Monoclonal antibodies reactive with components in serum-free conditioned medium from a human breast cancer cell line (MCF-7)

Approximately 10,000 primary hybridomas were generated after immunization with either serum-free conditioned medium (SFCM) or extracts from the human breast cancer cell line MCF-7. A total of 11 different monoclonal antibodies (MAbs; 8 generated against SFCM and 3 generated against cell extracts) were selected on the basis of high specificity in cell-binding ELISA for human breast cancer cell lines. The 8 different MAbs obtained by immunization with SFCM all reacted with secreted components in SFCM from MCF-7 cells and 4 of these MAbs reacted with glycolipids extracted from MCF-7 cells. 1 of these MAbs (S2) also recognized a secreted glycoprotein of approximately 77 kilodaltons (kDa). The remaining 4 MAbs did not show specificity solely for carbohydrate determinants. 1 of these MAbs (S7) recognized a secreted protein of approximately 41 kDa. The 3 MAbs raised against cell extracts from breast cancer cells reacted with cytoplasmic antigens in immunofluorescence but also reacted with a secreted component in SFCM from MCF-7 cells. Immunoblotting experiments with proteins from cell extracts and with proteins in SFCM showed that these antibodies all reacted with a protein of a molecular weight of approximately 40 kDa. Our results suggest that this component is cytokeratin 19 or proteolytically processed cytokeratin 19.     

Generation and Characterization of Novel Diagnostic Mouse Monoclonal Antibodies Against Human Estrogen Receptor Alpha and Progesterone Receptor

Background: Estrogen and progesterone regulate the growth and development of several human cells and tissues. Their corresponding receptors (ER and PR) are important diagnostic and prognostic indicators for cancers of the breast and reproductive organs. Immunohistochemical analysis of ER and PR is the current standard method for evaluating the expression of these receptors in different cancers. Nonetheless, there is a significant lack of reproducibility of IHC results in laboratories worldwide, necessitating to develop more sensitive and specific antibodies for ER and PR IHC staining. Methods: ER and PR-specific monoclonal antibodies (MAbs) were generated by immunizing mice with synthetic peptides from ERα and PR. The isotypes and affinity constants of the selected MAbs were determined, and their specificities were assessed by peptide-specific ELISA, IHC, Western-blot analysis, and flow cytometry. In addition, the reactivity of generated MAbs was compared with that of the commercially-available anti-ER and anti-PR antibodies in IHC using normal and cancerous tissue sections. Moreover, 200 breast cancer tissue samples were stained using the newly generated MAbs along with commercial antibodies by IHC, and the sensitivity, specificity and accuracy of our MAbs were evaluated. Results: Among different MAbs generated in this study, two anti-ER and one anti-PR MAbs specifically detected the target antigens in normal and cancerous tissues in IHC. Further analyses confirmed the specificity of the MAbs in Western blotting and flow cytometry using a panel of ER and PR positive cell lines. The sensitivity, specificity and accuracy calculated for clone 1B9 (anti-ER) were 92.3%, 94.8% and 93%, and for clone 3D6 (anti-PR) were 93.0%, 94.3% and 93.5%, respectively. Conclusion: Our novel anti-ER and PR MAbs could be considered as suitable tools for diagnostic and research purposes.     

Preparation and characterization of new anti-PSMA monoclonal antibodies with potential clinical use

Monoclonal antibodies with high specificity for prostate cancer tissue are of interest for diagnostic and therapeutic applications employing targeted therapy. The prostate-specific membrane antigen (PSMA) is a protein predominantly found in epithelial cells of prostate tissue origin and its expression correlates with tumor aggressiveness. Here, we report the development and characterization of new antibodies against PSMA. Murine monoclonal antibodies (MAb) were obtained by immunizing mice with a peptide corresponding to PSMA extracellular residues 490-500 -- GKSLYESWTKK (PSMA(490-500)). The MAbs react specifically to PSMA and to the prostate cancer cell line LNCaP with an affinity for PSMA in the low nanomolar range. This study also demonstrates the potential use of these antibodies for targeted drug delivery to prostate cancer cells. Nanomolar concentrations of PSMA-specific MAb in association with a molecule with cytotoxic potential were sufficient to allow for binding and uptake by LNCaP cells within minutes, leading to complete cell death within 3 days. These MAbs have potential clinical value in the development of diagnostic and therapeutic applications for prostate cancer.     

Evidence for homology of normal and neoplastic human placental alkaline phosphatases as determined by monoclonal antibodies to the cancer-associated enzyme

The HeLa TCRC-1 human adenocarcinoma cell line expresses a form of alkaline phosphatase that is similar to the common S-variant of placental alkaline phosphatase (PLAP) on the basis of electrophoretic mobility, catalytic properties, and reactivity with polyclonal antibodies. More sensitive probes of changes in protein structure than polyclonal antibodies are monoclonal antibodies (MAbs) which recognize individual antigenic sites on molecules. Therefore, we produced MAbs to HeLa TCRC-1 cells and selected those which bound to the alkaline phosphatase expressed by the cancer cells. Seven MAbs were obtained and characterized by (a) fine specificity analysis using allelic variants of PLAP and other human alkaline phosphatase isozymes, (b) immunoglobulin isotype, and (c) relative binding affinities to PLAP from two sources, placental tissue and HeLa TCRC-1 cells. The seven MAbs bind the enzymes from both sources with equal affinity indicating a high degree of structural homology if not identity between the normal S-variant of PLAP and its cancer-associated counterpart. We note that most of the MAbs to cancer cell surface-bound PLAP express either Ig (immunoglobulin) G2a or IgG2b heavy-chain isotypes, a higher incidence of these classes of IgG than has been observed with the purified and soluble PLAP immunogen which yields MAbs predominantly of the IgG1 isotype. Finally, some of these antibodies, like the ones prepared from purified PLAP, recognize differences between allelic variants.     

Characterization of 10 new monoclonal antibodies against prostate-specific antigen by analysis of affinity,specificity and function in sandwich assays

While prostate-specific antigen (PSA) is already an invaluable marker for prostate cancer, there is continuing demand for new anti-PSA antibodies with specific characteristics, e.g., high sensitivity and specificity and equimolar binding to free PSA (f-PSA) and the PSA-α-1-antichymotrypsin complex (PSA-ACT), as well as the ability to distinguish between these 2 immunoreactive forms of PSA. We have therefore generated and characterized 10 anti-PSA monoclonal antibodies (MAbs). Apparent dissociation constants (K_d) of MAbs were determined by direct ELISA yielding K_d-0.2-164.0 nM. Western blots suggested that 3 of the MAbs (60-1A2, 60-8A2 and 17-1A2) bind to linear epitopes. Sandwich assays identified 5 major antigenic regions as binding targets of the MAbs. Three combinations of MAbs recognize f-PSA and PSA-ACT in equimolar fashion with high sensitivity. Two of the MAb combinations are specific for f-PSA. Physical analysis of the new antibodies has allowed us to assign the MAbs to binding classes (based on their sandwiching capabilities) and to determine accurate apparent dissociation constants. Int. J. Cancer 71: 1019-1028, 1997. © 1997 Wiley-Liss Inc.     

Immunological analysis of mucin molecules expressed by normal and malignant mammary epithelial cells

Many existing MAbs raised against the human milk fat globule or against carcinoma cells recognise epitopes on high-molecular-weight glycoproteins. In a comparative ELISA assay a number of these antibodies have been shown to react with an extract of the human milk fat globule. In comparative immunoblots of cultured normal milk cells and breast cell lines, all were found to bind to large molecules which show some variation in molecular weight depending on the cell source. The HMFG-2 antibody, which is widely used in cancer diagnosis, also recognises epitopes on lower-molecular-weight components. In T47D cells these may be as small as 80,000 Mr, and with electron microscopy this cell line can be shown to accumulate HMFG-2-reactive components in the Golgi apparatus. Using an HMFG-2 affinity column we have immunopurified HMFG-2-reactive material from the 2 breast cancer cell lines MCF-7 and T47D and shown that all of the above antibodies react in a solid-phase ELISA with the purified material. In addition to high-molecular-weight components, the immunopurified material was found to contain lower-molecular-weight components including a glycoprotein of 68,000 Mr that was not, however, recognised by the HMFG-2 antibody on a Western blot. We have used this immunopurified material to generate new MAbs. All of these recognise the high-molecular-weight bands seen with the other antibodies, but 2 of them also recognise a band at 68,000 Mr in blots of MCF-7 and T47D. The second-generation antibodies show a spectrum of reactivity on tissues similar to HMFG-2 and one reacts at least as strongly as HMFG-2 with methanol-acetone-fixed sections of breast cancers.     

Generation and characterization of a high-affinity monoclonal antibody for MUC1 measurement in breast cancer

Breast mucin is secreted by breast tumor cells and serves as a marker for breast cancer. Thus, antibodies against breast mucin will be valuable in the development of immunotherapy and laboratory diagnostic tests. Monoclonal antibodies (MAbs) against breast cancer-associated antigen were generated and characterized. Balb/c mice were immunized with breast cancer-associated antigen CA15-3, and subsequently splenocytes from immunized mice were fused with myeloma cells. After fusion, culture supernatants from hybridomas surviving HAT medium were screened by enzyme-linked immunosorbent assay (ELISA). A total of eight hybridomas producing MAbs against breast cancer showed significant levels of antibody activity against CA15-3. Two selected stable hybridomas were adapted into CELLine CL 350 bioreactors, and the MAbs produced were characterized for their subclass, specificity, and affinity. The MAbs were of high specificity and affinity as shown by ELISA. The MAbs produced may represent a powerful tool and are considered promising reagents for use in diagnosis and detection of early stage of the disease.     

Characterization of monoclonal antibodies against stromelysin-3 and their use to evaluate stromelysin-3 levels in breast carcinoma by semi-quantitative immunohistochemistry

Stromelysin-3 (ST3) is a matrix metalloproteinase which is expressed in fibroblastic cells of most human invasive carcinomas and represents a potential new prognostic indicator. Expression of recombinant ST3 forms in Escherichia coli from cDNA constructs indicated that high levels of expression were achieved when the ST3 pro-domain was deleted. The putative mature form of ST3 thus produced and recovered from bacterial inclusion bodies was used to prepare monoclonal antibodies (MAbs) against ST3 by immunization of BALB/C mice. Ten hybridomas producing MAbs against ST3 were obtained and analyzed for their ability to detect endogenous ST3 in breast cancer and in conditioned media from human fibroblasts. One of these MAbs (5ST-4A9) was found to be suitable for the routine detection of ST3 on breast cancer tissue sections, thus opening the possibility to evaluate ST3 prognostic value in breast cancer using semi-quantitative immunohistochemistry. © 1995 Wiley-Liss, Inc.     

Progesterone receptor (PR) variants exist in breast cancer cells characterised as PR negative

Progesterone receptor (PR) expression is measured in breast cancer by immunohistochemistry using N-terminally targeted antibodies and serves as a biomarker for endocrine therapeutic decisions. Extensive PR alternative splicing has been reported which may generate truncated PR variant proteins which are not detected by current breast cancer screening or may alter the function of proteins detected in screening. However, the existence of such truncated PR variants remains controversial. We have characterised PR protein expression in breast cancer cell lines using commercial PR antibodies targeting different epitopes. Truncated PR proteins are detected in reportedly PR negative MDA-MB-231 cells using a C-terminally targeted antibody. Antibody specificity was confirmed by immunoblotting following siRNA knockdown of PR expression. We have further demonstrated that alternatively spliced PR mRNA is present in MDA-MB-231 cells and in reportedly PR-negative breast tumour tissue which could encode the truncated PR proteins detected by the C-terminal antibody. The potential function of PR variant proteins present in MDA-MB-231 cells was also assessed, indicating the ability of these PR variants to bind progesterone, interact with a nuclear PR co-factor and bind DNA. These findings suggest that alternative splicing may generate functional truncated PR variant proteins which are not detected by breast cancer screening using N-terminally targeted antibodies leading to misclassification as PR negative.     

The epidermal growth factor receptor as a target for therapy in breast carcinoma

Summary The epidermal growth factor (EGF) receptor and its ligands have an important regulatory role in breast carcinoma. We have produced a series of monoclonal antibodies (MAbs) directed against the external portion of the EGF receptor. These MAbs prevent the binding of the ligands to the receptor, block ligand-induced activation of the receptor, and can inhibit the growth of breast cancer cells both in tissue culture and in human tumor xenografts in nude mice. We have also shown that anti-EGF receptor antibodies greatly enhance the antitumor effects of chemotherapeutic agents active in breast cancer. Phase I clinical trials with single doses of MAb conducted in patients with tumors over-expressing EGF receptors demonstrated favorable pharmacokinetics, good tumor imaging, and a lack of toxicity. A human:murine chimeric antibody has been produced with comparable affinity and antitumor activity that will enable us to administer repeated doses of MAb either alone or in combination with chemotherapy. Our pre-clinical data support the concept that the EGF receptor may be an optimal target for treatment with receptor blocking antibodies, either alone or in combination with chemotherapy.     

Characterization of MUC1 glycoprotein on prostate cancer for selection of targeting molecules

MUC1 glycoprotein that is overexpressed in aberrant forms in epithelial cancers has been used for diagnosis, staging and therapy. As normal prostate and prostate cancer tissues express MUC1, it represents a potential target, but MUC1 epitopes specific to prostate cancer have not been well characterized. In order to assess MUC1 epitopes in prostate cancer, and their correlation with Gleason grades, binding of 7 well-characterized anti-MUC1 monoclonal antibodies (MAbs) (BrE-3, SM3, BC2, EMA, B27.29, HMFG-1 and NCL MUC1 core), were studied on a prostate tissue microarray. This microarray contained 197 prostate tissue cores representing: i) normal/benign prostate; ii) prostatic intraepithelial neoplasia and Gleason grades 1 and 2; and iii) Gleason grades 3-5. These MAbs bind the MUC1 extracellular domain, but have variable sensitivity to MUC1 glycosylation. To further characterize the effect of glycosylation on their binding, MAb reactivities with unglycosylated MUC1 core peptide and breast and prostate cancer cell lysates were compared. These studies demonstrated strong binding of BrE-3, BC2 and EMA to the peptide core and recognition by BrE-3, SM3, BC2 and EMA of hypoglycosylated MUC1. The results for the microarray indicated that higher Gleason grades were associated with markedly increased cellular staining by MAbs that preferentially recognize less glycosylated MUC1 (BrE-3, p<0.001; SM3, p<0.004; EMA, p=0.009; and BC2, p<0.001). Staining by MAbs that bind preferentially to hyperglycosylated MUC1 (B27.29, p=0.33; HMFG-1, p=0.89; and NCL MUC1 core, p=0.96) did not correlate with Gleason grade. These results demonstrated that hypoglycosylated MUC1 expression increased with Gleason grade, thus supporting the targeting of hypoglycosylated MUC1 epitopes in prostate cancer for more specific imaging and therapy applications.     

New monoclonal antibodies specific for human sarcomas

Monoclonal antibodies (MAbs) against sarcoma-associated cell membrane antigens were prepared by immunizing BALB/c mice with tumor cells from a human osteosarcoma, TPX, grown as a xenograft in athymic BALB/c nude mice. Spleen cells from immunized mice were hybridized with X-63 Ag. 8.653 mouse myeloma cells which yielded 260 growing hybridomas. Seven of these produced antibodies that bound to TPX cells and to cells from another osteosarcoma, but not to autologous skin fibroblasts. MAbs from 2 (TP-1 and TP-3) of these 7 clones did not cross-react with non-sarcomatous tumor cells or peripheral blood lymphocytes. Immunohistochemical studies on frozen tissue sections showed that the TP-1 (IgG-2a) and TP-3 (IgG-2b) antibodies had characteristic and identical specificity profiles. Binding of TP-1 (TP-3) was demonstrated to 15/15 (15/15) osteosarcomas, 3/3 (2/2) synovial sarcomas, 7/9 (6/8) malignant fibrous histiocytomas, 2/2 (1/1) malignant hemangiopericytomas, 1/2 (1/2) chondrosarcomas and 3/6 (1/3) unclassified sarcomas. The antibodies did not bind to any of 16 sarcomas belonging to other histological subtypes, including liposarcomas and leio- and rhabdomyosarcomas. Moreover, they failed to bind to sections of 66 different non-sarcomatous malignancies, or to any of a range of normal adult and fetal tissues, although some weak staining of proximal kidney tubules was seen. The restricted specificity of these antibodies to some major subtypes of human sarcomas makes them promising tools for identification and subclassification of sarcomas.     

Murine monoclonal antibodies raised against human non-small cell carcinoma of the lung: specificity and tumor targeting

The tumor targeting properties of murine monoclonal antibodies (MAbs) generated in our laboratory against non-small cell carcinoma of the lung have been investigated in nude mouse xenograft models. The MAbs selected for evaluation, RS5-4H6, RS7-3G11, and R511-51, have pancarcinoma reactivity, as shown by immunoperoxidase staining of the majority of tumors from the lung as well as breast, colon, kidney, and ovary. The localization of the three MAbs which bind to distinct antigens, and exhibit different levels of cross-reactivity with normal human epithelial tissues, are compared. The MAbs are of the IgG1 isotype. Since these MAbs were reactive with Calu-3, a human adenocarcinoma of the lung cell line grown as xenografts in nude mice, this system was selected as our initial tumor target. The MAbs were found to localize preferentially to the heterotransplanted tumors, with from 6.6 to 8.6% of the injected dose per gram accreting in the tumor at 7 days. Tumor/nontumor ratios of up to 9.7 were seen with one MAb at day 14. The targeting of MAb RS11-51 and F(ab')2 fragments of RS11-51 in GW-39, a human colon cancer grown in nude mice, was also studied. Accretion of intact RS11-51 and F(ab')2 fragments into GW-39 was greatly increased compared to Calu-3. In view of the high frequency of antigen expression on a wide variety of tumors, and the ability to target in vivo, these new MAbs may have potential use in the imaging and therapy of cancer.     

Generation and characterization of a panel of monoclonal antibodies specific for human fibroblast growth factor receptor 4 (FGFR4)

Fibroblast growth factor receptor 4 (FGFR4) is a member of the FGFR family of receptor tyrosine kinases, and plays important roles in a variety of biological functions such as cell proliferation, differentiation, migration, angiogenesis, tissue repair, and tumorigenesis. The human FGFRs share a high degree of sequence homology between themselves, as well as with their murine homologs. Consequently, it has been suggested that it may be difficult to prepare monoclonal antibodies (MAbs) that are specific for the individual receptor types. In this communication, we report on the development and characterization of a panel of anti-human FGFR4 MAbs that were generated in mice using a rapid immunization protocol. Using a modified rapid immunization at multiple sites (RIMMS) protocol with the soluble extracellular domain of human FGFR4 (FGFR4-ECD), the immunized mice developed high levels of polyclonal IgG to the immunogen within 13 days of the first immunization. The lymph node cells isolated from the immunized animals were then fused with mouse myeloma cells for hybridoma generation. Use of an efficient hybridoma cloning protocol in combination with an ELISA screening procedure allowed for early identification of stable hybridomas secreting antihuman FGFR4 IgG. Several identified MAbs specifically reacted with the FGFR4 protein without binding to the other human isoforms (FGFR1, FGFR2, and FGFR3). As evaluated by BIAcore analysis, most anti-FGFR4 MAbs displayed high affinities (8.6 x 10(8) approximately 3.9 x 10(10) M) to FGFR4. Furthermore, these MAbs were able to bind to FGFR4 expressed on human breast tumor cell lines MDA-MB-361 and MDA-MB-453. Taken together, the results demonstrate that the RIMMS strategy is an effective approach for generating class-switched, high-affinity MAbs in mice to evolutionarily conserved proteins such as human FGFR4. These MAbs may be useful tools for further investigation of the biological functions and pathological roles of human FGFR4.     

Internal antigens accessible in breast cancer: implications for tumor targeting

Proponents of monoclonal antibody (MAb)-mediated cancer therapy often assume that a major limitation in clinical application of MAbs is their lack of absolute specificity for malignant cells. In addition, the presence of surface target antigens is thought to be essential. These requirements may be more stringent than necessary for the clinical usefulness of MAbs. We have demonstrated selective localization of a MAb to keratin polypeptides in malignant breast epithelium under conditions of passive infusion of antibody in fresh surgical specimens of breast carcinoma. Although these proteins are normal intracellular constituents of epithelial cells throughout the body, localization of antikeratin antibodies only within the tumor population is most probably associated with the presence of cells permeable to macromolecules. This permeable tumor cell fraction could be recruited for targeting neighboring impermeable tumor cells with radioisotopes or other antitumor agents conjugated to antibodies directed against intracellular antigens.     

Differences in expression of SPan-1 and CA15-3 antigens in blood and tissues.

Pancreatic and mammary cancer cells are reported to have different oligosaccharides on the same apomucin, the MUCI gene product. A better understanding of the tissue specificity of these sugar structures may help in identifying the source of mucins when they are found in the sera. Serum levels of 3 pancreatic-cancer-associated carbohydrate epitopes identified by monoclonal antibodies (MAbs) SPan-1, 19-9 and DU-PAN-2, were compared to those of CA15-3 in a variety of malignant conditions. CA15-3 identifies both carbohydrate and peptide determinants associated with the MUC1 apomucin in breast tissues. SPan-1 antigen was elevated in a high percentage of patients with pancreatic, gastric and colorectal cancer but in only a few of the patients with malignancies of non-GI origin such as breast, ovary and lung. The 19-9 and DU-PAN-2 antigens had a similar pattern of much greater sensitivity for pancreatic cancer than for these non-gastrointestinal cancers. The levels of these 3 markers showed significant correlations in pancreatic cancer. In contrast, CA15-3 was elevated in a large number of patients with breast, lung, ovarian and pancreatic cancers. There was no correlation of CA15-3 with the 3 other markers in pancreatic cancer. SPan-1 and DF3/115D8 antigens in blood have different mobilities in SDS-PAGE and buoyant densities. Moreover, SPan-1 and DF3 antigenic determinants are localized in different regions of the same normal and malignant pancreas and breast tissues. Thus the SPan-1 determinant can be dissociated from the breast peptide and/or carbohydrate determinants.     

Normal tissue reactivity of four anti-tumor monoclonal antibodies of clinical interest

Normal tissue reactivity on frozen sections was examined with 4 monoclonal antibodies (MAbs) that were reported previously to be negative or weakly reactive with normal tissues, and strongly reactive with some types of carcinoma. All 4 antibodies reacted strongly with certain normal epithelial cells. The antibodies tested include 2 antibodies to ovarian cancer, MOv18 and MOv19, one antibody to breast cancer, H23, and one antibody reactive with a range of carcinomas, B72.3. MOv18, MOv19 and H23 reacted with many normal glandular and ductal epithelial cells, while B72.3 reacted most strongly with secretions of the small intestine epithelium and the suprabasal squamous epithelial cells of the esophagus. Since the tissue distribution of MOv19 was very similar to that of another antibody described previously, MW207, these two antibodies were compared by competitive binding inhibition, and found to recognize the same epitope. Our data emphasize the importance of repeated, independent tests of antibody specificity.     

Application of monoclonal antibodies in diagnosis of human breast cancer by fine-needle aspiration cytology

In the present study the authors have examined 100 breast nodules by fine-needle aspiration cytology (FNAC) using the avidin-biotin complex method of immunoperoxidase staining. The monoclonal antibodies (MAbs) employed, B 6.2 and B 72.3, recognize two distinct molecules expressed by transformed breast epithelium. The authors have compared this immunocytochemical reaction with the conventional cytology and the histological examination as well. The data of the MAbs alone or in combination have been evaluated in terms of sensitivity, specificity, predictive value and test efficiency. The results show that tumor cells are strongly reactive with the MAbs. So the authors believe that this method in conjunction with conventional cytology may be useful in the diagnosis of breast tumor, especially in doubtful FNAC.     

人乳腺珠蛋白检测与乳腺癌诊治

人乳腺珠蛋白(hMAM)是近年发现的乳腺组织特异性蛋白,具有较高的敏感性和特异性.hMAM在乳腺癌组织中的表达及与乳腺癌关系的研究表明,hMAM是一种新的具有临床应用前景的乳腺癌标记物,可诱导机体免疫反应产生细胞毒作用,有望用于乳腺癌治疗.     

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)