Abstract
Drug design is an iterative process requiring cycles of compound synthesis and testing, with each successive synthesis phase yielding molecules predicted to have improved characteristics over the previous set of compounds. In the field of cancer drug discovery, a key early-stage element of the drug design and testing process usually involves the screening of compounds in cell-based in vitro assays. One of the most frequent parameters assessed in cancer drug discovery is the impact of a given molecule on the proliferation of a cancer cell. The methyl-[3H]-thymidine incorporation assay is a widely used, gold standard, method for measuring inhibition of cell proliferation and has been used successfully to screen and optimize potential new cancer drugs. The assay is based on measuring incorporation of methyl-[3H]-thymidine (the radiolabeled form of the DNA precursor thymidine) into the DNA of dividing cancer cells. The screen is used to generate concentration effect relationships for test compounds and for the derivation of IC50 values. IC50 value is defined as the concentration of a test compound required to achieve half maximal inhibition of methyl-[3H]-thymidine incorporation, a parameter that is indicative of antiproliferative potency. IC50 values derived from cell-based assays help drive the medicinal chemistry efforts toward improved drug design, and it is, therefore, critical that the screen provides consistent, robust data over the lifetime of the project – a requirement that necessitates good-quality cell culture practices. The methyl-[3H]-thymidine incorporation assay has been adapted to high-throughput format to facilitate screening of large numbers of compounds. The detailed description of this method, exemplified using the COLO-205 colorectal cancer cell line in a 96-well format, should give the reader a thorough account of how to conduct proliferation assays, as well as some notes and tips on how to ensure success and avoid potential pitfalls.
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References
Friedman, H.M., and Glaubiger, D.L. (1982) Assessment of in vitro drug sensitivity of human tumor cells using thymidine incorporation in a modified human tumor stem cell. Cancer Res. 42, 4683–4689.
Naito, K., Skog, S., Tribukait. B., Andersson. L., Hisazumi. H. (1987) Cell cycle related [3H]-thymidine uptake and its significance for the incorporation into DNA. Cell Tissue Kinet. 20, 447–457.
Harrington, E.A., Bebbington, D., Moore, J., Rasmussen, R.K., Ajose-Abeogun, A., Nakayama, T., Graham, J.A., Demur, C., Hercend, T., Diu-Hercend, A., Su, M., Golec, J.M., Miller, K.M. (2004). VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases suppresses tumour growth in vivo. Nature Med. 10, 262–267.
Hennequin, L.F., Thomas, A.P., Johnstone, C., Stokes, E.S.E., Ple, P.A., Lohmann, J.M., Ogilve, D.J., Dukes, M., Wedge, S.R., Curwen, J.O., Kendrew, J., Lambert-van der Bempt, C. (1999). Design and structure-activity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors. J. Med. Chem. 42, 5369–5389.
Ogita, H., Isobe, Y., Takaku, H., Sekine, R., Goto, Y., Misawa, S., Hayashi, H. (2001). Synthesis and structure-activity relationship of diarylamide derivatives as selective inhibitors of the proliferation of human coronary artery smooth muscle cells. Bioorganic & Med. Chem. Lett. 11, 549–551.
Boss DS, Beijnen JH and Schellens JH (2009) Clinical experience with aurora kinase inhibitors: a review. Oncologist 14, 780–793.
Bebbington, D., Binch, H., Charrier, J.D., Everitt, S., Fraysse, D., Golec, J., Kay, D., Knegtel, R., Mak, C., Mazzei, F., Miller, A., Mortimore, M., O’Donnell, M., Patel, S., Pierard, F., Pinder, J., Pollard, J., Ramaya, S., Robinson, D., Rutherford, A., Studley, J., Westcott, J. (2009) The discovery of the potent aurora inhibitor MK-0457 (VX-680) Bioorg Med Chem Lett. 19, 3586–3592.
Zhang, J., Chung, D.Y., Oldenburg, K.R (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73.
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Griffiths, M., Sundaram, H. (2011). Drug Design and Testing: Profiling of Antiproliferative Agents for Cancer Therapy Using a Cell-Based Methyl-[3H]-Thymidine Incorporation Assay. In: Cree, I. (eds) Cancer Cell Culture. Methods in Molecular Biology, vol 731. Humana Press. https://doi.org/10.1007/978-1-61779-080-5_36
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DOI: https://doi.org/10.1007/978-1-61779-080-5_36
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