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Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivity to TS-
D B Longley1, P R Ferguson, J Boyer
1Department of Oncology, Cancer Research Centre, Queen's University of Belfast, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland.
Abstract:
Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.
Insights
Overexpressing thymidylate synthase (TS) increases resistance to TS-targeted drugs like 5-FU and Tomudex. This upregulation overcomes S-phase arrest, crucial for understanding chemotherapy effectiveness.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Thymidylate synthase (TS) is essential for DNA synthesis and repair.
- TS is a key target for antifolate and fluoropyrimidine chemotherapies.
- Understanding TS expression is vital for predicting drug resistance.
Purpose of the Study:
- To investigate the role of thymidylate synthase (TS) expression in mediating resistance to TS-targeted drugs.
- To develop a model system for studying inducible TS expression in breast cancer cells.
Main Methods:
- Developed a tetracycline-regulated TS-expressing MDA435 breast cancer cell line (MTS-5).
- Assessed drug sensitivity (IC50) to various chemotherapeutic agents upon TS induction.
- Performed cell cycle analysis and gene expression profiling of cell cycle regulators.
Main Results:
- Inducible TS overexpression increased IC50 values for 5-FU, Tomudex (TDX), and ZD9331 significantly.
- Multitargeted antifolate (MTA) toxicity was highly sensitive to TS levels, with no obtainable IC50 at overexpression.
- TS upregulation overcame drug-induced S-phase arrest and associated cyclin E/A and cdk2 expression.
- Non-TS-targeted agents (CPT-11, cisplatin, oxaliplatin, Taxol) efficacy was unaffected by TS overexpression.
Conclusions:
- Acute increases in TS expression levels are critical determinants of cellular sensitivity to TS-directed chemotherapy.
- TS modulation impacts S-phase arrest, influencing drug response.
- TS-targeted therapies' effectiveness is directly linked to thymidylate synthase expression levels.
