Western immunoblotting and enzymatic activity analysis of cathepsin D in human breast cancer cell lines of different invasive potential. Regulation by 17beta-estradiol, tamoxifen and ICI 182,780.

We used enzymatic activity and immunochemical quantifications to analyse the expression and secretion of cathepsin D by human breast cancer cell lines of different invasive potentials (MCF-7/6, MCF-7/AZ, MDA-MB-231). This study does not directly prove that cathepsin D or procathepsin D is involved in human breast cancer cell invasion and metastasis but it shows that the proportion of procathepsin D (activity and antigen) secreted by the human breast cancer cell lines tested correlates with their invasive potential. In the estrogen receptor-positive MCF-7 subclones, this proportion is increased by estradiol only in the invasive MCF-7/6 variant. The cell content in procathepsin D is increased by estrogens to a greater extent in MCF-7/6 cells as compared to non-invasive MCF-7/AZ cells. Tamoxifen appears to be an estrogen agonist concerning cathepsin D regulation, whereas ICI 182,780 is a true antagonist. Our results suggest that synthesis and secretion of cathepsin D are regulated at two distinct levels and differentially affected by estrogens. Synthesis only seems to be affected in non-invasive MCF-7/AZ cells, whereas in invasive MCF-7/6 cells, both synthesis and the efficiency of secretion are increased by estrogens. Our results also confirm that the key site of regulation leading to lysosomal enzyme oversecretion is the Golgi apparatus insulin-like growth factor-II/mannose 6-phosphate receptor

Intracellular levels and secretion of insulin-like-growth-factor-binding proteins in MCF-7/6, MCF-7/AZ and MDA-MB-231 breast cancer cells. Differential modulation by estrogens in serum-free medium.

The synthesis and secretion of insulin-like growth factor binding proteins (IGFBPs) were studied in MDA-MB-231 (estrogen-receptor-negative), MCF-7/6 (estrogen-receptor-positive, invasive) and in MCF-7/AZ (estrogen-receptor-positive, non-invasive) human breast carcinoma cell lines. Cells were grown or maintained in a chemically defined medium. Under these conditions, we found different patterns of IGFBPs in the three cell types. MDA-MB-231 cells secrete most of the IGFBPs they produce whereas MCF-7/6 and MCF-7/AZ cells maintain a high intracellular level. In MDA-MB-231 cells, the major IGFBP is IGFBP-4 which is the minor form in MCF-7/6 and MCF-7/AZ cells. IGFBP-2 and IGFBP-5 are predominant in MCF-7/6 cells while MCF-7/AZ cells produce far less IGFBPs and do not contain detectable amounts of 29-32-kDa forms (IGFBP-5). In MCF-7/6 cells, estradiol-17 beta specifically decreases both the intracellular content and secretion of IGFBP-2 and IGFBP-5. Estrogen regulation of IGFBPs cell content and secretion was found to be tamoxifen-resistant, and only slightly antagonized by ICI 182,780, a pure antiestrogen. The function of these regulations relative to the invasive phenotype and proliferation has now to be determined

Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria.

Two methods for harvesting osteoblast-like cell populations from newborn (10 days) rat calvaria were compared. The first one consisted in culturing the periosteum-free bones and then trypsinizing the cells on the bone surface. The second one involved the migration of the osteoblasts on glass fragments before trypsinization. Since the plating efficiency, the proportion of alkaline phosphatase-positive cells, the population doubling time, and the calcium deposition were more adequate, the second method was used to further characterize the behavior of the cultures. During the first week of culture, the cells featured shapes similar to those observed in vivo on the surface of periosteum-free calvaria. They formed multilayers and, in the presence of ascorbic acid, synthetized an organic matrix containing exclusively type I collagen. Later, small amounts of type III collagen appeared. The cells were embedded in the matrix and progressively acquired the morphologic phenotype of osteocyte-like cells. The matrix mineralized in the presence of beta-glycerophosphate. The technique of drop-inoculation (high concentration of cells in a small volume of medium) promoted the multilayer formation and the achievement of large mineralized plates (about 1 cm2) in 3 weeks of culture