Two-site immunoenzymometric assay for the 52-kDa cathepsin D in cytosols of breast cancer tissues.

We developed a solid-phase two-site immunoenzymometric assay (IEMA) of the estrogen-induced 52-kDa cathepsin D (EC 3.4.23.5) and its processed forms (48-kDa and 34-kDa proteins) in cytosols of breast cancer tissues, using two monoclonal antibodies directed against two different epitopes of these antigens. The first antibody is bound to a polystyrene microtiter well; the second is labeled with alkaline phosphatase. The assay involves a simultaneous incubation of the antigen with both antibodies, because we observed signal loss during sequential incubations. Alkaline phosphatase was chosen because other enzymes (peroxidase, beta-galactosidase) were inhibited by cytosol extraction buffers. The measurable range of 52-kDa-related proteins is from 0.3 to 6 nmol/L with precision (CVs) within and between runs of 3.9% and 15.8%, respectively. The sensitivity, accuracy, and rapid turnaround time of the two-site IEMA should facilitate the clinical evaluation of this new marker in oncology.

Effect of estradiol on nonmalignant human mammary cells in primary culture.

We have studied effects of estradiol on primary cultures of nonmalignant human mammary tissue collected surgically from fibroadenomas or during reduction mammoplasties. After enzymatic digestion, "organoids" made of epithelial cells organized in ductal or alveolar structure were grown in primary cultures (up to 12 days) on different substrata (glass, plastic, collagen-coated plastic, and floating collagen membranes). Transmission and scanning electron microscopy showed that these organoids were responsive to physiological concentrations of estradiol. Condensed chromatin of epithelial cells became dispersed following estrogen treatment. The plasma membrane of epithelial cells at the surface of the organoids was dramatically modified by estradiol, which increased the number and the length of the microvilli, as observed previously in the MCF7 breast cancer cell line (Vic et al., Cancer Res., 42: 667-673, 1982). This effect was not observed with the same concentrations of progesterone, dexamethasone, dihydrotestosterone, or 1 microM tamoxifen or in fibroblasts of the same tissue, demonstrating that epithelial mammary cells are specifically responsive to estradiol. By contrast, no effect of estradiol could be evidenced on the [35S]methionine-labeled proteins released into the medium by the organoids. The estrogen-regulated protein of Mr 52,000 was not found in the medium after purification by concanavalin A-sepharose or immunoprecipitation with specific antibodies to the Mr 52,000 protein from MCF7 cells. We conclude that nonmalignant mammary cells are responsive to estrogens in primary culture.