Molecular characterisation of two cell lines selected for resistance to the folate-based thymidylate synthase inhibitor, ZD1694
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S J Freemantle1, A L Jackman, L R Kelland
1Cancer Research Unit, Medical School, University of Newcastle-upon-Tyne, UK.
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.
Insights
Drug resistance in cancer is a major challenge. This study shows that even small increases in thymidylate synthase (TS) levels can lead to significant resistance to anti-cancer drugs targeting TS.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Drug resistance is a significant obstacle in cancer treatment.
- Folate-based thymidylate synthase (TS) inhibitors are crucial anti-cancer agents.
- Understanding resistance mechanisms is vital for improving therapeutic outcomes.
Purpose of the Study:
- To investigate the mechanistic basis of resistance to folate-based thymidylate synthase (TS) inhibitors.
- To analyze resistance in two distinct cell lines selected for resistance to ZD1694.
- To correlate drug resistance levels with changes in TS expression.
Main Methods:
- Selection of drug-resistant cell lines (W1L2:R and CH1:R).
- Quantitative reverse transcription-polymerase chain reaction (RT-PCR) to measure mRNA levels.
- Southern and Western blotting to assess gene copy number and protein levels.
- Enzyme activity assays.
Main Results:
- Both cell lines exhibited significant resistance to ZD1694, with W1L2:R showing >20,000-fold resistance and CH1:R showing ~14-fold resistance.
- Increased TS activity was observed in both resistant cell lines.
- W1L2:R cells had ~100-fold increase in TS gene copy number and mRNA, and 500-1000-fold increase in enzyme levels.
- CH1:R cells showed a ~2-2.5-fold increase in TS gene copy number, mRNA, and protein.
- Fold resistance significantly exceeded the fold increase in TS DNA, mRNA, or protein levels in both cell lines.
Conclusions:
- Elevated thymidylate synthase (TS) levels are a key mechanism of resistance to TS inhibitors.
- Even modest increases in TS expression can result in substantial drug resistance.
- These findings highlight the critical role of TS in determining sensitivity to folate-based anti-cancer drugs.