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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

Lookup NU author(s): Gordon Taylor, Professor John LunecORCiD, Professor Andrew Pearson, Professor Herbie Newell

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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:RC2(Tomudex). 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: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(Tomudex) cells, MTX polyglutamate formation was undetectable after exposure to 1 αM [3H]MTX for 24 h. After exposure to 0.1 αM raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2(Tomudex) cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM: RC2(Tomudex) 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: RC2(Tomudex) 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:RC2(Tomudex) 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.

Publication metadata

Author(s): Barnes, M., Estlin, E., Taylor, G., Aherne, G., Hardcastle, A., McGuire, J., Calvete, J., Lunec, J., Pearson, A.D.J., Newell, D.

Publication type: Article

Publication status: Published

Journal: Clinical Cancer Research

Year: 1999

Volume: 5

Issue: 9

Pages: 2548-2558

Print publication date: 01/09/1999

ISSN (print): 1078-0432

ISSN (electronic): 1557-3265

PubMed id: 10499632

Funding

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