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5-Fluorouracil-Methotrexate Synergy: Enhancement of By Proc. Natl. Acad. Sci. USA Vol. 77, No. 10, pp. 5663-5667, October 1980 Biochemistry 5-Fluorouracil-methotrexate synergy: Enhancement of 5-fluorodeoxyuridylate binding to thymidylate synthase by dihydropteroylpolyglutamates (vterovlvolvglutamates/ternary enzyme complexes/combination chemotherapy) DANIEL J. FERNANDES AND JOSEPH R. BERTINO Departments of Pharmacology and Medicine, Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Alfred Gilman, June 9, 1980 ABSTRACT Ternary complex formation of thymidylate FdUMP and purified L1210 thymidylate synthase because of synthase (5,10-methylenetetrahydrofolate:dUMP C-methyl- (i) the preponderance of reduced pteroyl and MTX polyglu- transferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP), and tamates in mammalian cells and their functions as the poly(I-glutamyl) conjugates of pteroate and methotrexate (MTX) probable has been examined as a basis for the sequence-dependent syn- active cofactors (13, 14) or inhibitors (15, 16) of thymidylate ergism of the 5-fluorouracil-MTX combination in inhibiting biosynthesis in vivo and (ii) the possible contribution of the viability of L1210 murine tumor cells. A 1.4-log (25-fold) increase formation of ternary complexes to the 'synergistic antitumor in the inhibition of soft agar colony formation was observed effect observed when FUra and MTX are used in combination when MTX preceded 5-fluorouracil, as compared to the reverse cancer chemotherapy. This synergistic effect has been noted sequence. L1210 cells converted 39% of the total intracellular when either tumor-bearing mice (17, 18) or tumor cells MTX into MTX poly(y-glutamate)s within 4 hr of exposure to only 1 gM MTX. MTX and MTX('y-glutamate) formed reversible in culture (19) are exposed to MTX prior to FUra. ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8 ihydropteroylpentaglutamate and MATERIALS AND METHODS 1-5,10-methylenetetrahydropteroylpentaglutamate stoichio- Materials. MTX (Na salt) was obtained from the Division metric binding of FdUMP to two sites on thymidylate synthase Institute. was observed. The dissociation constants for FdUMP in the of Cancer Treatment, National Cancer MTX(Glul) ternary complexes formed in the presence of MTX, MTX('y- was the generous gift of Samuel Jacobs, University of Pittsburgh glutamate), 7,8-dihydropteroylpentaglutamate, and 1-5-10- School of Medicine. MTX(Glu2) and MTX(Glu3) were kindly methylenetetrahydropteroylpentaglutamate were estimated provided by Charles Baugh, Univeristy of Alabama School of to be 370, 27, <10, and <10 nM, respectively, by equilibrium Medicine. FUra was obtained from Hoffmann-La Roche. dialysis. We propose that the sequence-dependent effect of MTX FdUMP and dUMP (Na salts), Triton X-100, and dithiothreitol plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase (te- were purchased from Sigma. [5-3H]dUMP was purchased from trahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ Amersham and had an estimated radiochemical purity of 95% oxidoreductase, EC 1.5.1.3) and consequently a trapping of in- by anion-exchange high-performance liquid chromatography tracellular folates as dihydropteroylpolyglutamates, which in- (HPLC). [6-3H]FdUMP was obtained from Moravek Bio- crease the extent of FdUMP binding to thymidylate syn- chemicals (City of Industry, CA); radiochemical purity of thase. various batches was estimated to be between 89% and 96% by The antitumor drug 5-fluorouracil (FUra) is converted intra- anion-exchange HPLC, and specific radioactivity between 12.5 cellularly to FdUMP, which in the presence of 5,10-meth- and 16.0 Ci/mmol (1 Ci = 3.7 X 1010 becquerels). dl-H4Pte- ylene-5,6,7,8-tetrahydropteroylglutamate (5,10-CH2-H4Pte- L-Glu prepared (20) and purified (21) as described was obtained Glu) is a tight-binding inhibitor of thymidylate synthase from John McGuire, Department of Pharmacology, Yale (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, University School of Medicine. H2PteGlu5 prepared by sodium EC 2.1.1.45) (1, 2). The antifolate methotrexate (MTX), a dithionite reduction of PteGlu5 and purified on DEAE-Seph- tight-binding inhibitor of dihydrofolate reductase (tetrahy- adex (22) was kindly provided by James Coward and A. R. drofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ Cashmore, Department of Pharmacology, Yale University, and oxidoreductase, EC 1.5.1.3) (3), also enhances binding of was desalted by Bio-Gel P-2 chromatography for use in equi- FdUMP to Lactobacillus casei thymidylate synthase, although librium dialysis. H2PteGlu5 was converted to l-H4PteGlu5 by not as effectively as does 5,10-CH2-H4PteGlu (1). However, reaction catalyzed by dihydrofolate reductase (22). The con- folates (4-6) and MTX (7-11) exist in most cells predominantly centrations of H2PteGlu5 stock solutions were calculated as poly(y-glutamyl) conjugates. Recently Washtien and Santi (12) have reported that the ternary complex isolated from Abbreviations: HPLC, high-performance liquid chromatography; FUra, 5-fluorouracil; FdUMP, 5-fluorodeoxyuridylate; MTX, meth- L1210 murine tumor cells was approximately 1000 daltons otrexate, 2,4-diamino-10-methylpteroylglutamate; MTX(Glul), higher in molecular mass than the complex formed by incu- methotrexate(y-glutamate), 2,4-diamino-10-methylpteroyldigluta- bation of FdUMP, 5,10-CH2-H4PteGlu, and cell cytosol, which mate; MTX(Glu2), methotrexate(y-glutamate-y-glutamate), 2,4- suggested the presence of a pteroylpolyglutamate in the ternary diamino-10-methylpteroyltriglutamate; H2PteGlu, 7,8-dihydropter- complex formed intracellularly. oylglutamate; H2PteGlu5, 7,8-dihydropteroylpentaglutamate; 5,10- Accordingly, we have attempted to characterize ternary CH2-H4PteGlu, dl-5,10-methylene-5,6,7,8-tetrahydropteroylgluta- mate, racemic mixture of the physiologically active diastereoisomer complexes of reduced pteroyl and MTX polyglutamates with (1) and the nonphysiological diastereoisomer (d); 5,10-CH2-H4PteGlu5, 1-5,10-methylene-5,6,7,8-tetrahydropteroylpentaglutamate. PteGlu, The publication costs of this article were defrayed in part by page is used as the general term for polyglutamyl conjugates of pteroic acid charge payment. This article must therefore be hereby marked "ad- or any mixture of them. All glutamic acid residues have the L absolute vertisement" in accordance with 18 U. S. C. §1734 solely to indicate configuration and are linked in peptide bonds through the y-carboxyl this fact. groups in MTX and pteroylpolyglutamates. 5663 Downloaded by guest on October 2, 2021 5664 Biochemistry: Fernandes and Bertino Proc. Natl. Acad. Sci. USA 77 (1980) spectrophotometrically. The purity of H2PteGlu5 and 1- used. Membranes having a 12,000-14,000 molecular weight H4PteGlu5 was checked on reversed-phase HPLC (23). No cut-off were prepared by boiling for 5 min in 5 mM Na2EDTA contamination of H2PteGlu5 with either H4PteGlus or 5,10- plus 16 mM NaHCO3. With the exception of enzyme, equi- CH2-H4PteGlu5 was detected either spectrally or by re- molar amounts of all compounds were added to both sides of versed-phase HPLC. the membrane in buffer A containing 0.1% (vol/vol) Triton The composition of buffers was as follows: buffer A, 50 mM X-100, which served to stabilize the enzyme activity (25). Di- Tris-HCl, pH 7.5/10% (vol/vol) glycerol/2 mM dithiothreitol; alysis was carried out at 40C for 24 hr at which time the [3H]- buffer B, 250 mM Tris-HCl, pH 7.5/10% glycerol/13 mM 2- FdUMP had equilibrated across the membrane. No loss of en- mercaptoethanol/6 mM formaldehyde; buffer C, 50 mM zyme activity was detected during dialysis of control samples Tris-HCl, pH 7.5/10% glycerol/20 mM 2-mercaptoethanol/3 lacking FdUMP. After 24 hr the entire contents of each mM formaldehyde/42 mM NaCl; buffer D, 50 mM Tris-HCI, chamber was removed with a syringe and combined with the pH 7.5/10% glycerol/20 mM 2-mercaptoethanol/168 mM volume obtained after two successive washings of each chamber NaCl. The concentrations of all drugs and folates were deter- with 200,l of buffer A. Radioactivity was measured as de- mined spectrophotometrically. scribed (8). After this procedure no more than 2% of the re- Soft Agar Cloning. The viability of logarithmically growing covered radioactivity remained associated with the membrane L1210 cells (doubling time 10.8 hr) after drug exposure was over the entire range of [3H]FdUMP concentrations employed. assayed by cloning in soft agar as described by Chu and Fischer Because of the low enzyme concentrations used, no corrections (24) with the following modification. Because of the large kill were made for Donnan effects. The amount of bound ligand produced by combination treatment it was sometimes necessary was calculated as the difference in radioactivity between the to clone between 104 and 106 cells per tube. However, the protein and ligand sides of the chamber. Scatchard analysis (30) cloning efficiency of untreated cells was found to decrease when of the data was performed by plotting (V/[Lf]) = (V/Kd) + they were cloned in the presence of greater than 104 drug- (n/Kd), in which V is the moles of ligand bound per mole of treated cells. Thus, as an attempt to avoid an overestimation of protein, [Lf] is the free ligand concentration, n is the number cell kill, untreated cells were added to parallel groups of treated of moles of ligand binding sites per mole of protein, and Kd is cells and cloned. The cloning efficiency of untreated cells in the intrinsic ligand dissociation constant of a binding site. Data the presence of drug-treated cells (corrected cloning efficiency) points were fitted by eye. was calculated according to the following formula: RESULTS Corrected cloning efficiency (E) = Ct+U Effect of FUra and MTX on Cell Viability. It was observed that, when L1210 cells were exposed to both FUra and MTX, in which Ct+ u is the number of clones formed in tubes con- the effect on L1210 cell viability was highly dependent upon taining both treated and untreated cells, Ct is the number of the sequence of drug administration (Table 1).
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