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Evaluation of a Tetrazolium-based Semiautomated Colorimetric Assay: Assessment of Radiosensitivity¹

NCI-Navy Medical Oncology Branch [J.C., A.F.G., J.D.M.] and Radiobiology Section, Radiation Oncology Branch [J.C., W.G.D., J.B.M.], National Cancer Institute and Naval Hospital, Bethesda, Maryland 20814

Radiation survival curves were generated for V79 Chinese hamster and two human lung cancer cell lines (NCI-H460 and NCI-H249) with doubling times of 10, 20, and 85 h, respectively, using a standard clonogenic assay, a dye exclusion assay, and a semiautomated colorimetric assay utilizing a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide. Comparable results for D₀ and extrapolation number (n) were observed for all assays in the lines with doubling times of 10 and 20 h. In these instances the tumor cell lines had undergone seven or more doublings after radiation. For the tumor line (H249) with an 80-h doubling time the D_es were comparable between the assays while the extrapolation number was increased in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay, a result probably related to the lower number of doublings (<4) after radiation. We then tested the ability of the assays to detect radiation protection and sensitization using known agents. We found that cysteamine treatment resulted in radioprotection (by a factor of 8 at 8 Gy) while 5-bromo-2-deoxyuridine incorporation caused enhancement of radiation sensitivity in all three assays. We conclude that, while optimal conditions for each cell line (cell number plated and doubling time) must be established, using characterized tumor cell lines, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay could be automated and thus be of great value in screening large numbers of potential radiosensitizers or protectors.

¹ The opinions or assertions contained herein are the private views of the authors and are not to be considered as official or as reflecting the views of the Department of the Navy or the Department of Defense.

² To whom requests for reprints should be addressed, at Radiation Oncology Branch, National Cancer Institute, Bldg. 10, Room B3-B69, Bethesda, MD 20892.

Received 4/25/86. Revised 9/18/86. Accepted 10/30/86.

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