y. & Coleman, M. S. (1978) Cancer Res. 38, 2. Lin, J. Y., Tserng, K. Y., Chen, C. C., Lin, L. 1. & Tung, T. C. 1617-1620. (1970) Nature (London) 227, 292-293. 21. IBrahmachary, R. L. & De, A. (1973) Cell Differ. 2, 151- 3. Shoham, J., Inhar, M. & Sachs, L. (1970) Nature (London) 227, 156. 1244-1246. 22. McCarty, K. S., Jr., Olive, K. E., Kaufman, B. & McCarty, K. S., 4. Burger, M. M. & Noonan, K. D. (1970) Nature (London) 228, Sr. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 1550. 512-515. 23. Burns, M. V. & Lozzio, B. B. (1973) J. Cell Physiol. 82, 277- 5. Inbar, M., Ben-Bassat, H. & Sachs, L. (1972) Int. J. Cancer 9, 283. 143-149. 24. Dent, P. B. (1971) J. NatI. Cancer Inst. 46, 763-773. 6. Caso, L. V. (1968) Anat. Rec. 162, 459-466. 25. Bhattacharva, P., Basu, S. & Saz, H. J. (1978) Fed. Proc. Fed. Am. 7. Gail, M. H. & Boone, C. W. (1972) Exp. Cell Res. 70,33-40. Soc. Exp. Biol. 37, 1304. 8. Ralph, P. & Nakoinz, 1. (1973) J. Natl. Cancer Inst. 51, 883- 26. Bhattacharya, P. & Basu, S. (1978) Proc. Natl. Acad. Sci. USA 890. 75, 1289-1293. 9. Nicolson, G. L., Lacorbiere, M. & Hunter, T. R. (1975) Cancer 27. Bhattacharva, P., Moskal, J. R. & Basu, S. (1977) Proc. Natl. Acad. Res. 35, 144-145. Sci. USA 74, 842-845. 10. Nicolson, G. L. (1974) Int. Rev. Cytol. 39, 89-190. 28. Duguet, M., Mechali, M. & Rossignol, J. M. (1978) Anal. Biochem. 11. Refsnes, K., Olsnes, S. & Pihl, A. (1974) J. Biol. Chem. 249, 88, 399-405. 3557-3562. 29. Saltvedt, E. (1976) Biochim. Biophys. Acta 451, 536-548. 12. Olsnes, S., Refsnes, K. & Pihl, A. (1974) Nature (London) 249, 30. Holley, R. W. (1972) Proc. Natl. Acad. Sci. USA 69, 2840- 627-631. 2841. 13. Olsnes, S. & Pihl, A. (1972) FEBS Lett. 28, 48-50. 31. Toyoshima, S., Fukuda, M. & Osawa, T. (1972) Biochemistry 11, 14. Nicolson, G. L., Blaustein, J. & Etzler, M. E. (1974) Biochemistry 4000-4005. 13, 196-204. 32. Holmes, A. H. & Johnston, I. R. (1975) FEBS Lett. 60, 233- 15. Olsnes, S., Saltvedt, E. & Pihl, A. (1974) J. Biol. Chem. 249, 243. 803-810. 33. Matsukage, A., Bohn, E. W. & Wilson, S. H. (1974) Proc. Natl. 16. Nowell, P. C. (1960) Cancer Res. 20, 462-466. Acad. Sci. USA 71, 578-582. 17. Abell, C. W., Kamp, C. W. & Johnson,L. D. (1970) Cancer Res. 34. Bollurm, F. J. (1975) Prog. Nucleic Acid Res. Mol. Biol. 15, 30, 717-723. 109-144. 18. Agarwal, S. S. & Loeb, L. A. (1972) Cancer Res. 32, 107-113. 35. Weissbach, A. (1977) Annu. Rev. Biochem. 46, 25-47. 19. Spadari, S., Villani, G. & Hardt, N. (1978) Exp. Cell Res. 113, 36. Bhattacharya, P., Simet, I. & Basu, S. (1979) Fed. Proc. Fed. Am. 57-62. Soc. Exp. Biol. 38, 485. Downloaded by guest on October 2, 2021