Walter Imagawa

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

Mammary glands are enzymatically dissociated and the resulting tissue digest enriched for epithelial cells by isopycnic banding on a density gradient of Percoll. The cells are embedded within a rat tail collagen gel matrix and fed with the appropriate medium. Growth and differentiation are superior in such a system when compared to culture on plastic, using identical media.

Role of polyunsaturated fatty acids as signal transducers: Amplification of signals from growth factor receptors by fatty acids in mammary epithelial cells

The growth, morphogenesis and differentiation of milk producing epithelial tissues in the developing mammary glands require interaction with extracellular matrices and stimulation by hormones, growth factors and essential fatty acids. In primary culture, the proliferation of mammary epithelial cells (MEC), induced by epidermal growth factor (EGF), is enhanced and sustained by linoleate and its eicosanoid metabolites. Since a combination of linoleic acid (18:2 omega 6) and prostaglandin E2 or cAMP has synergistic effect on EGF-stimulated growth, it is suggested that additional cAMP-dependent protein kinase A (PK-A) independent pathways may also contribute to the linoleate effect on EGF action. Possible involvement of Ca2+/phospholipid-dependent protein kinase C (PK-C) is explored. Both linoleate and arachidonate can activate Type-II and Type-III protein kinase-C in MEC and a PK-C inhibitor can block growth stimulation by EGF and fatty acids. Like 12-O-Tetradecanoly phorbol-13-acetate (TPA), a PK-C activator which also enhances EGF-stimulated growth of MEC, linoleate can phosphorylate a 40-42 KD protein. EGF itself can stimulate transient phosphorylation of the same protein in MEC cultures but when supplemented with linoleate, which does not influence the ligand binding affinity of EGF-receptors, the transient phosphorylation signal in 40-42 KD protein is sustained.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphogenesis of mouse mammary epithelial cells growing within collagen gels: ultrastructural and immunocytochemical characterization

Mammary epithelial cells from adult virgin mice have been cultured within collagen gels in totally serum-free medium containing either epidermal growth factor or the mammogenic hormones, progesterone and prolactin, or prolactin alone. The cellular organization, differentiation and cell-type composition of the colonies from the three culture conditions were assessed by transmission electron microscopy and light-microscope immunocytochemistry. The epithelial cells form branching duct-like structures and, when exposed to mammogenic hormones, assume a secretory morphology (including casein micelles) similar to that seen in the early to mid-pregnant mouse.

Basic fibroblast growth factor stimulates the growth and inhibits casein accumulation in mouse mammary epithelial cells in vitro

The effects of basic fibroblast growth factor (bFGF) on the growth and differentiation of mouse mammary epithelial cells in serum-free collagen gel culture were examined. Epithelial cells obtained from virgin or midpregnant mice grew when bFGF was added to medium containing either insulin at a concentration greater than or equal to 1 microgram/ml or somatomedin-C (Sm-C) at 150 ng/ml. This growth-promoting effect is of the same magnitude as, and additive with, the growth-promoting effect of epidermal growth factor (EGF) or mammogenic hormones. The sensitivity of the cells to EGF or mammogenic hormones was not altered by exposure to bFGF. The progeny cells resulting from growth stimulation by bFGF are capable of accumulating casein upon subsequent stimulation by prolactin (PRL), but accumulate less casein than cells grown in response to EGF. bFGF also appears to reduce casein accumulation if it is added to the cultures at the same time as PRL.

Collagen gel culture system and analysis of estrogen effects on mammary carcinogenesis

The results obtained to date from studies dealing with the role of hormones, including estrogen, on growth of mammary epithelial cells inside the collagen gel are described. The collagen gel matrix culture system appears to be a suitable system to obtain in vivo-like effects of hormones on mammary cell in vitro. The results thus far indicate that prolactin along with progesterone or cortisol can stimulate mammary cell proliferation. Thus far, estrogen has not been found to be mitogenic in our in vitro system.

Role of GTP-binding proteins in the polyunsaturated fatty acid stimulated proliferation of mouse mammary epithelial cells

Polyunsaturated fatty acids enhance the proliferation of mouse mammary epithelial cells stimulated by epidermal growth factor (EGF) by modulating the post-receptor signaling pathways. The growth stimulatory effect of these fatty acids is completely inhibited by pertussis toxin, whereas the inhibition of EGF and insulin stimulated growth is only partial. The treatment of cell cultures with 12-O-tetradecanoyl-phorbol-13 acetate (TPA) reverses the growth inhibitory effect of pertussis toxin and fully restores the growth as was in the control cultures untreated with the toxin suggesting a role for PKC in this reversal. It appears that the functions of Gi-proteins are required in the mediation of fatty acid effect on growth. The predominant types of Gi alpha in mammary epithelial cells are Gi alpha 1, Gi alpha 2, and Gi alpha 3. Among these, the levels of Gi alpha 1 and 2 appears to be regulated by steroid hormones. Linoleic acid raises the level of GTP-bound Ras in the cells above the levels induced by EGF. Pertussis toxin reduces the level of Ras-GTP and inhibits phosphorylation of MAP kinase by EGF. It has been speculated that Gi-proteins interact with the receptor bound nucleotide exchange factor and the membrane anchored Raf kinase and constitute two sites for pertussis toxin action. The phosphorylation by PKC may uncouple Gi-protein interaction with these effectors and enable the agonist-induced signals to bypass the inhibitory action of PT on growth.(ABSTRACT TRUNCATED AT 250 WORDS)

ω-3 and ω-6 fatty acids and PGE2 stimulate the growth of normal but not tumor mouse mammary epithelial cells: Evidence for alterations in the signaling pathways in tumor cells

Abstract The direct effect of ω-3 and ω-6 fatty acids on the proliferation of mouse mammary tumor cells (MTC) was examined in a serum-free cell culture system. While the EGF-induced proliferation of normal mammary epithelial cells was shown to be enhanced by ω-3 and ω-6 fatty acids and prostaglandins (PGs), a majority (75–80%) of primary mammary tumors were not stimulated by these agents. Compared to normal cells, some MTC cultures showed a higher susceptibility to inhibition by ω-3 fatty acids. The general lack of response of MTC cultures to PGE 2 and cyclic adenosine monophosphate (cAMP) suggests some alterations in the cAMP-mediated pathway. However, the PGE 2 -induced cAMP levels and cAMP-dependent protein kinase (PKA) activities in the tumor cells were comparable to normal cells. We conclude that the proliferation of mammary tumor cells either follow a cAMP-PKA-independent pathway or have some alterations in the serine/threonine kinase mediated signaling pathway.

Docosahexaenoic acid is neither synthesized nor retroconverted by the normal and tumor mouse mammary epithelial cells in primary culture

Metabolism of 1-14C-[18:3(n-3)] and 1-14C-[22:6(n-3)] were investigated in the primary cultures of normal and tumor mouse mammary epithelial cells. Analysis of endogenous fatty acid composition indicated a decreased proportion of total (n-6) PUFA in the cultured tumor cells compared to normal cells. These cells can synthesize significant amount of 20:5 (n-3) and 22:5 (n-3) but not 22:6 (n-3), from 18:3 (n-3). There was very little or no retroconversion of 22:6 (n-3) by these cells. It has been concluded that mammary epithelial cells may be deficient in 4-desaturase activity and also that exogenous 22:6 (n-3), instead of serving as a source of 20:5 (n-3), may actually counter the effects of both 20:4 (n-6) and 20:5 (n-3) in the mammary tissue.