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Generation and characterization of monoclonal antibodies against plasmin receptor from human carcinoma cells
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149
POSTER PRESENTATIONS P-4: Receptors
GENERATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST PLASMIN RECEPTOR FROM HUMAN CARCINOMA CELLS. LODeif-AlemanV R.. Mirshahi F.. Buttin P.. Soria C.. Soria N., Faure J.P. and Mirshahi M. INSERM U 66. 15 rue de I’Ecole de M&kcine, 75270 Paris Cedex
06, FRANCE We have previously shown the existence of a receptor for plasminogen (Pg) and plasmin (Pli) in a human breast cancer cell line, MCF7MF. This receptor has been characterized and purified: a high homology with aenolase was found. In this study, we have generated a panel of monoclonal antibodies against the plasmin receptor (Pli-R). Hybridomas were produced by fusion of P3lJl myeloma cells and immunized BALBlc mouse spleen cells, and clones were selected by ELISA and blots on immobilized purified PICR and by immunofluorescence. Seven mAbs were selected by their strong reactivity with Pli-R. All of them recognized human aenolase and rabbit 6-enolase, but with less intensity than Pli-R. Two mAbs, PRlOD6 and PRllGl were able to
inhibit l*%Pg binding to its receptor, as measured by dotblot. The same result was obtained in living cells, as
measured by an enzyme-substrate system. An immunofluorescence study was performed using mAbs against the Pli-R on MCF7MF cells and sections from human breast cancer and intestinal polyps. Several antibodies gave a difusse cytoplasmic labeling in all the cells, which could correspond to enolase present in the cytoplasm. This pattern of labeling was similar to that obtained with an anti-a-enolase serum (gift of Dr. Giallongo, Palermo, Italy). In contrast, one of the mAbs, PR7H8 showed a different distribution of the immunoreactivity. The label was localized at the cell surface, in form of contours and grains, and in some cases in the extracellular matrix. The brightest fluorescence was usually found in cancer cells. Thus this mAb may recognize an insoluble protein, present in the membrane of cancer cells, related to enolase, but with a different cellular localization. These findings confirm that Pli-R from carcinoma cells has a high homology with a-enolase, but that both proteins are not identical.
149
POSTER PRESENTATIONS P-4: Receptors
GENERATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST PLASMIN RECEPTOR FROM HUMAN CARCINOMA CELLS. LODeif-AlemanV R.. Mirshahi F.. Buttin P.. Soria C.. Soria N., Faure J.P. and Mirshahi M. INSERM U 66. 15 rue de I’Ecole de M&kcine, 75270 Paris Cedex
06, FRANCE We have previously shown the existence of a receptor for plasminogen (Pg) and plasmin (Pli) in a human breast cancer cell line, MCF7MF. This receptor has been characterized and purified: a high homology with aenolase was found. In this study, we have generated a panel of monoclonal antibodies against the plasmin receptor (Pli-R). Hybridomas were produced by fusion of P3lJl myeloma cells and immunized BALBlc mouse spleen cells, and clones were selected by ELISA and blots on immobilized purified PICR and by immunofluorescence. Seven mAbs were selected by their strong reactivity with Pli-R. All of them recognized human aenolase and rabbit 6-enolase, but with less intensity than Pli-R. Two mAbs, PRlOD6 and PRllGl were able to
inhibit l*%Pg binding to its receptor, as measured by dotblot. The same result was obtained in living cells, as
measured by an enzyme-substrate system. An immunofluorescence study was performed using mAbs against the Pli-R on MCF7MF cells and sections from human breast cancer and intestinal polyps. Several antibodies gave a difusse cytoplasmic labeling in all the cells, which could correspond to enolase present in the cytoplasm. This pattern of labeling was similar to that obtained with an anti-a-enolase serum (gift of Dr. Giallongo, Palermo, Italy). In contrast, one of the mAbs, PR7H8 showed a different distribution of the immunoreactivity. The label was localized at the cell surface, in form of contours and grains, and in some cases in the extracellular matrix. The brightest fluorescence was usually found in cancer cells. Thus this mAb may recognize an insoluble protein, present in the membrane of cancer cells, related to enolase, but with a different cellular localization. These findings confirm that Pli-R from carcinoma cells has a high homology with a-enolase, but that both proteins are not identical.