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Characterization and Properties of Two Monoclonal Antibodies Specific for the M_r 52,000 Precursor of Cathepsin D in Human Breast Cancer Cells¹

INSERM U 148, Unité Hormones et Cancer et UA Centre National de la Recherche Scientifique 59, 60 Rue de Navacelles, 34090 Montpellier, France

The M_r 52,000 estrogen-induced protein secreted by MCF7 cells has been identified as a procathepsin D (procath D), which increases cell growth in vitro, may stimulate invasiveness by digesting extracellular matrix and appears to be a tissue marker for predicting relapses in breast cancer patients. The protease is also present within mammary cells as M_r 48,000, 34,000, 14,000 mature forms that were also recognized by the previously described antibodies to the M_r 52,000 protein (M. Garcia, F. Capony, D. Derocq, D. Simon, B. Pau, and H. Rochefort, Cancer Res., 45: 709–716, 1985).

Using selective screening with a [³⁵S]methionine-labeled MCF7 cell lysate, we have now isolated two new monoclonal antibodies interacting exclusively with the M_r 52,000 procath D. The two monoclonal antibodies, M2E8* and D9H8*, purified from ascitic fluids are IgG1. Their respective K_ds for M_r 52,000 procath D are 0.96 and 0.18 nM. They are directed against two separate domains of the proenzyme that differ from the three domains of previously described antibodies. They both interact with the deglycosylated protein and recognize the autoactivated secreted proenzyme (M_r 51,000), which is devoid of the first part of the NH₂-terminal end.

By immunodetection of cathepsin D proteolytic activity in plasma, these two antibodies were found to recognize selectively human cathepsin D but not the cathepsin D of other species (rat, mouse, rabbit, goat, and horse) whereas antibodies to mature cathepsin D were less species specific. Using sequential passages on concanavalin A-Sepharose and Sepharose matrices coupled to antibodies to the precursor and antibodies to the mature cathepsin D, we separately purified to homogeneity the M_r 52,000 procath D form and its processed cellular forms, whose biological activities can now be assessed independently.

The two monoclonal antibodies were also shown to inhibit the uptake and processing of the M_r 52,000 cathepsin D in MCF7 cells (mostly D9H8*) and to decrease its proteolytic activity, mostly on extracellular matrix (M2E8*). These two monoclonal antibodies are therefore new tools for studying the function, regulation, cellular processing, and localization of procath D as well as the clinical significance of its concentration in normal and tumoral mammary epithelial cells.

¹ These studies were supported by the Institut National de la Santé et de la Recherche Médicale, the Association pour la Recherche sur le Cancer, and the University of Montpellier.

² Recipient of the Ministère de la Recherche et de l'Enseignement Supérieur Fellowship Grant.

³ To whom requests for reprints should be addressed.

Received 2/29/88. Accepted 3/29/88.

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				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]