Activity of progesterone and anti-progestins in a rat mammary primary cell culture system

A primary culture system of virgin rat mammary epithelial cells, grown in a serum-free medium, was developed as a means of assaying the efficacy of compounds with known anti-progestational properties. Cells were grown in 24-well plates on hydrated collagen gels and could be cultured for at least seven days. Experiments were routinely stopped three days after overnight attachment of cells using fibronectin (4 micrograms/ml). DNA synthesis, measured by thymidine incorporation, was significantly increased by the addition of ovine prolactin (43 nM; P < 0.01) or progesterone (0.15 microM; P < 0.05) or both (P < 0.01) to the basal medium. When added to medium containing progesterone plus prolactin (complete medium), RU486 (mifepristone) and ZK98734 (lilopristone) significantly depressed DNA synthesis in a dose-dependent manner using doses ranging from 0.015 microM to 15 microM. Maximum inhibition was achieved at 15 microM for both compounds. DNA synthesis was 24.5 +/- 2.6% (mean +/- SEM, n = 4) and 32.0 +/- 2.2% (n = 3) of that in complete medium for RU486 and ZK98734, respectively (both P < 0.001). There was no inhibitory effect of either compound in basal medium or basal medium plus prolactin, indicating the absence of toxicity and that the inhibitory effect is specific for a progesterone-mediated process.