Immunocytochemical localization of progesterone receptors in breast cancer with anti-human receptor monoclonal antibodies.

Monoclonal antibodies (MAbs), recently produced against human progesterone receptors (PR), were used for immunocytochemical localization of PR. The specificity of the immunocytochemical assay for PR was demonstrated by incubation with control MAbs, preabsorption of MAbs with highly purified human PR, and by the cell and tissue distribution of the immunostaining reaction. With human breast cancer cell lines, immunoreactivity was confined to cells that contain PR by steroid-binding assay. Moreover, immunostaining was induced by estradiol in estrogen-responsive cells, MCF-7 and ZR-75-1. In a preliminary study with 33 breast carcinomas, a good correspondence was obtained between immunocytochemical staining and PR content assessed by conventional steroid-binding assay. Immunoperoxidase localization was also obtained with other human target tissues. In normal breast and benign breast disease, immunoreactivity was observed with nuclei of ductal epithelial cells and hyperplastic epithelium. In uterus, immunostaining of endometrium was localized to nuclei of stromal and glandular epithelial cells and in myometrium to nuclei of smooth muscle cells. The effect of the progestin agonist, R5020, and antagonist, RU 486, on PR localization was investigated with the PR-rich T47D human breast cancer cell line. In the absence of hormone, immunostaining was exclusively nuclear. This was true under a number of cell culture conditions designed to eliminate endogenous progestins from the culture medium. Exclusive nuclear localization of PR was not due to a failure of the MAbs to recognize unoccupied PR, since each MAb bound equally well in vitro with different receptor forms. These included liganded and unliganded cytosol PR, molybdate stabilized PR, and nuclear-transformed receptors. Nor was failure to detect cytoplasmic staining due to a selective destruction or loss of unoccupied PR from the cytoplasmic compartment as a result of cell fixation. This was assessed by dot blot immunoassay of PR antigen distribution in subcellular fractions of fixed and unfixed cells. Continuous exposure of cells to R5020 resulted in a transient (30-60 min) increase in nuclear staining intensity (without change in cytoplasmic reactivity), followed by a progressive decline in immunoreactivity. By 24 h of R5020 treatment, the vast majority of cells displayed no immunostaining reaction. These immunocytochemical data are consistent with progestins down regulating their own receptors due to a loss in cellular PR content and not to inactivation of receptors.(ABSTRACT TRUNCATED AT 400 WORDS)