Method for culturing mammary epithelial cells in a rat tail collagen gel matrix

Richards, John O.; Larson, Lisa; Yang, Jason; Guzman, Raphaël; Tomooka, Yasuhiro; Osborn, Rebecca C.; Imagawa, Walter; Nandi, Satyabrata

doi:10.1007/bf01834632

Cited by 97 publications

(45 citation statements)

References 13 publications

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“…The cells were grown in multiwell dishes or T25 flasks (Corning, Corning, NY) and kept in an atmosphere of 5% CO 2 and 95% air at 37°C. Cells were grown on soft collagen type I gels, prepared from rat tails as described previously (34), or grown directly on tissue culture-treated plastic. Cells were seeded at high densities, 1 ϫ 10 6 cells/T25 and 2 ϫ 10 5 cells/well in 24-well dishes, unless otherwise indicated.…”

Section: Methodsmentioning

confidence: 99%

Evidence for a Role of the Gut Hormone PYY in the Regulation of Intestinal Fatty Acid-binding Protein Transcripts in Differentiated Subpopulations of Intestinal Epithelial Cell Hybrids

Halldén

Aponte

1997

Journal of Biological Chemistry

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Peptide tyrosine tyrosine (PYY) 1 is member of a 36-amino acid regulatory peptide family that includes neuropeptide Y (NPY) and pancreatic polypeptide (PP). PYY has greater than 70% sequence identity with NPY and shares a common structural motif consisting of two antiparallel helices, an aminoterminal polyproline helix, and a long amphipathic ␣ helix connected by a ␤ turn (1). PYY-secreting cells occur mainly in the distal small intestine and the large intestine, locations where dietary fatty acids can act as potent stimulants of PYY release into the circulation. PYY-induced effects on the gastrointestinal tract can be reproduced by infusions of the peptide at concentrations less than postprandial blood concentrations (2, 3). Specific receptors for NPY/PYY have been characterized in distinct gastrointestinal tissue, such as chief cells (4), and mucosa of the small and large intestine (5). Specific PYY receptors have also been reported in brain tissue (6, 7), spleen (8), vascular smooth muscle (9), and in several neuroendocrine cell lines (10). Such locations make it possible for PYY to act both as an endocrine and a paracrine agent. Many of the reported effects of PYY on the gut, such as the inhibition of intestinal secretion, motility, and gastric acid secretion, occur as interdigestive events coordinated with release of PYY after a meal. We and others have demonstrated that luminal oleic acid induces the release of PYY in the dog (11), rat (12), and in isolated primary cultured PYY cells from the canine mucosa (13). Although it has been proposed that NPY acts centrally to initiate feeding (14), PYY seems to modify digestive processes to ensure efficient utilization of ingested food. PYY acts to slow gastric emptying and intestinal transit, changes that increase the efficiency of nutrient digestion and absorption. In the central nervous system, PYY may act through specific receptors in the dorsal vagal complex to inhibit vagal tone (15). As a result, PYY establishes a negative feedback loop that could act centrally in the brain to inhibit neurally mediated pancreatic exocrine secretion. This negative feedback may serve as part of an "ileal brake" in response to excess dietary triglyceride. For example, triglyceride hydrolysis resulting in FFAs in the distal intestine would induce PYY secretion whenever the rate of triglyceride hydrolysis exceeded the rate of fatty acid absorption. This induction of PYY secretion would then inhibit intestinal motility, causing an increase in FFA absorption and a decrease in luminal FFAs. Therefore PYY, like other gut-regulatory peptides such as cholecystokinin and somatostatin, can act as both a hormone and a neuromodulator.Because PYY secretion can occur in direct response to luminal long-chain FFAs, we chose to examine the possibility that this peptide may act on the expression of an intestinal cytosolic fatty acid-binding protein, the intestinal fatty acid-binding protein (

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Section: Methodsmentioning

confidence: 99%

Evidence for a Role of the Gut Hormone PYY in the Regulation of Intestinal Fatty Acid-binding Protein Transcripts in Differentiated Subpopulations of Intestinal Epithelial Cell Hybrids

Halldén

Aponte

1997

Journal of Biological Chemistry

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“…Rat tail collagen (kindly provided by S. Nandi, University of California, Berkeley) was used as described (19). For the preparation ofcell aggregates, primary cultures ofCEFs were allowed to grow to confluency for =3 days.…”

Section: Methodsmentioning

confidence: 99%

The position of the microtubule-organizing center in directionally migrating fibroblasts depends on the nature of the substratum.

Schütze

Maniotis

Schliwa

1991

Proc. Natl. Acad. Sci. U.S.A.

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Immunofluorescence and confocal microscopy were used to monitor the positioning of microtubuleorganizing centers (MTOCs) during directional migration of chicken embryo fibroblasts on planar substrata and within three-dimensional collagen gels. Homologous assay conditions based on the radial emigration of cells from cell aggregates were used in both cases. Whereas =70% of the cells migrating directionally on glass and at least 60% on other planar substrata have their MTOCs anterior to the nucleus, MTOCs are randomly distributed around the nucleus in cells within collagen gels. The anterior location of the MTOC in cells on glass is attained gradually during the first 4 hr of directional migration. Cells on oriented planar substrata, manufactured by photolithographic etching of narrow parallel grooves into the glass surface, also have a random position of the MTOC, although the cells themselves assume a highly polarized cell shape parallel to the grooves. This environment mimics the partial orientation of the collagen fibers produced by the tractive forces of the cells within collagen networks. These findings demonstrate a difference in MTOC positioning between Jibroblasts on planar substrata and within a quasinatural environment.Microtubules play important roles in the life of a eukaryotic cell. Originating from the centrosome, the cell's microtubuleorganizing center (MTOC), they are involved in cell division, intracellular transport, the development and maintenance of cell asymmetry, and cell migration (1). An involvement in the expression of a locomotory phenotype is revealed in the experimental observation that microtubule depolymerization impairs cell locomotion (e.g., see refs. 2 and 3), and the morphological finding that, in certain migrating cells, the MTOC is located ahead of the nucleus and behind the advancing lamellipodium (4-6). This intriguing correlation was interpreted to mean that MTOC reorientation to the front of the cell not only accompanies the onset of cell migration but actually "may play a role in determining the direction of cell movement" (ref. 4; for reviews, see refs. 7-9).Fibroblasts cultured on glass or plastic substrata have served as important models for some of these studies (3,5,10). However, the advantage of two-dimensional surfaces for microscopic observation is counterbalanced by the disadvantage that these conditions are clearly unrepresentative of a fibroblast's natural environment, a three-dimensional collagen network (11, 12). Observations on fibroblasts in hydrated collagen gels in vitro and in situ demonstrate a number of differences in morphology and behavior from their counterparts on planar substrata (e.g., see refs. 11, and 13-17). It would seem important to ascertain that findings on cells cultured on planar substrata apply to cells in a more natural environment as well, particularly with respect to the question of MTOC positioning during cell migration.We have used a convenient assay for determining directional migration of large numbers of cells that does no...

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“…In this system, mammary epithelial cells were grown inside collagen gels (12,13) in serum-free medium (14,15). Transformation was assayed by transplanting cultured cells into cleared mammary fat pads of syngeneic female mice (16).…”

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confidence: 99%

Neoplastic transformation of mouse mammary epithelial cells by in vitro exposure to N-methyl-N-nitrosourea.

Miyamoto

Guzman²,

Osborn³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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High-efficiency neoplastic transformation of mouse mammary epithelial cells in primary collagen gel culture was induced by N-methyl-N-nitrosourea (MNU). Mammary epithelial cells, isolated from virgin BALB/c mice, were embedded within collagen gels and grown in a serum-free medium containing prolactin, progesterone, and linoleic acid.The cells were then treated with MNU on day 3 of culture and subsequently at weekly intervals for up to 4 weeks. Eleven to 14 days after the final carcinogen treatment, the cells were removed from the collagen gels and i jected into the cleared mammary fat pads of syngeneic hosts to assay for transformed cell populations. A single exposure or multiple exposures of these cells to MNU was effective in inducing tumorigenic cells that produced palpable tumors as early as 6 weeks after transplantation. Two treatments with MNU (100 ,ug/ml) were optimal for neoplastic transformation and produced tumors in 79% of the injected fat pads. All the tumors originated at the site of injection and had extensive central necroses. Histological examination indicated that the tumors were mammary carcinomas. Secondary transplantation of tumor pieces into intact mammary glands produced palpable carcinomas of the same histology within 1-S weeks. Control cells cultured for the same periods of time as MNU-treated cells produced only ductal outgrowths that were morphologically similar to those found in the mammary glands of adult virgin hosts. This system provides a distinct means to study the mechanism of mammary neoplastic transformation at cellular and molecular levels.Mammary neoplasias induced by chemical carcinogens have been studied extensively in animal model systems. These in vivo studies have provided invaluable information about effective carcinogens, susceptible strains, the sensitive age groups, hormonal and dietary influences, and the presence of preneoplastic stages in mammary tumor development (1-3). It has been difficult, however, to directly study the mechanisms underlying the neoplastic transformation of mammary cells because of inherent complexities present in vivo.In vitro transformation systems provide a distinct opportunity to directly study the mechanisms of transformation under well-controlled conditions because (i) mammary epithelial cells with or without their accompanying stroma can be treated with carcinogens, (ii) the direct effects of different hormones and growth factors on transformation can be studied, (iii) the molecular effects of carcinogens can be examined, and (iv) the cellular origin of various mammary tumors observed in vivo can be elucidated. Many attempts have been made to develop a suitable system to transform mammary cells in cell and organ cultures (4-10). In previous studies transformed mammary cells were identified by enhanced growth potential (4-6) or anchorage independence (7) in cell culture, induction of alveolar lesions (8, 9) in organ culture, and induction of preneoplastic lesions or carcinomas (9, 10) in animal hosts. Unfortunately, these systems...

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Method for culturing mammary epithelial cells in a rat tail collagen gel matrix

Cited by 97 publications

References 13 publications

Evidence for a Role of the Gut Hormone PYY in the Regulation of Intestinal Fatty Acid-binding Protein Transcripts in Differentiated Subpopulations of Intestinal Epithelial Cell Hybrids

Evidence for a Role of the Gut Hormone PYY in the Regulation of Intestinal Fatty Acid-binding Protein Transcripts in Differentiated Subpopulations of Intestinal Epithelial Cell Hybrids

The position of the microtubule-organizing center in directionally migrating fibroblasts depends on the nature of the substratum.

Neoplastic transformation of mouse mammary epithelial cells by in vitro exposure to N-methyl-N-nitrosourea.

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