Assessment of mitogenesis of the hormone-responsive NMU rat mammary tumor grown in culture in soft agar, using³H-thymidine incorporation into DNA

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.