Monolayers vs. Postconfluent Cultures 1573 zyxwvutsr 47. 48. 49. in the topical treatment of skin metastases in breast cancer patients. monolayers and spheroids to the novel indoloquinone E09. Ann Cancer Treat Rev 1990,1?, 243- 246. O ncal 1992,3 (Suppl. I), 106. Stratford IJ, Adams GE, Bremner JC, et al. The assessment of 50. bioreductive drug toxicity in witro and in experimental tumors in vim In Adams GE, Breccia A, Fielden EM, Wardman P, eds. Selective Activation of Lhgs by Redox Processes. New York, Plenum 5 1. Press, 1990,203-212. Phillips RM, Hulbert PB, Bibby MC, Sleigh NR, Double JA. In vitro activity of the novel indoloquinone E09 and the influence of 52. pH on cytotoxicity. BrJ Cancer 1992,65,359-364. Phillips RM, Bibby MC, Cronin BP, Ekebuisi M. Comparative in vitro chemosensitivity of DLD-1 human colon adenocarcinoma Hendriks HR, Plowman J, Berger DP, et al. Preclinical antitumor activity and animal toxicology of rhizoxin, a novel tubulin-inter- acting agent. Ann Oncol (in press). Kerr DJ, Bisset D, Graham M, Setanoians A. A phase I and pharmacokinetic study of rhizoxin. Ann Oncol 1992,3 (Suppl. l), 120. Hilgard P, Stekar J, Voegeli R, et al. Characterization of the antitumor activity of hexade-cylphosphocholine (D 18506). Eur J zyxwvutsr Cancer zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI Clin Oncoll988,24,1457-1461. zyxwvutsrqponmlkjihgfedcbaZY Eurf zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Cawn, VoJ. 29A,No. JJ,f$. J573-J577,1993 Printed m Grear Brimn 0964- 1947193$6.W f 0.00 @ 1993 PeTgalwon Press Ltd Genistein Inhibits Tumour Cell Growth in vitro but Enhances Mitochondrial Reduction of Tetrazolium Salts: A Further Pitfall in the Use of the MTT Assay for Evaluating Cell Growth and Survival M.C. Pagliacci, F. Spinozzi, G. Migliorati, G. Fumi, M. Smacchia, F. Grignani, C. Riccardi and I. Nicoletti zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON The natural isoflavone genistein inhibits the growth of a number of tumour ceU lines in vitro. During investigations on the antiproliferative effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) calorimetric assay consistently underestimated the growth inhibitory activity of the substance. Cell proliferation was markedly inhibited by genistein in three tumour ceU lines (MCF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929, mouse transformed fibroblasts) when cell number was evaluated by direct counting, whereas a 72-h MTT assay failed to reveal any growth-inhibitory effect. CeU cycle analysis by propidium iodide staining and flow-cytometry revealed a G2/M ceU cycle arrest after genistein treatment. Genistein-treated cells displayed an increase in ceU volume and in mitochondrial number and/or activity, as revealed by enhanced formazan generation and increased uptake of the vital mitochondrial dye rhodamine 123. These results suggest that alterations in cell cycle phase redistribution of tumour cells by genistein may significantly infiuence mitochondrial number and/or function and, consequently, MTT reduction to formazan. This may constitute an important bias in analysing the effects of genistein, and possibly other drugs that block the G2/M transition, on growth and viability of cancer cells in vitro by MTT assay. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF EurJ Camxr, Vol. 29A, No. 11, pp. 1573-1577,1993. INTRODUCTION zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA COLORIMETRX ASSAYS are extensively used for evaluating the effect of growth factors, hormones and drugs on growth and survival of both normal and tumour cells in culture [ 1, 21. Two reagents, tetrazolium (MTT) and formazan (XTT) [3] are commonly employed as indicators of cell number and viability, since they are converted to a coloured formazan derivative via Correspondence to I. Nicoletti. I. Nicoletti, M.C. Pagliacci, F. Spinozzi, G. Fumi, M. Smacchia and F. Grignani are at the Istituto di Clinics Medica 1; and G. Migliorati and C. Riccardi are at the Istituto di Farmacologia Medica, Perugia University School of Medicine, 06100 Perugia, Italy. Received 13 Jan. 1993; accepted 15 Feb. 1993. mitochondrial dehydrogenase activity by viable cells. Although a number of factors, such as pH, medium glucose content and age of cultures, influence MTT and XTT reduction by living cells [4, 51, there is, in general, a good concordance between the number of viable cells in the culture and the production of formazan, which can be easily measured by calorimetric methods after solubilisation in dimethylsulphoxide. Because of its sim- plicity, precision and low-cost, the MTT assay is currently used in cytotoxic drug screening protocols [3, 5, 61. It has, also, been proposed as a valid alternative to the [3H]thymidine uptake methods for analysing cell proliferation [7]. Our laboratory has successfully applied the test to investigating the growth- inhibitory effect of drugs and cytokines [S, 91. During the course of experiments aimed at analysing the