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

The aim of this study was to investigate the influence of folylpolyglutamyl synthetase (FPGS) activity on the cellular pharmacology of the classical a ntifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2(Tomudex). Cell growth inhibition and drug -induced inhibition of de novo thymidylate and purine biosynthesis were use d as measures of the cellular effects of the drugs. CCRF-CEM:RC2(Tomudex) cells had <11% of the FPGS activity of CCRF-CEM cells , whereas MTX uptake and TS activity were equivalent. In CCRF-CEM:RC2(Tomud ex) cells, MTX polyglutamate formation was undetectable after exposure to 1 mu M [H-3]MTX for 24 h. After exposure to 0.1 mu M raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2(Tomudex) c ells were 30- to 50- fold lower than in the CCRF-CEM cell line. CCRF-CEM: R C2(Tomudex) cells were >1000-fold resistant to raltitrexed and 6-fold resis tant to lometrexol but sensitive to MTX and nolatrexed when exposed to thes e antifolates for 96 h. After 6 h of exposure, CCRF-CEM cells retained sens itivity to MTX and raltitrexed but were less sensitive to lometrexol-mediat ed growth inhibition. In contrast, CCRF-CEM: RC2(Tomudex) cells were marked ly insensitive to raltitrexed, lometreuol, and to a lesser degree, MTX. Sim ultaneous measurement of de novo thymidylate and purine biosynthesis reveal ed 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 nh I raltitrexed induced >90% inhibition of TS activity in CCRF-CEM cells, whereas in CCRF-CEM:RC2(Tomude x) cells, there was no evidence of inhibition after exposure to 1000 nM ral titrexed. These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both raltitrexed and MTX, markedly influencin g potency in the case of raltitrexed and locus of action in the case of MTX .