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• W2000760541 abstract "Four L1210 murine leukemia cell lines resistant to 5,10-dideazatetrahydrofolate (DDATHF) and other folate analogs, but sensitive to continuous exposure to methotrexate, were developed by chemical mutagenesis followed by DDATHF selective pressure. Endogenous folate pools were modestly reduced but polyglutamate derivatives of DDATHF and ALIMTA (LY231514, MTA) were markedly decreased in these mutant cell lines. Membrane transport was not a factor in drug resistance; rather, folypolyglutamate synthetase (FPGS) activity was decreased by >98%. In each cell line, FPGS mRNA expression was unchanged but both alleles of the FPGS gene bore a point mutation in highly conserved domains of the coding region. Four mutations were in the predicted ATP-, folate-, and/or glutamate-binding sites of FPGS, and two others were clustered in a peptide predicted to be β sheet 5, based on the crystal structure of theLactobacillus casei enzyme. Transfection of cDNAs for three mutant enzymes into FPGS-null Chinese hamster ovary cells restored a reduced level of clonal growth, whereas a T339I mutant supported growth at a level comparable to that of the wild-type enzyme. The two mutations predicted to be in β sheet 5, and one in the loop between NH2- and COOH-terminal domains did not support cell growth. When sets of mutated cDNAs were co-transfected into FPGS-null cells to mimic the genotype of drug-selected resistant cells, clonal growth was restored. These results demonstrate for the first time that single amino acid substitutions in several critical regions of FPGS can cause marked resistance to tetrahydrofolate antimetabolites, while still allowing cell survival. Four L1210 murine leukemia cell lines resistant to 5,10-dideazatetrahydrofolate (DDATHF) and other folate analogs, but sensitive to continuous exposure to methotrexate, were developed by chemical mutagenesis followed by DDATHF selective pressure. Endogenous folate pools were modestly reduced but polyglutamate derivatives of DDATHF and ALIMTA (LY231514, MTA) were markedly decreased in these mutant cell lines. Membrane transport was not a factor in drug resistance; rather, folypolyglutamate synthetase (FPGS) activity was decreased by >98%. In each cell line, FPGS mRNA expression was unchanged but both alleles of the FPGS gene bore a point mutation in highly conserved domains of the coding region. Four mutations were in the predicted ATP-, folate-, and/or glutamate-binding sites of FPGS, and two others were clustered in a peptide predicted to be β sheet 5, based on the crystal structure of theLactobacillus casei enzyme. Transfection of cDNAs for three mutant enzymes into FPGS-null Chinese hamster ovary cells restored a reduced level of clonal growth, whereas a T339I mutant supported growth at a level comparable to that of the wild-type enzyme. The two mutations predicted to be in β sheet 5, and one in the loop between NH2- and COOH-terminal domains did not support cell growth. When sets of mutated cDNAs were co-transfected into FPGS-null cells to mimic the genotype of drug-selected resistant cells, clonal growth was restored. These results demonstrate for the first time that single amino acid substitutions in several critical regions of FPGS can cause marked resistance to tetrahydrofolate antimetabolites, while still allowing cell survival. folylpoly-γ-glutamate synthetase (6R)-5,10-dideazatetrahydrofolate (lometrexol) glycinamide ribonucleotide formyltransferase Chinese hamster ovary 5-formyltetrahydrofolate methotrexate pemetrexed disodium, MTA, LY231514 polymerase chain reaction Folate cofactors play an essential role in the biosynthesis of purines, thymidylate, glycine, and methionine by providing one-carbon moieties at a variety of oxidation levels. Folates are absorbed through the intestine as monoglutamates and are transported in that form through the circulation and into peripheral cells in mammals. Once in the cell, they are rapidly metabolized to folylpoly-γ-glutamates, the preferred substrates for many of the tetrahydrofolate cofactor-dependent enzymes (1McGuire J.J. Mini E. Hsieh P. Bertino J.R. Cheng Y.C. Goz B. Minkoff M. The Development of Target-oriented Anticancer Drugs. Raven Press, New York1983: 97-106Google Scholar, 2Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (290) Google Scholar). Folylpoly-γ-glutamate synthetase (FPGS)1 mediates the synthesis of these derivatives, and is distributed in both the cytoplasmic and mitochondrial compartments of mammalian cells (3Lin B.-F. Huang R.-F.S. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar, 4Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Crossref PubMed Scopus (59) Google Scholar). As polyglutamates, folates are retained in the cytosol and in mitochondria and accumulate to levels far higher than can be achieved for unmetabolized folate monoglutamates. Transport of the reduced folates into mammalian cells is mediated by the reduced folate carrier (5Sirotnak F.M. Tolner B. Annu. Rev. Nutr. 1999; 19: 91-122Crossref PubMed Scopus (260) Google Scholar, 6Sierra E.E. Goldman I.D. Semin. Oncol. 1999; 26 Suppl. 6: 11-23Google Scholar), although two other routes have been identified in some tissues, the folate receptor family of proteins (7Henderson G.B. Annu. Rev. Nutr. 1990; 10: 319-335Crossref PubMed Scopus (187) Google Scholar, 8Brigle K.E. Westin E.H. Houghton M.T. Goldman I.D. J. Biol. Chem. 1991; 266: 17243-17249Abstract Full Text PDF PubMed Google Scholar, 9Kamen B.A. Wang M.-T. Streckfuss A.J. Peryea X. Anderson R.G.W. J. Biol. Chem. 1988; 263: 13602-13609Abstract Full Text PDF PubMed Google Scholar) and a low-pH transporter (10Henderson G.B. Strauss B.P. Cancer Res. 1990; 50: 1709-1714PubMed Google Scholar, 11Sierra E.E. Goldman I.D. Biochem. Pharmacol. 1998; 55: 1505-1512Crossref PubMed Scopus (34) Google Scholar, 12Assaraf Y.G. Babani S. Goldman I.D. J. Biol. Chem. 1998; 273: 8106-8111Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). While polyglutamation occurs rapidly within the cell, available evidence indicates that folates are not channeled from the transport proteins to FPGS (13Spinella M.J. Brigle K.E. Freemantle S.J. Sierra E.E. Goldman I.D. Biochem. Pharmacol. 1996; 52: 703-712Crossref PubMed Scopus (14) Google Scholar). The first folate antimetabolites discovered, such as methotrexate (MTX), are inhibitors of dihydrofolate reductase that require both efficient transport and polyglutamation for maximal activity. Resistance of tumor cells to MTX can involve any of several mechanisms, including mutation or down-regulation of the reduced folate carrier (14Gorlick R. Goker E. Trippett T. Steinherz P. Elisseyeff Y. Mazumdar M. Flintoff W.F. Bertino J.R. Blood. 1997; 89: 1013-1018Crossref PubMed Google Scholar, 15Matherly L.H. Taub J.W. Ravindranath Y. Proefke S.A. Wong S.C. Gimotty P. Buck S. Wright J.E. Rosowsky A. Blood. 1995; 85: 500-509Crossref PubMed Google Scholar, 16Brigle K.E. Spinella M.J. Sierra E.E. Goldman I.D. J. Biol. Chem. 1995; 270: 22974-22979Crossref PubMed Scopus (106) Google Scholar, 17Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 7873-7879Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 19065-19071Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Roy K. Tolner B. Chiao J.H. Sirotnak F.M. J. Biol. Chem. 1998; 273: 2526-2531Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 20Zhao R. Sharina I.G. Goldman I.D. Mol. Pharmacol. 1999; 56: 68-76Crossref PubMed Scopus (58) Google Scholar, 21Jansen G. Mauritz R. Drori S. Sprecher H. Kathmann I. Bunni M. Priest D.G. Noordhuis P. Schornagel J.H. Pinedo H.M. Peters G.J. Assaraf Y.G. J. Biol. Chem. 1998; 273: 30189-30198Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), amplification or mutation of the dihydrofolate reductase gene (22Schimke R.T. J. Biol. Chem. 1988; 263: 5989-5992Abstract Full Text PDF PubMed Google Scholar, 23Dicker A.P. Waltham M.C. Volkenandt M. Schweitzer B.I. Otter G.M. Schmid F.A. Sirotnak F.M. Bertino J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11797-11801Crossref PubMed Scopus (40) Google Scholar), and/or decreased formation of polyglutamate derivatives by FPGS (24Pizzorno G. Mini E. Coronnello M. McGuire J.J. Moroson B.A. Cashmore A.R. Dreyer R.N. Lin J.T. Mazzei T. Periti P. Cancer Res. 1988; 48: 2149-2155PubMed Google Scholar). More recently, several classes of antimetabolites have been developed as antitumor agents which are targeted directly against the folate biosynthetic enzymes. These antimetabolites can be subdivided as compounds which are: 1) active against de novo purine synthesis due to inhibition of the third enzyme of this pathway, glycinamide ribonucleotide formyltransferase (GART), 2) active against thymidylate synthase, or 3) active against multiple folate biosynthetic enzymes. Clinically relevant compounds that fall into these three groupings are lometrexol (DDATHF) and L309887 (25Moran R.G. Baldwin S.W. Taylor E.C. Shih C. J. Biol. Chem. 1989; 264: 21047-21051Abstract Full Text PDF PubMed Google Scholar, 26Baldwin 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, 27Mendelsohn L.G. Shih C. Schultz R.M. Worzalla J.F. Invest. New Drugs. 1996; 14: 287-294Crossref PubMed Scopus (48) Google Scholar), raltitrexed (Tomudex, ZD1694) (28Jackman 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 ALIMTA (LY231514, MTA) (29Taylor 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 (292) Google Scholar, 30Shih C. Chen V.J. Gossett L.S. Gates S.B. MacKellar W.C. Habeck L.L. Shackelford 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; 57: 1116-1123PubMed Google Scholar), respectively. Most of these agents are strongly activated by polyglutamation, not only because they are better inhibitors of their target enzymes as polyglutamates, but also because they are retained in target cells and accumulate to high levels as these metabolites (26Baldwin 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, 27Mendelsohn L.G. Shih C. Schultz R.M. Worzalla J.F. Invest. New Drugs. 1996; 14: 287-294Crossref PubMed Scopus (48) Google Scholar, 28Jackman 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, 29Taylor 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 (292) Google Scholar, 30Shih C. Chen V.J. Gossett L.S. Gates S.B. MacKellar W.C. Habeck L.L. Shackelford 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; 57: 1116-1123PubMed Google Scholar, 31Sanghani S.P. Moran R.G. Biochemistry. 1997; 36: 10506-10516Crossref PubMed Scopus (28) Google Scholar, 32Jackman A.L. Gibson W. Brown M. Kimbell R. Boyle F.T. Rustum Y.M. Inhibition of Thymidylate Synthase by Pyrimidines and Folate Analogs: Therapeutic Implications for Cancer Therapy. Plenum Press, New York1993: 274-285Google Scholar). In this study, we investigate the mechanisms which allow resistance to develop to the prototypical GART inhibitor DDATHF after treatment with the mutagen N-methyl-N-nitrosourea. In several independent cell lines, point mutations in FPGSsubstantially decreased polyglutamation of DDATHF and several other folate antimetabolites, causing up to a 1700-fold decrease in the potency of these drugs. However, the in vivo function of polyglutamation of the naturally occurring folates was sufficient to sustain cell growth and replication. 3′,5′,7-3H]MTX and 3′,5′,7-3H]-(6S)-5-CHO-THF were purchased from Amersham Pharmacia Biotech and Moravek Biochemicals (Brea, CA), respectively. [3H]ALIMTA, [3H]DDATHF, unlabeled ALIMTA, LY309887, (6R)-DDATHF, and DDATHF polyglutamates were kindly provided by Eli Lilly Research Laboratories. ZD1694 and ZD9331 were obtained from ICI (United Kingdom). Tritiated folates were purified by high performance liquid chromatography prior to use (33Fry D.W. Yalowich J.C. Goldman I.D. J. Biol. Chem. 1982; 257: 1890-1896Abstract Full Text PDF PubMed Google Scholar, 34Tse A. Moran R.G. J. Biol. Chem. 1998; 273: 25944-25952Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). All other reagents were of the highest purity available from various commercial sources. L1210 murine leukemia cells and its sublines were grown in RPMI 1640 medium containing 2.3 μm folic acid, supplemented with 5% bovine calf serum (HyClone), 2 mm glutamine, 20 μm 2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere of 5% CO2. L1210 cells grown in complete RPMI 1640 medium were treated with 0.4 mm N-methyl-N-nitrosourea for 12 h to achieve about 10% cell survival (35Lee G.S. Blonsky K.S. Van On D.L. Savage E.A. Morgan A.R. von Borstel R.C. J. Mol. Biol. 1992; 223: 617-626Crossref PubMed Scopus (49) Google Scholar). After cells were washed to remove the mutagen they were placed in 24-well plates at a density of 2 × 105 cells/ml and allowed to grow for 3 days. Cells from each well were then seeded in fresh complete RPMI 1640 medium containing 400 nm DDATHF and grown for 2 additional weeks. The surviving cells were assayed for cross-resistance to MTX. Cells that were not resistant to MTX were plated in complete RPMI 1640 containing 0.5% soft agar. After an additional 2 weeks, individual clones were picked up and expanded in the absence of DDATHF. The clonal cell lines L7, L15, L44, and L51 were maintained thereafter in DDATHF-free RPMI 1640 medium. Cells in mid-log growth phase were grown in 96-well plates (1 × 105 cells/ml), exposed continuously to the appropriate concentrations of DDATHF, LY309887, MTX, ZD1694, ZD9331, and ALIMTA for 72 h following which cell numbers were determined by hemocytometer count and viability assessed by trypan blue exclusion. Prior to assessment of folate growth requirement, cells were grown for 1–2 weeks in folate-free RPMI 1640 medium supplemented with 200 μm glycine, 100 μm adenosine, and 10 μm thymidine (GAT) to deplete endogenous folates. Then these cells were exposed to different concentrations of folic acid and 5-CHO-THF for 72 h and cell numbers determined. For measurement of the total pool of folate derivatives, cells were grown in folate-free RPMI 1640 supplemented with 5% dialyzed bovine calf serum (HyClone) and either 2 μm [3H]folic acid or 25 nm[3H]5-CHO-THF. After 1 week of exponential growth in this medium, cells were harvested, washed twice with HBS, and processed for the measurement of intracellular radioactivity (36Zhao R. Seither R. Brigle K.E. Sharina I.G. Wang P.J. Goldman I.D. J. Biol. Chem. 1997; 272: 21207-21212Crossref PubMed Scopus (58) Google Scholar). For determination of total accumulation of ALIMTA and DDATHF, cells were grown with 50 nm [3H]ALIMTA or [3H]DDATHF for 3 days in RPMI 1640 medium with GAT to overcome the cytotoxicity of these drugs. The cells were harvested, washed, and intracellular radioactivity determined (36Zhao R. Seither R. Brigle K.E. Sharina I.G. Wang P.J. Goldman I.D. J. Biol. Chem. 1997; 272: 21207-21212Crossref PubMed Scopus (58) Google Scholar). To assess formation of DDATHF polyglutamate derivatives, cells were exposed to 0.2 μm [3H]DDATHF for 8 h in RPMI 1640 medium containing 10% dialyzed fetal calf serum, 100 μm hypoxanthine, and 10 μm thymidine. Cells were harvested, broken by sonication, and folates extracted by heating the sonicate at 100 °C for 3 min. Supernatants were filtered, mixed with 500–1500 pmol of DDATHF polyglutamate standards (glutamate chain lengths 1–6) and 100 μl injected onto a 10-cm 3-μm Luna C18 column (Phenomenex, Torrence, CA). Elution was achieved with a multiphase gradient of methanol and tetraethylammonium phosphate (PicA reagent, Waters Associates) (34Tse A. Moran R.G. J. Biol. Chem. 1998; 273: 25944-25952Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Influx measurements were performed by methods described previously (36Zhao R. Seither R. Brigle K.E. Sharina I.G. Wang P.J. Goldman I.D. J. Biol. Chem. 1997; 272: 21207-21212Crossref PubMed Scopus (58) Google Scholar). Briefly, exponentially growing cells were harvested, washed twice, and resuspended in HBS (20 mmHEPES, 140 mm NaCl, 5 mm KCl, 2 mmMgCl2, 5 mm glucose, pH 7.4) to a density of 1.5 × 107 cells/ml. Cell suspensions were incubated at 37 °C for 25 min following which uptake was initiated by the addition of radiolabeled folate and samples removed at the indicated times. Uptake was terminated by injection of 1 ml of the cell suspension into 10 ml of ice-cold HBS. Cells were collected by centrifugation, washed twice with ice-cold HBS and processed for measurement of intracellular radioactivity (36Zhao R. Seither R. Brigle K.E. Sharina I.G. Wang P.J. Goldman I.D. J. Biol. Chem. 1997; 272: 21207-21212Crossref PubMed Scopus (58) Google Scholar). The pentaglutamate of DDATHF labeled with tritium in the four terminal side chain glutamic acids was prepared for use as a substrate for γ-glutamyl carboxypeptidase assays. [3,4-3H]Glutamic acid (140 μCi, 46 Ci/mmol) was incubated with 28 μm DDATHF, 10 mm ATP, 20 mm MgCl2, 30 mm KCl, 20 mm 2-mercaptoethanol, 50 μg/ml bovine serum albumin, and 16 μg of pure recombinant human cytosolic FPGS (37Sanghani P.C. Moran R.G. Protein Expr. Purif. 2000; 18: 36-45Crossref PubMed Scopus (19) Google Scholar) in 100 mm Tris buffer, pH 8.9, for 30 min at 37 °C. The major product, DDATHF pentaglutamate for which 4 mol of [3H]glutamic acid were added per mole of DDATHF, was purified on a 10-cm column of 3-μm particle size C18 Luna HPLC column using a multiphase gradient of methanol in tetrabutylammonium phosphate (Pic A reagent) (38Sanghani S.P. Sanghani P.C. Moran R.G. J. Biol. Chem. 1999; 274: 27018-27027Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Pic A was removed by adsorption of the purified product on a 300-μl column of DEAE-Sephacel and elution with ammonium acetate. γ-Glutamylcarboxypeptidase was measured by incubating cellular protein with [3H]DDATHF pentaglutamate and separating unreacted substrate from product by adsorption onto activated charcoal. Cells were broken in a hand-held Dounce homogenizer in 50 mm Tris acetate buffer, pH 6.0, containing 50 mm 2-mercaptoethanol, and lysates were centrifuged for 10 min at 4 °C and 14,000 rpm in a Beckman Microfuge. Protein was incubated with 100 μm [3H]DDATHF pentaglutamate in a total volume of 25 μl of 50 mm Tris acetate buffer, pH 6.0, containing 50 mm 2-mercaptoethanol. Charcoal slurry (34Tse A. Moran R.G. J. Biol. Chem. 1998; 273: 25944-25952Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) was added, the mixture was centrifuged in a Microfuge, and radioactivity in the supernatant was determined on a liquid scintillation spectrometer. FPGS activity was measured using a microprocedure previously described (39Antonsson B. Barredo J. Moran R.G. Anal. Biochem. 1990; 186: 8-13Crossref PubMed Scopus (15) Google Scholar) in which cytosolic protein, prepared by a 110,000 ×g centrifugation step, was incubated with 10 μm (6S)-tetrahydrofolate in the presence of 5 mm ATP, 10 mm MgCl2, 30 mm KCl, and 1 mm [3H]glutamic acid in 200 mm Tris, pH 8.5, containing 36 mm2-mercaptoethanol. The product was isolated by Sephadex spin chromatography after conversion to a macromolecule in the presence of fluorodeoxyuridylate, pure Lactobacillus caseithymidylate synthase, and formaldehyde. The procedure used in these experiments was modified from the published procedure (39Antonsson B. Barredo J. Moran R.G. Anal. Biochem. 1990; 186: 8-13Crossref PubMed Scopus (15) Google Scholar) by increasing the amount of thymidylate synthase used from 60 to 300 pmol per assay. With this modification, the isolation of FPGS product was substantially more complete, and FPGS activity values agreed well with other literature procedures. GART activity was measured spectrophotometrically as described previously (26Baldwin 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, 40Smith G.K. Mueller W.T. Benkovic P.A. Benkovic S.J. Biochemistry. 1981; 20: 1241-1245Crossref PubMed Scopus (65) Google Scholar). Protein was determined using a dye binding assay (Bio-Rad). Total RNA was isolated using the TRIzol reagent (Life Technologies). RNA (20 μg) was resolved by electrophoresis on 1% agarose gels containing formaldehyde. Transfer and hybridization were performed as described previously (36Zhao R. Seither R. Brigle K.E. Sharina I.G. Wang P.J. Goldman I.D. J. Biol. Chem. 1997; 272: 21207-21212Crossref PubMed Scopus (58) Google Scholar). Transcripts were quantitated by PhosphorImager analysis of the hybridization signals and normalized to β-actin. Poly(A)+mRNA was purified using a Dynabeads mRNA DIRECT kit (Dynal). The first DNA strand synthesis was carried out with a Superscript Reverse Transcriptase according to the manufacturers protocol (Life Technologies). The coding sequence was amplified with PfuTurbo polymerase (Stratagene) utilizing oligonucletide primers which flank the coding region of cytoplasmic FPGS (upsteam primer: at nucleotide −10 from the cytosolic translation start codon 5′-GGAGCCGGGCATGGAGTA-3′ and downstream primer at nucleotide +36 from translation stop codon 5′-TGTGGAAAGGCGGACCGATG-3′) (41Roy K. Mitsugi K. Sirotnak F.M. J. Biol. Chem. 1996; 271: 23820-23827Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The PCR amplifications were performed for 35 cycles of 45 s at 95 °C, 45 s at 60 °C, and 4 min at 72 °C. The 1683-base pair long predicted PCR product was purified on an agarose gel (Qiagen) and cloned into a pCR-Blunt vector (Invitrogen). Both the whole cDNA population and cloned fragments were sequenced using the two primers described above for the PCR reaction and two additionalFPGS-based primers: 5′-CCTCTTACTTCCGCTTCCTC-3′ and 5′-CACCTGTGTTCCGCCCATCC-3′. pCR-Blunt vector-based primers: T7 and M13 R (−24) were also used for the cloned fragments. The sequence analysis was obtained on Applied Biosystems models 373A and 377 Sequencers in the DNA Sequencing Facility of the Albert Einstein College of Medicine Comprehensive Cancer Center. cDNAs were recloned into pcDNA3 (Invitrogen) and plasmids were purified after growth in XL1-Blue cells (Stratagene). Sixteen hours prior to transfections, 5 × 104 AUXB1 cells were plated per 100-mm dish and incubated overnight in minimal essential medium α containing 10% fetal calf serum. For each dish, calcium phosphate/DNA co-precipitates were generated by mixing 5 μg of plasmid DNA and 20 μg of sheared human liver genomic carrier DNA in a final volume of 0.5 ml of 0.27 m CaCl2, then slowly adding 0.5 ml of 2 × HEPES-buffered saline (4Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Crossref PubMed Scopus (59) Google Scholar, 42Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6490) Google Scholar). Dual transfection plates were treated with 5 μg of each plasmid and 15 μg of carrier DNA. After 30 min, the microprecipitates were added to cells and transfected cells were osmotically shocked with 10% Me2SO in complete medium the next morning. After 2 days incubation at 37 °C in non-selective conditions, the medium was changed to either minimal essential medium α containing 10% fetal calf serum and 1 mg/ml G418 as a transfection efficiency control, or minimal essential medium α formulated without nucleosides containing 10% dialyzed fetal calf serum and G418 to test for the ability of a construct to confer FPGS activity to the cells. Plates were fixed and stained after 2 weeks of selection, and macroscopic colonies were counted. L1210 cells were exposed to a mutagenic concentration ofN-methyl-N-nitrosourea, followed by selective pressure with 0.4 μm (6R)-DDATHF. This selection strategy minimized the likelihood of acquired resistance due to changes in the level of expression of genes critical to drug action. A total of 53 DDATHF-resistant clones were initially identified; the majority were cross-resistant to MTX and were not studied further. Six clones which were not cross-resistant to continuous exposure to MTX exhibited stable resistance to DDATHF after passage in drug-free medium for 6 months. These clones maintained a doubling time similar to that of the parent L1210 cells. Four of these cell lines had very similar biochemical and pharmacological phenotypes and are the subject of this report. The levels of DDATHF required to inhibit these four cell lines were 13–40-fold higher than those inhibitory to wild-type L1210 cells (TableI). In contrast, the IC50 for MTX was unchanged or slightly decreased compared with parental cells. All four cell lines were cross-resistant to the second generation GART inhibitor LY309887. Interestingly, the resistance of the L15 cell line to LY309887 was only 4-fold greater than that of L1210 cells, whereas these cells were 40-fold more resistant to DDATHF than the parental L1210 cells. A much higher level of cross-resistance to the TS-inhibitor D1694 was observed in several of these cell lines; for instance, the L7 cells were nearly 2000-fold less sensitive to ZD1694 than were L1210 cells, but only 17-fold resistant to DDATHF, the agent used in the original selection procedure. However, none of these cell lines were resistant to a non-polyglutamatable TS inhibitor, ZD9331 (43Jackman A.L. Kimbell R. Aherne G.W. Brunton L. Jansen G. Stephens T.C. Smith M.N. Wardleworth J.M. Boyle F.T. Clin. Cancer Res. 1997; 3: 911-921PubMed Google Scholar); this suggested alterations in polyglutamation as the mechanism of resistance. All four resistant cell lines displayed moderate (15–60-fold) resistance to ALIMTA, similar in each case to that for DDATHF.Table IGrowth inhibition by continuous exposure to antifolates (IC 50 ) and growth requirements (EC 50 ) in DDATHF-resistant L1210 cell linesCell lineIC50EC50DDATHFMTXLY309887ZD1694ZD9331ALIMTAFolic acid5-CHO-THFnmnmL121027 ± 6 (1)12 ± 0.5 (1)8.5 ± 1.6 (1)3.6 ± 0.2 (1)12 ± 1 (1)10 ± 1 (1)206 ± 23 (1)1.7 ± 0.3 (1)L7450 ± 100 (17)9.5 ± 0.5 (0.8)93 ± 7 (11)6250 ± 250 (1700)11 ± 1 (0.9)600 ± 100 (60)1500 ± 0 (7)13.2 ± 6.4 (8)L151070 ± 220 (40)6.1 ± 0.1 (0.5)32 ± 2 (3.8)2530 ± 290 (700)8.7 ± 1 (0.7)700 ± 60 (70)2730 ± 180 (13)23 ± 8 (14)L44340 ± 30 (13)5.3 ± 0.3 (0.4)143 ± 24 (17)69 ± 7 (19)13 ± 1 (1.1)150 ± 23 (15)1770 ± 230 (9)10.1 ± 3.6 (6)L51700 ± 120 (26)5.3 ± 0.4 (0.4)147 ± 12 (17)930 ± 60 (260)11 ± 4 (0.9)230 ± 35 (23)2030 ± 270 (10)19.4 ± 5.4 (11)L1210 and mutant cells grown in RPMI 1640 medium were exposed continuously to the appropriate concentrations of drugs for 72 h following which cell numbers were determined by hemocytometer count. For assessment of folic acid and 5-CHO-THF growth requirement, cells were first grown for 1–2 weeks in folate-free RPMI 1640 medium supplemented GAT to deplete endogenous folates and then exposed to different concentration of folic acid or 5-CHO-THF for 72 h and cell numbers determined. The numbers in parentheses are fold increase as compared to wild-type L1210 cells. The data are the mean ± S.E. from three separate experiments. Open table in a new tab L1210 and mutant cells grown in RPMI 1640 medium were exposed continuously to the appropriate concentrations of drugs for 72 h following which cell numbers were determined by hemocytometer count. For assessment of folic acid and 5-CHO-THF growth requirement, cells were first grown for 1–2 weeks in folate-free RPMI 1640 medium supplemented GAT to deplete endogenous folates and then exposed to different concentration of folic acid or 5-CHO-THF for 72 h and cell numbers determined. The numbers in parentheses are fold increase as compared to wild-type L1210 cells. The data are the mean ± S.E. from three separate experiments. The growth requirement of these mutant cell lines for folic acid and for 5-CHO-THF was examined. All mutant cells required higher levels of either folate source for half-maximal growth (Table I); the increased requirement of each of the cell lines for folic acid was nearly identical to that for growth on 5-CHO-THF. The increased growth requirement for exogenous folates of these mutant cell lines was much lower than the degree of resistance to DDATHF or ALIMTA, and differed from the degree of resistance to ZD1694 by factors of as much as 250. The accumulation of folates and antifolates was studied in these mutant cell lines. Cells were grown in folate-free medium supplemented with either 2 μm[3H]folic acid or 25 nm[3H]5-CHO-THF. After 1 week of exponential growth, cells were harvested and the total intracellular folate content determined. Total cellular folates were lower in the mutant cell lines than in parental L1210 cells after growth on either folic acid or 5-CHO-THF (Fig. 1 A). Typically, the intracellular folate pools were decreased by 45–55% in L7, L44, and L51 cells, and by 60–75% in L15 cells grown on either folic acid or 5-CHO-THF. The accumulation of DDATHF and ALIMTA was studied over a period of 3 days in medium supplemented with nucleosides protective of the antifolate effects of these d" @default.
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• W2000760541 title "Molecular Analysis of Murine Leukemia Cell Lines Resistant to 5,10-Dideazatetrahydrofolate Identifies Several Amino Acids Critical to the Function of Folylpolyglutamate Synthetase" @default.
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• W2000760541 doi "https://doi.org/10.1074/jbc.m002580200" @default.
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