Abstract
Resistance to anti-cancer drugs has proved to be a major barrier in the clinical management of neoplastic disease. We have investigated the mechanistic basis for resistance to folate-based thymidylate synthase (TS) inhibitors using two cell lines selected for resistance to ZD1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N -methylamino]-2 - thenoyl)-L-glutamic acid), a drug currently in phase III clinical trial. The degree of resistance was > 20,000 for the human lymphoblastoid cell line W1L2:R and approximately 14 for the ovarian carcinoma cell line CH1:R. In both cases resistance was associated with increased TS activity. The W1L2:R cell line had an approximately 100-fold increase in TS gene copy number and mRNA levels and a 500- to 1000-fold increase in enzyme levels determined using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern and Western blotting. The CH1:R cell line had an approximately 2- to 2.5-fold increase in TS gene copy number, mRNA and protein levels. In both cell lines the fold resistance determined was significantly higher than the fold increase in target enzyme DNA, mRNA or protein levels. Small changes in TS levels may therefore translate to clinically significant alterations in drug sensitivity.
Highlights
S_inminary Resistance to anti-cancer drugs has proved to be a major barrier in the clinical management of neoplastic disease
We have investigated the mechanistic basis for resistance to folate-based thymidylate synthase (TS) inhibitors using two cell lines selected for resistance to ZD1694
Results are expressed as target mRNA levels relative to the internal reference standards: 1-actin mRNA and 18S rRNA
Summary
RPMI-1640 [containing 20 mM 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid buffer and lacking sodium bicarbonate and L-glutamine] was from Flow Laboratories (Irvine, UK). Folinic acid (calcium leucovorin 3 mg ml-') was from David Bull laboratories (Warwick, UK). (±)-L-Tetrahydrofolic acid (HCI) (97% pure) was from Fluka (NewUlm, Germany). [5-3H]dUMP, [3'2P-dATP (-30 TBq mmol-') and '"I-labelled protein A fragments (> 1.1 GBq mg-') were supplied by the Radiochemical Centre (Amersham, Buckinghamshire, UK). [6-3HJdUMP was from Moravek Biochemicals (Brea, CA, USA). Taq polymerase was purchased from Perkin Elmer Cetus (Norwalk, CT, USA). Moloney murine leukaemia virus (M-MLV) reverse transcriptase and placental ribonuclease inhibitor were from Gibco BRL (Gaithersburg, MD, USA). ZD1694 was synthesised and supplied by ICI Pharmaceuticals PLC (Macclesfield, Cheshire, UK). All other reagents were purchased from Fisons (Loughborough, UK), Sigma (London, UK) or British Drug Houses (BDH) (UK)
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