Inhibition of Mammalian and Oncornavirus Nucleic Acid Polymerase Activities by Alkoxybenzophenanthridine Alkaloids I

[CANCER RESEARCH 36, 2390-2395, July 1976]

V. Sagar Sethi Department of Virology and Cell Biology, Litton Bionetics, Inc,, Kensington, Maryland 20795

SUMMARY useful prophylactic agents in cancer. With a view to establish in vitro test systems for potential The alkoxybenzophenanthridine alkaloids (coralyne ace- antitumor activity, we are examining synthetic compounds tosulfate, fagaronine chloride, and nitidine chloride) have and natural products for the inhibition of virus transforma- been reported to possess antileukemic activity in mice. tion of normal cultured cells, viral and cellular replication, These compounds were tested for inhibition of reverse tran- and transcription enzymes (11, 15). scriptase activity of an RNA tumor virus and DNA polymer- A number of alkoxybenzophenanthridine alkaloids exhibit ase, RNA polymerase, and polyadenylic acid polymerase interesting biological and pharmacological activities. activities of NIH-Swiss mouse embryos. Reverse transcrip- Among these, fagaronine chloride (NSC 157995), isolated tase and DNA polymerase activities were strongly inhibited from Fagara zanthoxyloides, has shown good activity by these antileukemic alkaloids, whereas RNA polymerase against P388 leukemia in mice (19). Nitidine chloride (NSC and polyadenylic acid polymerase activities were only mod- 146397) isolated from Fagara macrophylla (23), is highly erately affected. Viral and cellular DNA polymerase activi- cytotoxic and has shown activity in L1210 and P388 leu- ties were potently diminished by the alkaloids when kemias and Lewis lung carcinomas (16, 23, 25). Coralyne poly[d(A-T)], poly(dA)-oligo(dT), and poly(rA).oligo(dT) acetosulfate (NSC 154890) has also displayed inhibitory template primers were used in the reaction mixture; how- activity against both L1210 and P388 leukemias in mice ever, no inhibition of enzyme activity was obtained with (25). This report describes the inhibition of mammalian and poly(rC).oligo(dG) as template primer. These results sug- oncornavirus nucleic acid-polymerizing enzyme activities gest that alkoxybenzophenanthridine alkaloids inhibit DNA by these alkoxybenzophenanthridine alkaloids. polymerase activity by interaction with A:T base pairs of the template primer. MATERIALS AND METHODS INTRODUCTION Reagents and Templates Nucleocapsids of RNA tumor viruses contain RNA-directed DNA polymerase (reverse transcriptase) activity that Tritium-labeled nucleotides were obtained from Schwarz/ catalyzes the formation of DNA from an RNA template (13, Mann, Orangeburg, N. Y. Unlabeled nucleotides were pur- 18, 20). This enzyme is involved in viral DNA (provirus) chased from Worthington Biochemical Corp., Freehold, N. formation (7, 13, 18, 20) which, on integration into the host J., or from P-L Biochemicals, Milwaukee, Wis. Radioactive genome, can lead to cell transformation (13, 18, 20). Be- nucleotides were evaporated to dryness under vacuum to sides viral reverse transcriptase, other host enzymes and remove ethanol and dissolved in water. The specific activity factors may also be involved in provirus formation (13, 18, of each nucleotide was adjusted to the desired level by the 20). Host DNA-directed RNA polymerase is involved in for- addition of unlabeled nucleotide. Phosphocreatine (diso- mation of viral genome (2, 12, 14). Viral RNA at its 3' end dium), creatine phosphokinase, and calf liver tRNA, type IV, contains poly(A) 2 sequences (4, 5, 8), and host poly(A) po- were purchased from Sigma Chemical Co., St. Louis, Mo. lymerase activity is probably responsible for the addition of Calf thymus DNA and DNase I were obtained from Worthing- these sequences. If RNA tumor viruses are involved in hu- ton. Crystalline bovine serum albumin and poly(A) were man neoplasia (3, 17), viral and host nucleic acid-polymeriz- from Miles Laboratories, Elkhart, Ind. Poly(dA)-oligo(dT), ing enzymes become important for oncogenesis. Specific poly(rA).oligo(dT), and poly[d(A-T)] were purchased from P- inhibitors of these enzyme activities might, therefore, be L Biochemicals. Poly(rC).oligo(dG) was obtained from Schwarz/Mann. DEAE-cellulose filter discs (Whatman DE ' This work was supported by Contract NOI-CM-33741 from the Division of 81) were obtained from H. Reeve Angel & Co., Inc., Clifton, Cancer Treatment, National Cancer Institute, Department of Health, Educa- N. J. Polyethylene glycol 6000 was purchased from tion and Welfare, Bethesda, Md. Schwarz/Mann. All other reagents were of analytical grade. 2 The abbreviations used are: poly(A), polyadenylicacid; AMV, avian mye- Ioblastosis virus; SSV-1, simian sarcoma virus type 1. All templates were dissolved in 0.01 M Tris-HCI (pH 7.0), Received October 31, 1975; accepted March 25, 1976. 0.1 mM EDTA, and 0.1 M NaCI. Denatured calf thymus DNA

2390 CANCER RESEARCH VOL. 36

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1976 American Association for Cancer Research. Nucleic Acid Polymerases and Alkaloids was prepared by heating the DNA in a boiling water bath for mination. Nucleic acid concentrations were determined 15 min followed by quick chilling in ice water. Activated calf spect ro p h oto metrical ly. thymus DNA was prepared by incubating 6 mg DNA in 10 ml buffer containing 0.05 M Tris-HCI (pH 7.0), 5 mM MgCI2, and Purification of Enzyme Activities bovine serum albumin (500/~g/ml) with 0.01 /~g crystalline pancreatic DNase for 15 min at 37 ~ DNase was inactivated Reverse Transcriptase from Oncornaviruses. SSV-1, de- by incubating at 70 ~ for 5 min. All templates were stored at rived from tissue culture fluids of chronically SSV-I-in- 4 ~ fected NC-37 cells, was obtained from Pfizer, Inc., May- wood, N. J. Chicken plasma containing AMV was obtained Enzyme Assays from Life Sciences Research Laboratories, St. Petersburg, Fla., through the courtesy of Dr. J. Beard. Viral DNA polym- RNA Polymerase. The assay mixture of 100/~1 contained erase was purified by a modification of the procedure of 5/~moles Tris-HCI (pH 8.0); 25 nmoles each ATP, CTP, and Abrell and Gallo (1). Five mg virus were sedimented by GTP; 2.5 nmoles [:~H]UTP (200 to 300 cpm/pmole); 5 /~g centrifugation, and the pellet was extracted with Buffer A phosphocreatine kinase; 0.4/~mole creatine phosphate; 25 (0.05 M Tris-HCI (pH 7.9), 0.05 M each NaCI and KCI, 0.02 M /~g bovine serum albumin; 0.2 pmole MnCI2; 0.2 /~mole dithiothreitol, 1 mM EDTA, and 0.25% Triton X-100). The MgCI2; 0.1 /~mole NaF; 0.4 /~mole dithiothreitol; 10% (v/v) viral extract was applied to a DEAE-cellulose column and glycerol; 15/~g denatured calf thymus DNA; and the enzyme eluted with Buffer B (0.05 M Tris-HCI (pH 7.9), 1 mM EDTA, 1 fraction. The reaction mixture was incubated at 37 ~ for 30 mM dithiothreitol, 20% (v/v) glycerol, 0.025% Triton X-100, min. and 0.3 M KCI). The eluate was exhaustively dialyzed against DNA Polymerase. The assay mixture of 100/~1 contained Buffer B without KCI and applied to a phosphocellulose 100 /~moles sodium glycinate (pH 8.8); 10 nmoles each column that was previously saturated with bovine serum dATP, dCTP, and dGTP; 2 nmoles [:~H]TTP (200 to 300 cpm/ albumin. Enzyme activity was eluted from the column by a pmole); 1 /~mole MgCI2; 0.1 /~mole NaF; 10% (v/v) glycerol; linear KCI gradient. The peak enzyme activity eluted at 0.25 15/~g activated calf thymus DNA; and enzyme. The reaction to 0.3 M KCI. Active enzyme fractions were pooled and con- mixture was incubated for 30 min at 37 ~ centrated by dialysis against Buffer B without KCI, but with Poly(A) Polymerase. The assay mixture of 100 /~1 con- 30% polyethylene glycol 6000. The enzyme preparation was tained 10 /~moles Tris-HCI (pH 8.0), 0.1 /~mole MnCI2, 0.4 stored at -80 ~. The amount of protein in the enzyme prepa- /~mole dithiothreitol, 0.5/~mole sodium fluoride, 0.4/~mole ration, due to its very low content, could not be determined. phosphocreatine, 5/~g phosphocreatine kinase, 25 nmoles The activities of SSV-1 and AMV DNA polymerases were 4.5 [~H]ATP (20 to 40 cpm/pmole), 200 /~g tRNA, 10% (v/v) and 6 nmoles dTMP incorporated per 30 min per ml, respec- glycerol, 25/.~g bovine serum albumin, and enzyme fraction. tively. The reaction mixture was incubated at 37 ~ for 60 rain. Purification of Poly(A) Polymerase. Poly(A) polymerase Viral DNA Polymerase (Reverse Transcriptase). The as- was purified from the cytoplasmic fraction of 12- to 16-day say mixture of 100/~1 contained 5/~moles Tris-HCI (pH 7.3), old NIH-Swiss mouse embryos according to a procedure 8/~moles KCI, 0.1 /~mole MnCI=, 0.5/~mole dithiothreitol, 2 established in this laboratory (6). In this study, poly(A) po- nmoles [:~H]TTP (300 to 400 cpm/pmole), 2 ~g lymerase purified by phosphocellulose column chromatog- poly(rA).oligo(dT), 20 /~g bovine serum albumin, 10% (v/v) raphy was used. The specific activity of the enzyme was 60 glycerol, and enzyme fraction. The reaction mixture was nmoles AMP incorporated per hr per mg protein. incubated at 37 ~ for 30 min. Purification of RNA and DNA Polymerases. RNA polymerases I and II from the nuclei of NIH-Swiss mouse embryos Termination of Enzyme Reactions and Determination of were purified by procedures established in our laboratory Activity, (14). 3 The specific activity of RNA polymerases I and II purified by DEAE-cellulose column chromatography were Enzyme reactions were terminated by chilling in ice and 100 pmoles UMP incorporated per 30 min per mg protein by the addition of 25/~1 0.1 M EDTA. One hundred/~1 of each and 3 nmoles UMP incorporated per 30 min per mg protein, reaction mixture were spotted uniformly onto a 2.5-cm cir- respectively. DNA polymerases ~ and /3 from the cytoplas- cular Whatman DE-81 filter, kept at room temperature for 15 mic fraction of NIH-Swiss mouse embryo homogenate were min and washed batchwise by swirling 4 times in 10 ml of purified according to a procedure developed in this labora- 5% Na2HPO4.7 H=O per filter, followed by 2 washings each of tory. 3 The specific activities of DNA polymerases ot and /3 water and 95% ethanol. Filters were dried, and radioactivity purified by phosphocellulose column chromatography were was counted in a toluene-based scintillation fluid~ 2.5 and 1.7 nmoles dTMP incorporated per 30 min per mg protein, respectively. Determination of Protein and Nucleic Acid. Testing of the Alkaloids for Inhibition of Enzyme Activi- ties, Two types of reaction mixture, one containing enzyme, Protein content was determined by the method of Lowry buffer, and bovine serum albumin (Mix 1) and the other et al. (10) using crystalline bovine serum albumin as a having template, substrates, metal ions, dithiothreitol, and standard. Protein in Tris-HCI buffer or in salt solution was glycerol (Mix 2), were made. The final concentrations of the precipitated with 5% trichloroacetic acid, centrifuged to a ingredients of the reaction mixture were the same as de- pellet, and dissolved in the alkali reagent for protein deter- 3 v. s. Sethi and P. Okano, submitted for publication.

JULY 1976 2391

i i i i scribed above for enzyme assays. An appropriate concen- Ioo tration of the alkaloid was added to Mix 1 and placed in ice for 10 min before adding Mix 2. Control experiments contained an equivalent amount of dimethylsulfoxide:water (9:1) mixture in which alkaloids were dissolved. The reac- 75 tion mixtures were incubated at 37 ~ for the desired time, after which the reactions were terminated and the enzyme activity was determined. Inhibition results were calculated and expressed as percentage of control activity. 50 The amounts of various enzymes used in each assay mixture were: reverse transcriptase from SSV-1 or AMV, 5 /.d; DNA polymerase o~, 11.5 ~g; DNA polymerase/3, 7.5/.Lg; RNA polymerase I, 80 /.Lg; RNA polymerase II, 20 /.~g; and poly(A) polymerase, 10 /~g. 25

RESULTS

Inhibition of SSV-1 Reverse Transcriptase, Mouse Em- 0 25 50 75 I00 bryo DNA Polymerase, RNA Polymerase, and Poly(A) Po- CORALYNE (jJg/mi )

lymerase Activities by Coralyne, Nitidine, and Fagaron- I I I I ine. Benzophenanthridine alkaloids (Chart 1) were exam- I00 ined for their effects on the enzyme activities of SSV-1 DNA polymerase, mouse embryo cytoplasmic DNA polymerases o~ and /3, mouse embryo nRNA polymerases I~ and II, and mouse embryo cytoplasmic poly(A) polymerase at increas- ~- 75 ing concentration of the alkaloids. Viral and cellular DNA l-

J OH 0

z 5o H3CO ~ OCH3 8 I-- Z w CH 3 U.I " 25

B I I I II 0 25 50 75 I00 CORALYNE (~,g/ml ) Chart 2. A, effect of coralyne acetosulfate on viral and cellular DNA polymerase activities. In the standard enzyme assay mixtures of 0.10 ml (see "Materials and Methods"), 5/~1 of SSV-1 reverse transcriptase (/x), 16.2/~g of mouse embryo DNA polymerase a (9 or 12.0 /~g of mouse embryo DNA poymerase /3 (O) and different concentrations of the alkaloid were used for inhibition of enzyme activities. The control enzyme activities assay of reverse transcriptase and DNA polymerase (x and ,8 were 11.9,254, and 107 pmoles, ]I respectively, after subtracting the blank (no enzyme) values. Blank values were in the range of 40 to 60 cpm (0.20 pmole). B, influence of coralyne acetosulfate on mouse embryo RNA polymerase and poly(A) polymerase activities. In the standard enzyme assay mixtures of 0.10 ml (see "Materials and Methods"), 80 #g of RNA polymerase I (O), 20/~g of RNA polymerase II (1), or: 10 /~g of poly(A) polymerase (/x), and different concentrations of the OCH 3 alkaloid were used for enzyme inhibition. The control enzyme activities assay of RNA polymerase I, RNA polymerase !1, and poly(A) polymerase were 7, 19, OCH 3 and 560 pmoles, respectively, after deducting the blank (no enzyme) values. Blank values were in the same range as in A. H3CO~ ~ polymerase activities were strongly inhibited by coralyne H3CO (Chart 2A). Similar results were obtained with nitidine chloride (data not given). The 50% enzyme-inhibitory doses of OH3 CH3CO2SO 3- 11[ coralyne and nitidine for DNA polymerase activities were 20 to 25 and 30 to 45 /~g/ml, respectively. RNA polymerase Chart 1. Chemical structure of antileukemic alkoxybenzophenanthridine alkaloids. I, fagaronine chloride; II, nitidine chloride; III, coralyne acetosul- activity was inhibited by these alkaloids at higher dose fate. levels, and poly(A) polymerase activity was only moderately

2392 CANCER RESEARCH VOL. 36

inhibited (Chart 2B). Results similar to those described IOO above were obtained with fagaronine chloride and nitidine chloride (data not shown). Mechanism of DNA Polymerase Inhibition by Benzo- 80 84 phenanthridine Alkaloids. In order to elucidate the mechanism of enzyme inhibition, different template primers [poly(rA)-oligo(dT), poly(dA).oligo(dT), and poly(rC)-oligo(dG)] were used for AMV reverse transcriptase inhibition 60 studies. The enzyme activity with poly(rA).oligo(dT) and poly(dA).oligo(dT) was strongly inhibited by coralyne and nitidine, but with poly(rC).oligo(dG) there was no inhibition (Table 1). These results suggest that inhibition was not due to interaction of the alkaloid with the enzyme pro- 4 tein. Moreover, the inhibition was also not due to inter- 3 action or competition with KCI or substrate ([3H]TTP or 2 I ~______--~ [:~H]dGTP), since these were present in the reaction mixture in large excess relative to the alkaloid. It was also observed O that enzyme activity remained unchanged by increasing AMV- REVERSE TRANSCRIPTASE (~1/assay) MnCI2 in the assay mixture (data not shown), indicating that Chart 3. Effect of increasing concentration of AMV reverse transcriptase the metal ions did not interact with the alkaloids. The data on coralyne- and nitidine-inhibited reaction mixtures. The standard reaction of Table 1 further suggested that the alkaloids had less mixture of 0.10 ml contained 5 /~g of poly(rA).oligo(dT). Five/~g of coralyne acetosulfate (O) or nitidine chloride (@) per assay were used. The control affinity for (rC),, or C:G pairs. The strong inhibition of values of 5, 10, 20, and 30/~1 of the enzyme per assay were 21.7, 58.5, 192, enzyme activity with poly(rA)-oligo(dT) and poly(dA).ol- and 262 pmoles dTMP incorporated per assay, respectively. igo(dT) as template primers suggested that inhibition was due to interaction of the alkaloids with (rA),, (dT),, or A:T 10o I , , , ,_ pairs. Similar results have been reported for fagaronine chloride (15).

When the amount of AMV reverse transcriptase was in- >- creased from 5 to 30/~1 in the reaction mixture inhibited by I-- _~ 75 coralyne and nitidine, the level of enzyme inhibition re- b- O mained unaffected (Chart 3). These data support the con- ._J O clusion that the alkaloids did not interact with the enzyme e~ protein (Table 1). However, when poly(rA).oligo(dT) con- z 50 centration was increased from 2/~g to 40/.Lg/assay mixture, o t.-- inhibition of reverse .transcriptase by coralyne and nitidine z was completely overcome (Chart 4). Similar data have been 1.1.1 reported for fagaronine chloride (15). Inhibition of the cellu- w lar DNA polymerase activity by coralyne and nitidine was a_ 25 also reversed by increase of the excess of poly[d(A-T)] (Chart 5). These results further substantiate our previous

Table 1 o Io 20 30 40 Inhibition of AMV DNA polymerase activity by coralyne and nitidine with different template primers Poly (rAl.oligo (dT) (~,g) Chart 4. Influence of increasing concentration of poly(rA).oligo(dT) on the Enzyme ac- Enzyme ac- alkaloid-inhibited AMV DNA polymerase reaction. The standard reaction Coralyne tivity tivity mixture of 0.10 ml contained 5 /~1 of the enzyme. Five P-g each of coralyne Template acetosul- (pmoles/as- Nitidine (pmoles/as- acetosulfate (O) or nitidine chloride (t) per assay were used. The control primer fate say) chloride say) values at 5, 10, 20, 30, and 40 ~g of the template primer per assay were 36.0, 36.5, 41.0.45.6, and 39.0 pmoles dTMP incorporated per assay, respectively. (rA),-(dT),2_,," - 28.3 (100) ~' - 28.3 (100) (KA),.(dT),=_,," + 0.68 (2.4) + 0.93 (3.3) (dA),.(dT),~_,," - 4.5 (100) - 4.5 (100) findings that the benzophenanthridine alkaloids exhibit an (dA)..(dT),~_,,' + 0.25 (5.5) + 0.16 (3.6) affinity for A:T base pairs (15) .... (rC),.(dG),2_,8' - 8.55 (100) - 8.55 (100) (rC),.(dG),2_,s" + 11.88 (139) + 8.44 (98.8) In the above-described experiments, the alkaloid was added to the reaction mixture before incubation at 37 ~ and " Standard assay conditions as described in "Materials and Methods," except 5/~g of the template primer, were used. Five/~g inhibitor effects were directed to free template primer and of the alkaloid per assay were used. its complexes with enzyme, substrate, arid metal ions. In t, Numbers in parentheses, percentage. another situation, where the enzyme molecules were elon- ' Standard assay mixture contained 5/zg of the template primer. gating the polynucleotide chain, the effect of addition of Five /~g of the inhibitor per assay were used. '~ Standard assay mixture contained 5 /~g of the template primer coralyne acetosulfate was examined. Addition of coralyne and 2.2 nmoles of ['~H]dGTP (340 cpm/pmole). Five /,g of the acetosulfate during AMV DNA polymerase reaction at 5, 10, alkaloid per assay were used. and 20 min stopped [:~H]dTMP incorporation instantly, and

JULY 1976 2393

I I I the enzyme product did not take place in the presence of IOO the alkaloid. Similar results were obtained with nitidine chloride (data not shown) and fagaronine chloride (15).

~_ 8o DISCUSSION

60 Cellular and oncornavirus nucleic acid-polymerizing enzymes (DNA-directed RNA polymerase, DNA polymerase, or

o reverse transcriptase), which require a double-stranded DNA ~z 40 as template or template primer, are more potently inhibited by alkoxybenzophenanthridine alkaloids (coralyne, faga-

Q. ronine, or nitidine) than is poly(A) polymerase, which re- 20 quires a single-stranded RNA primer with free 3' terminus. The alkaloid inhibition of viral or cellular DNA potymerase reaction is reversed by the addition of excess template primer. Increase of enzyme, metal ions, or substrate has no iiO 20I 50I effect on the alkaloid inhibition. These results indicate that Poly d (A-T) (.~ug/ossoy) the alkaloids inhibit the enzyme reaction by interacting with Chart 5. Effect of increasing concentration of polyd(A-T) on the alkaloid the template primer. inhibited mouse embryo DNA polymerase I reaction. The standard assay mixture of 0.10 ml contained 16.2/~g of the enzyme and no dCTP and dGTP. Among the synthetic templates [poly[d(A-T)], Five p.g each of coralyne acetosulfate (O) or nitidine chloride (0) per assay poly(rA).oligo(dT), poly(dA).oligo(dT), and poly(rC).ol- were used. The control values at 5, 10, 20, and 30 ~g poly[d(A-T)] per assay igo(dG)] used for examining inhibition of viral DNA polym- were 8.8, 7.5, 7.2, and 3.9 pmoles dTMP incorporated per assay, respectively. erase by the alkaloids, the incorporation of [:~H]dGMP by the enzyme with poly(rC)-oligo(dG) as template primer is not inhibited, whereas the incorporation of [3H]dTMP 40 using other template primers is strongly inhibited. These results indicate a preferential interaction of these alkaloids with A:T pairs of the DNA or DNA-RNA hybrids. In- deed a change in the absorption spectrum of coralyne acetosulfate when added to a calf thymus DNA solution has 3o been reported (24), suggesting a binding of the alkaloid to the DNA. Experiments conducted in this laboratory show that the absorption maxima of coralyne acetosulfate and t~ nitidine chloride are shifted toward higher wavelengths on addition of synthetic polynucleotides (V. S. Sethi, unpub- 2o lished results). These results confirm the previous report nt- O (24) and indicate an interaction of the polynucleotides with _z o_ coralyne acetosulfate and nitidine chloride. I- The preferential inhibition by the alkoxybenzophenanthri-

9~, IO dine alkaloids of DNA synthesis by viral reverse transcriptase or mammalian DNA polymerases from an A:T template primer may have some implication for the biology of RNA tumor viruses. The RNA of RNA tumor viruses, at its 3' end, contains poly(A) sequences similar to mammalian mRNA (4, I L , , 1 o io 20 50 5, 8). The exact role of poly(A) sequences is still unknown, TIME (MINUTES) but one possibility is that these sequences may be the Chart 6. Addition of coralyne acetosulfate during AMV reverse transcrip- attachment site for an initiator polynucleotide. If RNA tumor tase kinetic reaction. Two ml of standard assay mixture containing 50 /~1 of viruses are involved in the etiology of human cancer, in- enzyme per ml were made and distributed into 0.6-ml (A), 0.5-ml (8), 0.5-ml (C), and 0.4-ml (D) aliquots. From A, 0.10-ml sample was withdrawn at 0 min. hibitors that interfere with the initiation of viral RNA tran- A to D were incubated at 37~ Samples (0.10 ml) from A were withdrawn at 5, scription could be effective agents in cancer chemotherapy. 10, 20, and 30 min postincubation. Coralyne acetosulfate (50 /~g/ml) was Other antitumor drugs, such as adriamycin, daunomycin, added to B, C, and D at 5, 10, and 20 min, respectively. One rain after addition of the alkaloid, 0.10-ml samples were withdrawn from B, C, and and acridinylaniside inhibit DNA polymerase reactions more D at 5-min intervals. The enzyme activity of each sample was determined. strongly when template primers with A:T pairs, such as Control, 9 alkaloid treated, Q. Arrows, addition of drug at various times. poly(dA).oligo(dT), poly(rA).oligo(dT), or poly[d(A-T)] are used. 4 These findings suggest a stronger or preferential there was no decrease of the synthesized product on further binding of these drugs to A:T regions of the DNA, and thus incubation (Chart 6). These data clearly indicate that cora- may be related to the differential fluorescent staining of lyne halted the elongation of nascent polynucleotide chromosomes by these agents (9). Furthermore, preferen- chains, probably by interacting with the template primer. Moreover, these results further indicate that degradation of " v. s. Sethi, submitted for publication.

2394 CANCER RESEARCH VOL. 36

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1976 American Association for Cancer Research. Nucleic Acid Polymerases and Alkaloids tial inhibition by drugs of DNA polymerase activity utilizing 11. O'Connor, T. E., Stansly, P., Sethi, V. S., Hadidi, A. and Okano, P. Antibiotic Control of Infection of Human or Mouse Cells with Oncornavi- A:T template primers may prove to be a useful in vitro test rus. In: Pharmacological Basis of Cancer Chemotherapy, M. D. Ander- for the screening of antitumor compounds. son Hospital and Tumor Institute at Houston, pp. 319-327. Baltimore: The Williams & Wilkins Co., 1975. 12. Rymo, L., Parson, J. T., Coffin, J. M., and Weissmann, C. In vitro Synthesis of Rous Sarcoma Virus-Specific RNA Is Catalyzed by a DNA- ACKNOWLEDGMENTS dependent RNA Polymerase. Proc. Natl. Acad. Sci. U. S., 71: 2782-2786, 1975. I thank Dr. Vincent H. Bono, Jr., Dr. Timothy E. O'Connor, and Dr. Mary K. 13. Sarin, P., and Gallo, R. C. RNA-directed DNA Polymerase. In: K. Burton Wolpert for their interest and encouragement. I am grateful to Dr. Harry B. (ed.), International Review of Science Series in Biochemistry, Vol. 6, Wood, Jr., for providing the alkaloids. I also express my sincere thanks to Dr. pp. 219-254. London: Butterworth & Co., (Publishers) Ltd., 1974. C. D. Aldrich for a critical reading of the manuscript. The technical assist- 14. Sethi, V. S., and Gallo, R. C. DNA-dependent RNA Polymerases from ance of J. Shilling, L. MacDowell, and A. Claysmith is gratefully acknowl- Spleen of Uninfected and Rauscher Murine Leukemia Virus Infected NIH- edged. Swiss Mice. Biochim. Biophys. Acta, 278: 269-281, 1975. 15. Sethi, V. S:, and Sethi, M. L. Inhibition of Reverse Transcriptase Activity of RNA-Tumor Viruses by Fagaronine. Biochem. Biophys. Res. Com- mun., 63: 1070-1076, 1975. REFERENCES 16. Stermitz, F. R., Larson, K. A., and Kim, D. K. Some Structural Relation- ships among Cytotoxic and Anti-tumor Benzophenanthridine Alkaloid 1. Abrell, J. W., and Gallo, R. C. Purification, Characterization and Com- Derivatives. J. Med. Chem., 16, 939-940, 1973. parison of the DNA Polymerases from Two Primate RNA Tumor Viruses. 17. Temin, H. M. Introduction to Virus-caused Cancers. Cancer, 34: 1347- J. Virol., 12: 431-439, 1973. 1352, 1974. 2. Dinowitz, M. Inhibition of Rous Sarcoma Virus by ~-amanitin: Possible 18. Temin, H. M., and Baltimore, D. RNA-directed DNA Synthesis and RNA Role of Cell DNA-dependent RNA Polymerase Form I1. Virology, 66: 1-9, Tumor Viruses. Advan. Virus Res., 17: 129-188, 1972. 1975. 19. Tin-Wa, M., Bell, C. L., Bevelle, C., Fong, H. H. S., and Farnsworth, N. R. 3. Gallagher, R. E., and Gallo, R. C. Type C RNA Tumor Virus Isolated Potential Anticancer Agents I: Confirming Evidence for the Structure of from Cultured Human Acute Myelogenous Leukemia Cells. Science, 187: Fagaronine. J. Pharm. Sci., 63: 1476-1477, 1974. 350-353, 1975. 20. Tooze, J. The Molecular Biology of Tumor Viruses. Cold Spring Harbor, 4. Gillespie, D., Marshall, S., and Gallo, R. C. RNA of RNA Tumor Viruses N. Y.: Cold Spring Harbor Laboratory, 1973. Contains Poly(A). Nature New Biol., 236: 227-231, 1972. 21. Varmus, H. E., Guntaka, R. V., Fan, W. J. W., Heasley, S., and Bishop, J. 5. Green, M., and Cartas, M. The Genome of RNA Tumor Virus Contains M. Synthesis of Viral DNA in the Cytoplasm of Duck Embryo Fibroblasts Poly (A) Sequences. Proc. Natl. Acad. Sci. U. S., 69: 791-194, 1972. and in Enucleated Cells after Infection by Avian Sarcoma Virus. Proc. 6. Hadidi, A., and Sethi, V. S. Polyadenylate Polymerase from Cytoplasm Natl. Acad. Sci. U. S., 71: 3874-3878, 1974. and Nuclei of NIH-Swiss Mouse Embryos. Biochim. Biophys. Acta, 425: 22. Verma, I. M., Mason, W. S., Drost, S. D., and Baltimore, D. DNA Polymer- 95-109, 1976. ase Activity from Two Temperature Sensitive Mutants of Rous Sarcoma 7. Hanafusa, H., and Hanafusa, T. Noninfectious RSV Deficient in DNA Virus Is Thermolabile. Nature, 251: 27-31, 1974. Polymerase. Virology, 43: 313-316, 1971. 23. Wall, M. E., Wani, M. C., and Taylor, H. L. Plant Anti-Tumor Agents. VIII. 8. Lai, M. M. C., and Duesberg, P. H. Adenylic Acid-Rich Sequence in RNAs Isolation and Structure of Anti-Tumor Alkaloids from Fagara macro- of Rous Sarcoma Virus and Rauscher Mouse Leukemia Virus. Nature, phylla. Abstracts of the American Chemical Society Meeting, Washing- 235: 383-386, 1972. ton, D. C., September 1971. 9. Lin, C. C., and Van de Sande, J. H. Differential Fluorescent Staining of 24. Zee-Chang, K. Y., and Cheng, C. C. Interaction between DNA and Cora- Human Chromosomes with Daunomycin and Adriamycin--The D Bands. lyne Acetosulfate, an Antileukemic Compound. J. Pharm. Sci., 62:1572- Science, 190: 61-63, 1975. 1573, 1973. 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein 25. Zee-Chang, R. K. Y., and Cheng, C. C. Preparation and Antileukemic Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265- Activity of Some Alkoxybenzophenanthridinium Salts and Correspond- 275, 1951. ing Dihydro Derivatives. J. Med. Chem., 18: 66-71, 1975.

JULY 1976 2395

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1976 American Association for Cancer Research. Inhibition of Mammalian and Oncornavirus Nucleic Acid Polymerase Activities by Alkoxybenzophenanthridine Alkaloids

V. Sagar Sethi

Cancer Res 1976;36:2390-2395.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/36/7_Part_1/2390

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/36/7_Part_1/2390. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.