Estrogen-insensitive progesterone receptors in a human breast cancer cell line: characterization of receptors and of a ligand exchange assay.
In modified culture conditions, T47D human breast cancer cells synthesize extraordinary amounts of progesterone receptors (PgR), but, unlike other progesterone target cells, the PgR are entirely independent of estrogen controls. In the present studies we characterize some physicochemical properties of the PgR in T47D cells. We also describe an exchange assay for cytoplasmic and nuclear forms of the receptors which has enabled us to demonstrate that after progesterone treatment, translocation is stoichiometric. Despite the anomalous regulation of PgR levels, these receptors are typical of steroid receptors; they sediment at 7-8S on sucrose density gradients, they bind ligands with high affinity (Kd approximately 4 nM for R5020; Kd approximately 2 nM for progesterone), they bind only progestins specifically, and they are thermolabile (t1/2 at 37 C is approximately 15 min). Receptor levels range from 15-40 pmol/mg DNA, or more than 300,000 sites/cell. The ability of ligands to dissociate from and rebind to the receptors was measured and used in an exchange assay for nuclear PgR. The synthetic progestin R5020 dissociates readily from receptors (t1/2 approximately 3 h at 0 C and 1.5 h at 10 C), and the dissociation of progesterone is even faster (t1/2 approximately 30 min at 0 C). To quantify steroid exchange, receptor levels were measured in mixtures of hormone-filled and unfilled cytosols. These studies assess ligand dissociation and subsequent ligand rebinding. At 0 C for 4-18 H or at 10 C for 4 h, unlabeled progesterone dissociates from receptors, and R5020 rebinds all sites, resulting in 100% exchange. In contrast, despite the use of a variety of incubation times and temperatures, no more than 50% of receptors previously filled with R5020 can exchange for [3H]R5020. The progesterone to [3H]R5020 exchange assay was used to measure salt-extracted nuclear progesterone receptors. In cells treated for 5 min with 0.1 microM progesterone, all depleted cytoplasmic sites were quantitatively recovered from nuclei. These cells provide a new model system to study the molecular biology of human PgR.
D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011374:Progesterone / Q000378:metabolism; D011397:Promegestone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000187:drug effects / Q000378:metabolism*
D004967:Estrogens; D008024:Ligands; D011980:Receptors, Progesterone; D011374:Progesterone; D011397:Promegestone