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On the Action of Fluorouracil on Leukemia Cells1

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On the Action of Fluorouracil on Leukemia Cells1 [CANCER RESEARCH 26 Part 1, 1611-1615,August 1966] On the Action of Fluorouracil on Leukemia Cells1 ALLAN R. GOLDBERG, JOHN H. MACHLEDT, JR., AND ARTHUR B. PARDEE Department of Biology, Princeton University, Princeton, New Jersey Summary In the present study the lymphoid leukemia L1210 of the mouse and a FU-resistant line were investigated. The problem The uptake and metabolism of radioactive uracil, uridine, posed was to discover a site of FU inhibition in the sensitive phosphate, and 5-fluorouracil by the mouse L1210 leukemic cells. The results suggest that the "salvage" pathway of pyrimi leukocytes and a fluorouracil-resistant variant were investigated. dine synthesis (see Chart 1) is sensitive to FU, with a resulting The dual aims of the research were to locate a metabolic differ inhibition of nucleic acid synthesis in the sensitive cells. The re ence responsible for resistance, and to define the site of action of the inhibitor. The resistant cells possess a much less active "sal sistant cells do not depend on this pathway, and hence are not vage" pathway, from uracil to nucleic acids, owing to a weaker susceptible to the inhibitor. uridine phosphorylase activity. They depend on the de novo pathway for a supply of pyrimidine nucleotides. Also, the con Materials and Methods version of fluorouracil to phosphorylated derivatives and its Uracil-3H and uridine-3H were obtained from the New Eng incorporation into RNA is somewhat reduced. Fluorouracil is land Nuclear Corporation, 5-FU-3H from Schwarz BioResearch, postulated to be less effective against these cells because its main Inc., and Na2H32PO4from Volk Radiochemical Co. Nonradio- site of action is in the salvage pathway, on which the resistant active FU and FUR were gifts of Hoffman-LaRoche Corp. cells do not depend. Perhaps the sensitive cells are only moder Both L1210/V (sensitive to FU) and L1210/FU-XIII (re ately affected by fluorouracil because they can utilize the de novo sistant) cells were generously supplied by Dr. Doris Hutchison. pathway for growth, as can other tissues lacking an active sal They were maintained in ascitic form by serial transfers in 6- vage pathway. The relations of these results to other proposed 10-week-old BDFi mice weighing 20-25 gm (Jackson Memorial mechanisms of fluorouracil action and resistance are discussed. Laboratory)- To maintain FU resistance, the L1210/r cells were periodically exposed to FU (25 mg/kg), beginning 1 day after Introduction transplant. Fluoropyrimidines are able to inhibit the growth of both bac For in vivo incorporation studies, 32P was injected i.p. into terial and mammalian cells (1, 7, 14, 15, 21, 22). Several modes 7-day leukemic mice. Fluorinated pyrimidines and sodium of inhibition have been proposed, the principal ones being: (a) orotate were injected 30 min prior to phosphate. Leukemia formation of TMP2 is inhibited by FdUMP, thereby blocking cells were withdrawn after 2.5 hr, and fractionated as described DNA synthesis (5, 11); (6) abnormal RNA is produced by incor below. poration of FU in place of uracil, and this produces a toxic en For in vitro incorporation studies cells were centrifuged several zyme pattern (4, 16); (c) nucleotide synthesis is inhibited, result times in cold Krebs-Ringer-bicarbonate containing 60 mg/ml ing in a reduced rate of RNA or DNA synthesis, or both (24). heparin and 10 mg/ml glucose, until free of blood cells and as Considerable controversy exists as to which of these mechanisms citic fluid. The leukemia cells were then weighed in a tared cen is most important for inhibition in vivo. Extensive work has been trifuge tube, and were resuspended in cold Krebs-Ringer solution done with mutant cell lines that arise spontaneously after treat containing glucose. Fluorinated compounds, when present, were ment with FIT, owing to their importance in cancer chemother preincubated with the cells for 15 min at 37°C.The cells were apy. Several modes of resistance have been reported, but no then added to 50 ml Ehrlenmeyer flasks containing radioactive single basis takes precedence. These problems are treated fur compounds. They were subsequently gassed with 5% C02 in air ther in the discussion. and were equilibrated for a few minutes at 37°.Incubations were carried out for 45 min at 37°with slow shaking. The reac tion mixtures were transferred to test tubes immersed in ice 1This investigation was supported by Grants 5T1-GM-457 and water, were centrifuged, and the pellet was washed in cold Krebs- Al-04409 from the USPHS. 2The following abbreviations are used: FU, 5-fluorouraeil; Ringer to remove most of the extracellular radioactivity and FUR, 5-fluorouridine; FUDR, 5-fluorodeoxyuridine; FUMP, then was weighed. 5-fluorouridylic acid; FdUMP, 5-fluorodeoxyuridylic acid; UR, The cells were fractionated according to a modified Schmidt- uridine; UMP, uridylic acid; UDP, uridine diphosphate; UTP, Thannhauser procedure (23). Protein and nucleic acids were uridine triphosphate; TMP, thymidylic acid; L1210/S, L1210cells precipitated with cold 0.6 N perchloric acid. The precipitate was sensitive to FU (D. J. Hutchison's L1210/V strain); L1210/r, washed once with perchloric acid. The supernatant fractions L1210 cells resistant to FU (D. J. Hutchison's L1210/FU-XIII were pooled and neutralized with 4 N KOH. Nonlabeled carriers strain). (uracil, UR, UMP, UDP, UTP, or the corresponding fluorinated Received for publication November 19, 1965. compounds) were added. The pyrimidine derivatives were sepa- AUGUST 1966 1611 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1966 American Association for Cancer Research. Allan R. Goldberg, John H. Machledt, Jr., and Arthur B. Pardee dsportic a.->—»—>orotic a.- OMP TABLE 1 INFLUENCEOF FLUOROUHACILONPHOSPHATEINCORPORATION INTORNA OF L1210/S CELLS ANDL1210/R CELLS UMP RNA Each value represents the mean of triplicate determinations on pooled cells from 5 animals. Percentage changes from control uridine uridine kinase values are given in parentheses. Each animal received i.p. 1.0 phosphorylase gniole phosphate (1.5 X IO6cpm) in 0.1 ml 2.5 hr prior to sacrifice. uracil uridine + ribose-l-P Fluorouracil was also injected i.p. (25 mg/kg) in 0.1 ml isotonic CHART1. The "salvage" and de novo pathways of pyrimidine NaCl. biosynthesis. OMP, orotidine phosphate; UMP, uridine phos OF PHOSPHATEINTORXA phate; ATP, adenosine triphosphate. protein)L1210/S(m/imoles/mg EXPERIMENTi2GÕOUPControlFluorouracilControlFluorouracilUPTAKE cells13.70.3 cells9.58.9 rated by descending paper chromatography in a solvent system of 100 ml isobutyric acid plus 60 ml 0.5 N NH4OH (3). Spots were located with an ultraviolet lamp, cut out, and counted with (-54)13.25.1 (-6)9.38.5 a Packard liquid scintillation counter. The perchloric acid precipitate was washed twice with 95% (-61)L1210/F (-9) ethanol and once with 1:1 ethanol-ether to remove lipids. RXA was extracted from the precipitate by hydrolysis in 1.0 N KOH for 16 to 20 hr at 37°,andthen was neutralized with perchloric acid. TABLE 2 The precipitate was washed once with perchloric acid. Superna- INFLUENCEOFOROTICACIDANDFLUOHOURACILONPHOSPHATE tants were combined and counted to determine the amount of INCORPORATIONINTORNA OF L1210/S CELLS radioactive precursor incorporated into RNA. Counts incor Each value represents the mean of triplicate determinations on porated into the residue, in part DNA, were also determined, pooled cells from 5 animals. Percentage changes from control but too few counts were found for conclusive results. values are given in parentheses. Each animal received i.p. 1.0 /imole phosphate (4 X 10scpm) in 0.1 ml 2.5 hr prior to sacrifice. Results Fluorouracil (25 mg/kg) and orotic acid (10 mg/kg) were injected i.p. in 0.1 ml isotonic NaCl. RNA synthesis in vivo was much more strongly inhibited by of phosphate FU in L1210/S than in L1210/r cells (Table 1). FU decreased AdditionNoneOrotic intoR.VA(m(<moles/mg 32Pincorporation by 54-61 % in the former and negligibly in the protein)6.383.32 latter. Since FU entered both types of cells equally well (see below), the difference cannot be attributed to changed permea bility to FU. Therefore, the decreased 32P incorporation should (-48)8.756.77 be due to inhibition in the sensitive cells of 1 or both of the path acidGroupControlFluorouracilControlFluorouracilUptake ways of pyrimidine synthesis. (-22) The de novo pathway was studied in vivo.Attempts to measure incorporation into RNA of the precursors aspartate-14C, or "CO»,were unsuccessful. Negligible incorporation in the case of salvage pathway was much more active in the sensitive than in the former compound was probably due to poor penetration the resistant cells. The latter appeared from the metabolic pat into the cells. Data obtained with orotic acid are summarized in tern, to have a reduced activity of uridine phosphorylase, since Table 2. Orotic acid stimulated phosphate incorporation into uridine and all of the phosphorylated intermediates, as well as RXA by about 35%. This indicates that the supply of UMP RNA, were poorly labeled. limited the rate of RNA synthesis; also, the rate-limiting step Uridine metabolism was studied similarly (Table 4). This in de novo synthesis preceded orotic acid. The stimulation by compound was found in equal amounts in both types of cells, orotic acid was even greater, approximately 100%, in the pres and so its transport was not altered. Its intracellular concentra ence of FU. This result suggests that FU does not inhibit the tion was about 10 times higher in the cells than in the medium, conversion of orotic acid to UMP, nor later steps in the path indicating an active transport mechanism. Uridine metabolism way to RNA. It is, however, consistent with the hypothesis, was similar in all respects in both types of cells, in contrast to proposed below, that FU inhibits the salvage pathway but not uracil metabolism.
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