title: Impact of polyglutamation on sensitivity to raltitrexed and methotrexate in relation to drug-induced inhibition of de novo thymidylate and purine biosynthesis in CCRF-CEM cell lines.
abstract: The aim of this study was to investigate the influence of folylpolyglutamyl synthetase (FPGS) activity on the cellular pharmacology of the classical antifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2Tomudex. Cell growth inhibition and drug-induced inhibition of de novo thymidylate and purine biosynthesis were used as measures of the cellular effects of the drugs. CCRF-CEM:RC2Tomudex cells had <11% of the FPGS activity of CCRF-CEM cells, whereas MTX uptake and TS activity were equivalent. In CCRF-CEM:RC2Tomudex cells, MTX polyglutamate formation was undetectable after exposure to 1 microM [3H]MTX for 24 h. After exposure to 0.1 microM raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2Tomudex cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM: RC2Tomudex cells were >1000-fold resistant to raltitrexed and 6-fold resistant to lometrexol but sensitive to MTX and nolatrexed when exposed to these antifolates for 96 h. After 6 h of exposure, CCRF-CEM cells retained sensitivity to MTX and raltitrexed but were less sensitive to lometrexol-mediated growth inhibition. In contrast, CCRF-CEM: RC2Tomudex cells were markedly insensitive to raltitrexed, lometrexol, and to a lesser degree, MTX. Simultaneous measurement of de novo thymidylate and purine biosynthesis revealed 90% inhibition of TS activity by 100 nM MTX in both cell lines, whereas inhibition of de novo purine synthesis was only observed in CCRF-CEM cells, and only after exposure to 1000 nM MTX. Ten nM raltitrexed induced >90% inhibition of TS activity in CCRF-CEM cells, whereas in CCRF-CEM:RC2Tomudex cells, there was no evidence of inhibition after exposure to 1000 nM raltitrexed. These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both raltitrexed and MTX, markedly influencing potency in the case of raltitrexed and locus of action in the case of MTX.
authors.affiliation: Cancer Research Unit, University of Newcastle, Newcastle upon Tyne, United Kingdom.
authors.forename: M J
authors.initials: MJ
authors.lastname: Barnes
authors.forename: E J
authors.initials: EJ
authors.lastname: Estlin
authors.forename: G A
authors.initials: GA
authors.lastname: Taylor
authors.forename: G W
authors.initials: GW
authors.lastname: Aherne
authors.forename: A
authors.initials: A
authors.lastname: Hardcastle
authors.forename: J J
authors.initials: JJ
authors.lastname: McGuire
authors.forename: J A
authors.initials: JA
authors.lastname: Calvete
authors.forename: J
authors.initials: J
authors.lastname: Lunec
authors.forename: A D
authors.initials: AD
authors.lastname: Pearson
authors.forename: D R
authors.initials: DR
authors.lastname: Newell
mesh_terms: D001692:Biological Transport; D002455:Cell Division / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007938:Leukemia / Q000188:drug therapy* / Q000201:enzymology / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011687:Purines / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D011799:Quinazolines / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D013876:Thiophenes / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D013938:Thymidine Monophosphate / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D005493:Folic Acid Antagonists; D006131:Growth Inhibitors; D011687:Purines; D011799:Quinazolines; D012333:RNA, Messenger; D013876:Thiophenes; D013938:Thymidine Monophosphate; D013940:Thymidylate Synthase; D010453:Peptide Synthases; C068874:raltitrexed; D008727:Methotrexate