Gautam K. Bandyopadhyay

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)

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)

Effect of reproductive states on lipid mobilization and linoleic acid metabolism in mammary glands

Effects of pregnancy and lactation on lipid metabolism in mouse mammary fat pads and nonmammary adipose tissues have been studied. In order to address the question whether the influence of hormonal milieu on lipid metabolism in mammary epithelial cells during pregnancy and lactation is the same as in fat cells, we have studied the mobilization of lipids and metabolism of fatty acids in the intact mammary glands, parenchyma-free mammary fat pads and in the perimetrial fat tissues of virgin, pregnant and lactating mice. Compared to parenchyma-free mammary fat pads, the perimetrial adipose tissues accumulated 5-fold higher levels of triglycerides during pregnancy. Mammary fat cells maintained overall lipid levels during pregnancy and lactation (16–20 μg/fat pad). In contrast, lactation depleted total lipid stores from 108 ± 5 to 24 ± 4.5 μg/fat pad in perimetrial fat pads. Results of comparative analysis of fatty acid composition of mammary fat pads, with and without epithelial tissue, from virgin and lactating mice showed stimulation of 18∶2ω6 metabolism leading to 130% increase in the ratio 20∶4ω6 to 18∶2ω6 in the epithelial compartment. Pregnancy and lactation resulted in the elevation of 20∶4ω6 levels probably due to a 4-fold increase in Δ5 desaturase activity and a decrease in oxidative degradation of 18∶2ω6. These results suggest that, unlike other adipose tissues, the metabolic pathways in mammary fat cells are not dedicated to sequestration and accumulation of dietary lipids during pregnancy. Lactation favors mammary epithelial cell-stimulated production of precursors of eicosanoids which are known to have agonist-like effect on mammary epithelial cells.

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.

Phenotypic characterization of collagen gel embedded primary human breast epithelial cells in athymic nude mice

We have developed a method to characterize the phenotypes and tumorigenicity of dissociated human breast epithelial cells. The dissociated cells were first embedded in collagen gels and subsequently transplanted subcutaneously in vivo in athymic nude mice. The transplantation of dissociated epithelial cells from reduction mammoplasties, presumed to be normal, always resulted in normal histomorphology. Epithelial cells were arranged as short tubular structures consisting of lumina surrounded by epithelial cells with an occasional more complex branching structure. These outgrowths were surrounded by intact basement membrane and were embedded in collagen gel that, at termination, contained collagenous stroma with fibroblasts and blood vessels. In contrast, transplantation of dissociated breast epithelial cells from breast cancer specimens resulted in outgrowths with an invasive pattern infiltrating the collagen gel as well as frank invasion into vascular space, nerves and muscles. These observations were made long before the subsequent palpable stage which resulted if left in the mouse for a long enough time. The dissociated human breast epithelial cells thus retained their intrinsic property to undergo morphogenesis to reflect their original phenotype when placed in a suitable environment, the collagen gel.

Adenosine-mediated inhibition of casein production by mouse mammary glands in culture

The present study was carried out to examine whether activation of adenosine receptors by adenosine analogues will affect casein production by mouse mammary epithelial cells. The morphogenesis and functions of epithelial tissue in the mammary gland are influenced by their surrounding adipocytes. Adipocytes are known to release adenosine into the extracellular fluid which can modulate cyclic‐AMP levels in surrounding cells through binding to their adenosine receptors. To examine a possible paracrine effect of adenosine, the modulation of casein production in mammary explant culture and mammary epithelial cell (MEC) culture by adenosine receptor agonists has been investigated. We have observed that activation of the A1‐adenosine receptor subtype in mammary tissue by an adenosine analogue (—)N6‐(R‐phenyl‐isopropyl)‐adenosine (PIA) raised cAMP levels. PIA and another adenosine receptor agonist, isobutylmethylxanthine (IBMX), inhibited casein accumulation both in explants and in MEC cultures in the presence of lactogenic hormones, which suggests that PIA or adenosine can act directly on the epithelial cells. This inhibition does not appear to be caused by elevation of cAMP levels or phosphodiesterase activity. The inhibition of intracellular casein accumulation by PIA and IBMX in explant cultures can be reversed via treatment of pertussis toxin which is known to ADP‐ribosylate GTP‐binding Gαi‐proteins, indicating that a Gi‐protein‐dependent pathway may be involved in this inhibition. The results also suggest that local accumulation of adenosine in the extracellular fluids of mammary glands is likely to inhibit the lactogenic response of mammary epithelial cells.

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.