Suramin Affects DNA Synthesis in Hela Cells by Inhibition of DNA Polymerases1

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[CANCER RESEARCH 50, 7754-7757, December 15. 1990] Suramin Affects DNA Synthesis in HeLa Cells by Inhibition of DNA Polymerases1

Hitesh K. ,1Â¡miai,Christopher W. Anderson, Randall G. Davis, and Jamboor K. Vishwanatha2

Department of Biochemistry, University of Nebraska Medical Center, Omaha, Nebraska 68198-4525

ABSTRACT DNA synthesis in vivo, on SV403 DNA replication in vitro, and on the activities of various cellular DNA polymerases. Our Suramin, a polysulfonated naphthylurea widely used in the treatment results suggest that the inhibition of DNA synthesis by suramin of trypanosomiasis and onchocerciasis, is currently being investigated as is due to the inhibition of replicative polymerases. an antitumor agent for the treatment of advanced cancer. Suramin exerts a wide variety of biological effects. We have shown that suramin inhibits cell proliferation and DNA synthesis in cultured HeLa cells. The repli MATERIALS AND METHODS cation in vitro of SV40 DNA is completely abolished by 40 MMsuramin. The inhibition of DNA replication is due to inhibition of DNA polymer- Materials. Suramin was generously provided by the Drug Synthesis ases a and 6, the replicative enzymes in eukaryotic cells. DNA polymerase and Chemistry Branch of the National Cancer Institute. Pancreatic DNase I-activated calf thymus DNA, heat-denatured DNA, and syn a is sensitive to lower concentrations of suramin (concentration to achieve 50% inhibition (!<'<â€ž)of8 MM]than is DNA polymerase Â¿(!('â€ž,36MM), thetic template-primers were prepared as described previously (16). whereas DNA polymerase ÃŸisrelatively insensitive to the drug (ICM of Plasmid pUCHSO containing SV40 orÃwas provided by Dr. Marc Wold, Johns Hopkins University Medical School, and SV40 large T- 90 MM).Suramin inhibits other replicative DNA polymerases such as I:\cherichia coli polymerase I (Klenow fragment) and Thermits aquaticus antigen was generously provided by Dr. Bruce Stillman, Cold Spring polymerase. Suramin is noncompetitive with both substrate deoxyribo- Harbor Laboratories. nucleotides and template-primers with respect to DNA polymerase inhi Measurement of DNA Synthesis in Vivo. Logarithmically growing HeLa cells were seeded at 2 x 10' cells/well in 6-well plates (Falcon) bition. Much lower concentrations (8-30 MM)of the drug are required for containing 2 ml of Dulbecco's minimal essential medium supplemented 50% inhibition of DNA polymerases than for 50% inhibition of other enzymes such as protein kinase C and reverse transcriptase. These results with 5% (v/v) fetal bovine serum (Hyclone). Various amounts of sura min were added to different wells, and the plates were incubated for 24 show an important biological effect of this drug and indicate the need for h. ['HjThymidine (1CN Radiochemicals) was added (6 MCi/well), and more studies before its clinical use as an antitumor agent. incorporation was allowed to proceed for an additional 30 min. After removal of medium, the cell layer was washed twice with 1 ml of cold INTRODUCTION Hanks' balanced salt solution and the cells were dislodged by trypsini- zation. Cells were collected in microcentrifuge tubes and washed twice Suramin, the hexasodium salt of 8,8'-|carbonylbis[imino-3,l- with 1 ml of cold phosphate-buffered saline, and then 1 ml of cold 10% phenylenecarbonylimino(4-methyl-3 , 1-phenylene)carbon- trichloroacetic acid was added. Acid-precipitable radioactivity was col ylimino]]bis-l,3,5-naphthalenetrisulfonic acid (Fig. 1), is used lected on a glass fiber filter (Whatman Grade GF/C), and radioactivity in the treatment of African trypanosomiasis (sleeping sickness) was determined in a liquid scintillation spectrometer (Packard). and onchocerciasis (1). Although suramin was tested in the SV40 in Vitro DNA Replication Assay. Replication of pUCHSO chemotherapy of acquired immunodeficiency syndrome (2, 3), DNA containing the replication origin sequence of SV40 was measured by a modification of previously published procedures (17, 18). The its host toxicity has prevented use of this drug in treatment of reaction (25-Mlvolume) contained 30 HIMA'-2-hydroxyethylpiperazine- acquired immunodeficiency syndrome. More recently, the drug jV'-2-ethanesulfonic acid-HCl, pH 7.2; 7 mM MgCl2; 0.5 IHMdithio- has been reported to have antitumor activity in a number of threitol; 100 MMeach dATP, dGTP, and dTTP; 50 MM[a-32P]dCTP systems of advanced cancer (4, 5). (4000 cpm/pmol); 4 mM ATP; 200 MMeach CTP, GTP, and UTP; 40 Suramin has a wide variety of biological effects. It is taken mM phosphocreatine; 100 Mg creatine phosphokinase; 100 ng of up in cells by endocytosis and inhibits phagosome-lysosome pUCHSO DNA; HeLa cell extract (prepared as described in Ref. 19); fusion (6, 7). Suramin is a potent inhibitor of reverse transcrip- and 0.2 Mgof T-antigen. Incubation was at 35Â°Cfor 2 h. The reaction tases of a number of animal retroviruses (8) and protects T- was terminated by the addition of 0.2% sodium dodecyl sulfate, 15 mM cells in vitro against infectivity and cytopathic effects of HTLV- EDTA, and 0.2 mg/ml proteinase K followed by incubation at 35Â°C for 1 h. After phenol-chloroform extraction, the DNA was precipitated III (9). Suramin inhibits protein kinase C and induces differ with isopropyl alcohol in the presence of 2 Mammonium acetate. DNA entiation in neuroblastoma cell clone NB2A (10, 11). The precipitate was resuspended in 10 mM Tris-HCl, pH 7.5, and 1 mM binding of platelet-derived growth factor to its receptor is EDTA and was digested by the addition of 8 units of Dpn\ (to digest inhibited by suramin (12-15), and it appears to revert the methylated input DNA) and 20 units of Sail (to linearize replicated transformed phenotype of fibroblasts infected with simian sar DNA). DNA was resolved on a 1.2% agarose gel in 89 mM Tris-89 mM coma virus (14), presumably by the neutralization of an exter boric acid I mM EDTA buffer, pH 7.8). The agarose gel was soaked in nalized v-sis product. Although many of these studies reported 95% ethanol for 1 h and dried under vacuum. The dried gel was scanned the inhibition of cell growth and growth factor-induced mho- in a Betascope 603 (Belagen) radioanalytical imager and exposed to Kodak XAR-5 film at â€”80Â°Cwithan intensifying screen. genesis by suramin, the effect of suramin on DNA replication machinery has not been examined. Enzymes and Enzyme Assays. DNA polymerase a was purified by a In the present study, we examined the effects of suramin on previously published procedure (20). Partially purified HeLa DNA polymerase ÃŸandDNA polymerase 6 were obtained following published Received 7/26/90; accepted 9/18/90. protocols (21, 22). EscherÃ¬chiacoli DNA polymerase I (Klenow frag The costs of publication of this article were defrayed in part by the payment ment) was purchased from Pharmacia-LKB. Thermits aquaticus DNA of page charges. This article must therefore be hereby marked advertisement in polymerase was purified and provided to us by Dr. Robert Ramaley of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This study was supported by grants to Dr. J. K. Vishwanatha from the this department. Leukemia Research Foundation, Inc., and the Nebraska Cancer and Smoking DNA polymerase a activity with activated and primed single- Diseases Program. J To whom requests for reprints should be addressed, at Department of 3The abbreviations used are: poly(dA)-(dT)i0, polymer of deoxyadenosine Biochemistry, University of Nebraska Medical Center, 600 South 42nd Street, associated with deoxyribosylthymine decapeptide; ICso, concentration to achieve Omaha, NE 68198-4525. 50% inhibition; dNTP, deoxynucleotide triphosphate. 7754

tinued incubation of cells with 200 ng/m\ of suramin resulted NaCbS in complete inhibition of DNA synthesis after 2 d. Inhibition of SV40 DNA Replication in Vitro. To test the possibility that the inhibition of cell growth and DNA synthesis is due to the inhibitory effect of suramin on DNA replication machinery, we utilized the SV40 in vitro DNA replication system. In this in vitro system, DNA replication is dependent NaOsS SMii on a functional replication apparatus from cell extracts and on SV40 large T-antigen. The effect of various concentrations of suramin on the SV40 in vitro DNA replication is presented in Fig. 3. It is seen that, in the absence of T-antigen, there is no replication (Lane A) and that increasing concentrations of suramin inhibit replication of the input plasmid DNA (Lanes B to E). Prior to autoradiography, the gel was scanned in the Betascope 603 radioanalytical imager. Data from the scanner Fig. 1. Structure of suramin. showed that at 10, 20, and 40 JIM suramin, there was 20, 66, and 100% inhibition, respectively, of linearized plasmid DNA 20 (denoted by arrow in Fig. 3). Thus, the concentration of suramin 6- needed for 50% inhibition of SV40 in vitro replication is be tween 10 and 20 UM.This concentration is significantly lower S- than the concentration of suramin needed for 50% inhibition 4 -10 of protein kinase C, platelet-derived growth factor binding to 3- its receptor, retroviral reverse transcriptase, or cyclic AMP- 2- dependent protein kinase activities. O Effect of Suramin on DNA Polymerase a. Since DNA polym 0 erase a is believed to be the principal replicative enzyme, we 0 100 200 300 400 500 examined the effect of suramin on DNA polymerase a activity. Suramin ( u g/ml) Although suramin inhibited DNA polymerase Â«activity on Fig. 2. Effect of suramin on cell number and DNA synthesis. Cells (2 x IO*/ well) were seeded in Dulbecco's minimal essential medium containing 5% fetal each of the different DNA templates examined (Fig. 4), we bovine serum and indicated concentrations of suramin and were incubated for 24 observed significant differences among the templates. Suramin h. ['HJThymidine (6 nCi/well) was added for the last 30 min, following which the was a potent inhibitor of polymerase a when natural DNA cells were washed and collected. Cell number was determined prior to the addition templates such as DNase I-activated DNA and heat-denatured of 10% trichloroacetic acid. The acid-precipitable counts were determined by liquid scintillation spectrometry. Each point represents an average of four obser DNA were used. The IC50 on activated DNA template was 8 vations. The cell number is presented as cell number x 10"', and DNA synthesis MMand on heat-denatured DNA was 17 /UM.We also measured is presented as cpm x 10~3. synthetic template primers such as poly(dA)-(dT)10, poly(dA)- (dT),2_,8, and poly[d(A-T)]. On poly(dA)-(dT),0 template, there stranded DNA templates was assayed according to previously published procedures (16, 20). DNA polymerase ÃŸactivitywas assayed on acti vated DNA template in the presence of 10 mivi yV-ethylmaleimide, at ABCDE which concentration polymerase a is inactive. DNA polymerase 6 activity on poly(dA)-(dT)10 template was assayed according to a pub lished procedure (22). E. coli polymerase 1 (Klenow fragment) and T. aquaticus polymerase were assayed on activated DNA template at 35Â°C. One unit of enzyme activity corresponds to 1 nmol of deoxynucleotide monophosphate incorporated per h at 35Â°C. Â«- RESULTS Effect of Suramin on DNA Synthesis and Cell Growth. The effect of various concentrations of suramin on cell growth and *â€¢- ongoing DNA synthesis was measured, and the data are pre sented in Fig. 2. Concentrations of suramin of up to 100 Mg/ml did not have any effect on cell division. However, at 200 Â¿ig/ml of suramin, there was a 28% reduction in cell number; at higher concentrations there was no cell division and the viability of cells was affected. Continued growth of cells in 200 ng/m\ suramin for 5 d resulted in complete inhibition of growth and loss of viability (data no shown). There was a drastic effect of SURAMIN O O 10 20 40 various suramin concentrations tested on the ongoing DNA (uM) synthesis as measured by [3H]thymidine incorporation (Fig. 2). Fig. 3. Effect of suramin on SV40 DNA replication in vitro. Mil. cell extract (25 //g protein) was incubated in a SV40 DNA replication assay, as described While cell number and viability were not affected by incubation under "Materials and Methods," including various indicated amounts of suramin. of cultures for 24 h with up to 100 /Â¿g/mlof suramin, DNA Arrow, position of linearized plasmid DNA. Lane A, products from a reaction without added T-antigen; Lanes B-E contain indicated concentrations of suramin. synthesis was reduced by 50% at 100 Me/ml. At 200 Me/ml, The dried gel was exposed to a Kodak XAR-5 film with an intensifying screen at there was greater than 75% inhibition of DNA synthesis. Con -80'Cfor 12h. 7755

0.10 -

0.08 -

0.06-

10 20 30 Suramin (u M) Fig. 4. Template specificity of DNA polymerase a (Pola) inhibition by sura- iiiin. DNA polymerase a (0.2 unit, 2 UKprotein) was incubated with indicated amounts of suramin and various template-primers and assayed as described under "Materials and Methods." The various template-primers used (and corresponding 100% values in parentheses) are: â€¢ activated DNA (200 pmol/h); A, heat- denatured DNA (180 pmol/h); O, poly(dA)-(dT)10 (600 pmol/h); Â».poly(dA). (dT),2_â€ž(180 pmol/h); D, poly[d(A-T)] ( 134 pmol/h). The results represent means of four observations. -0.05 0.05 0.15 0.25

1/dNTP was a linear reduction of polymerase activity with increasing suramin concentrations and the IC50 was about 30 Â¿IM.How Fig. 5. Double-reciprocal kinetic plot demonstrating noncompetitive inhibi tion relative to dNTPs by suramin. DNA polymerase a assays were performed ever, on the other two templates, significant stimulation was on activated DNA template in the presence of various concentrations of substrate observed at low concentrations of suramin (<20 Â¿Â¿M),andIC50 dNTPs and without (Q) or with (*) 10 /IM suramin. Reactions were carried out values were 35-40 Â¿*Monthese templates. Stimulation of pro in triplicate. Inset, the Michaelis-Menten plot of V versus S, without (â€¢)orwith (O) suramin. tein kinase C at lower concentrations of suramin and inhibition at higher concentrations have been reported previously (11). 150- Protein concentration of polymerase a (0.2 to 6 Â¿tg)inthe assay did not affect the extent of inhibition by suramin (data not shown). The concentration of template DNA in the reaction had no effect on the inhibition of polymerase activity by suramin (data not shown). Polymerase activity was inhibited to the same extent at 50, 100, and 200 ng/m\ of activated DNA template in the reaction. Incubation time did not have any effect on 80 100 polymerase inhibition. Incubation of polymerase a with 20 /UM Suramin (fiM) suramin and activated DNA template for as little as 2 min Fig. 6. Differential inhibition of various DNA polymerases by suramin. DNA resulted in complete loss of activity. Addition of ATP at various polymerase .>'(- /

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. INHIBITION OF DNA POLYMERASES BY SURAMIN of suramin on HTLV-III/LAV infection presenting as Kaposi's sarcoma or Although suramin is known to exhibit a variety of effects on AIDS-related complex: clinical pharmacology and suppression of virus rep cell cultures, extreme caution must be observed in relating these lication in vivo. Lancet, 2: 627-630, 1985. to inhibition of DNA synthesis and cell proliferation. 3. Collins, J. M., Klecker, R. W., Yarchoan, R., Lane, H. C, Fauci, A. S., Redfield, R. R., Broder, S. B., and Myers, C. E. Clinical pharmacokinetics The mechanism of DNA synthesis inhibition by suramin was of suramin in patients with HTLV HI/LAV infection. J. Clin. Pharmacol., through inhibition of cellular DNA polymerases. DNA polym- 26: 22-26, 1986. 4. Stein, C. A., LaRocca, R. V., McAtee, T. N., and Myers, C. E. Suramin: an erase a, the principal replicative enzyme in eukaryotic cells anticancer drug with a unique mechanism of action. J. Clin. Oncol., 7: 499- (19), is very sensitive to the drug. The time course of inhibition 508, 1989. indicates that the drug prevents the initiation stage of DNA 5. LaRocca, R. V., Meer, J., Gilliat, R. W., Stein, C. A., Cassidy, J., Myers, C. E., and Dalakas, M. C. Suramin-induced polyneuropathy. Neurology, 40: synthesis. Suramin is a polyanionic compound and presumably 954-960, 1990. binds and forms stable complexes with proteins (23). The 6. Hart, P. D., and Young, M. R. Interference with normal phagosome-lysosome noncompetitive inhibition of polymerase a by suramin with fusion in macrophages, using ingested yeast cells and suramin. Nature (Lond.), 256:47-49, 1975. respect to dNTPs suggests that suramin binds to polymerase a 7. Kielian, M. C., Steinman, R. M., and Cohn, Z. A. Intralysosomal accumu or the polymerase a-dNTP complex. lation of polyanions. I. Fusion of pinocytic and phagocytic vacuoles with Several prokaryotic and eukaryotic DNA polymerases were secondary lysosomes. J. Cell Biol., 93: 866-874, 1982. 8. DeClercq, E. Suramin: a potent inhibitor of the reverse transcriptase of RNA inhibited by suramin. DNA polymerases involved in DNA tumor viruses. Cancer Lett., S: 9-22, 1979. replication were more sensitive to the drug. Thus, the toxic 9. Mitsuya, H., Popovic, M., Yarchoan, R., Matsushita, S., Gallo, R. C., and Broder, S. Suramin protection of T cells in vitro against infectivity and effect of suramin on prokaryotic and eukaryotic cells may be cytopathic effect of HTLV-III. Science (Washington DC), 226: 172-174, due mainly to inhibition of DNA polymerases and secondarily 1984. to the other effects of suramin. 10. Hensey, C. E., Boscoboinik, D., and Azzi, A. Suramin, an anti-cancer drug, inhibits protein kinase C and induced differentiation in neuroblastoma cell Suramin has been investigated as an antitumor drug in ad clone NB2A. FEBS Lett., 25Â«:156-158, 1989. vanced cancers (4, 5). The serum suramin levels were positively 11. Mahoney, C. W., Azzi, A., and Huang, K.-P. Effects of suramin, an anti- correlated with patient response. However, only 4 of 15 patients human immunodeficiency virus reverse transcriptase agent, on protein kinase C. J. Biol. Chem., 265: 5424-5428, 1990. had partial oncological response, even though their serum sur 12. Williams, L. T., Tremble, P. M., Lavin, M. F., and Sunday, M. E. Platelet- amin levels were 200 to 400 ng/m\. Because of the polyanionic derived growth factor receptors from a high affinity state in membrane preparations. Kinetic and affinity cross-linking studies. J. Biol. Chem., 259: nature of suramin, it may be heavily protein bound in the 5287-5294, 1984. serum; consequently, the suramin concentration available to 13. Hosang, M. Suramin binds to platelet-derived growth factor and inhibits its the intracellular machinery is low. In another study (5), paren- biological activity. J. Cell. Biochem., 29: 265-270, 1985. 14. Betsholtz, C., Johansson, A., Heldin, C.-H., and Westermark, B. Efficient teral suramin therapy caused a severe polyneuropathy, includ reversion of simian sarcoma virus-transformation and inhibition of growth ing generalized flaccid paralysis with bulbar and respiratory factor-induced mitogenesis by suramin. Proc. Nati. Acad. Sci. USA, 83: involvement, and an estimated 40% risk of developing neuro- 6440-6444, 1986. 15. Sjolund, M., and Thyberg, J. Suramin inhibits binding and degradation of toxicity in patients whose maximal plasma levels of suramin platelet-derived growth factor in arterial smooth muscle cells but does not were 350 /Â¿g/mlor greater. Thus, life-threatening toxicity may interfere with autocrine stimulation of DNA synthesis. Cell Tissue Res., 256: result if larger doses of suramin are given to patients, and these 35-43, 1989. 16. Jinda!, H. K., and Vishwanatha, J. K. Purification and characterization of studies indicate the necessity for careful monitoring of plasma primer recognition proteins from HeLa cells. Biochemistry, 29: 4767-4773, suramin concentrations during suramin therapy. 1990. 17. Li, J. J., and Kelly, T. J. Simian virus 40 DNA replication in vitro. Proc. Nati. Acad. Sci. USA, */: 6973-6977, 1984. ACKNOWLEDGMENTS 18. Wold, M. S., and Kelly, T. J. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc. Nati. Acad. Sci. USA, 85: 2523-2527, 1988. The authors wish to acknowledge the Drug Synthesis and Chemistry 19. Barii, E. F., Malkas, L. M., Hickey, R. H., Li, C.-J., Vishwanatha, J. K., and Branch, Division of Cancer Treatment, National Cancer Institute, for Coughlin, S. A multiprotein DNA polymerase a complex from HeLa cells: supplying suramin. We thank Drs. Stanley Cox and William Vaughn interaction with other proteins in DNA replication. Cancer Cells (Cold for critical review of this manuscript and Pamela Walter for preparation Spring Harbor), 6: 373-384, 1988. 20. Vishwanatha, J. K., Coughlin, S. A., Owen, M. W., and Baril, E. F. A multi- of this manuscript. protein form of DNA polymerase a from HeLa cells. J. Biol. Chem., 26/: 6619-6628, 1986. 21. Yamaguchi, M., Tanabe, K., Taguchi, Y. N., Nishizawa, M., Takahashi, T., REFERENCES and Matsukage, A. J. Biol. Chem., 255:9942-9948, 1980. 22. Lee, M. Y. W. T., and Toomey, N. L. Human placental DNA polymerase S: 1. Hawking, F. Suramin: with special reference to onchocerciasis. Adv. Phar- identification of a 170-kilodalton polypeptide by activity staining and im macol. Chemother., 15: 289-322, 1978. munoblotting. Biochemistry, 26: 1076-1085, 1987. 2. Broder, S., Collins, J. M., Markham, P. D., Redfield, R. P., Hoth, D. F., 23. Marzouk, H. F. A. I., Zuyderwijk, J., Vitterlinden, P., deJong, F., and Groopman, G. E., Gallo, R. C., Yarchoan, R., Lane, H. C., Klecker, R. W., Lamberts, S. W. J. Suramin prevents ACTH-stimulated corticosterone re Mitsuya, H., Gelmann, E., Resnick, L., Myers, C. E., and Fauci, A. S. Effects lease by dispersed adrenocortical cells. Endocrinology, /26:666-668, 1990.

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Hitesh K. Jindal, Christopher W. Anderson, Randall G. Davis, et al.

Cancer Res 1990;50:7754-7757.

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