The Influence of Methotrexate Pretreatment on 5-Fluorouracil Metabolism in L1210 CelW

Pretreatment of L1210 cells with methotrexate in concentrations which produced free intracellular methotrexate and near maximal inhibition of dihydrofolate reductase resulted in an enhancement of intracellular 5-fluorouracil (FUra) accumulation. This enhancement of FUra accumulation was maximum (5-fold increase) after a 6-h exposure to 100 pM methotrexate. The nu- cleotide derivatives of FUra, including 5-fluoro-2’-deox-yuridylate, and 5-fluorouridine-5’-triphosphate were also increased nearly &fold following methotrexate treatment. In cells pretreated with methotrexate, there was an increase in intracellular 5-phosphoribosyl-1-py- rophosphate pools which ranged from 2 to 8 times control values following concentrations of methotrex- ate between 0.1 p~ and 10 p ~ . Both the increase in 5-phosphoribosyl-1-pyrophosphate and FUra accumula- tion could be prevented by the addition of Leucovorin (W-formyltetrahydrofolate) at concentrations which rescued cells from the inhibitory effects of methotrex- ate. Pretreatment with 6-methylmercaptopurine riboside, which inhibits amidophosphoribosyltransferase, the first committed step in de nouo purine synthesis, also resulted in a similar elevation in 5-phosphoribosyl- 1-pyrophosphate pools and enhancement of FUra accumulation. If the 5-phosphoribosyl-1-pyrophosphate was determined performing simultaneous with known quantities of

Pretreatment of L1210 cells with methotrexate in concentrations which produced free intracellular methotrexate and near maximal inhibition of dihydrofolate reductase resulted in an enhancement of intracellular 5-fluorouracil (FUra) accumulation. This enhancement of FUra accumulation was maximum (5-fold increase) after a 6-h exposure to 100 pM methotrexate. The nucleotide derivatives of FUra, including 5-fluoro-2'-deoxyuridylate, and 5-fluorouridine-5'-triphosphate were also increased nearly &fold following methotrexate treatment. In cells pretreated with methotrexate, there was an increase in intracellular 5-phosphoribosyl-1-pyrophosphate pools which ranged from 2 to 8 times control values following concentrations of methotrexate between 0.1 p~ and 10 p~. Both the increase in 5phosphoribosyl-1-pyrophosphate and FUra accumulation could be prevented by the addition of Leucovorin (W-formyltetrahydrofolate) at concentrations which rescued cells from the inhibitory effects of methotrexate. Pretreatment with 6-methylmercaptopurine riboside, which inhibits amidophosphoribosyltransferase, the first committed step in de nouo purine synthesis, also resulted in a similar elevation in 5-phosphoribosyl-1-pyrophosphate pools and enhancement of FUra accumulation. If the 5-phosphoribosyl-1-pyrophosphate pools were reduced following methotrexate pretreatment by the addition to the cultures of hypoxanthine, which utilizes 5-phosphoribosyl-1-pyrophosphate for the conversion to IMP, the intracellular accumulation of FUra was not enhanced. Also, if the inhibitor of 5phosphoribosyl-1-pyrophosphate synthetase, 7-deazaadenosine, was given to cultures with methotrexate, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of FUra accumulation. In addition, when 5-fluoro-Z'-deoxyuridine was added with the methotrexate to cell cultures, there was no increase in 5-phosphoribosyl-1-pyrophospbate pools, nor enhancement of intracellular FUra accumulation.
These results indicate that the ability of methotrexate to enhance FUra accumulation was probably the consequence of the antipurine effect of methotrexate which resulted in a reduction of the complex feedback inhibition on 5-phosphoribosyl-1-pyrophosphate synthesis and utilization. The resultant increased 5-phosphoribosyl-1-pyrophosphate pools were then capable of being utilized for the conversion of FUra to 5-fluorouridylate, the possible rate-limiting step in FUra intra-cellular metabolism and the major determinant of the rate of intracellular FUra accumulation. When methotrexate preceded FUra, there was synergistic cell killing as determined by soft agar cloning. The exact mechanism of this sequential synergistic antitumor activity may be the result of the enhanced incorporation of FUra into RNA, since the increased 5-fluoro-2'-deoxyuridylate which is formed is unlikely to increase substantially the inhibition of dTMP synthesis induced by methotrexate pretreatment.
Methotrexate and 5-fluorouracil are frequently used in combination for the treatment of cancer, especially in women with breast cancer (1). Antagonism between these two drugs, regardless of drug sequence, on their respective ability to inhibit dTMP synthesis (2, 3) have resulted in concern for the continued concurrent use of these drugs (4). Synergistic antitumor activity on experimental tumors in rodents, however, has been observed when methotrexate preceded FUra' (5-7). These conflicting reports indicated that methotrexate and FUra interactions could also be occurring at metabolic sites other than those which influence the formation of dTMP.
The presumed major antitumor derivative of FUra, 5-fluoro-2'-deoxyuridylate, requires the reduced folate, N5,'"-methylenetetrahydrofolate, for covalent binding to thymidylate synthetase (EC 2. 1.1.45). The result is inhibition of dTMP synthesis (8)(9)(10). Because the inhibition of dihydrofolate reductase (EC 1.5.1.3) by methotrexate pretreatment prevents the regeneration of Ns,'o-methylenetetrahydrofolate, FdUMP is unable to form the ternary complex necessary for prolonged inhibition of dTMP synthesis. Ullman et al. (3) has proposed this mechanism as evidence against the use of this sequence for the treatment of malignancy. Methotrexate, which is a folate analogue, can promote binding of FdUMP to thymidylate synthetase, but the maximal effect is only 21% as efficient as N5~1Q-methylenetetrahydrofolate (11). This methotrexateformed ternary complex has not yet been identified in vitro, and is unlikely to be the total explanation for the in vivo synergism observed in animals when methotrexate is given before FUra.
Bowen et al.

1695
the effect of the direct inhibition of thymidylate synthetase by FdUMP diminished, there would, however, be 1) increased dUMP pools and 2) available N5310-methylenetetrahydrofolate pools which could be used for dTMP synthesis in spite of the methotrexate-induced inhibition of dihydrofolate reductase. Therefore, the in vivo synergistic antitumor effects observed when methotrexate is administered prior to FUra is not explicable by the known biochemical interactions that occur with thymidylate synthetase and must be due to other interactions not previously described. We have found that methotrexate at doses which maximally inhibit dihydrofolate reductase and, subsequently, dTMP formation lead to a 5-fold increase in the intracellular accumulation of FUra, primarily as the nucleotide derivatives, and that this sequence results in an augmented incorporation of FUra derivatives into RNA. This enhanced intracellular metabolism of FUra that follows methotrexate treatment is a consequence of the intracellular increase of 5-phosphoribosyl-1-pyrophosphate pools and the result of purine synthesis inhibition that occurs with these doses of methotrexate. Portions of this work have been reported in preliminary form elsewhere (13-18).  (24.5 Ci/mmol) which was used for the 5-phosphoribosyl-I-pyrophosphate assay, and [l-'4C]glycine (20 mCi/mmol) which was used to evaluate purine synthesis, were procured from New England Nuclear. The methotrexate and N-(phosphonacetyl)-L-aspartate were provided by the National Cancer Institute, Bethesda, MD. Pyrazofurin was a tgft of Eli Lilly Co., Indianapolis, IN. All other nonradiolabeled compounds were purchased from Sigma.

Drug~
Cells-L1210 murine leukemia cells, with a doubling time of 10 to 12 h, were maintained as stationary suspension cultures in Fischer's medium plus 10% horse serum, transferred twice weekly, and kept at 37°C in a 5% COZ atmosphere. Tests performed monthly were negative for mycoplasma contamination. All experiments were performed with cells which had been inoculated at 1 to 3 X IO4 cells/ml and had been in the logarithmic phase of growth for 48 h, which corresponded :,o 2 to 5 X lo5 cells/&. Enumeration of cells was performed with a model ZBI Coulter Counter (Coulter Electronics, Inc., Hialeah, FL).
Cloning-The biological antitumor effect of methotrexate and FUra was determined by cloning L1210 cells in soft agar by the technique we previously reported (19,20). Following the indicated single drug exposure to logarithmically growing cell cultures, the second drug was added for the specified time. The drug-containing medium was then removed after centrifuging at lo00 X g for 5 min at 37°C. The cell pellet was resuspended in drug-free medium and then recentrifuged as before. This washing procedure was repeated twice to remove any extracellular drug before cloning.
Fifty cells were pipetted into 10-ml culture tubes which contained 2 ml of liquified agar (37°C) and 3 ml of drug-free Fischer's medium plus 15% horse serum. The tubes were capped and placed upright and incubated at 37°C in a 5% COa atmosphere. The amount of agar in the culture medium was 0.088 g/100 ml; the consistency of this mixture does not allow cell settling but does permit cell growth. Cells that remain viable after the drug exposure, as defined by having the continued capability to divide and produce progeny, will form individual cell colonies after 10 days of incubation. All clones were counted with an inverted microscope. Single cells, which can be visualized in suspension cultures, were not observed in the cloning medium after the 10-day incubation indicating all viable cells had developed into clones. The per cent viability is the ratio of clones formed from drug-treated cultures to clones formed from untreated cultures, multiplied by 100. The cloning efficiency of L1210 cells in this system was 85 to 90%. All experiments were done in triplicate on three separate occasions. Mean values are shown; the maximum range was *5%.
Total Intracellular FUra Accumulation-Logarithmically growing Cells (2 to 5 X IO5 cells/&) were exposed to methotrexate in concentrations from 0.01 to 100 PM for 1, 3, and 6 h. After the appropriate methotrexate exposure time, 50 ml of the cell suspension was centrifuged at Lo00 X g for 5 min at 37°C. The drug-containing medium was removed and the cell pellet gently resuspended in 2 ml of the initial drug-containing supernatant to which was added 0.1 ml of 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (pH 7.4). This concentrated cell suspension was placed in a 25-ml stoppered flask and incubated in a 37°C shaker water bath. FUra (2 Ci/mmol) was then added to a concentration of 3 p~. Over the course of an hour, 0.1-ml aliquots of this cell suspension were gently placed in a 0.4-ml plastic microfuge tube which contained 0.04 ml of 5% HC10, at the tip, which was overlayed with 0.1 ml of a silicone/mineral oil mixture of 84:16 proportion (the silicone fluid used was Hi-phenyl 125, DC550, from Wm. F. Nye, Inc., Bedford, MA; the mineral oil used was light mineral oil from Invenex, Chagrin Falls, OH). The tube was then immediately centrifuged at 1O,o00 rpm for 15 s. The silicone/mineral oil interphase allowed the cells to pass into the HClO,-containing tip but kept the drug-containing medium at the top of the tube. The time necessary for complete separation of cells was determined by microscopic examination of the medium and was 15 s. The tubes were then quickly frozen in an ethyleneglycol/dry ice bath, cut at the liquid interfaces into three fractions: the medium, the oil, and the cell pellet. Radioactivity in each fraction was quantitated; radioactivity was only present in the cell pellet and medium fractions. All experiments were done in duplicate three times. The maximum range of the mean values shown was f8%. A separate experiment was designed to evaluate the effectiveness of the silicone/oil interphase to prevent drug-containing cell culture medium from entering the tip of the microfuge tube.
[3H]inulin (1 pCi/l00 pg) was added to the medium containing cells. Radioactivity was only detected in the mediumcontaining section of the microfuge tube and not in the cell pellet following centrifugation. Therefore, the radioactivity in the cell pellet fraction is an accurate representation of the total intracellular FUra which includes the base as well as nucleotide derivatives.
Identical cloning procedures as outlined above as well as trypan blue exclusion determinations were performed on a control group of cells which had been concentrated from 50 ml to 2 ml as was done for the FUra-accumulation studies. There was no reduction in cloning efficiency, and greater than 99% of the cells excluded the dye, indicating the high concentration of the cells for the duration of these experiments did not adversely affect cell viability.
Nucleotide Pools-Fifty milliliters of cells under identical conditions as outlined above were exposed first to 1 p~ methotrexate for 3 h and then 3 p~ FUra (2 Ci/mmol) for an additional hour. The cells were then centrifuged at 100 g for 5 min, the supernatant was discarded, and the cells were resuspended in phosphate-buffered 0.9% NaCl at 4°C to remove any extracellular drug and then recentrifuged as before. The final cell pellet was precipitated in 1 ml of 0.5 N HCIO, and recentrifuged at lo00 X g. The supernatant which contained the nucleotides was neutralized with 4 N KOH, the salt was removed by centrifugation, and the final supernatant was stored at -20°C. All ribonucleotide assays were evaluated consecutively by high pressure liquid chromatography (Altex model 110 A pump microprocessercontrolled gradient system, Altex Scientific, Inc., Berkeley, CA) with a linear gradient (0.01 to 1.0 M) of NaH2P04 (pH 3.31) at a flow rate of 0.9 ml/min on a Whatman Partisil SAX (particle size, 10 pm) column 25 cm X 4.6 mm. Absorbance was recorded at 254 and 280 nm and 0.5-ml fractions were collected of the entire column flow. FUra, FUrd, FdUrd, FdUMP, FUMP, UMP, UDP, UDP-Glc, and UTP were used as unlabeled markers.
Acid-soluble fractions were also prepared for FdUMP analysis from cells under identical conditions and treatment, except that FUra of higher specific activity was used (25 Ci/mmol). Periodate oxidation of this fraction (12 PM NaI0, at 37'C for 30 min, followed by 0.4 M CH3NHz plus 0.01 N NaOH for 15 min) cleaved the base and phosphates from the ribonucleotides but not the deoxynucleotides. On the SAX column, the FUMP, and FdUMP were inseparable and eluted within the fvst 5 min prior to periodate treatment. Separation of these two nucleotides, however, was achieved on a polystyrene column (BA-X4, James Benson, Reno, NV) eluted at 50°C with CH3COONH4 (pH 7.0, 0.5 M) at a rate of 1 ml/min. The only radioactivity remaining after periodate treatment was in the FdUMP region and void volume; no radioactivity appeared in the FUMP region. When the periodate-treated sample was rechromatographed on the SAX column, radioactivity was only present in the void volume and the monophosphate region, indicating the oxidation process had efficiently eliminated the FUDP and FUTP derivatives below detectability. [2-14C]FUMP (20 mCi/mmol) at 70,000 cpm was reduced to background counts (40 cpm) by periodate oxidation, indicating the reaction was greater than 99.94% complete, whereas [2-'4ClFdUMP (18 mCi/mmol) at 70,000 cpm was unaffected by this oxidation process.
FUra Incorporation into RNA-The acid-precipitable fraction of the cells prepared for ribonucleotide analysis was washed repeatedly with 2 ml of 0.5 N HC104 until no radioactivity was detected in 1 ml of this supernatant (six washes). The precipitate was then separated into the RNA, DNA, and protein fractions by the methods of Traketelliis and Axelrod (21) and the radioactivity in the DNA and RNA fractions was determined and related to.micrograms of D-ribose and deoxyribose by the orcinol (22) and diphenylamine reactions (23), respectively.
dUrd Incorporation-Cells were prepared as before for the FUraaccumulation studies and, at designated times after adding [6-3Hl-dUrd (21.9 Ci/mmol) to a concentration of 1 pM, 0.025 ml of the cell suspension was placed in harvesting plates (Limbro Scientific Inc., Hamden, CT) and automatically sucked onto Reeve angle 934AH glass fdter strips (Whatman) by use of a MASH cell harvester (Microbiological Associated, Walkersville, MD). The acid-insoluble fraction was then precipitated on the fdter strips with 10 ml of 10% trichloroacetic acid and washed twice with 10 ml of distilled Hz0 to remove any nonprecipitated label. After drying at room temperature, the cell-precipitated areas were placed in Omni counting vials, (Wheaton Scientific, MillviUe, NJ), 3 ml of Omni counting solution was added (Amersham), and radioactivity was quantitated with a Packard Tricarb liquid scintillation spectrometer (Packard Instument Co.).
Free Intracellular Methotrexate-Suspension cultures were exposed to methotrexate (0.01 to 100 pM) for 3 h during logarithmic growth (250 ml at 2 to 5 X lo5 cells/ml). The cells were separated from the drug-containing medium by centrifuging at 1,000 X g for 10 min at 4°C. Then 1.0 ml of a Tris/KCl solution (0.05 ~: 0 . 1 M, pH 7.0). which contained 10% glycerol, was added to the cell pellet. The cells in this suspension were lysed by freeze thawing three times, which released intracellular methotrexate into the solution. The cellular debris was sedimented by centrifuging at 10,000 X g for 10 min. The supernatant was removed, and the quantity of non-dihydrofolate reductase-bound or free intracellular methotrexate was determined as previously described by the enzymatic inhibition of dihydrofolate reductase (24). The same enzymatic method was used to determine the specific activity of dihydrofolate reductase. Protein content was established by the method of Lowry et al. (25).
Purine Synthesis-The incorporation of [l-'4C]glycine (20 mCi/ mmol) into purine bases of nucleic acid was used as a measure of de nouo purine synthesis. Cells under identical conditions as outlined in the previous sections were divided into 100-ml aliquots and either exposed to 10 p~ methotrexate, 10 p~ FdUrd, or both drugs together for 2 h, or they were used as controls. After the 2-h drug exposure, [l-'4C]glycine was added to a concentration of 10 pM; 2 h later the cells were centrifuged at 1000 X g for 5 min, the supernatanL was discarded and the cell pellet was precipitated in 1.5 ml of 1 N HC104. The samples were then kept at l00'C for 1 h to depurinate the nucleic acid. The supernatant, following centrifugation at lo00 X g, was neutralized with 4 N KOH. The soluble extract was then analyzed on the chromatography system previously described but using an ODS-2 column (Whatman) eluting with 0.1 M sodium acetate, pH 5.5, and an acetonitrile gradient from 0 to 7.5% over 30 min. Nonradiolabeled adenine and guanine were used as markers, absorbance was recorded at 254 and 280 nm, 0.5-ml fractions were collected during the column flow, and radioactivity was quantitated. Glycine appeared in the void volume and did not interfere with the analysis.

5-Phosphoribosyl-I-pyrophosphate
Assay-Intracellular 5-phosphoribosyl-1-pyrophosphate of L1210 cells after variable drug and exposure time was quantitated by modifications of a previously reported technique (26). Adenine phosphoribosyltransferase (EC 2.4.2.7.) was extracted from the 40 to 70% (NH4)&04 fraction of a cell homogenate prepared from 0.6 liter of an L1210 culture at a density of 6 X lo5 cells/ml. Following centrifugation at 10,ooO X g for 20 min, this supernatant was placed on a G-150 Sephadex column and eluted with 0.2 M Tris, pH 7.4, 20 mM MgSO,, to separate the adenine phosphoribosyltransferase from the 5"nucleotidase activity. Twenty milliliters of each cell culture from which 5-phosphoribosyl-l-pyrophosphate was to be determined was centrifuged at 4°C for 8 min at 1,200 rpm. The cell pellet was resuspended at 4°C in 5 ml ofNa2HPo4, 0.9% NaCl (pH 7.4); 0.1 ml was removed to compute cell density; and then the pellet was recentrifuged as before. The cell pellet was resuspended in 0.5 m10.2 M Tris, pH 7.4, and placed in a boiling water bath for 90 s to extract the 5-phosphoribosyl-1-pyrophosphate. Extractions were performed a t 15-s intervals to 5 min; the optimal time for maximum 5-phosphoribosyl-1-pyrophosphate quantitation was 90 s. The tubes were then placed on ice and centrifuged for 4 min at 2,000 rpm, the supernatant was removed and stored at -20°C until multiple determinations could be performed simultaneously within 3 weeks. The quantity of the 5-phosphoribosyl-1-pyrophosphate in these extracts exceeded 90% of the original values following storage at -2O' C for 3 weeks; by 6 weeks the quantitation had decreased to 75% of these initial values.
To the reaction mixture, which contained 0.1 ml of 0.2 M Tris, pH 7.4, 0.02 of 20 m~ MgCL, and 0.005 ml of [2-3H]adenine (24.5 Ci/ mmol) to achieve 30 p~, was added 0.1 ml of the 5-phosphoribosyl-lpyrophosphate cell extract and then 0.1 ml of the adenine phosphoribosyltransferase extract. The adenine phosphoribosyltransferase converts [2-3H]adenine to [3H]AMP in the presence of 5-phosphoribosyl-1-pyrophosphate. After 20 min at 19'C, the reaction was stopped by adding 0.265 ml of 3 mM citric acid in methanol and the 3-ml tube containing this assay mixture was placed in ice. Onto DE81 discs (Whatman) was spotted 0.05 ml of this reaction mixture. The discs were then washed twice with 2 mM citric acid in 60% methanol and then with absolute methanol. This washing procedure removed greater than 99% of [3H]adenine. The discs were dried at room temperature and then placed in scintillation vials, the ['HIAMP was eluted with 1 ml of 0.5 M NaCl in 1 N HCI, and then 8 ml of scintillant was added (15 g of 2,5-diphenyloxazole, 0.3 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene, 1 liter of Triton X-100, and 2 liters of toluene) and the amount of E3H]AMP formed was quantitated. The amount of 5phosphoribosyl-1-pyrophosphate used in the conversion of adenine to AMP was determined by performing simultaneous controls with known quantities of 5-phosphoribosyl-1-pyrophosphate.

RESULTS
FUra Intracellular Accumulation-The intracellular FUra accumulation was enhanced following methotrexate exposure and was dependent on the pretreatment concentration of methotrexate (Fig. 1). The maximum enhancement of FUra accumulation was after a 6-h exposure for all methotrexate concentrations. The enhancement of FUra accumulation following methotrexate occurred with concentrations of FUra as high as 1 mM. Leucovorin (N'"formy1tetrahydrofolate) at a concentration which reversed the methotrexate-induced inhibition of dTMP synthesis as measured by the return to normal of the rate of ['HIdUrd incorporation into the acidprecipitable cell fraction also prevented the augmentation of intracellular FUra accumulation (Fig. 2).
Several experiments were performed to investigate the relationship between the inhibition of dihydrofolate reductase and the enhanced intracellular FUra accumulation which occurred in methotrexate-treated cell cultures. The maximum inhibition of [3H]dUrd-incorporation into the acid-precipitable cell fraction following methotrexate treatment occurred only after free non-dihydrofolate reductase-bound methotrexate was present within these cells (Fig. 3). A similar relationship existed between the rate of intracellular FUra accumulation and free nonenzyme-bound methotrexate (Fig. 4).
Free intracellular methotrexate does not result until nearly all of the dihydrofolate reductase is bound with methotrexate. White and Goldman (27) and Sirotnak and Donsbach (28) demonstrated that the greatest inhibition of dihydrofolate reductase activity requires free intracellular methotrexate. Our data is consistent with this concept and, in addition, suggests that the optimum enhancement of FUra accumulation which follows methotrexate exposure is also correlated with maximum dihydrofolate reductase inhibition. To substantiate the latter assertion, FUra-accumulation studies were performed with two methotrexate resistant LE10 cell lines. These mutant lines were developed by continuous exposure to methotrexate. The R line had normal methotrexate transport, but the specific activity of dihydrofolate reductase was methotrexate; nor did methotrexate enhance the rate of FUra accumulation in this cell line (Fig. 5). Therefore, free intracellular methotrexate must be present for enhanced FUra accumulation to occur.
Cloning-Synergistic killing of LE10 cells resulted when methotrexate preceded FUra, but not if the sequence was reversed ( Table I). If cells were exposed to 100 p~ Leucovorin 2 h after being exposed to 1 p~ methotrexate but 1 h before being treated with 1 p~ FUra, the viability was equivalent to nondrug-treated cells. 6-Methyhercaptopurine riboside also resulted in a synergistic killing of cells when given before FUra. The possible explanation of this observation is examined in subsequent experiments.
Nucleotide Pools-The ribonucleotides and FdUMP were increased &fold following a 3-h exposure to 1 PM methotrexate (Table 11). No radioactivity was detected following periodate oxidation in the regions which would correspond to FdUDP and FdUTP.
Incorporation of FUra into RNA-Because of the increased quantities of FUTP in response to methotrexate, it was not unexpected to find that the FUra content of RNA from methotrexate-treated cells was 5-fold greater than that present in control cells (Table 111). Radioactivity was not detected in the DNA or protein cell fractions. It is unlikely

([MTX],) and the per cent inhibition of the incorporation of
[3H]dUrd into the acid-precipitable fraction of L1210 cells were determined in L1210 cells after a 3-h exposure to doses of methotrexate ranging from 0.1 to 100 pM. that a 5-fold increase in RNA synthesis is occurring in response to methotrexate treatment. To evaluate this possibility [ ~-'~C]glycine (20 mCi/mmol, 10 p~) incorporation into purine bases of nucleic acid was determined, and found to be reduced by 85% following identical methotrexate treatment. The salvaging of performed purines could account for some dilution of the glycine incorporation into RNA. Because purines are incorporated into both DNA and RNA, the reduction in glycine incorporation into nucleic acid in these experiments is only an indication that RNA synthesis, as well as DNA synthesis, is reduced. The precise magnitude of this reduction of RNA synthesis cannot be accurately ascertained from this data.

5-Phosphoribosyl-1-pyrophosphate Levels and FUra
Metabolism-An increase in 5-phosphoribosyl-1-pyrophosphate levels did occur in cells exposed to methotrexate and was related to the dose and time of methotrexate exposure (Table concentrations of methotrexate ranging from 0.1 to 1 0 0 PM was determined and correlated with the rate of intracellular 13HJFUra (2 Ci/mmol, 3 p~) accumulation that resulted from this methotrexate pretreatment.

I "
A C IV). The increase in 5-phosphoribosyl-1-pyrophosphate levels was also correlated with the increased rate of intracellular FUra accumulation (Fig. 6). These results suggested that the enhancing effect of methotrexate on FUra accumulation was related to an increased conversion of FUra to FUMP by the transfer of the 5-phosphate ribose moiety from 5-phosphoribosyl-1-pyrophosphate. Several experiments were performed to evaluate this possibility. Hypoxanthine requires 5-phosphoribosyl-1-pyrophosphate for conversion to the nucleotide derivative, IMP. Therefore, hypoanthine would be expected to decrease intracellular 5-phosphoribosyl-1-pyrophosphate pools and reduce the methotrexate effect on both 5-phosphoribosyl-1-pyrophosphate levels and FUra accumulation. Table IV shows that 10 p~ hypoxanthine for l h following 3 h of 1 p~ methotrexate reduced the previously elevated 5-phosphoribosyl-1-pyrophosphate levels that were present after a 3-h exposure of 1 PM methotrexate. This concentration of TABLE I

Viability of L1210 cells after drug treatment
Viability of L1210 cells after drug treatment. Logarithmically growing cells were exposed to methotrexate and 6-methylmercaptopurine riboside at the indicated concentrations for 3 h before cloning in drugfree medium. FUra was given only for 1 h. When methotrexate or 6methylmercaptopurine riboside preceded FUra, there was a synergistic reduction of cell viability. When FUra preceded methotrexate, antagonism resulted. MTX, methotrexate, MMPR, 6-methylmercaptopurine riboside. Mlnutes hypoxanthine given after 3 h of 1 PM methotrexate also reduced the intracellular FUra accumulation to rates observed in control cells (Table V). These interactions among 5-phosphoribosyl-1-pyrophosphate, FUra, and hypoxanthine pro-    vides an explanation for the recent observation that hypoxanthine and other purine bases which utilize 5-phosphoribosyl-I-pyrophosphate for their metabolic conversions to nucleotides can prevent or reduce FUra cytotoxicity (29,30).
Pyrazofurin, which, as the monophosphate, is a tight-binding inhibitor of orotidylate decarboxylase (K, = 5 m, EC 4.1.1.23) (31,32), did not affect 5-phosphoribosyl-1-pyrophosphate values (Table LV), but this drug did prevent FUra accumulation in both the methotrexate-treated and control cells (Table V). The orotate accumulation that occurs following pyrazofurin treatment could be competing with FUra for the available 5-phosphoribosyl-1-pyrophosphate and, therefore, conversion to their respective monophosphorylated nucleotide derivatives. This is unlikely to be the entire cause of the marked reduction in FUra accumulation which followed pyrazofurin treatment, however, because the 5-phosphoribosyl-1-pyrophosphate levels which would be expected to be depleted in the presence of excess orotate were unaffected. These persistent 5-phosphoribosyl-1-pyrophosphate levels could be the result of the antipurine effect of pyrazofurin (33). N-(phosphonacety1)-L-asparatate, which inhibits aspartate transcarbamylase (EC 2.1.3.2) results in reduced orotate levels (34); therefore, it was not unexpected that this drug resulted in some augmentation of FUra accumulation (Table V). Uridine a t 100 p~ also resulted in a modest enhancing effect of FUra accumulation of a similar magnitude as that which followed N-(phosphonacety1)-L-aspartate treatment. This, too, is not unexpected, since this concentration of uridine inhibits de nouo pyrimidine synthesis and also reduces orotate formation (35). Fluorouridine accumulation studies were performed to determine if this nucleoside might be responsible for the enhanced FUra accumulation which followed methotrexate. This nucleoside would fiist need to be formed by uridine phosphorylase (EC 2.4.1.3) acting on FUra (36) and then subsequently be phosphorylated by uridine-cytidine kinase (EC 2.7.1.48) (37) to form FUMP. Following methotrex-

TABLE V Effect of various inhibitors of purine and pyrimidine synthesis on total FUra accumulation into L1210 cells
The effect of various inhibitors of purine and pyrimidine synthesis on the total intracellular accumulation of FUra into L1210 cells was examined. After the indicated exposure time, [3H]FUra (2 Ci/mmol) was added to a concentration of 3 PM and the rate of intracellular accumulation was determined as outlined under "Materials and Methods." The rates of FUra accumulation were linear for the hour during which the experiments were performed. All experiments were performed twice in duplicate; the mean values are shown, the greatest range was 28%. The arrow (4) and plus (+) are explained in Table  IV  ate exposure, the accumulation of fluorouridine was reduced, not enhanced. This has been reported by us previously (18). The results of these experiments indicate that the initial metabolism for FUra in L1210 cells is as indicated in Fig. 7. The rate of FUra accumulation was linear for the duration of these studies; mean values are shown. The range was &8%.

Pyrlmdont S y n t h t w
5-Phosphoribosyl-1 -pyrophosphate-The purine synthetic rate, as measured by the incorporation of [l-'4C]glycine into the adenine and guanine bases of nucleic bases of nucleic acid was reduced by 85% following a 2-h exposure to 10 PM methotrexate. The likely explanation of the mechanism by which methotrexate leads to an elevation of 5-phosphoribosyl-1-pyrophosphate is the result of the inhibition of de nouo purine synthesis. The following experiments were performed to test this hypothesis.
7-Deazaadenosine (tubercidin) is a nucleoside analogue which does not require 5-phosphoribosyl-1-pyrophosphate for metabolic conversion but does inhibit 5-phosphoribosyl-1-pyrophosphate synthetase (EC 2.7.6.1) (38). The 5-phosphori-  bosyl-1-pyrophosphate levels following 10 ~L M of this drug for 1 h were less than control values, even when the cells were pretreated for 2 h with 10 ~L M methotrexate. FUra accumulation was likewise not enhanced following methotrexate when 7-deazaadenosine was also added to the cell cultures in the dose and time intervals above (Tables IV and V).
6-Methylmercaptopurine riboside is a nucleoside analogue which is phosphorylated by adenosine kinase (EC 2.7.1.20), and in the nucleotide forms inhibits amidophosphoribosyltransferase (EC 2.4.2.14) (39). The consequence of this reaction is inhibition of de novo purine synthesis and increased levels of 5-phosphoribosyl-1-pyrophosphate (Table IV). 6-Methylmercaptopurine riboside like methotrexate, also resulted in enhanced intracellular FUra accumulation (Table v) and probably accounts for the synergistic cell killing when this drug preceded FUra (Table I).

DISCUSSION
These findings demonstrate that the metabolism of FUra by L1210 cells can be altered with methotrexate pretreatment. The increased amounts of the phosphorylated derivatives of FUra which are formed indicate that either the entry of FUra into cells has increased or the activity of a metabolic process has been enhanced. Since the quantity of the base, FUra, within methotrexate-treated cells was unchanged from control cells, it is unlikely that the effect being observed is related to the transport of FUra into the cells.
FUra conversion to nucleotides can occur via three metabolic pathways. FUra can react with ribose-l-phosphate in the presence of uridine phosphorylase to form FUrd (30) which is then phosphorylated to FUMP by uridine-cytidine kinase (37). The intracellular accumulation of ['HIFUrd into L1210 cells was reduced in response to methotrexate pretreatment and previously reported by us (18). Therefore, these sequential metabolic conversions of FUra are not responsible for the enhanced metabolism of FUra to nucleotides observed in methotrexate-treated cells. FUra can also be converted to FdUrd by the deoxyribose phosphorylases (36). This nucleoside is then phosphorylated to FdUMP by thymidine kinase (40). However, since FdUDP was not observed and because ribonucleotide reductase cannot convert FdUDP to FUDP (41), which was increased in the methotrexate-treated cells, this pathway is inconsistent with our findings. The only metabolic pathway which is consistent with our data is the conversion of FUra directly to FUMP by orotate phosphoribosyltransferase (42). This reaction requires 5-phosphoribosyl-lpyrophosphate as a co-substrate for the source of the 5-phosphate ribose moiety. The FUMP can then be converted to the other nucleotide forms, including FUTP and FdUMP, the cytotoxic metabolites (Fig. 7).
The proposed mechanism by which methotrexate influences this metabolic conversion of FUra is the increased 5-phosphoribosyl-1-pyrophosphate levels which occur in methotrexate-treated cells. Reyes (42) has shown that the rate of FUMP formation from FUra by orotate phosphoribosyltranferase is increased with increasing concentrations of 5-phosphoribosyl-1-pyrophosphate. Reyes also demonstrated that this conversion of FUra to FUMP was nearly completely inhibited by orotate, which also requires the same enzyme and co-substrate to form the nucleotide, OMP. We have examined the K , values of FUra and orotate conversion to the 5"phosphate nucleotide derivative by orotate phosphoribosyltransferase in these L1210 cells and found them to be 520 ~L M and 12 p~, respectively. (The enzymatic changes following methotrexate treatment will be published in detail elsewhere.) The large difference between these K , values indicate that small changes in orotate levels within the cell could greatly affect the initial metabolic step of FUra. This may explain the modest enhancement of intracellular FUra accumulation within cells exposed to N-(phosphonacety1)-L-aspartate which can reduce intracellular orotate levels (34). The inhibitory effects of pyrazofurin, an inhibitor of OMP decaroxylase, on FUra intracellular accumulation is probably the result of the increased orotate levels in pyrazofurin-treated cells (31). Although OMP cannot be decaroboxylated to UMP in the presence of this drug, this nucleotide is converted to the nucleoside, orotidine, which is increased (31). Therefore, even though the levels of 5-phosphoribosyl-1-pyrophosphate are not depleted in response to pyrazofurin, there is a constant utilization of this co-substrate by the preferred substrate orotate.
Our studies indicate that the modulating effect of methotrexate of FUra metabolism is the result of the inhibition of de novo purine synthesis. Methotexate's influence on purine synthesis is not a new concept (43,44), although the cell killing from methotrexate has generally been considered to be secondary to the effect of this drug on the formation of dTMP (44). An increase in the concentration of the purine nucleotides can reduce 5-phosphoribosyl-1-pyrophosphate generation (45); therefore, a reduction in these modulating nucleotides might lead to an increased synthesis of 5-phosphoribosyl-1-pyrophosphate. Although we did not quantitate the concentrations of purine nucleotides in our studies, we did demonstrate that the synthesis of adenine and guanine bases which were incorporated into nucleic acid was reduced in the methotrexate-treated cells. The other possible mechanism by which 5-phosphoribosyl-1-pyrophosphate levels are increased in response to inhibition of the purine synthetic rate is from a decreased utilization of 5-phosphoribosyl-1-pyrophosphate which is normally used for purine synthesis. Cells which were deficient in hypoxanthine-guanine phosphoribosyltransferase and were unable to salavage hypoxanthine or guanine had increased levels of 5-phosphoribosyl-1-pyrophosphate even though the synthesis of 5-phosphoribosyl-1-pyrophosphate was normal (46). The elevation of 5-phosphoribosyl-1-pyrophosphate in these cells was thought to be the result of 5phosphoribosyl-1-pyrophosphate underutilization. The activity of 5-phosphoribosyl-1-pyrophosphate synthetase, however, was increased 3-fold in human fibroblasts exposed to the antifolate, aminopterin (47). In this study, as in ours, the increase in 5-phosphoribosyl-1-pyrophosphate levels was prevented if hypoxanthine, which utilizes 5-phosphoribosyl-1-pyrophosphate for conversion to IMP, was also present in the cell cultures exposed to the antifolate. The FUra metabolism of cells exposed to 6-methylmercaptopurine riboside, which inhibits the fEst committed enzymatic step in de nouo purine synthesis, was identical with that which followed methotrexate treatment. This observation is consistent with what would be expected if inhibition of de novo purine synthesis is the major determinate of 5-phosphoribosyl-1-pyrophosphate levels.
The enhanced FUra metabolism and the reduction in purine synthesis in cells exposed to methotrexate can be prevented if FdUrd is given with methotrexate to the cell cultures. This suggests that if the utilization of the tetrahydrofolate pools for dTMP synthesis can be prevented by a direct inhibitor of thymidylate synthetase, FdUMP, purine synthesis can continue for the duration of these studies in the presence of methotrexate. In normal cells N5~'o-methylenetetrahydrofolate is oxidized to a metabolically inactive dihydrofolate derivative only during the methylation of dUMP to dTMP, a reaction catalyzed by thymidylate synthetase. Dihydrofolate is then reduced by dihydrofolate reductase to a tetrahydrofolate derivative which can now undergo conversion to one of several metabolically active tetrahydrofolate compounds. The tetrahydrofolate compounds used for de novo purine synthesis are not oxidized and therefore can be converted to metabolically active derivatives independent of dihydrofolate reductase.
The consequence of the inhibition of dihydrofolate reductase by methotrexate on folate metabolism would be a gradual depletion or alteration of the tetrahydrofolate pool sizes from the continued utilization of N5*'0-methylenetetrahydrofolate for dTMP synthesis without an equivalent regeneration of a tetrahydrofolate. Eventually, the purine synthetic rate would decrease because of this alteration in the tetrahydrofolate pools. However, the alteration of the tetrahydrolfolate pools could be prevented by the direct inhibition of the process which converts N5~10-methylenetetrahydrofolate to the inactive dihydrofolate form. The known inhibitory effects of the major nucleotide form of FdUrd, FdUMP, on thymidylate synthetase and the ability of this drug to prevent the inhibitory effects of methotrexate on purine synthesis in our studies is consistent with this hypothesis.
Leucovorin (-X5-formyltetrahydrolfolate) can reduce the effectiveness of the methotrexate-induced inhibition of dihydrofolate reductase (44, 48) presumably by replenishing the tetrahydrofolate pools. A recent report by Moran et al. (49) has shown that concentrations of Leucovorin (0. 1 ~L M ) which are unable to rescue L1210 cells from the lethal effects of 10 ~L M methotrexate are capable of preventing this cytotoxicity if 3 PM FdUrd and 5.6 p~ dThd were also present in the cell cultures. Presumably, the FdUrd reduced the utilization of N5310-methylenetetrahydrofolate and, therefore, less exogenous tetrahydrofolate was needed to maintain the tetrahydrofolate pools at a level required to support purine synthesis. The dThd provided an exogenous source for dTMP, the synthesis of which was inhibited by the FdUrd metabolite, FdUMP. These studies illustrate the dynamic relationship among the folate pools, purine synthesis, and dTMP synthesis, and are concepts consistent with our proposed complex interactions between methotrexate and FUra metabolism.
The exact mechanism responsible for the synergistic cell killing observed in cell cultures treated with methotrexate followed by FUra can not be answered from our studies. Legitimate reasons why there might be less ternary complex formed among FdUMP, N5~'o-methylenetetrahydrofolate and dTMP synthetase have already been discussed. However, the increased incorporation of FUra into RNA of cells pretreated with methotrexate could be contributing to this enhanced cell killing, perhaps by altering further the funtional RNA defects that occur with FUra (50-53).
Further investigations need to be performed to determine if the biochemical modulations that were observed in L1210 cells also occur in human neoplastic and normal cells. In addition, evaluation of tissue selectivity to this drug sequence is important if a major therapeutic benefit is to be expected without increasing toxicity to normal tissues.