Human Breast Cancer Progesterone Receptors Research Articles

Up-regulation of estrogen receptor by tamoxifen in human breast cancer

Influence of tamoxifen treatment on estrogen receptor (ER) and progesterone receptor (PR) levels in human breast cancer has not been fully elucidated in vivo. This problem was studied in 20 postmenopausal patients with ER-positive and PR-positive primary breast cancer. Each patient underwent two fine-needle aspiration (FNA) biopsies. Between two FNA, 10 patients received no treatment (control group) and the other 10 patients were given tamoxifen (20 mg/day) for an average of 8 days (range, 6-10 days) (TAM group). Total ER and PR values (cytosolic plus nuclear fraction) in FNA samples were determined by enzyme immunoassay (EIA) for detecting ER and PR regardless of their occupancy with corresponding ligands. In the control group, no significant difference was found in ER and PR values between the first and second FNA samples. In the TAM group, ER and PR values (mean +/- standard error of the mean [SEM] fmol/mg DNA) in the second FNA samples were 605 +/- 186 and 1130 +/- 344, respectively, and were significantly higher (P < 0.05) than ER and PR values in the first FNA samples, which were 312 +/- 74 and 639 +/- 159, respectively. ER and PR values increased by 201 +/- 27% and 163 +/- 23%, respectively, on an individual basis after tamoxifen treatment. These results demonstrated that tamoxifen up-regulates ER and PR in human breast cancer.

Immunologic analysis of human breast cancer progesterone receptors. 2. Structure, phosphorylation, and processing.

We have used a monoclonal antibody (MAb) directed against chick oviduct progesterone receptors (PR), that cross-reacts with human PR, to analyze PR structure and phosphorylation. This MAb, designated PR-6, interacts only with B receptors (Mr 120,000) of T47D human breast cancer cells; it has no affinity for A receptors (Mr 94,000) or for proteolytic fragments from either protein. The antibody immunoprecipitates native B receptors and was used to study the structure of native untransformed 8S and transformed 4S receptors, using sucrose density gradient analysis, photoaffinity labeling, and gel electrophoresis. On molybdate-containing low-salt gradients, PR-6 complexes with 8S B receptors, causing their shift to the bottom of the gradient while A receptors remain at 8 S. Therefore, A and B receptors form separate 8S complexes, and we conclude that A and B do not dimerize in the holoreceptor. Similar gradient studies using salt-containing, molybdate-free buffers show that there are two forms of salt-transformed 4S receptors, comprising either A proteins or B proteins, suggesting that A and B are also not linked to one another in transformed PR. The independence of A- and B-receptor complexes was confirmed by the finding that purified, transformed B receptors bind well to DNA-cellulose. Since PR-6 cross-reacts with nuclear PR, it was used to analyze nuclear PR processing--a down-regulation step associated with receptor loss as measured by hormone binding. Insoluble nuclear receptors and soluble cytosol receptors were measured by immunoblotting following treatment of T47D cells for 5 min to 48 h with either R5020 or progesterone. From 8 to 48 h after R5020 treatment, immunoassayable receptors decreased in nuclei and were not recovered in cytosols. Nuclear receptors also decreased after progesterone treatment but replenished in cytosols between 8 and 24 h after the start of treatment. Thus, processing involves a true loss of nuclear receptor protein, and not just loss of hormone binding activity, and occurs after progesterone or R5020 treatment. This loss is chronic, however, only in R5020-treated cells. Additional studies focused on the covalent modifications of receptors. We previously described shifts in apparent molecular weight of nuclear PR following R5020 treatment using in situ photoaffinity labeling. To show whether these shifts can be explained by receptor phosphorylation, untreated cells and hormone-treated cells were metabolically labeled with [32P]orthophosphate, and the B receptors were isolated by immunoprecipitation with PR-6 and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunologic analysis of human breast cancer progesterone receptors. 1. Immunoaffinity purification of transformed receptors and production of monoclonal antibodies.

A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors [Sullivan, W. P., Beito, T. G., Proper, J., Krco, C. J., & Toft, D. O. (1986) Endocrinology (Baltimore) 119, 1549-1557] cross-reacts with the Mr 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with [3H]promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. A second purification step by diethylaminoethyl chromatography gives further enrichment to 3720 pmol/mg of protein (or 44% purity) to yield essentially two proteins, 120-kilodalton (kDa) B receptors and a 76-kDa non-steroid binding protein, each in approximately equivalent amounts. B receptors purified under these conditions are transformed and biologically active. They were maintained as undegraded 120-kDa doublets and retained both hormone and DNA binding activities. Isolated B receptors were free of the 90-kDa non-steroid binding protein observed to be associated with 8S untransformed receptors in other systems and were free also of the non-hormone binding 105-108-kDa B antigen described previously to copurify with chick PR. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG1), B-64 (IgG1), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG1), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology. These new MAbs are valuable reagents for further studies of human receptor structure and function and for clinical immunodetection of PR in breast tumors.

The subunit structure of human breast cancer progesterone receptors: characterization by chromatography and photoaffinity labeling.

We have partially purified the progesterone receptors (PR) from T47D human breast cancer cells and show that they consist of at least two hormone-binding proteins of dissimilar size and unequal DNA-binding forms with electrophoretic properties analogous to the A and B subunits of chick oviduct PR. Cytosols were labeled with the progestin [3H]R5020 in the presence or absence of unlabeled R5020, and samples of the parallel incubations were chromatographed on DEAE-cellulose. A large heterogeneous peak of specific R5020 binding eluted between 0.1 and 0.2 M KCl. Pooled fractions of this peak were resolved on phosphocellulose into two peaks: an acidic moiety eluting at 0.15 M KCl, and a more basic one eluting at 0.2 M KCl. Both sediment at approximately 4.1S on sucrose density gradients. To test DNA binding capacity, two studies were performed. First, ammonium sulfate-precipitated receptors were applied to DNA-cellulose; approximately half of the specific radioactivity was retained. Second, separate DNA-cellulo...