Induction of differentiation in a rat mammary epithelial stem cell line by dimethyl sulphoxide and mammotrophic hormones.

Rama 25 is a clonal epithelial cell line derived from a dimethylbenzanthracene-induced rat mammary adenocarcinoma. In the presence of the mammotrophic 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 sulphoxide 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 two 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 two markers, are characterised by two phases, a fixed period of 8 h (lag phase), the length of which is independent of the dimethyl sulphoxide concentration and a second phase where their rates are dependent on the concentration of dimethyl sulphoxide. Rama 25 cells do not become committed to differentiate during this lag phase but increasing numbers of cells do so after this period. We suggest that the differentiation processes occur in two stages. The first stage, involving the inducer, commits Rama 25 cells to a new differentiated state. The second 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. Thus, 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.