This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Garcia, M. Articles by Rochefort, H. Search for Related Content PubMed PubMed Citation Articles by Garcia, M. Articles by Rochefort, H.

Characterization of Monoclonal Antibodies to the Estrogen-regulated M_r 52,000 Glycoprotein and Their Use in MCF7 Cells¹

Unité d'Endocrinologie Cellulaire et Moléculaire, U 148 INSERM, 60, rue de Navacelles, 34100 Montpellier [M. G., F. C., D. D., H. R.] and Centre de Recherches Clin-Midy (Sanofi), Rue du Professeur Joseph-Blayac, 34082 Montpellier [D. S., B. P.], France

The M_r 52,000 glycoprotein is regulated by estrogen and released by breast cancer cells in culture (B. Westley and H. Rochefort, Cell, 20: 352–362, 1980). This rare protein was partially purified from 25 liters of medium conditioned by MCF7 cells and injected into Biozzi's selected mice. The spleen lymphocytes of one immunized mouse was fused with the murine myeloma P3-X63-Ag8-653. Sixteen hybridomas producing monoclonal antibodies to the M_r 52,000 protein were isolated, and seven of them were cloned and purified. The seven monoclonal antibodies were all of the immunoglobulin G1 isotype, and their dissociation constants ranged from 0.35 to 2.3 nM. The antibodies specifically recognized the secreted M_r 52,000 protein as evidenced by double immunoprecipitation and by immunoblotting after electrophoretic separation and transfer. Double-determinant immunoradiometric assay indicated that the seven purified monoclonal antibodies recognized three distinct regions of the M_r 52,000 protein, and it was used to assay the M_r 52,000 protein in biological fluids. These antibodies did not react with the external plasma membrane of MCF7 cells, as shown by immunofluorescence analysis. By contrast, the cytoplasm of MCF7 cells (but not T47D and RBA cells) was stained by the peroxidase-immunoperoxidase complex after plasma membrane permeation, indicating that the protein is secreted by exocytosis rather than shed from the plasma membrane.

¹ This work was supported by INSERM-Sanofi Contract 81039, the University of Montpellier 1, and the Fondation pour la Recherche Médicale Française.

² To whom requests for reprints should be addressed.

Received 6/26/84. Accepted 11/ 1/84.

This article has been cited by other articles:

				K. El Messaoudi, L. F. Thiry, C. Liesnard, N. Van Tieghem, A. Bollen, and N. Moguilevsky A Human Milk Factor Susceptible to Cathepsin D Inhibitors Enhances Human Immunodeficiency Virus Type 1 Infectivity and Allows Virus Entry into a Mammary Epithelial Cell Line J. Virol., January 1, 2000; 74(2): 1004 - 1007. [Abstract] [Full Text]

				V Laurent-Matha, M. Farnoud, A Lucas, C Rougeot, M Garcia, and H Rochefort Endocytosis of pro-cathepsin D into breast cancer cells is mostly independent of mannose-6-phosphate receptors J. Cell Sci., January 9, 1998; 111(17): 2539 - 2549. [Abstract] [PDF]