Natalie Pourreau-Schneider

Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems.

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.

Immunohistochemical detection of laminin in 98 human breast carcinomas: A light and electron microscopic study

The distribution of laminin was studied in 98 breast carcinomas with antilaminin and the avidin-biotin-peroxidase complex method. Laminin was observed within vascular and epithelial basement membranes. Laminin displayed a continuous linear pattern in intraductal carcinomas, and it was heterogeneously distributed, with a discontinuous linear pattern, in invasive carcinomas. No intracellular laminin staining was detected. Electron microscopic study showed laminin immunostaining in the lamina densa of basement membranes in nonneoplastic breast tissue. In tumors, laminin immunostaining frequently revealed multilayered basement membranes and abnormal multilayered basement membranes in blood vessels in the tumor stroma. These data suggest that laminin immunostaining, as a new approach to the heterogeneous basement membrane changes occurring in carcinomas, should permit better understanding of cell diffusion processes and of stroma-tumor cell interactions. The consistent extracellular distribution of laminin in contact with the stroma indicates that the latter plays an important role in the assembly of basement membrane components.

Localization of lactoferrin and nonspecific cross-reacting antigen in human breast carcinomas. An immunohistochemical study using the Avidin—biotin—peroxidase complex method

A retrospective immunocytochemical study was performed on 67 human breast carcinomas to determine whether the epithelial cell‐associated antigens, lactoferrin and nonspecific cross‐reacting antigen (NCA), could be used as markers in the prognostic assessment of breast cancers. Fixed paraffin sections were tested with anti‐lactoferrin and anti‐NCA. Lactoferrin and NCA were found in 7.5% and 19% of the cases, respectively. Furthermore, the association between these two antigens in tumor cells was significant (P < 0.05). Kappa‐casein was observed in all antigen‐positive cases. These antigens were observed more often in low‐grade ductal carcinomas that had positive estrogen and progestin receptors, but no relationship could be established between lactoferrin or NCA and other prognostic indicators, such as histologic type and grade of the tumor, stromal elastosis, or steroid receptors. Although more antigen might have been detected in unfixed, frozen specimens, the results indicate that lactoferrin and NCA possess minimal value as epithelial cell markers and no prognostic value when detected on routinely fixed, paraffin‐embedded samples.

How culture conditions modulate the morphofunctional differentiation of the human estradiol-sensitive mammary cell line (MCF-7)

The MCF-7 cell line grown on plastic surfaces is widely accepted as a model for hormone sensitivity in molecular biology. However, in vitro results concerning estrogen sensitivity remain controversial. In search of culture conditions most closely simulating the in vivo microenvironment we cultured MCF-7 cells on diverse substrates and in suspension culture. The different factors of the contact environment: (A) influence of diffusive medium, (B) influence of cell to cell contacts, and (C) influence of cell to substrate contacts were considered. Using morphological criteria:phase contrast microscopy, scanning and transmission electron microscopy we observed MCF-7 morphofunctional differentiation under the different culture conditions. Plastic, corneal endothelial cell extracellular matrix, and attached collagen gels imposed a planar medium-aggregate interface. The impermeability of the free surface and the intense basal tension antagonized epithelial polarization. Only at post-confluence did domes and clusters appear above the monolayer. On floating collagen gels and in suspension culture the cells established intimate cell-cell contacts over large surfaces and reconstituted tissular architecture. Three-dimensional growth conditions which approach the in vivo contact environment of epithelial cells should be used instead of the traditional monolayer cultures for assessing hormonal and pharmacological responses of human breast carcinomas.

Estrogen response of MCF-7 cells grown on diverse substrates and in suspension culture: Promotion of morphological heterogeneity, modulation of progestin receptor induction; cell-substrate interactions on collagen gels☆

In this study we observed the incidence of hormone sensitivity in the response of MCF-7 cells to estrogen stimulation when the cells were cultured in different contact environments (hydrophilic plastic, bovine corneal extracellular matrix, type I collagen and in suspension culture). The major purpose was to describe the influence of cell to cell and cell to substrate contacts on the morphological response to estrogen treatment. However, other parameters including growth and induction of progestin receptor were also explored, keeping in mind that the MCF-7 cell line, although representative of normal mammary epithelium in that it contains a similar hormone receptivity, was selected in vitro from a metastatic population in a pleural effusion. Although substrate conditions did not modify growth enhancement by estrogens, progestin receptor levels were significantly higher in three-dimensional spheroid cultures in which cell to cell contacts were optimal due to elimination of basal contact. A careful morphological survey of large surfaces lead to an objective opinion of the overall effect of the hormone treatment on the non-cloned cell line in which a marked heterogeneity in the response of individual cells was observed. In terms of morphofunctional differentiation, the edification of acini with dense microvillus coating was best in suspension culture. When sections were made perpendicular to the plane of cultures on collagen gel rafts two other phenomena were noted: decrease in intercellular junctions, resulting in reduced cell to cell cohesion, and accumulation biodegradation products in the collagen lattice. This suggested a hormone-mediated interaction between the metastatic cells and the fibrillar substrate, collagen I, one of the major constituents of tissue stroma. This estrogen response might be related to the metastatic phenotype and must be distinct from their hormone sensitivity in terms of growth and differentiation since hormone receptivity is generally considered to be a favorable prognosis for breast cancer.

Estrogen receptor immunocytochemical assay (ER-ICA) in human endometrium

An estrogen receptor immunocytochemical assay (ER-ICA) was applied to 15 tissue samples from human endometrium: five proliferative, five secretory, three carcinomas, and two atypical hyperplasias. A monoclonal anti-ER (H 222 SP gamma, Abbott Lab.) and peroxidase antiperoxidase method were applied on frozen sections, 5 micron thick for light microscopy (LM), 100 micron thick for electron microscopy (preembedding). Positive ER staining was quantitated on tissue sections (LM) using a computerized system of image analysis referred to as SAMBA 200 (Thomson TITN). Positive immunostaining was observed in the nuclei of both epithelial and stromal cells in normal and disordered endometrium. SAMBA 200 quantitative analysis permitted an accurate quantification of ER-positive staining. From this preliminary study it is concluded that (a) ER-ICA constitutes a reliable method to study the ER heterogeneous distribution in tissues and the precise intracellular ER localization, which is not feasible by ER biochemical binding assays; (b) SAMBA 200 analysis of the immunostained tissue sections permits an accurate and reproducible method of evaluating the results to quantitate the staining intensity and to determine the percentage of positive cells and the distribution of positive staining of the various tissue structures (glands and stroma).

Early alterations at the plasma membrane of breast cancer cell lines in response to estradiol and hydroxytamoxifen

The time course of the early stage of estradiol-17 beta (E2) and hydroxytamoxifen (OHTAM) action at the plasma membrane of hormone-responsive MCF-7 and non-responsive MDA-MB-231 (MDA) breast cancer cell lines was investigated using scanning electron microscopy (SEM), electron probe X-ray microanalysis and microelectrophysiology analysis. SEM showed a marked increase in the density and the length of microvilli (MV) on MCF-7 cells treated with 1 nM estradiol for 1 min. This membrane response disappeared at 5 min. No early effect was obtained with OHTAM, but both compounds produced a similar surge of heterogeneous MV at 15 min of treatment. The morphological change induced by E2 subsided at 60 min, whereas that of OHTAM persisted. X-ray microanalysis and computer determination of peak/background ratios permitted the demonstration that these morphological alterations were concomitant with a rise in the intracellular level of potassium. Microelectrophysiology analysis showed a sharp transitory decrease in the membrane potential of MCF-7 cells in response to estradiol. In the estrogen-insensitive MDA cells, the hormone did not modify the membrane potential and K levels decreased at 1 and 5 min before rising again to control levels at minute 15 when MV appeared. With OHTAM, potassium decreased significantly at 60 min of treatment. These initial and transitory changes in surface morphology paralleled by alterations in potassium level may be consistent with the occurrence of estrogen membrane receptors on target cells, a new aspect of steroid hormone action.