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

Abstract

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.

Heat and Chemical Shock Potentiation of Glucocorticoid Receptor Transactivation Requires Heat Shock Factor (HSF) Activity

“…Although it is interesting to speculate that one of the major known heat shock proteins may serve the function of product X in our system, to date no direct evidence for this possibility has been found. However, there is evidence that HSPs can affect the DNA-binding properties of steroid receptors, since the glucocorticoid (49), progesterone (50,51), and estrogen receptors (52) have been shown to remain tightly complexed to HSP70 even after hormone-induced transformation of these receptors to the DNA-binding state. Moreover, purified HSP70 has been shown to enhance the in vitro DNA binding properties of the estrogen receptor (52).…”

Transcriptional Hyperactivity of Human Progesterone Receptors Is Coupled to Their Ligand-Dependent Down-Regulation by Mitogen-Activated Protein Kinase-Dependent Phosphorylation of Serine 294

“…Lactacystin and calpain inhibitor I (N-acetyl-Leu-Leu-norleucinal [LLnL]) were purchased from Calbiochem (La Jolla, Calif.). The anti-PR monoclonal antibodies, AB-52 and B-30, were produced in the K. B. Horwitz laboratory (9). c-Myc antibodies were a kind gift of S. R. Hann (42).…”

An N-terminal Inhibitory Function, IF, Suppresses Transcription by the A-isoform but Not the B-isoform of Human Progesterone Receptors

“…The expressed PR fragments were resolved by electrophoresis on 7.5% or 10% SDS-containing denaturing polyacrylamide gels and transferred to nitrocellulose. Protein blots were probed with our anti-PR monoclonal antibodies, AB-52 and B-30 (24), and the anti-DBD polyclonal antibody ␣266 (25) provided by D. Toft (Rochester, MN). For detection of ER or IF-ER, the anti-ER antibody SRA 1,000 was used (StressGen, Victoria, BC).…”