Action of 1-β-d -Arabinofuranosylcytosine on mammalian tumor cells—1.

Europ. J. Cancer Vol. 8, pp. 391-396. Pergamon Press 1972. Printed in Great Britain

Action of 1-fl-D-Arabinofuranosylcytosine on Mammalian Tumor Cells l. Incorporation Into DNA RUDOLF K. ZAHN, WERNER E. G. MI~LLER, WOLF FORSTER, ARMIN MAIDHOF and RUDOLF BEYER Institut fiir Physiologische Chemie, Johannes Gutenberg UniversitJt, 65 Mainz, Johann Joachim Becher Weg 13, West Germany Abstract--Synchronized mouse lymphoma cells were treated with ara-C-3H at the beginning of the S-phase with twice the EDso concentration. In D N A isolated from these cells ara-C could be traced. It could be shown that the radioactivity in nucleotideand nucleosidefractions obtained after enzymic digestion of ara-C-DNA was due for more than 87 % to ara-C-3 H. The radioactivity of ara-C-3 H can be almost quantitatively attributed to cytosine and arabinose. An incorporation of ara-C into RNA could not be detected.

INTRODUCTION

In this paper the question whether ara-C molecules as such are incorporated in vivo into the cellular DNA during the DNA synthesis is therefore studied again. The experiments were performed with intact mouse lymphoma cells. It has been definitively established that ara-C3H is incorporated into the nucleotide sequences of cellular DNA. It was possible to prove that D-arabinose is associated with the labeled cytosine in the nucleoside- and nucleotide fractions.

1-~-D-ARABINOFURANOSYLCYTOSINE(ara-C) can inhibit cell proliferation and DNA synthesis of mammalian cells [1, 2] and of DNA viruses [3]. Two reports have shown that ara-C is able to inhibit proliferation of malignant tumors [4, 5]. About the main target of ara-C different views exist [survey: 6]. In one hypothesis [1] it is suggested that the incorporation of ara-C into DNA is the cause for the inhibition of DNA synthesis and thus for the inhibition of tumors. Data on the in vivo incorporation of ara-C into the DNA of mammalian cells have been given by several authors [7, 8, 1]. In vitro incorporation of ara-C into DNA by a DNA-synthesizing system has been described [9]. Up to now the results indicating an incorporation of ara-C into DNA cannot be considered as definitive evidence in the light of the cautions stressed by Cohen [10].

MATERIAL AND M E T H O D S 1. Sources of materials The following materials were obtained: Unlabeled ara-C, D-ribose, deoxy-D-ribose and v-arabinose from H. Mack, Illertissen; caesium chloride, anilinephthalate from E. Merck, Darmstadt; insta-gel from Packard Instrument, Ztirich; Sephadex G-25 from Deutsche Pharmacia, Frankfurt; thin layer plates (MNpolygramm, eel 300) from Macherey and Nagel, Dtiren; ara-C-3H labeled generally from Schwarz Bioresearch, Orangeburg (The

Accepted 17 J a n u a r y 1972. The abbreviations used are: ara-C: 1-fl-D-arabinofuranosylcytosine; a r a - C - D N A : ara-C- SH-labeled D N A ; DNase : deoxyribonuclease; dCyd : deoxyribosylcytosine; Cyt : cytosine.

391

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Rudolf K. Zahn, Werner E. G. M~ller, Wolf Forster, Armin Maidhof and Rudolf Beyer

ara-C-3H has been purified by thin layer chromatography in butanol-NH4OH, prior to use described later. The ara-C-3H, 12 ~mole per ml, had a specific activity of 1.1 Ci per mmole); DNase I (2000 units/mg) and phosphodiesterase (venom, potency -- 0.3-0.2 % AMP/0.25 mg) from Worthington, Freehold; alkaline phosphatase (calf intestine, activity -- 2500 /~mole of phosphate liberated per mg per 30 min, 37°C, pH 9.0) from Mann, New York.

2. Cells L5178y cells [11] were grown in Fischer's medium for leukemic cells from mice (Grand Island Biological Comp., Grand Island) in suspension cultures [12]. In a batch of 250 ml cells were inoculated at a concentration of 6 x 103/ml and incubated at 37°C in roller tubes. After 120 hr the cells reached the stationary phase and are, according to the data given by Tobey et al. [13], partially synchronized. The cells were transferred by centrifugation (800 × g, 20°C, 3 min) into 420 ml of fresh medium, containing 2.5raM thymidine. After 17hr 80 % of the cells were at the beginning of the S-phase. These cells after transfer into thymidine-free medium, containing purified 53 ng ara-C-3H/ml with a specific activity of 1.1 Ci/mmole were incubated; during 24 hr the cells run through a complete cell cycle. The cells were washed three times with 0.07 M K-phosphate buffer (pH 7.2) by centrifugation. 3. Extraction of D NA DNA from the cells was extracted by the method of Kirby [14]. The ratios of optical densities of the following wavelengths (mp) were 2 6 0 : 2 8 0 = 2.17, 2 6 0 : 2 4 0 = 1.58 and 260 : 230 = 1-93. 4. Digestion of ara-C-DNA The enzymic digestion of the DNA was performed in a pH-stat (Dosimat, Metrohm, Herisau). First the DNA was degraded by DNase I under limiting substrate conditions at 37°C and pH 7.0. The reaction mixture contained: 1-0 ml DNA solution (usually 200/~g/ ml) 0-1 ml 0.1 M MgC12 and 0.1 ml DNase I (1 mg/ml). The reaction was terminated when the enzyme reaction stopped, usually after 30 rain. The resulting DNA fragments were further hydrolyzed by the addition of phosphodiesterase and alkaline phosphatase at 37°C and pH 9-0 for 90 min. The reaction mixture contained the following components: 1.0 ml DNA products resulting from DNase I digestion, 0.1 ml 0.1 M MgC12, 0.15 ml phospho-

diesterase (1 rag/m1) and 0.1 ml alkaline phosphatase (1 mg/ml). For a complete hydrolysis the DNA fragments obtained by enzymic treatment of ara-CDNA were incubated in 2 N K O H at 100°C for 2 hr. The hydrolysate was neutralized with conc. HC104 and the resulting KC104 removed by centrifugation.

5. Analytical methods DNA was determined by the method of Kissane et al. [15], pentose in solution by the orcinol reaction described by I-San Lin et al. [16], pentose on thin layer plates with anilinephthalate [17]. The nucleotides, nucleosides and bases were located in ultra-violet light (Mineralight-u.v. lamp). Thin layer chromatography was performed in an ascending system on cellulose plates with water saturated nbutanol-15 N NH4OH solvent (100 : 1) [18]. In this solvent system the different RI values are: ara-C 0.32, dCyd 0.40, Cyt 0.38, D-arabinose 0.19, ~-ribose 0.31 and deoxy-D-ribose 0.45. For spotting the radioactivity, the developed chromatograms were cut into pieces (0.5 cm by 1.0 cm). Each strip was eluted with 0.4 ml of 0.1 N HC1. For determination of radioactivity samples of 0.05-0.2 ml were dissolved in 10 ml of insta-gel and counted in a scintillation counter (TriCarb Packard Inst., La Grange). Preparative centrifugation was performed at 20°C in a 40.3 rotor of the Spinco model L2-65 for 90 hr at 30 000 rev/min. The initial density of the CsC1 was adjusted to 1.700g/cm3; CsC1 was dissolved in 15raM tris (pH 8.0). Gradients were analyzed by collecting 25 fractions by displacement with saturated CsC1 solution. The optical density was measured in a Zeiss PMQ. 11 spectrophotometer. For concentrating of DNA, the samples were sedimented at 2°C in 0.1 M CsC1 solution in a 40.3 rotor at 30 000 rev/min for 12 hr. RESULTS

To synchronized mouse lymphoma cells (L5178y) ara-C was added 0.5hr before entering the S-phase. 53 ng ara-C per ml were added; this concentration is twice the EDs0 concentration (EDs0 is the concentration which induces in a dose-response experiment a 50% inhibition of cell proliferation). After a complete cell cycle cells were harvested and the DNA extracted. From 1.4 × 108 cells 810/~g of DNA could be obtained on the average. This corresponds to a yield of about 85 % (DNA content per lymphocyte: 6.8×10 -12 g; [19]). The

Incorporation into D NA specific activity for ara-C incorporation was 5-05 x 10-4 moles ara-C per mole DNA nucleotide. By this it can be calculated that about 2000 DNA nucleotides correspond to one molecule of ara-C. From the resulting DNA preparation the DNA was chromatographically separated on a Sephadex G-25 column (diameter: 6.8 mm; height: 77.5 cm; elution with 0.01 N NI-I4HCO3) from oligonucleotides and other

393

low molecular DNA components. The DNA fraction (Kd: 0-0.068) was subsequently purified from RNA residues by CsC1 densitygradient-centrifugation. The banding profile of the ara-C-DNA in the CsC1 gradient is shown in Fig. 1. A buoyant density of the ara-C-DNA of 1.7025 (g/cm -3) is found. The total radioactivity accompanies the DNA (range of buoyant densities from 1-67 to 1.75). This indicates that the radioactivity may be associated with

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Fig. 1. Buoyant density of ara-C-DNA. 50 ltl D N A isolated from ara-Co3H incubated L5178y cells, were transferred into a CsCl solution and centrifuged (see under methods). 25 fractions (0.4 ml) were collected and buoyant density (.--.) and optical density ( ~ ) were determined. For radioactivity (x - - x) 50 ltl aliquots were counted in a scintillation counter (see under Methods). x-axis : number of the fractions; yl-axis : optical density at 260 mp ; y2-axis : radioactivity, in dpm/5O ltl ; y3-axis : buoyant density in g/cm 3.

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4O XI Fig. 2. Chromatographic separation of D N A fragments, digested by D Nase 1, phosphodiesterase and alkaline phosphatase on Sephadex G-25 (as described uder methods). 0.5 ml of the hydrolysate corresponding to 25 pg ara-C-DNA have beenfractionated. Column: 12 mm ×81.7 cm; volume per fraction : 4 ml. Elution was performed with distilled water. xl-axis : fraction number; x 2-axis : RFvalues, pyrimidine-nucleotides : 0.68, purine nueleotides : 0"97, pyrimidine-nucleosides : 1.14, purinenudeosides 1-67. yz-axis: optical density at 260nm ( - - ) ; y2-axis: radioactivity (x - -) (dpm per 50 ld).

394

R u d o l f K. Zahn, Werner E. G. MiJller, W o l f Forster, Armin M a i d h o f and R u d o l f Beyer

the DNA. The RNA fraction (buoyant densities greater than 1.8) is free from radioactivity. The DNA fraction (density from 1.67-1.75 g x c m -3) was pooled and concentrated by sedimentation. Subsequently the DNA was digested enzymically with DNase I, phosphodiesterase and alkaline phosphatase. The hydrolysate when fractionated on Sephadex G-25 (Fig. 2), shows two symmetric peaks of radioactivity, one in the pyrimidine nucleotide and one in the nucleoside fraction. The volume of these two fractions was reduced by evaporation in vacuo at 20°C. Samples of 50 #1, containing 870 +80 dpm were chromatographed on thin layer plates (Fig. 3). Practically the total amount of radioactivity (747 zk80 dpm) of the applied fraction could be detected on the ara-C

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Separation of Cyt, D-arabinose and ara-C by thin layer chromatography on cellulose. xl-axis: line of sample application; 1 : KOH-hydrolysate of ara-C-3H-DNA, 2: Cyt, 3: D-arabinose and 4: ara-C. yl-axis: direction of development of chromatogram; trace 1 was cut into strips and eluted. The position of the other substances is indicated; x 2-axis : radioactivity in dpm ; y2-axis : number of strips. The two maxima of radioactivity in the y-direction coincide with the positions of cytosine and arabinose.

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Fig. 3. Chromatographic separation of ara-C and dCyd by thin layer chromatography on cellulose. Separation conditions as described under methods. xl-axis : line of sample application to chromatogram, 1 : 50/ll pyrimidine-nucleotide fraction from Sephadex G 75 separation, 2 : 5 itg of ara-C in 50 ld of water, 3 : 5 Izg of dCyd in 50 Ill of water; yx-axis : direction of development of chromatogram. Trace 1 was cut into strips and eluted; the position of ara-C (2) and dCyd (3) is indicated for comparison, x2-axis : radioactivity in dpm ; y 2-axis : number of strip measuredfrom trace 1. Maximum radioactivity in the y-direction coincides with the ara-C position.

spot while only 50 d p m + 1 0 were eluted from the dCyd spot. This fact indicates that at least ca. 90 % of the radioactivity incorporated into DNA is due to ara-C. The pyrimidine-nucleotide fraction (from Sephadex G-25) was also pooled and hydrolyzed again with DNase I, phosphodiesterase and alkaline phosphatase (the incubation time in this

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X.2~ Fig. 5. Separation of ara-C-DNA hydrolysate. xl-axis: line of sample application to chromatogram, 1: K OH-hydrolysate of ara-C-3H-D NA, 2 : arabinose, 3 : ribose, 4: deoxyribose, 5 : cytosine, yl-axis : direction of development. Trace 1 was cut into strips and eluted; x~-axis : radioactivity in dpm; y2-axis : number of strip measuredfrom trace 1. The two maxima of radioactivity in they-direction coincide with the positions of cytosine and arabinose, as in the test chromatogram Fig. 4.

Incorporation into D N A

case was 16 hr). After concentration in vacuo the hydrolyzed DNA products were fractionated on Sephadex G-25. Radioactivity could only be found in the nucleoside fractions (K a = 1.1-1.3). As described above this radioactivity is clearly due to ara-C. Other aliquots of enzymically digested ara-C-DNA fragments were hydrolyzed with K O H . By this method an almost complete cleavage of nucleosides into Cyt and D-arabinose is caused (Fig. 4). This K O H hydrolyzed sample was analyzed by thin layer chromatography. In the solvent used Cyt and D-arabinose can be separated from D-ribose and deoxyD-ribose. As shown in Fig. 5 almost the total amount of radioactivity could be recovered on the arabinose- and Cyt-spot.

DISCUSSION

The data presented in this paper show, that the suggested incorporation of ara-C into DNA [7, 8, 1] can be demonstrated thoroughly. The argument against this incorporation raised by Cohen [10] could be eliminated by the evidence that in the nucleoside- as well as in the nucleotide fraction arabinose is associated with labeled cytosine. We found that at least 87 % of the radioactivity incorporated into the D N A are due to ara-C. Only 6 % can be attributed to dCyd. Therefore we suggest that from all the ara-C molecules incorporated into DNA, less than 6 % are metabolically altered.

395

We found that per 1.98 x 103 moles of DNA nucleotide 1 mole of ara-C is incorporated into DNA. This ratio is much higher than that (2.7 x 104 : l) found by Silagi [1]. In contrast to the results of Silagi [1] and Chu et al. [20] we could not detect any appreciable ara-C incorporation into RNA. This divergence, in our opinion, is due to the different methods used for separation of R N A from DNA. The isolation method applied by us seems to be more selective for large R N A molecules of possibly slow metabolism. As ara-C incorporation into DNA is a fact the question arises if the inhibition of DNA synthesis in mammalian cells [1, 2] and viruses [3] is caused by this incorporation. It may be that by incorporation of ara-C into DNA a change in the structure of the DNA molecule results due to the more space filling trans C 2' O H groups. By this the proper function of the DNA polymerase may be affected. O n the other hand it can be expected that at least some inhibition observed in isolated DNA polymerizing enzyme systems [21, 22] via the triphosphate stage occurs also in intact biological systems. This will be discussed in another paper [23]. Acknowledgements--We are indebted to Miss Helga Taschner for excellent technical assistance. Our thanks are due to H. Mack GmbH, Illertissen for gifts of highly purified ara-C and pentoses. We gratefully acknowledge loans from the Deutsche Forschungsgemeinschaft.

REFERENCES

1. S. SILAGI,Metabolism of 1-fl-D-arabinofuranosylcytosine in L-cells. Cancer Res. 25, 1446 (1965). 2. A. DOEHRING,J. KELLER and S. S. COHEN, Some effects of D-arabinofuranosyl Nucleosides on polymer synthesis in mouse fibroblasts. Cancer Res. 26, 2444 (1966). 3. D.A. BUTHALA, Cell culture studies on antiviral agents: 1. Action of cytosine arabinoside and some comparison with 5-Jodo-2-deoxyuridine. Proc. Soc. exp. Biol. (N.Y.) 115, 69 (1964). 4. J. B. HOWARD, N. CEVlZ and M. L. MORPI-Iy, Cytosine arabinoside (NSC63878) in acute leukemia in children. Cancer Chemother. Rep. 50, 287 (1966). 5. I. WODINSKY and C. J. KELLER, Activity of cytosine arabinoside (NSC63878) in a spectrum of rodent tumors. CancerChemother. Rep. 47, 65 (1965). 6. F.L. GgAI-IA~t,G. F. WI-IITMORE,The Effect of 1-fl-D-arabinofuranosylcytosine on growth, viability, and DNA synthesis in mouse L-cells. Cancer Res. 30, 2627 (1970). 7. M.Y. CHU and G. A. FISCHER, The incorporation of 3H-cytosine arabinoside and its effect on murine leukemic cells (L5178y). Biochem. Pharmacol. 17, 753 (1968). 8. W.A. CREASY,R.. J. PAPAC,M. E. MARKIW,P. CALABRESIand A. D. WELCH, Biochemical and pharmacological studies with 1-fl-D-arabinofuranosylcytosine in man. Biochem. Pharmacol. 15, 1417 (1966). 9. R. L. MOMPARLER, Effect of cytosine arabinoside 5'-triphosphate on mammalian DNA polymerase. Biochem. Biophys. Res. Commum. :$4, 465 (1969).

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Rudolf K. Zahn, Werner E. G. Miiller, Wolf Forster, Armin Maidhof and Rudolf Beyer 10. S.S. COHEN,Introduction to the biochemistry of D-arabinosyl nucleotides. In Progress in Nucleic Acid Research and Molecular Biology (Edited by J. N. DAVIDSON and W. E. COHN) Vol. 5 pp. 1-88; Academic Press, New York (1966). 11. E. E. HALEY, G. A. FISCHER and A. D. WELCH, The Requirement for Lasparagine of mouse leukemic cells L5178y in Culture. Cancer Res. 21~ 532 (1961). 12. R . K . ZAHN,E. TIESLER,B. HEICKE,W. HANSKE,W. FORSTER,W. HOLLSTEIN and H. WALTER, Cellular division and cellular volume distribution in the presence of 2-phenylethanol and some of its derivatives. Nature (Lond.) 212, 298 (1966). 13. R . A . TOBEY and K. D. LEY, Regulation of initiation of DNA synthesis in Chinese hamster cells I. Production of stable, reversible Gx-arrested populations in suspension culture. J. Cell. Biol. 46~ 151 (1970). 14. K. S. KXRBY and E. A. CooK, Isolation of deoxyribonucleic acid from mammalian tissues. Biochem. J. 104~ 254 (1967). 15. J. M. KISSANEand E. ROBINS, The Fluorometric measurement of deoxyribonucleic acid in animal tissue with special reference to the central nervous system. J. Biol. Chem. 233~ 184 (1958). 16. R. I-SAN LIr~ and O. A. SCHJErDE, Micro estimation of RNA by the cupric ion catalyzed orcinol reaction. Anal. Biochem. 27~ 473 (1969). 17. W. PESCHKE, Beitrag zur Dtinnschicht-Chromatographie der Anionen von Halogensauerstoffs~uren. J. Chromatog. 20~ 572 (1965). 18. SCHWARZBIORESEARCH,Orangeburg, Technical Service, without year. 19. P. HAUSEN,H. STEINand H. PETERS, On the synthesis of RNA in lymphocytes stimulated by phytohemaglutinin. The activity of deoxyribonucleoproteinbound and soluble RNA polymerase. Europ. J. Biochem. 90~ 542 (1969). 20. M. Y. CHU and G. A. FISCHER, Comparative studies of leukemic cells sensitive and resistant to cytosine arabinoside. Biochem. Pharmacol. 14n 333 (1965). 21. F.L. GRAHAMand G. F. WHITMORE,Studies in mouse L-cells on the incorporation of 1-fl-D-arabinofuranosylcytosine into DNA and on inhibition of DNA polymerase by 1-fl-D-arabinofuranosylcytosine 5'-triphosphate. Cancer Res. 30, 2636 (1970). 22. J . j . FURTH and S. S. COHEN, Inhibition of mammalian DNA polymerase by the 5'-triphosphate of 1-fl-D-arabinofuranosylcytosine and the 5'-triphosphate of 9-fl-D-arabinosuranosyladenine. Cancer Res. 28~ 2061 (1968). 23. W . E . G . MT3LLER,Z. YAMAZAKr,H. H. S~OTROP and R. K. ZAHN, Action of 1-fl-D-arabinofuranosylcytosine on mammalian tumor cells. 2. Inhibition of mammalian and oncogenic viral polymerases. Europ. J. Cancer, 8~ 421-428 (1972).