Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells.

Abstract

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial ceils in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.In confluent, high-density cell culture, normal or neoplastic epithelial cells from several mammalian sources spontaneously form multicellular, blisterlike, hollow domes (2, 9, 14, 19, 24, 32, 39, 40, 48 50, 58). Leighton et al. (32,33) recorded by time-lapse photography of MDCK (canine kidney cell line) cultures the growth, rupture, and collapse of domes, "like gently boiling oatmeal." McGrath et al. (39,41), using primary cultures of mouse mammary tumor cells, found that replication and release of mammary tumor virus (MTV) were highest under conditions that also promoted dome formation and that these conditions included, besides high cell density, insulin and hydrocortisone in the medium. The authors suggested for both the kidney and mammary cells that fluid pressure in the dome cavity was maintained by active secretion, presumably related to the process 316

Mechanical tension induces lateral movement of intramembrane components of the tight junction: studies on mouse mammary cells in culture.

“…Where overlap occurs, junctional membranes usually lie in the same plane as the adjacent apical and lateral membranes of the overlapped cell and are approximately parallel to the apical surface of the overlapping cell. As described elsewhere (26), the pattern of intramembrane strands of tight junctions of mammary cells in primary culture generally resembles that typical of non.lactating normal mammary gland in situ. Strands are sinuous, but their predominant orientation usually is roughly parallel to the apical edge of the junction belt, which itseff undulates gently ( Fig.…”

“…Mammary (26,31) and other (32,33) epithelial cell sheets spread on a fixed substrate undergo spontaneous contraction if released from the substrate, suggesting that spread epithelial cells are chronically under tension. If so, increased cell spreading might be expected to increase the tension.…”

“…Under these conditions, the cells undergo one to two rounds of DNA synthesis at most; they form a confluent pavement in 2-4 d. In cultures seeded on plastic at densities of 5 × l0 s cells/era 2 or higher, domes (hemicysts) begin to appear at 4-5 d. Their presence is proof of the presence and integrity ofzonuiae occludentes (6,(26)(27)(28).…”

Transepithelial transport in cell culture.

“…For light microscopy, the cells were fixed and stained with hematoxylin (12). Preparation for transmission and freeze-fracture electron microscopy was as described (13 was measured as the progression of the meniscus along the capillary tubes. Statistical Methods.…”

Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes.

“…The amount of DNA in each sample was measured by a fluorometric assay modified from Hinegardner (14). The (7,12). However, intracellularly, they lack morphological properties that would identify them as mammary secretory cells.…”