Cellular Folates Prevent Polyglutamation of 5,10-Dideazatetrahydrofolate
1998; Elsevier BV; Volume: 273; Issue: 40 Linguagem: Inglês
10.1074/jbc.273.40.25944
ISSN1083-351X
Autores Tópico(s)HIV/AIDS drug development and treatment
ResumoMouse L1210 cell variants were selected for resistance to 5,10-dideazatetrahydrofolate, a potent inhibitor of the first folate-dependent enzyme in de novo purine synthesis, glycinamide ribonucleotide formyltransferase. The drug-resistant phenotype selected was conditional to the folate compound used to support growth: grown on folic acid cells were 400-fold resistant, whereas they were 2.5-fold more sensitive to 5,10-dideazatetrahydrofolate than wild-type L1210 cells when grown on folinic acid. In folic acid-containing media, polyglutamation of 5,10-dideazatetrahydrofolate was markedly reduced, yet folylpolyglutamate synthetase activity was not different from that in parental L1210 cells. Resistance was due to two changes in membrane transport: a minor increase in the K m for 5,10-dideazatetrahydrofolate influx, and a major increase in folic acid transport. Enhanced folic acid transport resulted in an expanded cellular content of folates which blocked polyglutamation of 5,10-dideazatetrahydrofolate.We propose that polyglutamation of 5,10-dideazatetrahydrofolate is limited by feedback inhibition by cellular folates on folylpolyglutamate synthetase, an effect which reflects a mechanism in place to control the level of cellular folates. Although the primary alteration causative of resistance is different from those reported previously, all 5,10-dideazatetrahydrofolate resistance phenotypes result in decreased drug polyglutamation, reflecting the centrality of this reaction to the action of 5,10-dideazatetrahydrofolate. Mouse L1210 cell variants were selected for resistance to 5,10-dideazatetrahydrofolate, a potent inhibitor of the first folate-dependent enzyme in de novo purine synthesis, glycinamide ribonucleotide formyltransferase. The drug-resistant phenotype selected was conditional to the folate compound used to support growth: grown on folic acid cells were 400-fold resistant, whereas they were 2.5-fold more sensitive to 5,10-dideazatetrahydrofolate than wild-type L1210 cells when grown on folinic acid. In folic acid-containing media, polyglutamation of 5,10-dideazatetrahydrofolate was markedly reduced, yet folylpolyglutamate synthetase activity was not different from that in parental L1210 cells. Resistance was due to two changes in membrane transport: a minor increase in the K m for 5,10-dideazatetrahydrofolate influx, and a major increase in folic acid transport. Enhanced folic acid transport resulted in an expanded cellular content of folates which blocked polyglutamation of 5,10-dideazatetrahydrofolate. We propose that polyglutamation of 5,10-dideazatetrahydrofolate is limited by feedback inhibition by cellular folates on folylpolyglutamate synthetase, an effect which reflects a mechanism in place to control the level of cellular folates. Although the primary alteration causative of resistance is different from those reported previously, all 5,10-dideazatetrahydrofolate resistance phenotypes result in decreased drug polyglutamation, reflecting the centrality of this reaction to the action of 5,10-dideazatetrahydrofolate. 5,10-dideazatetrahydrofolate folylpoly-γ-glutamate synthetase glycinamide ribonucleotide formyltransferase methotrexate high performance liquid chromatography phosphate-buffered saline 2-mercaptoethanol N-{4-[2-(2-amino-4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-l-glutamic acid fetal calf serum Chinese hamster ovary 5-fluoro-2′-deoxyuridine 5′-monophosphate 5-fluorodeoxyuridine. Development of classes of folate antimetabolites inhibitory to target enzymes other than dihydrofolate reductase has offered new therapeutic agents for the treatment of human malignancies, and has also provided new biochemical probes for studying folate metabolism and the linkages between cell proliferation and survival. The prototypical members of three of these classes are (6R)-5,10-dideazatetrahydrofolate ((6R)-DDATHF, lometrexol)1 (1Taylor E.C. Harrington P.J. Fletcher S.R. Beardsley G.P. Moran R.G. J. Med. Chem. 1985; 28: 914-921Crossref PubMed Scopus (176) Google Scholar), the quinazoline-based compound, ZD-1694 (tomudex) (2Jackman A.L. Taylor G.A. Gibson W. Kimbell R. Brown M. Calvert A.H. Judson I.R. Hughes L.R. Cancer Res. 1991; 51: 5579-5586PubMed Google Scholar), and N-{4-[2-(2-amino-4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-l-glutamic acid (LY231514, MTA) (3Taylor E.C. Kuhnt D. Shih C. Rinzel S.M. Grindey G.B. Barredo J. Jannatipour M. Moran R.G. J. Med. Chem. 1992; 35: 4450-4454Crossref PubMed Scopus (293) Google Scholar). (6R)-DDATHF is a tight-binding inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) (4Sanghani S.P. Moran R.G. Biochemistry. 1997; 36: 10506-10516Crossref PubMed Scopus (28) Google Scholar, 5Moran R.G. Baldwin S.W. Taylor E.C. Shih C. J. Biol. Chem. 1989; 264: 21047-21051Abstract Full Text PDF PubMed Google Scholar, 6Baldwin S.W. Tse A. Gossett L.S. Taylor E.C. Rosowsky A. Shih C. Moran R.G. Biochemistry. 1991; 30: 1997-2006Crossref PubMed Scopus (109) Google Scholar), the first folate-dependent enzyme in de novo purine synthesis, ZD-1694 is an inhibitor of thymidylate synthase (2Jackman A.L. Taylor G.A. Gibson W. Kimbell R. Brown M. Calvert A.H. Judson I.R. Hughes L.R. Cancer Res. 1991; 51: 5579-5586PubMed Google Scholar), and MTA has multiple targets within folate metabolism (7Shih C. Chen V.J. Gossett L.S. Gates S.B. MacKellar W.C. Habeck L.L. Schackelford K.A. Mendelsohn L.G. Soose D.J. Patel V.F. Andis S.L. Bewley J.R. Rayl E.A. Moroson B.A. Beardsley G.P. Kohler W. Ratnam M. Schultz R.M. Cancer Res. 1997; 15: 1116-1123Google Scholar). All three of these drugs have been shown to be very active against several animal tumors and also against a spectrum of human carcinoma xenografts (2Jackman A.L. Taylor G.A. Gibson W. Kimbell R. Brown M. Calvert A.H. Judson I.R. Hughes L.R. Cancer Res. 1991; 51: 5579-5586PubMed Google Scholar, 7Shih C. Chen V.J. Gossett L.S. Gates S.B. MacKellar W.C. Habeck L.L. Schackelford K.A. Mendelsohn L.G. Soose D.J. Patel V.F. Andis S.L. Bewley J.R. Rayl E.A. Moroson B.A. Beardsley G.P. Kohler W. Ratnam M. Schultz R.M. Cancer Res. 1997; 15: 1116-1123Google Scholar, 8Alati T. Worzalla J.F. Shih C. Bewley J.R. Lewis S. Moran R.G. Grindey G.B. Cancer Res. 1996; 56: 331-335Google Scholar, 9Jackman A.L. Marsham P.R. Moran R.G. Kimbell R. O'Connor B.M. Hughes L.R. Calvert H.A. Adv. Enzyme Regul. 1991; 31: 13-27Crossref PubMed Scopus (50) Google Scholar). Clinical trials have demonstrated therapeutic activity of these drugs against advanced human cancers (10Ray M.S. Muggia F.M. Leichman C.G. Grunberg S.M. Nelson R.L. Dyke R.W. Moran R.G. J. Natl. Cancer Inst. 1993; 85: 1154-1159Crossref PubMed Scopus (81) Google Scholar, 12Cunningham D. Zalcberg J.R. Rath U. Oliver I. van Cutsem E. Seitz J.F. Harper P. Kerr D. Perez-Manga G. Ann. Oncol. 1996; 7: 961-965Abstract Full Text PDF PubMed Scopus (239) Google Scholar,13Rinaldi D.A. Burris H.A. Dorr F.A. Woodworth J.R. Kuhn J.G. Eckardt J.R. Rodriguez G. Corso S.W. Fields S.M. Langley C. J. Clin. Oncol. 1995; 13: 2842-2850Crossref PubMed Scopus (155) Google Scholar). 2J. Roberts, M. Tombes, B. Mitchell, and R. G. Moran, submitted for publication. Compared with methotrexate (MTX), the classical antifolate targeted toward dihydrofolate reductase, (6R)-DDATHF, ZD-1694, and MTA are metabolized more rapidly and extensively to long chain polyglutamates by the enzyme folylpolyglutamate synthetase (FPGS) (2Jackman A.L. Taylor G.A. Gibson W. Kimbell R. Brown M. Calvert A.H. Judson I.R. Hughes L.R. Cancer Res. 1991; 51: 5579-5586PubMed Google Scholar,9Jackman A.L. Marsham P.R. Moran R.G. Kimbell R. O'Connor B.M. Hughes L.R. Calvert H.A. Adv. Enzyme Regul. 1991; 31: 13-27Crossref PubMed Scopus (50) Google Scholar, 14Pizzorno G. Sokoloski J.A. Cashmore A.R. Moroson B.A. Cross A.D. Beardsley G.P. Mol. Pharmacol. 1990; 39: 85-89Google Scholar, 15Jackman A.L. Gibson W. Brown M. Kimbell R. Boyle F.T. Rustum Y. Inhibition of Thymidylate Synthase by Pyrimidines and Folate Analogs: Therapeutic Implications for Cancer Therapy. Plenum Press, New York1993: 274-285Google Scholar, 16Habeck L.L. Mendelsohn L.G. Shih C. Taylor E.C. Colman P.D. Gossett L.S. Leitner T.A. Schultz R.M. Andis S.L. Moran R.G. Mol. Pharmacol. 1995; 48: 326-333PubMed Google Scholar). The metabolism of these drugs to their polyglutamate derivatives is essential for their cellular retention and these polyglutamates have been reported to be substantially more potent inhibitors of their respective target enzymes (4Sanghani S.P. Moran R.G. Biochemistry. 1997; 36: 10506-10516Crossref PubMed Scopus (28) Google Scholar, 6Baldwin S.W. Tse A. Gossett L.S. Taylor E.C. Rosowsky A. Shih C. Moran R.G. Biochemistry. 1991; 30: 1997-2006Crossref PubMed Scopus (109) Google Scholar, 7Shih C. Chen V.J. Gossett L.S. Gates S.B. MacKellar W.C. Habeck L.L. Schackelford K.A. Mendelsohn L.G. Soose D.J. Patel V.F. Andis S.L. Bewley J.R. Rayl E.A. Moroson B.A. Beardsley G.P. Kohler W. Ratnam M. Schultz R.M. Cancer Res. 1997; 15: 1116-1123Google Scholar, 14Pizzorno G. Sokoloski J.A. Cashmore A.R. Moroson B.A. Cross A.D. Beardsley G.P. Mol. Pharmacol. 1990; 39: 85-89Google Scholar, 15Jackman A.L. Gibson W. Brown M. Kimbell R. Boyle F.T. Rustum Y. Inhibition of Thymidylate Synthase by Pyrimidines and Folate Analogs: Therapeutic Implications for Cancer Therapy. Plenum Press, New York1993: 274-285Google Scholar). These new antifolates, therefore, probably function as “pro-drugs,” with the synthesis of polyglutamates being a requisite step for the development of cytotoxicity (17Moran, R. G., Shih, C., Taylor, E. C., and Grindey, G. B. (1992) in NCI-EORTC Symposium on New Drugs in Cancer Therapy, March 17–20, 1992, Amsterdam, Vol. 7, p. 91Google Scholar). Biochemical and molecular analysis of the mechanisms of acquired resistance of tumor cells to a drug has historically been a powerful source of information on which steps in the action of a drug are necessary for cytotoxicity. Several tumor cell lines have been selected for resistance to (6R)-DDATHF (18Matherly L.H. Angeles S.M. McGuire J.J. Biochem. Pharmacol. 1993; 46: 2185-2195Crossref PubMed Scopus (26) Google Scholar, 19Rhee M.S. Wang Y. Nair M.G. Galivan J. Cancer Res. 1993; 53: 2227-2230PubMed Google Scholar, 20Pizzorno G. Moroson B.A. Cashmore A.R. Russello O. Mayer J.R. Galivan J. Bunni M.A. Priest D.G. Beardsley G.P. Cancer Res. 1995; 55: 566-573PubMed Google Scholar). To date, tumor cell resistance to (6R)-DDATHF has been attributed to decreased drug transport (18Matherly L.H. Angeles S.M. McGuire J.J. Biochem. Pharmacol. 1993; 46: 2185-2195Crossref PubMed Scopus (26) Google Scholar), decreased FPGS activity (20Pizzorno G. Moroson B.A. Cashmore A.R. Russello O. Mayer J.R. Galivan J. Bunni M.A. Priest D.G. Beardsley G.P. Cancer Res. 1995; 55: 566-573PubMed Google Scholar), and increased γ-glutamylcarboxypeptidase (hydrolase) activity (19Rhee M.S. Wang Y. Nair M.G. Galivan J. Cancer Res. 1993; 53: 2227-2230PubMed Google Scholar), all of which result in the reduction in the steady state level of cellular (6R)-DDATHF polyglutamates. In this report, we describe a unique mechanism by which murine leukemic L1210 cells develop resistance to (6R)-DDATHF (and cross-resistance to ZD-1694): an expansion of the intracellular folate pool with consequent blockade of the synthesis of (6R)-DDATHF polyglutamates. This unexpected mutant phenotype appears due to a suspected but heretofore unproven feedback control mechanism on the synthesis of cellular (anti)folate polyglutamates, presumably by direct effects of cellular folates on FPGS. 10-CHO-5,8-dideazafolic acid was a gift from Dr. Homer Pearce of Lilly Research Laboratories (Indianapolis, IN) and was subsequently obtained from Dr. John Hynes of the Medical University of South Carolina (Charleston, SC); α,β-GAR was also from Dr. Pearce. ZD-1694, 1-chloro-3,5-dimethoxytriazene, (6R,6S)-, (6R)-DDATHF, and their chemically synthesized polyglutamates, and (6S)-DDATHF were gifts from Dr. Chuan Shih of Lilly Research Laboratories. [3′,5′,7,9-3H]-(6S)-5-formyltetrahydrofolate, [3′,5′,7,9-3H]folic acid, and [6-3H]fluorodeoxyuridylate (FdUMP) were purchased from Moravek Biochemicals (Brea, CA),l-[3,4-3H]glutamic acid from either NEN Life Science Products (Boston, MA) or Amersham, 5-fluorodeoxyuridine (FUdR), MTX, and folinic acid ((6R,6S)-5-formyltetrahydrofolate) from Sigma. [3H]Folic acid and [3H]5-formyltetrahydrofolate were purified by reverse phase HPLC eluted by a linear gradient of methanol and 0.03m sodium acetate (4–12% methanol over 12 min for folic acid; 3–15% methanol over 15 min for 5-formyltetrahydrofolate) and were stored at −20 °C for no more than 2 weeks prior to use. [3H]FdUMP was purified by DEAE column chromatography (21Moran R.G. Spears C.P. Heidelberger C. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1456-1460Crossref PubMed Scopus (150) Google Scholar). (6S)-H4PteGlu and 10-CHO-H4PteGlu were prepared as described previously (22Moran R.G. Werkheiser W.C. Zakrzewski S.F. J. Biol. Chem. 1976; 251: 3569-3575Abstract Full Text PDF PubMed Google Scholar). (6R)-DDATHF-[3,4-3H]Glu3was synthesized by incubating 0.145 mm[3H]glutamic acid (200 μCi) at 37 °C with 200 μm (6R)-DDATHF, 20 mm2-mercaptoethanol, 10 mm ATP, 20 mmMgCl2, and 30 mm KCl, and 2.8 μg of recombinant cytosolic human folylpolyglutamate synthetase (23Sanghani P.C. Sanghani S.P. Moran R.G. Proc. Am. Assoc. Cancer Res. 1997; 38: 98Google Scholar) in a total volume of 30 μl of 0.2 m Tris, pH 9.0, containing 0.2 mg/ml bovine serum albumin. The reaction was stopped after 2 h by heating at 100 °C for 3 min. The major product was the tetraglutamate derivative, which was identified by cochromatography with authentic DDATHF polyglutamate standards on a reverse phase, paired-ion HPLC column. Product was purified, first on a Sep-Pak C18 column (Waters Associates, Milford, MA), then on a 10 × 0.46-cm Luna 3 μm C-18 (Phenomenex, Torrance, CA) eluted with a multiphase gradient of methanol in acqueous tetrabutylammonium hydrogen sulfate (Pic A reagent, Waters Associates) run at 0.6 ml/min. To generate this gradient, mobile phase methanol concentration was initially 27%, then was increased to 35% in a linear gradient over 10 min; subsequently, a less steep linear gradient was initiated which reached a methanol concentration of 42% after an additional 15 min; the methanol concentration was then held at 42% for the next 10 min. Pooled HPLC fractions containing (6R)-[3H]DDATHF tetraglutamate were dried under vacuum, dissolved in 100 μl of water, and passed through a 1-ml column of AG 50W-X8 (Bio-Rad) to remove the ion-pairing reagent; the product was concentrated and stored at −20 °C in 20% ethanol. The amount of tetraglutamate product in an HPLC run was quantitated against the area of a known amount of monoglutamate standard. A procedure for the synthesis of (6R)-[3H]DDATHF was developed with the advice of Dr. Chuan Shih of Eli Lilly Research Laboratories, which involved the coupling of [3,4-3H]glutamic acid diethyl ester to (6R)-5,10-dideazapteroic acid. (6R)-5,10-Dideazapteroic acid was prepared by hydrolyzing (6R)-DDATHF in 6 n HCl at 100 °C in a sealed tube for 4 h, followed by purification on a column of DEAE cellulose. The diethyl ester of [3H]glutamic acid was prepared by reacting 18 μmol of [3,4-3H]glutamic acid (1 mCi; 55 Ci/mmol) (Amersham) with 0.1 m ethyltosylate in dry ethanol under reflux for 24 h. Crude diethyl ester of [3H]glutamic acid was purified on a column of silica gel and the pooled fractions were dried under N2. Typically, 3.4 μmol of 5,10-dideazapteroic acid was added to an equimolar amount of 1-chloro-3,5-dimethoxytriazene in 30 μl of anhydrous dimethyl sulfoxide containing 36 μmol of 4-methylmorpholine and allowed to react for 30 min at room temperature. This mixture was added to the dried [3H]diethylglutamate and reaction was allowed to proceed for 6 h at room temperature. (6R)-[3H]DDATHF was obtained by hydrolysis of the [3H]diethyl ester of (6R)-DDATHF with 1n sodium hydroxide for 6 h at room temperature. The product was purified by chromatography on a column of DEAE-cellulose and further purified on a paired ion-reverse phase HPLC system and the tetrabutylammonium ion present in the mobile phase was removed as described above. The product was stored at −20 °C in 33% ethanol. The purity and specific activity of the final product was determined by paired ion HPLC and scintillation counting; coinjection of (6R)-[3H]DDATHF with standard (6R)-DDATHF allowed purity to be estimated at >98%. (6R)-[3H]DDATHF was stable (<2% impurities detected by HPLC) for at least 2 weeks when stored at −20° C. The stability of the radiolabel was studied by HPLC after incubation at 37 °C in transport buffer (see below); purity was 95% after 24 h, but dropped to about 70% after 72 h. Mycoplasma-free mouse L1210 cells were passaged in RPMI 1640 medium supplemented with 10% dialyzed FCS in the presence of increasing concentrations of (6R,6S)-DDATHF until the culture resumed the same growth rate as a parallel culture maintained without drug. Initial drug concentration was 0.05 μm; thereafter, drug concentration was increased 2-6-fold every 3–4 weeks. Continuous passage of cells resistant to 3 μm (6R,6S)-DDATHF for an additional 5 months in 10 μm drug did not allow the emergence of a phenotype which could grow rapidly at that concentration of selective agent. Sublines of L1210 cells which grew in 0.5, 3.0, and 10 μm (6R,6S)-DDATHF were initiated from clones grown in soft agarose (24Keyomarsi K. Moran R.G. Cancer Res. 1986; 46: 5229-5235PubMed Google Scholar), and used for all comparisons against similarly cloned wild-type L1210 cells. Exponentially growing cells were transferred to drug containing medium in 24-well plates at an initial density of 2 × 104/ml in a total volume of 1.5 ml. Culture density was determined after 72 h at 37 °C and compared with the density of wells determined at time 0. For short term exposure to MTX, cells were incubated with drug for 6 h, washed with prewarmed PBS, and resuspended in drug-free medium; cell density was determined after a total of 72 h of growth. IC50values were determined by interpolation (24Keyomarsi K. Moran R.G. Cancer Res. 1986; 46: 5229-5235PubMed Google Scholar). GARFT was purified to electrophoretic homogeneity as described previously (6Baldwin S.W. Tse A. Gossett L.S. Taylor E.C. Rosowsky A. Shih C. Moran R.G. Biochemistry. 1991; 30: 1997-2006Crossref PubMed Scopus (109) Google Scholar). This procedure is based on affinity chromatography on a 3-ml column of Sepharose 4B to which 10-formyl-5,8-dideazafolate was attached via an ethylene linker. GARFT was then eluted with a 3 mm solution of 10-formyl-5,8-dideazafolic acid and was passed through a 10-ml column of Sephadex immediately prior to kinetic experiments. GARFT activity was assayed using a spectrophotometric assay which followed the rate of conversion of 10-CHO-5,8-dideazafolic acid to 5,8-dideazafolate (6Baldwin S.W. Tse A. Gossett L.S. Taylor E.C. Rosowsky A. Shih C. Moran R.G. Biochemistry. 1991; 30: 1997-2006Crossref PubMed Scopus (109) Google Scholar, 25Smith G.K. Meuller W.T. Benkovic P.A. Benkovic S.J. Biochemistry. 1981; 20: 1241-1245Crossref PubMed Scopus (65) Google Scholar). Assays contained 11 μm 10-formyl-5,8-dideazafolic acid and 10 μm α,β-glycinamide in a 1-cm cuvette. Cellular FPGS activity was determined using (6S)-tetrahydrofolate as a substrate (26Antonsson B. Barredo J. Moran R.G. Anal. Biochem. 1990; 186: 8-13Crossref PubMed Scopus (15) Google Scholar). For some experiments, FPGS was partially purified as described previously and kinetic experiments were performed using a charcoal-adsorption based assay with either aminopterin or DDATHF as substrates (27Moran R.G. Colman P.D. Anal. Biochem. 1984; 140: 326-342Crossref PubMed Scopus (41) Google Scholar). For determination of FPGS and GARFT activities, enzyme assays were performed on a high speed supernatant fraction produced by centrifugation for 30 min at full speed in a Beckman Airfuge (about 200,000 × g) or for 1 h in a Beckman Ti50 rotor at 160,000 × g. γ-Glutamylcarboxypeptidase (conjugase) activity was measured by the release of glutamic acid from (6R)-DDATHF tetraglutamate, which was tritium-labeled in the second through fourth side chain glutamate moieties. For conjugase assays, 8 × 107cells were suspended in 1 ml of 50 mm Tris acetate buffer, pH 6.0, containing 50 mm 2-ME, and the cells were broken by 3 × 20 strokes of a hand-held Dounce homogenizer. The lysate was centrifuged for 20 min at 14,000 rpm at 4 °C in a Microfuge, and the supernatant was used for assays. Protein (0–30 μg) was incubated with 100 μm(6R)-DDATHF-[3,4-3H]Glu3 (0.08 μCi/assay) in lysate buffer for up to 30 min and the reactions were stopped by the addition of 500 μl of a suspension of activated charcoal in 10 mm glutamate, 10 mm 2-ME, and 150 mm KH2PO4, pH 5.0. The charcoal had been pretreated with Dextran T-70 (27Moran R.G. Colman P.D. Anal. Biochem. 1984; 140: 326-342Crossref PubMed Scopus (41) Google Scholar). The reaction mixtures were centrifuged in a Microfuge for 5 min and the supernatant added to scintillation fluid for determination of radioactivity. Log-phase cells, grown in RPMI 1640 medium at a final concentration of 2.0 μm folic acid, were treated with (6R)-[3H]DDATHF for 16 h in the presence of 32 μm hypoxanthine and 5.6 μmthymidine. In some experiments, cells were depleted of intracellular folates by 6 days of exponential growth in RPMI 1640 medium formulated without folic acid and supplemented with 10% dialyzed FCS, 32 μm hypoxanthine, and 5.6 μm thymidine. Cellular folates were reduced to less than 0.02% of control by this procedure (22Moran R.G. Werkheiser W.C. Zakrzewski S.F. J. Biol. Chem. 1976; 251: 3569-3575Abstract Full Text PDF PubMed Google Scholar). After incubation with (6R)-[3H]DDATHF, cells were harvested by centrifigation and washed twice with 10 ml of ice-cold PBS containing 5% FCS. A known number of cells were resuspended in 100 μl of 5 mm tetrabutylammonium hydrogen sulfate (Pic A reagent, Waters Instruments, Milford, MA), sonicated for 8–10 1-min pulses in a Heat Systems Ultrasonics sonic disruptor using a cup horn attachment, and the broken cell suspensions were subsequently boiled for 3 min. The samples were filtered through a Microcon 10 filter (Amicon Corp.) and an aliquot was analyzed by HPLC. Recoveries from filtration were noted and used to adjust subsequent calculations. (6R)-DDATHF polyglutamates were separated on a 10 × 0.32-cm column of 3-μm pelicular C18 reverse phase column (Applied Biosystems) as described above. Fractions of 200 μl were collected, radioactivity was determined by liquid scintillation counting, and the identity of labeled peaks was determined from the retention times of chemically synthesized polyglutamate derivatives of (6R)-DDATHF. For measurement of the total pool of folate derivatives, cells were cultured in RPMI 1640 medium formulated without folic acid to which was added 10% dialyzed FCS and either 2.0 μm [3H]folic acid or 60 nm folinic acid spiked with high specific activity (6S)-[3H]5-formyltetrahydrofolate. After 1 week of exponential growth in labeled folate, the specific activity of the intracellular folates becomes equivalent to that in the medium, and pool size can be estimated by the level of intracellular radioactivity (22Moran R.G. Werkheiser W.C. Zakrzewski S.F. J. Biol. Chem. 1976; 251: 3569-3575Abstract Full Text PDF PubMed Google Scholar). Cells were harvested by centrifigation, and the washed cell pellet was dissolved in 0.5 ml of 1 n NaOH. The cell density of an aliquot of the third wash was determined electronically. The lysate was neutralized with HCl and radioactivity was determined by scintillation counting. Standard labeled compound was counted under the same conditions to allow counts/min to be directly converted to pmol of intracellular folates. Cellular levels of 10-CHO-H4PteGlun, and the sum of the H4PteGlun and 5,10-CH2-H4PteGlun pools were estimated using a modification of procedures (28Kesavan V. Doig M.T. Priest D.G. J. Biochem. Biophys. Methods. 1986; 12: 311-317Crossref PubMed Scopus (12) Google Scholar, 29Schmitz J.C. Grindey G.B. Priest D.G. Biochem. Pharmacol. 1994; 48: 319-325Crossref PubMed Scopus (44) Google Scholar) based on the entrapment of 5,10-CH2-H4PteGlun by excess thymidylate synthase and [3H]FdUMP as a macromolecular ternary complex. The content of H4PteGlun and 5,10-CH2-H4PteGlun was determined by this method, then the cellular levels of 10-CHO-H4PteGlun were determined indirectly by quantitating the additional H4PteGlun formed in the presence of excess GARFT and glycinamide ribonucleotide. Briefly, cell pellets containing 4 × 106 cells were resuspended in 200 μl of boiling 10 mm sodium phosphate buffer, pH 7.5, containing 0.1% Triton X-100, 1% 2-ME, and 1% freshly prepared sodium ascorbate. After 3 min, the suspensions were brought to 0 °C, and centrifuged at 14,000 rpm for 2 min in a refrigerated Microfuge. The pellets were extracted again and the extracts were combined. Aliquots of extract were added to 125 μl of a solution containing 10 mIU of pure L. casei thymidylate synthase, 0.16 μm [3H]FdUMP, and 20 μmformaldehyde in 10 mm sodium phosphate buffer, pH 7.4, containing 1% 2-ME and 0.2 mg/ml bovine serum albumin, or 200 μl of this same solution containing, in addition, 1 mIU of recombinant mouse GARFT (4Sanghani S.P. Moran R.G. Biochemistry. 1997; 36: 10506-10516Crossref PubMed Scopus (28) Google Scholar) and 10 μm α,β-glycinamide ribonucleotide. After incubation at 30 °C for 2 h, the tubes were boiled for 10 min to denature the ternary complex formed, 200 μl of 1% bovine serum albumin was added as carrier, and protein was precipitated by the addition of 4 ml of 8% ice-cold trichloroacetic acid. The final pellets were dissolved in 100 μl of 1 n NaOH, neutralized with 750 μl of 0.1 n HCl, 0.2 m KCl, and radioactivty was determined by liquid scintillation counting. Standard curves were simultaneously run using both (6S)-H4PteGlu and 10-CHO-H4PteGlu as controls. RNA from cell lines was extracted using the Triazol reagent (Life Technologies, Inc., Gaithersburg, MD), denatured with glyoxal, and separated by size on a 1.2% denaturing agarose gel in 10 mm sodium phosphate buffer, pH 7.0. The RNA was transferred onto nylon membranes (Biotrans, ICN, Irvine, CA) and hybridized with either a 1.7-kilobase probe representing the downstream sequences of the mouse L1210 cell FPGS cDNA (30Spinella M.J. Brigle K. Goldman I.D. Biochem. Biophys. Acta. 1996; 1305: 11-14Crossref PubMed Scopus (7) Google Scholar) or an 800-base pair probe corresponding to the glycinamide ribonucleotide synthetase domain of the polyfunctional GARFT mRNA (probe pQ0.8 (31Kan J.L.C. Jannatipour M. Taylor S.M. Moran R.G. Gene (Amst.). 1993; 137: 195-202Crossref PubMed Scopus (18) Google Scholar)). Probes were random labeled to a specific activity of about 2 × 109 cpm/μg and were used at a concentration of 1 μCi/ml hybridization solution (0.5 m sodium phosphate, pH 7.0, 7% SDS, 1% bovine serum albumin, and 1 mm EDTA). All blots were hybridized at 65 °C and filters were washed to a stringency of 0.2 × SSC and 0.1% SDS at 65 °C; a human glyceraldehyde-3-phosphate dehydrogenase probe was used to normalize for RNA loading. Exponentially growing cells were harvested by centrifugation, washed once with prewarmed PBS containing 5% FCS, and once with transport buffer (107 mm NaCl, 20 mm Tris-HCl, 26.2 mm NaHCO3, 5.3 mm KCl, 1.9 mm CaCl2, 1 mm MgCl2 and 7 mmd-glucose, pH 7.4, at 37 °C) (32Sirotnak F.M. Goutas L.J. Jacobsen D.M. Mines L.S. Barrueco J.R. Gaumont Y. Kisliuk R.L. Biochem. Pharmacol. 1987; 36: 1659-1667Crossref PubMed Scopus (36) Google Scholar). For studies on influx rate, cells were resuspended in 0.25 ml of transport buffer at a density of 107 cells/ml at 37 °C. Transport was initiated by the forceful addition of 0.25 ml of radiolabeled folates in prewarmed transport buffer and quenched at the indicated times by the addition of 10 ml of ice-cold PBS containing 5% FCS. Cells were then washed an additional two times with 10 ml of ice-cold PBS containing 5% serum, and the final pellets were dissolved in 0.5 ml of 1 n NaOH, neutralized with HCl, and radioactivity was determined by liquid scintillation counting. For measurement of folic acid transport, cells were treated with 10 μmtrimetrexate at 37 °C for 10 min prior to harvest, to inhibit dihydrofolate reductase (33Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1997; 272: 17460-17466Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). For studies on (6R)-DDATHF efflux, cells were preloaded with radiolabeled drug for 20 min, washed twice with ice-cold PBS containing 5% serum, resuspended in drug-free medium at 37 °C, and aliquots of cell suspension were withdrawn with time and processed as above for determination of intracellular label. Sublines of murine leukemic L1210 cells were selected by continuous exposure to stepwise increments in concentration of (6R,6S)-DDATHF. Whereas the parental L1210 cells were half-maximally inhibited by 2.8 × 10−8m (6R,6S)-DDATHF, L1210/D0.5 and/D3 grew in 0.5 and 3 μm selecting agent, respectively, with no detectable change in growth rate (doubling time of 10–12 h) (Fig. 1). Despite several months of additional selection in 10 μm (6R,6S)-DDATHF, more highly resistant cells did not emerge, although a cell line with a slower growth rate, the L1210/D10 cell, was isolated (Fig. 1). The resistance of L1210/D10 cells to (6R,6S)-DDATHF was found to be stable for at least 9 months during continued growth in the absence of drug; the sensitivity of L1210/D3 cells to (6R)-DDATHF was also found to be stable after growth for 6 months in drug-free media. The highly resistant L1210/D3 cell line
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