Monoclonal antibody to chicken oviduct progesterone receptor.

Abstract

Antibodies to molybdate-stabilized chicken oviduct progesterone receptor were raised in a Wistar rat and detected by interaction with homogeneous radioiodinated progesterone receptor. Spleen cells of this rat were then fused with mouse Sp2/0-Agl4 myeloma cells and the antibodies produced by the hybrid cells were detected by double immunoprecipitation using the '5I-labeled receptor. Cells of one of the positive cultures were then cloned by limiting dilution and one hybridoma cell line was studied. The monoclonal antibody produced was an IgG2b, and it reacted with the molybdate-stabilized "8-9S" form of the chicken oviduct progesterone receptor, labeled with either [H] Although the chicken oviduct progesterone receptor (PR) has been extensively studied (for review, see ref. 1), initial attempts to obtain PR antibodies were unsuccessful (2). Now, however, a homogeneous molybdate-stabilized "8-9S" form of PR has become available (3) and we have obtained anti-PR antibodies in a rabbit and a goat (4). The goat antiserum crossreacted not only with mammalian PRs (4) but, more surprisingly, with chicken glucocorticosteroid receptor (5). The latter unexpected result prompted us to obtain monoclonal antibodies (6) Exponentially growing mouse myeloma cells were fused with spleen cells by using 50% (wt/vol) polyethylene glycol 4000 and 5% dimethyl sulfoxide according to a published procedure (10) with minor modifications: 5 x 107 myeloma cells were exposed to 4 x 108 spleen cells and plated in selective medium (50 pkM hypoxanthine/10 tuM azaserine). The next day, surviving cells were counted and 0.1-ml aliquots containing 104 cells were plated in the presence of 4 X 105 rat macrophages. The medium was changed once a week. Two to 3 wk later, when the cell concentration had reached 105/ml, growing cells were tested for antibody production.The antibody-secreting hybrids were cloned by limiting dilution in the presence of 2 x 106 mouse thymocytes per ml.Wells containing a single cell cluster were assayed for anti-PR antibody production. Labeling of Chicken Oviduct Progesterone Receptor. Iodination. Highly purified (>90% pure) molybdate-stabilized PR (6 Ag) was labeled with 1 mCi of 125I (specific activity, -2,200Ci/mmol; 1 Ci = 37 GBq; New England Nuclear) by using the Bolton and Hunter reagent (11). Human Mammary Cancer Cytosol. PR-containing cytosol was prepared from MCF-7 cells (12) and total tumor, as described (4 2854The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Antihormone-Steroid Hormonal Activity, Heat-Shock Protein hsp 90 and Receptors

“…In all cases, 8.6S complexes were shifted to ~ 1 IS after interaction with the BF4 mono clonal antibody [16], confirming the pres ence of hsp 90 [21]. TA-GR complexes, in absence of molybdate, shifted to 4.4S after exposure to transforming ionic strength or temperature.…”

“…If the extrac tion is performed with high ionic strength buffer (usually >0.25 M KC1), or if second arily the 8S-R is exposed to such a salt-con taining medium, the hormone-binding peak sediments with a ~4 S sedimentation coeffi cient (4S-R). Hormone affinity chromatogra phy purification has been performed on this premise, and thanks to the stabilizing effect of molybdate ions [11][12][13], the 8S-R form of the progesterone receptor, not binding to DNA-cellulose, was isolated [14], while the purified hormone-binding, DNA-binding 4S-R was also obtained in relatively good yield [15], A monoclonal antibody to 8S-PR [16] was found to detect a nonhormone binding, non-DNA-binding, MW ~ 90,000 superscript asterisk indicates that the receptor is ac tive. We have shown, by drawing a one-way arrow, that a practically irreversible transconformalion takes place after release of H R' from hsp, which might explain the difficulty to recombine H-R* with hsp.…”

Transformation of glucocorticoid and progesterone receptors to the DNA‐binding state

“…Radanyi et a1 [24] prepared a monoclonal antibody (BF4) to the purified, molybdate-stabilized chicken oviduct progesterone receptor and showed that it reacted with the 90 kD nonsteroid-binding protein but not with the 80 kD or the 110 kD progesterone-binding proteins [25]. Similarly, antiserum raised against the purified 110 kD progesteronebinding protein recognizes the 80 and 110 kD steroid-binding peptides but not the 90 kD peptide [26].…”

Nuclear progesterone receptor is mainly heat shock protein 90-free in vivo.

“…The second biotinylated antibody, PR22 (PR-IEMA) or 7Da (PR-hsp90 complex IEMA), and peroxidase-labeled avidin were added. During all incubations, 20 both receptor-free and receptor-bound chsp90 (19,20,25). Staining of chsp90 was specific, since no staining was seen when the antibody was replaced by either buffer or nonspecific antibodies or when the specific antibodies were presaturated with an excess of affinity-purified chsp90 (Fig.…”

“…Fixation with p-benzoquinone or formaldehyde vapor gave essentially similar results. Polyclonal antibody IgGRB (17); monoclonal antibodies PR6, PR13, and PR22 (18) against affinity-purified PR; and monoclonal antibodies BF4, 7Da, and 4F3 against hsp90 (19,20) Cytosols and Nuclear Extracts. These were prepared as described (10,11).…”