Summary
We have recently shown that the soft agar clonogenic assay is suitable for growing the hormone-responsive N-nitrosomethylurea (NMU) rat mammary tumor and for evaluating the factors affecting its growth. In order to improve our assessment of tumor mitogenesis beyond simple colony counting, we have validated and characterized in our system the use of3H-thymidine (3H-ThD) incorporation of DNA. In time-course studies, we observed that in most tumors an initial peak of3H-ThD uptake occurred on day 1 after plating followed by a decline in counts on day 2 and a second peak on day 3.3H-ThD incorporation was markedly diminished on day 4 and 6 when maximal colony formation occurred. In experiments where NMU rat mammary tumors were plated at different cell concentrations, we observed that increasing the number of cells plated resulted in a parallel increase in counts on day 1 and colonies on day 6. Overall, a highly significant correlation was observed between3H-ThD uptake and colony formation (r = 0.86,p<0.001). These results demonstrate that the3H-ThD incorporation assay is a fast and effective method for assessing mitogenesis of hormoneresponsive NMU mammary tumors grown in the soft agar clonogenic assay.
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References
Salmon SE, Hamburger AW, Soehnlen B, Durie BGM, Alberts DS, Moon TE: Quantitation of differential sensitivity of human tumor stem cells to anticancer drugs. N Engl J Med 298:1321–1327, 1978
Manni A, Wright C: Effect of tamoxifen and α-difluoromethylornithine on clones of nitrosomethylurea-induced rat mammary tumor cells grown in soft agar culture. Cancer Res 43:1084–1086, 1983
Tanigawa N, Kern DH, Hikasa Y, Morton DL: Rapid assay for evaluating the chemosensitivity of human tumors in soft agar culture. Cancer Res 42:2159–2164, 1982
Roobol C, Naaktgeboren N, Theunisen J, Sips H, Bernheim JL: Application of3H-thymidine incorporation for growth assessment in the human tumor stem cell clonogenic assay (Abstract). Proc Am Soc Clin Oncol 1:22, 1982
Callahan SK, Forseth B, Coltman CA, Von Hoff DD: A rapid radioisotope endpoint for assessment of clonogenic results in a soft agar system (Abstract). Proc Am Assoc Cancer Res 23:186, 1982
Hamburger AW, Salmon SE: Primary bioassay of human myeloma stem cells. J Clin Invest 60:846–854, 1977
Von Hoff DD, Casper J, Bradley E, Trent JM, Hodach A, Reichert C, Makuch R, Altman A: Direct cloning of neuroblastoma cells in soft agar culture. Cancer Res 40:3591–3597, 1980
Burton K: A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315–323, 1956
Manni A, Wright C: Estrogens may stimulate mitogenesis of experimental breast cancer through the polyamine pathway (Abstract). 65th Ann Mtg of the Endocrine Society, San Antonio, Texas, June, 1983, p 141.
Selby P, Buick RN, Tannock I: A critical appraisal of the human tumor stem-cell assay. N Engl J Med 308:129–134, 1983
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Address for reprints: Dr Andrea Manni, Division of Endocrinology, P.O. Box 850, The Milton S. Hershey Medical Center, Hershey, PA 17033, USA.
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Manni, A., Wright, C. Assessment of mitogenesis of the hormone-responsive NMU rat mammary tumor grown in culture in soft agar, using3H-thymidine incorporation into DNA. Breast Cancer Res Tr 3, 287–292 (1983). https://doi.org/10.1007/BF01806702
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DOI: https://doi.org/10.1007/BF01806702