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Action of 1-β-d -Arabinofuranosylcytosine on mammalian tumor cells—2
Europ. J. Cancer Vol. 8, pp. 421-428. Pergamon Press 1972. Printed in Great Britain
Action of 1-.8-D-Arabinofuranosylcytosine on Mammalian Tumor Cells 2. Inhibition of Mammalian and Oncogenic Viral Polymerases W E R N E R E. G. MI3LLER, ZEN-I. YAMAZAKI, HEINZ H. S O G T R O P and R U D O L F K. ZAHN Institut fiir Physiologische Chemie, Johannes Gutenberg Universit~t, 65 Mainz, Johann Joachim Becher Weg 13, West Germany
A b s t r a e t - - ( l ) 1-[3-D-Arabinofuranosylcytosinetriphosphate (ara-C TP ) is an inhibitor of: D NA-dependent D NA polymerase from mammalian cells, RNA-dependent D NA and D NA-dependent D NA polymerasefrom Rauscher murine leukemia virus (R M L V ) and R NA-dependent D NA polymerasefrom mouse lymphoma cells. (2) All these inhibitions are of the competitive type. The RNA-dependent DNA polymerasesfrom oncogenic RNA viruses and from lymphoma cells are found to be most sensitive. These enzymes are approximately 200 times as sensitive to ara-CTP as D NAdependent D NA polymerases.
that drug action may follow as a consequence of this incorporation. Several investigations explain ara-C action through inhibition of D N A synthesis at the level of DNA-dependent D N A polymerases [survey: 9]. Yet to explain antitumor action of ara-C [10], especially in leukemias [7, 11], other enzymes, specific for oncogenic R N A viruses [12, 13], should be considered [14]. One of the typical enzymes of such viruses [15-17] is RNA-dependent D N A polymerase. In some cases direct evidence for presence of virus has not been achieved while reverse transcriptase could be demonstrated. Thus Gallo et al. [18] and Bosmann [19] have shown human leukemic cells and L 5 1 7 8 y mouse lymphoma cells to contain RNA-dependent D N A polymerases. In view of these facts we discuss in this paper whether antitumor action of ara-C might be caused by inhibition of such an enzyme.
INTRODUCTION
IT IS known that ara-C inhibits the D N A synthesis of mammalian cells [1, 2], of D N A viruses [3, 4] and of malignant tumors [5-7]. The main target of ara-C is not yet known [review: 14]. In a preceding paper [8] we were able to show that ara-C molecules can be incorporated into D N A of L5178 y mouse lymphoma cells to a relatively large extent without any metabolic alteration. This suggests
Accepted 15 February 1972. The abbreviations used are: ara-C: 1-p-D-arabinofuranosyleytosine ara-CTP: 1-fl-D-arabinofuranosylcytosine-5'-triphosphate RMLV: Rauscher murine leukemia virus KM: Michaelis constant K~: Inhibitor constant 421
422
Werner E. G. Miiller, Zen-L Yamazaki, Heinz H. Sggtrop and Rudolf K. Zahn MATERIAL AND METHODS
(1) Source of materials The following materials were purchased: unlabeled deoxyribonucleoside triphosphates (dATP, d G T P and dTTP) from Schwarz Bio Research, Orangeburg; 3H-dGTP with a specific activity of 9-1 Ci/mmole from The Radiochemical Centre, Amersham; Nonidet NP 40 from Deutsche Shell Chemic Ges., Hamburg; dithiothreitol from Calbiochem, Los Angeles; micrococcal nuclease and deoxyribonuclease 1 (pancreas) from Worthington Biochemical Corp., Freehold and dialysis tubes (diameter 21 mm) from Visking Comp., Chicago. Ara-CTP was a gift from Dr. D. Gauchel (Universit~itsklinik, Dtisseldorf), who synthesized it chemically from ara-C [20]. The sample was chromatographically pure. The herring DNA was isolated according to Zahn et al. [21]. RNA from L 5178y cells was extracted by a method described by Joel et al. [22]. R M L V was obtained from spleens of infected N M R I mice and purified as described before [24]. The L 5178 y mouse lymphoma cells were cultured as described previously [25].
by a preincubation with micrococcal nuclease [27]. The extracts from mammalian cells contained 3 to 6 mg protein/ml, the virus preparation 0.3 mg protein/ml. (3) D N A polymerase assays The DNA-dependent DNA polymerase reaction mixture (0-1 ml) contained the following components: 40 m M Tris-HC1 (pH 7.8), 60 m M KC1, 2 m M (CH3COO)2Mg, 2 m M dithiothreitol, 0,1 m M each of dATP, dTTP, 3H-dGTP (5000 cpm/pmole), varying amounts of d C T P and ara-CTP, 20 pg native herring DNA (template saturating conditions) and 10 pl mammalian or R M L V enzyme preparation. The reaction mixture was incubated at 37°C for 15 min. The acid-precipitable radioactivity was collected and counted as previously described [24]. The assay conditions for RNA-dependent DNA polymerase were the same as described for DNA dependent DNA polymerase with the exception that DNA was replaced by RNA. For template saturation 200 pg RNA per ml were used. (4) Determination of protein, D N A and R N A
(2) Isolation of polymerases For preparation of crude extracts from mammalian ceils a modified method of Scolnick et al. [26] was used. Five grams of mammalian ceils were pulverized in a mortar together with dry ice, which was ultimately evaporated. The disrupted cells were suspended in 5 ml saturated (NH4)2SO~ containing 50 m M Tris-HCl, pH 7.8 and 0.1 m M EDTA and homogenized with 1 g of glass beads (diameter: 0.450.55 ram) in a cell homogenizer (type MSK, B. Braun, Melsungen) for 2 min. This preparation was centrifuged at 15 000 x g for 1 hr at 2°C. The pellet was resuspended in 5 ml buffer: 50 m M Tris-HC1, pH 7.8, 50 m M KCI and 1 m M dithiothreitol. A diluted solution of "Triton X-100" was added by drops to a final concentration of 0.3 % (v/v). This preparation was kept at 37°C for 15 min and centrifuged at 15 000 x g for 1 hr at 2°C. The supernatant was dialyzed for 12 hr at 2°C against 2000 ml buffer 1. The dialyzed extract was then assayed for enzyme activity. For observing full activities of the R M L V polymerases, the purified virus preparation was preincubated for 10min at O°C with 0"2 % Nonidet NP 40 and 0" 1 M dithiothreitol. In all experiments DNA and RNA, resident in the enzyme preparations, were destroyed
Protein determinations were performed according to Lowry et al. [28]. DNA and RNA were determined by the methods of Kissane et al. [23] respectively I-San-Lin et al. [29]. (5) Determination Of KM and Ki The calculation of the Michaelis constant of dCTP and the inhibitor constant of ara-CTP was based on double reciprocal plots [30] at two different inhibitor concentrations. Incorporation in the absence of d C T P during the polymerase reaction was subtracted from the data before plotting. In the presence of dATP, d G T P and dTTP, with d C T P missing, incorporation was 15 to 30 % of that obtained in'the presence of all four deoxynucleoside triphosphates. The Michaelis constants and the inhibitor constants (KM and Ki) for dCTP and ara-CTP were calculated according to Dixon et al. [31].
RESULTS
(1) Determination of Michaelis constants The experiments reported in Figs. 1 to 5 show linear relationships for the controls and araCTP-inhibited enzyme reactions. The double
Action of 1-fl-I)-Arabinofuranosylcytosine on Mammalian Tumor Cells--2
(2) Inhibition of D NA-dependent D NA polymerases
reciprocal plot of DNA synthesis versus d C T P concentration shows that the inhibitory action of ara-CTP can be abolished at high d C T P concentrations (Figs. 1 to 5 ) - - t h e straight lines intersect on the vertical axis--indicating that ara-CTP exerts its effect through competitive enzyme inhibition.
DNA-dependent DNA polymerases, extracted from three different sources: normal mouse liver, mouse lymphoma cells and R M L V , were tested for ara-CTP inhibition. Their plots are shown in Figs. 1 to 3. The ara-CTP inhibitor constant (Table 1) for the R M L V polymerase is about twice as high as the one for the mammalian enzymes. The Michaelis constant for d C T P of the virus enzyme is three to six times higher than that of the other two DNA-dependent DNA polymerases.
The KM and the Ki values calculated from the plots (Figs. 1 to 5) correspond to the equilibrium constants for the enzyme substrate and the enzyme inhibitor association reaction. T h e y are presented in Table 1. Table 1.
Quantitative comparison o f the Michaelis constants o f different D N A polymerases f o r d C T P and ara-CTP
Enzymes
Source of the enzyme
DNAodependent DNA polymerases
Mouse liver Mouse lymphoma cells L 5178 y Rauscher murine leukaemia virus
RNA-dependent DNA polymerases
Michaelis constant for d C T P (/tM)
Mouse lymphoma cells L 5178 y Rauscher murine leukemia virus
Inhibitor constant for ara-CTP (/tM)
10.2 + 5.9
10.7 _+6.7
21.6+ 10.5
18.4_+ 7.9
65.2 _+31 '9
25.7 _+ 14.0
15-7 _+8-5
0.05 + 0.03
19.3 _+9-1
0.09 _+0-05
x/
/ /
/
2-
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dcTP(pm~) Fig. 1. Competitive inhibition of DNA-dependent D N A polymerase from normal mouse liver by ara-CTP. Plot according to Lineweaver et al. [30]• The reaction conditions were as described in methods. The determination of the Ki of ara-CTP was carried out with 3 0 / t M ara-CTP (©) and 60 I~M ara-CTP ( × ) added to the reaction mixture. Control incubation was without ara-CTP ( 0 ) . x-axis: reciprocal values of dCTP concentration; y-axls: reciprocal values of the reaction velocity.
424
Werner E. G. Miiller, Zen-L Yamazaki, Heinz H. S6gtrop and Rudolf K. Zahn
7
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Fig. 2. Competitive inhibition of D NA-dependent D NA polymerasefrom mouse lymphoma cells L 5178y. Data for the control reaction ( 0 ) and for ara-CTP-inhibited reactions with 10 ,uM (©) and with 30 p M ( x ) have been plotted•
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Fig. 3. Competitive inhibition of DNA-dependent D N A polymerase from R M L V. Data for the control reaction ( 0 ) and for ara-CTP-inhibited reactions with 10/,t./l,1 (O) and with 3 0 / j M ( x ) have been plotted•
Action of l-p-D-Arabinofuranosylcytosine on Mammalian Tumor Cells 2
425
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Fig. 4. Competitive inhibition of RNA-dependent D N A polymerase from mouse lymphoma cells L 5178y. Data for the control reaction ( ~ ) and for ara-CTP-inhibited reactions with 0.I p M ( © ) and with 0"5 p M ( × ) have been plotted.
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Fig. 5. Competitive inhibition of RNA-dependent D N A polymerase from R M L V . Data for the control reaction ( 0 ) and for ara-CTP-inhibited reactions with 0.1 p M ( © ) and with 0.3 IJM ( × ) have been plotted.
426
Werner E. G. MiiUer, Zen-L Yamazaki, Heinz H. Sb'gtrop and Rudolf K. Zahn
(3) Inhibition of RNA-dependent D NA polymerases The activity of the R N A dependent DNA polymerase from mouse lymphoma cells and from R M L V can be competitively inhibited by ara-CTP also (Figs. 4, 5). The Michaelis constants of these two enzymes for d C T P are in the range (Table 1) of those determined for the mammalian DNA-dependent DNA polymerases. O n the other hand a large difference exists between the inhibitor constants of the t w o classes of polymerases for ara-CTP. The RNA-dependent DNA polymerases are about 200 times as sensitive for ara-CTP as the DNAdependent D N A polymerases. In normal mouse liver no RNA-dependent DNA polymerase activity could be detected. DISCUSSION
While there does not seem to exist any influence of ara-CTP on DNA-dependent R N A polymerases [32-34], DNA-dependent DNA polymerases from mammalian tissues are to a certain extent inhibited, with constants found to agree with G r a h a m et al. [33], but higher than those of Furth et al. [32]. The R M L V polymerase shows a twice higher inhibitor constant. In contrast the R M L V RNA-dependent DNA polymerase shows a remarkably high sensitivity towards ara-CTP: it is 200 times as sensitive. This m a y cause a corresponding susceptibility in R M L V and in mouse lymphoton cells. Since oncogenic viruses [ 12, 13, 15] as well as mammalian tissues including h u m a n ones infected by such viruses are known to contain the enzyme [14], the possibility to inhibit it seems quite interesting. Though the presence of RNA-dependent DNA polymerase in our L 5 1 7 8 y cells agrees with the results of Bosmann [19], for more conclusive evidence criteria of Spiegelman et al. [35] and of Gallo [14] have been applied by us: (a) the dependence of enzyme activity on R N A template, (b) the sensitivity of the reaction towards RNase, (c) the simultaneous requirement of all four deoxyribonucleoside triphosphates and (d) the proof that the product is DNA. In oncogenic R N A viruses a DNA-
dependent DNA polymerase is associated with a RNA-dependent DNA polymerase [16]. With the techniques applied in this study it was not possible to detect in extracts from mammalian cells this virus-specific DNAdependent DNA polymerase. The data given in this paper indicate that the Michaelis constant for d C T P measured in the DNA polymerizing system from mouse lymphoma cells is significantly higher than the value found for the one extracted from mouse liver. The data suggest that this higher constant might be caused by a virus DNA-dependent DNA polymerase possibly coexisting with the host DNAdependent DNA polymerase of the lymphoma cells, since the Michaelis constant for virus DNA-dependent DNA-polymerase was found to be considerably higher than the one for the host enzyme. Our results suggest that the RNA-dependent DNA polymerase, which in infection of mammalian cells by oncogenic RNA viruses possibly catalyzes one of the first steps of cell transformation or virus multiplication [14], could be inhibited by quite low concentrations of ara-CTP. Eidinoff et al. [36] have shown that the proliferation of the Rous sarcoma virus, which contains also the RNA-dependent DNA polymerase [13] can be inhibited by ara-C. This can be understood in view of the experiments of Momparler et al. [37], who showed that the ara-C molecules taken up by cells are readily phosphorylated to ara-CTP. O n the basis of these results and the data, found in the in vitro assays, presented here, it could be concluded that the cytostatical action of ara-C for mammalian cells, infected by oncogenic R N A viruses, is mainly due to a competitive inhibition of the RNA-dependent DNA polymerase by ara-CTP. This conclusion would suppose that the RNA-dependent DNA polymerase has become a necessary enzyme for multiplication of transformed cells.
A c k n o w l e d g e m e n t s - - T h e authors are most grateful to Fa. H. Mack, Illertissen for generous gifts of herring
DNA and 1-fl-v-arabinofuranosylcytosine.
REFERENCES
1. A. DOEHRING,J. KELLERand S. S. COHEN,Some effects ofD-Arabinosyl nucleosides on polymer synthesis in mouse fibroblasts. Cancer Res. 26, 2444 (1966). 2. S. SILAGI,Metabolism of 1-fl-D-arabinofuranosylcytosine in L-cells. Cancer Res. 25, 1446 (1965). 3. D.A. BUTHALA,Cell culture studies on antiviral agents. 1. Action of cytosine arabinoside and some comparison with 5-iodo-2-deoxyuridine. Proc. Soc. exp. Biol. Med. 115, 69 (1964).
Action of 1-fl-D-Arabinofuranosylcytosine on Mammalian Tumor Cells--2 4. G. E. UNDERWOOD,Activity of 1-fl-D-arabinofuranosylcytosine hydrochloride against herpes simplex keratitis. Proc. Soc. exp. Biol. Med. 105, 350 (1960). 5. C. HEIDELBEROER,Fluorinated pyrimidines. Progr. nucleic acid Res. 4, 1 (1965). 6. I. WODINSKYand C. J. KENSLER,Activity of cytosine arabinoside (NSC-63878) in a spectrum of rodent tumors. Cancer Chemother. Rep. 47, 65 (1965). 7. J.S. EVANS, E. A. MUSSER, G. D. MENOEL, K. R. FORSBLAD and J. H. HUNTER, Antitumor activityof l-fl-D-arabinofuranosylcytosinehydrochloride. Proc. Soc. exp. Biol. Med. 106, 350 (1960). 8. R. K. ZAHN, W. E. G. MOLLER, W. FORSTER, A. MAIDHOF and R. BE'e~R, Action of 1-fl-D-arabinofuranosylcytosine on mammalian tumor cells--1. Incorporation into DNA. Europ. or. Cancer. 8, 391-396 (1972). 9. F . L . GRAHAMand G. F. WHIT~ORE, The effect of l-fl-D-arabinofuranosylcytosine of growth viability, and DNA synthesis of mouse L-cells. Cancer Res. 30, 2627 (1970). 10. D. M. GOLDENBERG, V. BIRO, K. ELSTER, K. TH. SCHRICKER and H. H. S6OTROP, ExperimenteUe und klinische Erfahrungen mit Cytosin-Arabinosid bei soliden Tumoren und Haemoblastosen (Edited by S. Wr,-,'~ and R. K. ZAHN), p. 31, Editio Cantor Aulendorf (1968). 11. J. BROOK,J. L. SCOTTand R. E. MASS, Intermittent cytosine arabinoside in the treatment of acute leukemia. Proc. Amer. Ass. Cancer Res. 9, 32 (1968). 12. D. BALTIMORE,RNA-dependent DNA Polymerase in virions of RNA tumor viruses. Nature 226, 1209 (1970). 13. H . M . TEmN and S. MIZUTANI,RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226, 1211 (1970). 14. R . C . CTALLO,RNA-dependent DNA polymerase in viruses and cells: views on the current state. Blood. In press. 15. S. SPIEOELMAN, A. BURNY, M. R. DAS, J. KEYDAR, J. SCHLOM, M. TRAVNICEK and K. WATSON, Characterization of the products of RNA-directed D N A polymerases in oncogenic R N A viruses.Nature 227, 563 (I970). 16. S. SPIEGELMAN,A. BURNY, M. R. DAS,J. KEYDAR,J. SCHLOM,M. TRAVNICEK and K. WATSON, DNA-directed DNA polymerase activity in oncogenic RNA viruses. Nature 227, 1029 (1970). 17. R. C. GALLO, P. S. SARXN,P. T. ALLEN, W. A. NEWTON, E. S. Pmom, J. M. BOWEN and L. DMOCHOWSKI,Reverse transcriptase in type C virus particles of human origin. Nature, New Biol. 232, 140 (1971). 18. R . C . GALLO,S. S. YANOand R. C. TINO, RNA dependent DNA polymerase of human acute leukaemia cells. Nature 228, 927 (1970). 19. H.B. BOSMANN,RNA directed DNA synthesis : Identification in L 5178 y mouse leukemic cells and distribution of the polymerase in a synchronized L 5178 y cell population. FEBS Lett. 13, 121 (1971). 20. D. GAUCHEL,Die Synthese yon 1-fl-D-Arabinofuranosylcytosin-5' Triphosphat. Diplomarbeit der naturwissenchaftlichen FakultAt, Mainz (1968). 21. R.K. ZAHN,E. TIESLER,A. K. KLEINSCHMIDTund D. LANO,Ein Konservierungsund Darstellungs-Verfahren ffir Desoxyribonucleinsituren und ihre Ausgangsmaterialien. Biochem. Z. 336, 281 (1962). 22. P. B. JOEL and D. D. HAOERMAN,Extraction of RNA from rat uterus. Biochim. biophys. Acta 195, 328 (1969). 23. J. M. KlSSANE and 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). 24. W . E . G . MOLLER,R. K. ZAI-INand H. J. SEIDEL,Inhibitors acting on nucleic acid synthesis in an oncogenic RNA virus. Nature, New Biol. 232, 143 (1971). 25. R . K . ZAHN, E. TIESLER, B. HEmKE, W. HANSr,~, W. FORSaXR, H. HOLLSTEIN and H. WALTER,Cellular division and cellular volume distribution the presence of 2-phenylethanol and some of its derivatives. Nature 212, 298 (1966). 26. E. M. SCOLNICK, S. A. AARONSON, G. J. TADARO and W. S. PARKS, RNA dependent DNA polymerase activity in mammalian cells. Nature 229, 318 (1971). 27. W . E . G . MULLER,Z. YAMAZAKI,R. K. ZAHN, G. BREI-IMand G. KORTINO, RNA dependent DNA polymerase in cells ofxeroderma pigmentosum. Biochem. biophys. Res. Commun. 44, 433 (1971). 28. O. H. LOWRY, N. J. ROSEBROUOH, A. L. FARR and R. S. RANDALL,Protein measurements with the folin phenol reagent. J. Biochem. 193, 265 (1951).
427
428
Werner E. G. Miiller, Zen-L Yamazaki, Heinz H. SSgtrop and Rudolf K. Zahn 29. R. I°SAN-LIN and O. A. S(~HJEIDE,Micro estimation of RNA by the cupric ion catalyzed orcinol reaction. Anal. Biochem. 279 473 (1969). 30. H. LINEWEAVER and D. BURK, The determination of enzyme dissociation constants. J. Amer. chem. Soc. 569 408 (1934). 31. M. DIXON and E. C. WEBB,Enzyme. Longmans Green and Co., London (1966). 32. J.J. FURTH and S. S. COHEN, Inhibition of mammalian DNA polyrnerase by the 5'-triphosphate of 1-fl-D-arabinofuranosylcytosine and the 5'-triphosphate of 9-fl-D-arabinofuranosyladenine. Cancer Res. 211, 2061 (1968). 33. F . L . GRAHAMand G. F. WHITMORE,Studies on mouse L-cells on the incorporation of 1-fl-D-arabinofuranosylcytosine 5'-triphosphate. Cancer Res. 302 2636 (1970). 34. P . T . CARDEILHACand S. S. COHEN,Some metabolic properties of nucleotides of 1-fl-D-arabinofuranosylcytosine. Cancer Res. 249 1595 (1964). 35. J. SCHLOM,S. SPIEOELMANand D. MOORE,RNA-dependent DNA polymerase activity in virus-like particles isolated from human milk. Nature, New Biol. 231, 97 (1971). 36. M . L . EIDINOFF, B. BATESand M. STEINOLASS,Requirement for DNA synthesis in proliferation of Rous sarcoma virus (RSV). Proc. Am. Assoc. Cancer Res. 69 17 (1965). 37. R . L . MOMPARLER, M. Y. CHU and G. A. FISCHER, Studies on a new mechanism of resistance of L 5178 y murine leukemia cells to cytosine arabinoside. Biochim. biophys. Acta 1619 481 (1968).
Action of 1-.8-D-Arabinofuranosylcytosine on Mammalian Tumor Cells 2. Inhibition of Mammalian and Oncogenic Viral Polymerases W E R N E R E. G. MI3LLER, ZEN-I. YAMAZAKI, HEINZ H. S O G T R O P and R U D O L F K. ZAHN Institut fiir Physiologische Chemie, Johannes Gutenberg Universit~t, 65 Mainz, Johann Joachim Becher Weg 13, West Germany
A b s t r a e t - - ( l ) 1-[3-D-Arabinofuranosylcytosinetriphosphate (ara-C TP ) is an inhibitor of: D NA-dependent D NA polymerase from mammalian cells, RNA-dependent D NA and D NA-dependent D NA polymerasefrom Rauscher murine leukemia virus (R M L V ) and R NA-dependent D NA polymerasefrom mouse lymphoma cells. (2) All these inhibitions are of the competitive type. The RNA-dependent DNA polymerasesfrom oncogenic RNA viruses and from lymphoma cells are found to be most sensitive. These enzymes are approximately 200 times as sensitive to ara-CTP as D NAdependent D NA polymerases.
that drug action may follow as a consequence of this incorporation. Several investigations explain ara-C action through inhibition of D N A synthesis at the level of DNA-dependent D N A polymerases [survey: 9]. Yet to explain antitumor action of ara-C [10], especially in leukemias [7, 11], other enzymes, specific for oncogenic R N A viruses [12, 13], should be considered [14]. One of the typical enzymes of such viruses [15-17] is RNA-dependent D N A polymerase. In some cases direct evidence for presence of virus has not been achieved while reverse transcriptase could be demonstrated. Thus Gallo et al. [18] and Bosmann [19] have shown human leukemic cells and L 5 1 7 8 y mouse lymphoma cells to contain RNA-dependent D N A polymerases. In view of these facts we discuss in this paper whether antitumor action of ara-C might be caused by inhibition of such an enzyme.
INTRODUCTION
IT IS known that ara-C inhibits the D N A synthesis of mammalian cells [1, 2], of D N A viruses [3, 4] and of malignant tumors [5-7]. The main target of ara-C is not yet known [review: 14]. In a preceding paper [8] we were able to show that ara-C molecules can be incorporated into D N A of L5178 y mouse lymphoma cells to a relatively large extent without any metabolic alteration. This suggests
Accepted 15 February 1972. The abbreviations used are: ara-C: 1-p-D-arabinofuranosyleytosine ara-CTP: 1-fl-D-arabinofuranosylcytosine-5'-triphosphate RMLV: Rauscher murine leukemia virus KM: Michaelis constant K~: Inhibitor constant 421
422
Werner E. G. Miiller, Zen-L Yamazaki, Heinz H. Sggtrop and Rudolf K. Zahn MATERIAL AND METHODS
(1) Source of materials The following materials were purchased: unlabeled deoxyribonucleoside triphosphates (dATP, d G T P and dTTP) from Schwarz Bio Research, Orangeburg; 3H-dGTP with a specific activity of 9-1 Ci/mmole from The Radiochemical Centre, Amersham; Nonidet NP 40 from Deutsche Shell Chemic Ges., Hamburg; dithiothreitol from Calbiochem, Los Angeles; micrococcal nuclease and deoxyribonuclease 1 (pancreas) from Worthington Biochemical Corp., Freehold and dialysis tubes (diameter 21 mm) from Visking Comp., Chicago. Ara-CTP was a gift from Dr. D. Gauchel (Universit~itsklinik, Dtisseldorf), who synthesized it chemically from ara-C [20]. The sample was chromatographically pure. The herring DNA was isolated according to Zahn et al. [21]. RNA from L 5178y cells was extracted by a method described by Joel et al. [22]. R M L V was obtained from spleens of infected N M R I mice and purified as described before [24]. The L 5178 y mouse lymphoma cells were cultured as described previously [25].
by a preincubation with micrococcal nuclease [27]. The extracts from mammalian cells contained 3 to 6 mg protein/ml, the virus preparation 0.3 mg protein/ml. (3) D N A polymerase assays The DNA-dependent DNA polymerase reaction mixture (0-1 ml) contained the following components: 40 m M Tris-HC1 (pH 7.8), 60 m M KC1, 2 m M (CH3COO)2Mg, 2 m M dithiothreitol, 0,1 m M each of dATP, dTTP, 3H-dGTP (5000 cpm/pmole), varying amounts of d C T P and ara-CTP, 20 pg native herring DNA (template saturating conditions) and 10 pl mammalian or R M L V enzyme preparation. The reaction mixture was incubated at 37°C for 15 min. The acid-precipitable radioactivity was collected and counted as previously described [24]. The assay conditions for RNA-dependent DNA polymerase were the same as described for DNA dependent DNA polymerase with the exception that DNA was replaced by RNA. For template saturation 200 pg RNA per ml were used. (4) Determination of protein, D N A and R N A
(2) Isolation of polymerases For preparation of crude extracts from mammalian ceils a modified method of Scolnick et al. [26] was used. Five grams of mammalian ceils were pulverized in a mortar together with dry ice, which was ultimately evaporated. The disrupted cells were suspended in 5 ml saturated (NH4)2SO~ containing 50 m M Tris-HCl, pH 7.8 and 0.1 m M EDTA and homogenized with 1 g of glass beads (diameter: 0.450.55 ram) in a cell homogenizer (type MSK, B. Braun, Melsungen) for 2 min. This preparation was centrifuged at 15 000 x g for 1 hr at 2°C. The pellet was resuspended in 5 ml buffer: 50 m M Tris-HC1, pH 7.8, 50 m M KCI and 1 m M dithiothreitol. A diluted solution of "Triton X-100" was added by drops to a final concentration of 0.3 % (v/v). This preparation was kept at 37°C for 15 min and centrifuged at 15 000 x g for 1 hr at 2°C. The supernatant was dialyzed for 12 hr at 2°C against 2000 ml buffer 1. The dialyzed extract was then assayed for enzyme activity. For observing full activities of the R M L V polymerases, the purified virus preparation was preincubated for 10min at O°C with 0"2 % Nonidet NP 40 and 0" 1 M dithiothreitol. In all experiments DNA and RNA, resident in the enzyme preparations, were destroyed
Protein determinations were performed according to Lowry et al. [28]. DNA and RNA were determined by the methods of Kissane et al. [23] respectively I-San-Lin et al. [29]. (5) Determination Of KM and Ki The calculation of the Michaelis constant of dCTP and the inhibitor constant of ara-CTP was based on double reciprocal plots [30] at two different inhibitor concentrations. Incorporation in the absence of d C T P during the polymerase reaction was subtracted from the data before plotting. In the presence of dATP, d G T P and dTTP, with d C T P missing, incorporation was 15 to 30 % of that obtained in'the presence of all four deoxynucleoside triphosphates. The Michaelis constants and the inhibitor constants (KM and Ki) for dCTP and ara-CTP were calculated according to Dixon et al. [31].
RESULTS
(1) Determination of Michaelis constants The experiments reported in Figs. 1 to 5 show linear relationships for the controls and araCTP-inhibited enzyme reactions. The double
Action of 1-fl-I)-Arabinofuranosylcytosine on Mammalian Tumor Cells--2
(2) Inhibition of D NA-dependent D NA polymerases
reciprocal plot of DNA synthesis versus d C T P concentration shows that the inhibitory action of ara-CTP can be abolished at high d C T P concentrations (Figs. 1 to 5 ) - - t h e straight lines intersect on the vertical axis--indicating that ara-CTP exerts its effect through competitive enzyme inhibition.
DNA-dependent DNA polymerases, extracted from three different sources: normal mouse liver, mouse lymphoma cells and R M L V , were tested for ara-CTP inhibition. Their plots are shown in Figs. 1 to 3. The ara-CTP inhibitor constant (Table 1) for the R M L V polymerase is about twice as high as the one for the mammalian enzymes. The Michaelis constant for d C T P of the virus enzyme is three to six times higher than that of the other two DNA-dependent DNA polymerases.
The KM and the Ki values calculated from the plots (Figs. 1 to 5) correspond to the equilibrium constants for the enzyme substrate and the enzyme inhibitor association reaction. T h e y are presented in Table 1. Table 1.
Quantitative comparison o f the Michaelis constants o f different D N A polymerases f o r d C T P and ara-CTP
Enzymes
Source of the enzyme
DNAodependent DNA polymerases
Mouse liver Mouse lymphoma cells L 5178 y Rauscher murine leukaemia virus
RNA-dependent DNA polymerases
Michaelis constant for d C T P (/tM)
Mouse lymphoma cells L 5178 y Rauscher murine leukemia virus
Inhibitor constant for ara-CTP (/tM)
10.2 + 5.9
10.7 _+6.7
21.6+ 10.5
18.4_+ 7.9
65.2 _+31 '9
25.7 _+ 14.0
15-7 _+8-5
0.05 + 0.03
19.3 _+9-1
0.09 _+0-05
x/
/ /
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2-
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-OI.l
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dcTP(pm~) Fig. 1. Competitive inhibition of DNA-dependent D N A polymerase from normal mouse liver by ara-CTP. Plot according to Lineweaver et al. [30]• The reaction conditions were as described in methods. The determination of the Ki of ara-CTP was carried out with 3 0 / t M ara-CTP (©) and 60 I~M ara-CTP ( × ) added to the reaction mixture. Control incubation was without ara-CTP ( 0 ) . x-axis: reciprocal values of dCTP concentration; y-axls: reciprocal values of the reaction velocity.
424
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7
P
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Fig. 2. Competitive inhibition of D NA-dependent D NA polymerasefrom mouse lymphoma cells L 5178y. Data for the control reaction ( 0 ) and for ara-CTP-inhibited reactions with 10 ,uM (©) and with 30 p M ( x ) have been plotted•
1
¢:
/'x
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Fig. 3. Competitive inhibition of DNA-dependent D N A polymerase from R M L V. Data for the control reaction ( 0 ) and for ara-CTP-inhibited reactions with 10/,t./l,1 (O) and with 3 0 / j M ( x ) have been plotted•
Action of l-p-D-Arabinofuranosylcytosine on Mammalian Tumor Cells 2
425
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/
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Fig. 4. Competitive inhibition of RNA-dependent D N A polymerase from mouse lymphoma cells L 5178y. Data for the control reaction ( ~ ) and for ara-CTP-inhibited reactions with 0.I p M ( © ) and with 0"5 p M ( × ) have been plotted.
/
o
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Fig. 5. Competitive inhibition of RNA-dependent D N A polymerase from R M L V . Data for the control reaction ( 0 ) and for ara-CTP-inhibited reactions with 0.1 p M ( © ) and with 0.3 IJM ( × ) have been plotted.
426
Werner E. G. MiiUer, Zen-L Yamazaki, Heinz H. Sb'gtrop and Rudolf K. Zahn
(3) Inhibition of RNA-dependent D NA polymerases The activity of the R N A dependent DNA polymerase from mouse lymphoma cells and from R M L V can be competitively inhibited by ara-CTP also (Figs. 4, 5). The Michaelis constants of these two enzymes for d C T P are in the range (Table 1) of those determined for the mammalian DNA-dependent DNA polymerases. O n the other hand a large difference exists between the inhibitor constants of the t w o classes of polymerases for ara-CTP. The RNA-dependent DNA polymerases are about 200 times as sensitive for ara-CTP as the DNAdependent D N A polymerases. In normal mouse liver no RNA-dependent DNA polymerase activity could be detected. DISCUSSION
While there does not seem to exist any influence of ara-CTP on DNA-dependent R N A polymerases [32-34], DNA-dependent DNA polymerases from mammalian tissues are to a certain extent inhibited, with constants found to agree with G r a h a m et al. [33], but higher than those of Furth et al. [32]. The R M L V polymerase shows a twice higher inhibitor constant. In contrast the R M L V RNA-dependent DNA polymerase shows a remarkably high sensitivity towards ara-CTP: it is 200 times as sensitive. This m a y cause a corresponding susceptibility in R M L V and in mouse lymphoton cells. Since oncogenic viruses [ 12, 13, 15] as well as mammalian tissues including h u m a n ones infected by such viruses are known to contain the enzyme [14], the possibility to inhibit it seems quite interesting. Though the presence of RNA-dependent DNA polymerase in our L 5 1 7 8 y cells agrees with the results of Bosmann [19], for more conclusive evidence criteria of Spiegelman et al. [35] and of Gallo [14] have been applied by us: (a) the dependence of enzyme activity on R N A template, (b) the sensitivity of the reaction towards RNase, (c) the simultaneous requirement of all four deoxyribonucleoside triphosphates and (d) the proof that the product is DNA. In oncogenic R N A viruses a DNA-
dependent DNA polymerase is associated with a RNA-dependent DNA polymerase [16]. With the techniques applied in this study it was not possible to detect in extracts from mammalian cells this virus-specific DNAdependent DNA polymerase. The data given in this paper indicate that the Michaelis constant for d C T P measured in the DNA polymerizing system from mouse lymphoma cells is significantly higher than the value found for the one extracted from mouse liver. The data suggest that this higher constant might be caused by a virus DNA-dependent DNA polymerase possibly coexisting with the host DNAdependent DNA polymerase of the lymphoma cells, since the Michaelis constant for virus DNA-dependent DNA-polymerase was found to be considerably higher than the one for the host enzyme. Our results suggest that the RNA-dependent DNA polymerase, which in infection of mammalian cells by oncogenic RNA viruses possibly catalyzes one of the first steps of cell transformation or virus multiplication [14], could be inhibited by quite low concentrations of ara-CTP. Eidinoff et al. [36] have shown that the proliferation of the Rous sarcoma virus, which contains also the RNA-dependent DNA polymerase [13] can be inhibited by ara-C. This can be understood in view of the experiments of Momparler et al. [37], who showed that the ara-C molecules taken up by cells are readily phosphorylated to ara-CTP. O n the basis of these results and the data, found in the in vitro assays, presented here, it could be concluded that the cytostatical action of ara-C for mammalian cells, infected by oncogenic R N A viruses, is mainly due to a competitive inhibition of the RNA-dependent DNA polymerase by ara-CTP. This conclusion would suppose that the RNA-dependent DNA polymerase has become a necessary enzyme for multiplication of transformed cells.
A c k n o w l e d g e m e n t s - - T h e authors are most grateful to Fa. H. Mack, Illertissen for generous gifts of herring
DNA and 1-fl-v-arabinofuranosylcytosine.
REFERENCES
1. A. DOEHRING,J. KELLERand S. S. COHEN,Some effects ofD-Arabinosyl nucleosides on polymer synthesis in mouse fibroblasts. Cancer Res. 26, 2444 (1966). 2. S. SILAGI,Metabolism of 1-fl-D-arabinofuranosylcytosine in L-cells. Cancer Res. 25, 1446 (1965). 3. D.A. BUTHALA,Cell culture studies on antiviral agents. 1. Action of cytosine arabinoside and some comparison with 5-iodo-2-deoxyuridine. Proc. Soc. exp. Biol. Med. 115, 69 (1964).
Action of 1-fl-D-Arabinofuranosylcytosine on Mammalian Tumor Cells--2 4. G. E. UNDERWOOD,Activity of 1-fl-D-arabinofuranosylcytosine hydrochloride against herpes simplex keratitis. Proc. Soc. exp. Biol. Med. 105, 350 (1960). 5. C. HEIDELBEROER,Fluorinated pyrimidines. Progr. nucleic acid Res. 4, 1 (1965). 6. I. WODINSKYand C. J. KENSLER,Activity of cytosine arabinoside (NSC-63878) in a spectrum of rodent tumors. Cancer Chemother. Rep. 47, 65 (1965). 7. J.S. EVANS, E. A. MUSSER, G. D. MENOEL, K. R. FORSBLAD and J. H. HUNTER, Antitumor activityof l-fl-D-arabinofuranosylcytosinehydrochloride. Proc. Soc. exp. Biol. Med. 106, 350 (1960). 8. R. K. ZAHN, W. E. G. MOLLER, W. FORSTER, A. MAIDHOF and R. BE'e~R, Action of 1-fl-D-arabinofuranosylcytosine on mammalian tumor cells--1. Incorporation into DNA. Europ. or. Cancer. 8, 391-396 (1972). 9. F . L . GRAHAMand G. F. WHIT~ORE, The effect of l-fl-D-arabinofuranosylcytosine of growth viability, and DNA synthesis of mouse L-cells. Cancer Res. 30, 2627 (1970). 10. D. M. GOLDENBERG, V. BIRO, K. ELSTER, K. TH. SCHRICKER and H. H. S6OTROP, ExperimenteUe und klinische Erfahrungen mit Cytosin-Arabinosid bei soliden Tumoren und Haemoblastosen (Edited by S. Wr,-,'~ and R. K. ZAHN), p. 31, Editio Cantor Aulendorf (1968). 11. J. BROOK,J. L. SCOTTand R. E. MASS, Intermittent cytosine arabinoside in the treatment of acute leukemia. Proc. Amer. Ass. Cancer Res. 9, 32 (1968). 12. D. BALTIMORE,RNA-dependent DNA Polymerase in virions of RNA tumor viruses. Nature 226, 1209 (1970). 13. H . M . TEmN and S. MIZUTANI,RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226, 1211 (1970). 14. R . C . CTALLO,RNA-dependent DNA polymerase in viruses and cells: views on the current state. Blood. In press. 15. S. SPIEOELMAN, A. BURNY, M. R. DAS, J. KEYDAR, J. SCHLOM, M. TRAVNICEK and K. WATSON, Characterization of the products of RNA-directed D N A polymerases in oncogenic R N A viruses.Nature 227, 563 (I970). 16. S. SPIEGELMAN,A. BURNY, M. R. DAS,J. KEYDAR,J. SCHLOM,M. TRAVNICEK and K. WATSON, DNA-directed DNA polymerase activity in oncogenic RNA viruses. Nature 227, 1029 (1970). 17. R. C. GALLO, P. S. SARXN,P. T. ALLEN, W. A. NEWTON, E. S. Pmom, J. M. BOWEN and L. DMOCHOWSKI,Reverse transcriptase in type C virus particles of human origin. Nature, New Biol. 232, 140 (1971). 18. R . C . GALLO,S. S. YANOand R. C. TINO, RNA dependent DNA polymerase of human acute leukaemia cells. Nature 228, 927 (1970). 19. H.B. BOSMANN,RNA directed DNA synthesis : Identification in L 5178 y mouse leukemic cells and distribution of the polymerase in a synchronized L 5178 y cell population. FEBS Lett. 13, 121 (1971). 20. D. GAUCHEL,Die Synthese yon 1-fl-D-Arabinofuranosylcytosin-5' Triphosphat. Diplomarbeit der naturwissenchaftlichen FakultAt, Mainz (1968). 21. R.K. ZAHN,E. TIESLER,A. K. KLEINSCHMIDTund D. LANO,Ein Konservierungsund Darstellungs-Verfahren ffir Desoxyribonucleinsituren und ihre Ausgangsmaterialien. Biochem. Z. 336, 281 (1962). 22. P. B. JOEL and D. D. HAOERMAN,Extraction of RNA from rat uterus. Biochim. biophys. Acta 195, 328 (1969). 23. J. M. KlSSANE and 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). 24. W . E . G . MOLLER,R. K. ZAI-INand H. J. SEIDEL,Inhibitors acting on nucleic acid synthesis in an oncogenic RNA virus. Nature, New Biol. 232, 143 (1971). 25. R . K . ZAHN, E. TIESLER, B. HEmKE, W. HANSr,~, W. FORSaXR, H. HOLLSTEIN and H. WALTER,Cellular division and cellular volume distribution the presence of 2-phenylethanol and some of its derivatives. Nature 212, 298 (1966). 26. E. M. SCOLNICK, S. A. AARONSON, G. J. TADARO and W. S. PARKS, RNA dependent DNA polymerase activity in mammalian cells. Nature 229, 318 (1971). 27. W . E . G . MULLER,Z. YAMAZAKI,R. K. ZAHN, G. BREI-IMand G. KORTINO, RNA dependent DNA polymerase in cells ofxeroderma pigmentosum. Biochem. biophys. Res. Commun. 44, 433 (1971). 28. O. H. LOWRY, N. J. ROSEBROUOH, A. L. FARR and R. S. RANDALL,Protein measurements with the folin phenol reagent. J. Biochem. 193, 265 (1951).
427
428
Werner E. G. Miiller, Zen-L Yamazaki, Heinz H. SSgtrop and Rudolf K. Zahn 29. R. I°SAN-LIN and O. A. S(~HJEIDE,Micro estimation of RNA by the cupric ion catalyzed orcinol reaction. Anal. Biochem. 279 473 (1969). 30. H. LINEWEAVER and D. BURK, The determination of enzyme dissociation constants. J. Amer. chem. Soc. 569 408 (1934). 31. M. DIXON and E. C. WEBB,Enzyme. Longmans Green and Co., London (1966). 32. J.J. FURTH and S. S. COHEN, Inhibition of mammalian DNA polyrnerase by the 5'-triphosphate of 1-fl-D-arabinofuranosylcytosine and the 5'-triphosphate of 9-fl-D-arabinofuranosyladenine. Cancer Res. 211, 2061 (1968). 33. F . L . GRAHAMand G. F. WHITMORE,Studies on mouse L-cells on the incorporation of 1-fl-D-arabinofuranosylcytosine 5'-triphosphate. Cancer Res. 302 2636 (1970). 34. P . T . CARDEILHACand S. S. COHEN,Some metabolic properties of nucleotides of 1-fl-D-arabinofuranosylcytosine. Cancer Res. 249 1595 (1964). 35. J. SCHLOM,S. SPIEOELMANand D. MOORE,RNA-dependent DNA polymerase activity in virus-like particles isolated from human milk. Nature, New Biol. 231, 97 (1971). 36. M . L . EIDINOFF, B. BATESand M. STEINOLASS,Requirement for DNA synthesis in proliferation of Rous sarcoma virus (RSV). Proc. Am. Assoc. Cancer Res. 69 17 (1965). 37. R . L . MOMPARLER, M. Y. CHU and G. A. FISCHER, Studies on a new mechanism of resistance of L 5178 y murine leukemia cells to cytosine arabinoside. Biochim. biophys. Acta 1619 481 (1968).