Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2218-2221, May 1979 Biochemistry Inhibition of human neuroblastoma DNA polymerase activities by plant lectins and toxins (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 concanavalin A and ricin (RCAII, human neuroblastoma IMR-32 cells by the highly toxic plant Mr 65,000) on [3Hlthymidine incorporation into human neuro- protein 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 toxin 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 kidney bean were purchased from E. Y. Laboratories (San Mateo, CA). agglutinin (PHA-P). Wheat germ agglutinin (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 glycoprotein; Mr blastoma clone IMR-32 cells were purchased from the Ameri- 65,000) and abrin (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 lymphocyte 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 lymphocytes (18), Con A-treated polymerase a appeared between fractions 7 and 9, and the rabbit spleen 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, Canavalia ensiformis agglutinin or concana- thesis in cultured Gross ascites lymphoma cells at concentrations valin A; APA, Abrus precatorius agglutinin; RCAI, Ricinus 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 mitogen; 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 "Lectin," as used in this paper, denotes sugar-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 affinity chromatography 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 proteins. Ricin and abrin originate in the seeds 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 (disulfide 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. 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