Anti-steroid action in cultured L-929 mouse fibroblasts

L-929 mouse fibroblast growth is arrested by the glucocorticosteroid dexamethasone (dex), which also decreases adhesiveness to culture plates. Both dex effects were abolished when RU 486, a new synthetic anti-hormonal steroid, was added to the culture medium. Using [3H]RU 486, binding studies revealed that RU 486 bound approximately 25,000 sites/cell of the glucocorticosteroid receptor (GR) with affinity higher than that of dex and translocated GR to the nucleus. However, the distribution of steroid-receptor complexes between cytosol and nuclei was different for the agonist and the antagonist, with more nuclear accumulation in the case of dex. Estradiol increases L-929 cell growth and adhesiveness to culture plates, but not if the anti-estrogen tamoxifen (tam) was added. These observations initially made in serum containing medium, were confirmed in serum-free, chemically defined culture medium (SF). In SF medium, tam (1 microM) provoked death of most L-929 cells after 10 days of culture, leading to the selection of a variant clone LB of tam-resistant cells. Tam binds to the estrogen receptor (ER), but with less affinity than estradiol. ER concentration, estimated by the binding of 4-hydroxytamoxifen (OH-tam) and of estradiol was lower in LB cells than in original tam-sensitive L-929 cells. The concentration of specific anti-estrogen binding sites in the particulate fraction of the cells, measured by OH-tam binding, non competed by estradiol, was also significantly diminished in tam-resistant LB cells.