Induction of Differentiation in a Rat Mammary Epithelial Stem Cell Line by Dimethyl Sulphoxide and Mammotrophic Hormones

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Abstract
Rama 25 is a clonal epithelial cell line derived from a dimethylbenzanthracene-induced rat mammary adenocarcinoma. In the presence of the mammotropic hormones, insulin, hydrocortisone, estrogen and prolactin, Rama 25 produces small amounts of casein and forms domes at a low rate. The rates of both these processes can be greatly increased by the addition of dimethyl sulfoxide or hexamethylenebisacetamide, which are also known to induce the differentiation of Friend erythroleukemia cells. Other compounds which stimulate the differentiation of Rama 25 cells include linoleic acid and 6-thioguanine. The intracellular pathways triggering changes in the 2 markers of differentiation are partially separable using different combinations of hormones, prolactin and hydrocortisone being the most important for the production of casein and the formation of domes, respectively. The kinetics of differentiation, as judged by the appearance of these 2 markers, are characterized by 2 phases, a fixed period of 8 h (lag phase), the length of which is independent of the dimethyl sulfoxide concentration, and a 2nd phase in which their rates are dependent on the concentration of dimethyl sulfoxide. Rama 25 cells do not become committed to differentiation during this lag phase but increasing numbers of cells do so after this period. The differentiation processes evidently occur in 2 stages. The 1st stage, involving the inducer, commits Rama 25 cells to a new differentiated state. The 2nd stage, involving the hormones, modulates the expression of different markers of this state. Both casein production and dome formation can be blocked by inhibitors of DNA synthesis and show reciprocal changes with the rates of cellular DNA synthesis. In its hormonal and DNA synthetic requirements for differentiation, Rama 25 cells appear to resemble some of the mammary epithelial cells of mature virgin rats.