Transporters dynamically expressed at the mammary gland transport critical nutrients into the breast milk of nursing mothers to meet the nutritional demands of the suckling infant. However, xenobiotics may interact with these transporters to potentially alter the nutrient composition of milk and compromise neonatal nutrition. The aim of the present study was to quantitatively evaluate the constitutive expression of various nutrient transporters in whole mammary gland tissue and mammary epithelial organoids (MEO) isolated from female Sprague-Dawley rats at various stages of pregnancy, lactation, and involution. Furthermore, the studys aim was to determine if appropriately cultured mammary epithelial organoids (MEO) maintain in vivo transporter expression to lay down critical groundwork for the development of an in vitro screening tool assessing xenobiotic-nutrient transporter interactions. The following transporters were evaluated using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR): multidrug resistance protein (Mdr) 1a, 1b; multidrug resistance-like protein (Mrp) 1; organic cation transporter (Oct) 1; organic cation/carnitine transporter (Octn) 1, 2, and 3; concentrative nucleoside transporter (Cnt) 1, 2, and 3; equilibrative nucleoside transporter (Ent) 1, 2, and 3; nucleobase transporter (Ncbt) 1 and 2; oligopeptide transporter (Pept) 1 and 2; methotrexate carrier (Mtx) 1; divalent metal transporter (Dmt) 1; and the milk protein ?-casein. Transporter expression patterns in MEO differed from whole tissue for ?-actin, Mdr1a, Mdr1b, Oct1, Octn3, Ent3, Cnt1, Cnt3, Ncbt1, Pept2, Mtx1, and ?-casein. This brings into question whether whole mammary gland tissue is truly appropriate for an understanding of transporter expression in the mammary epithelium. Nevertheless, four general transporter expression patterns emerged in isolated MEO: decline throughout lactation (Mdr1a, Mdr1b, Mrp1 & Dmt1), increase throughout lactation (Cnt1 & Octn3), increase in early lactation (Oct1, Octn2, Ent1, Cnt2, Cnt3, Pept2 & Mtx1) and constant expression throughout lactation (Octn1, Ent2, Ent3, Ncbt1, Ncbt2 & Pept1). These expression patterns will provide insight into the critical windows of nutrient delivery to the breast milk to provide adequate nutritional stimuli to the suckling infant. Furthermore, MEO cultured in an extracellular matrix-rich environment maintained transporter expression at the mRNA level, which underscores the potential of the primary MEO in vitro model system as a screening tool for xenobiotic-transporter interactions at the mammary gland. Transporter expression patterns in MEO were unique for each transporter evaluated. This information accompanied by an in vitro screening tool may allow for predictions of xenobiotic interference with breast milk composition to help safeguard infant health.

Transporters dynamically expressed at the mammary gland transport critical nutrients into the breast milk of nursing mothers to meet the nutritional demands of the suckling infant. However, xenobiotics may interact with these transporters to potentially alter the nutrient composition of milk and compromise neonatal nutrition. The aim of the present study was to quantitatively evaluate the constitutive expression of various nutrient transporters in whole mammary gland tissue and mammary epithelial organoids (MEO) isolated from female Sprague-Dawley rats at various stages of pregnancy, lactation, and involution. Furthermore, the studys aim was to determine if appropriately cultured mammary epithelial organoids (MEO) maintain in vivo transporter expression to lay down critical groundwork for the development of an in vitro screening tool assessing xenobiotic-nutrient transporter interactions. The following transporters were evaluated using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR): multidrug resistance protein (Mdr) 1a, 1b; multidrug resistance-like protein (Mrp) 1; organic cation transporter (Oct) 1; organic cation/carnitine transporter (Octn) 1, 2, and 3; concentrative nucleoside transporter (Cnt) 1, 2, and 3; equilibrative nucleoside transporter (Ent) 1, 2, and 3; nucleobase transporter (Ncbt) 1 and 2; oligopeptide transporter (Pept) 1 and 2; methotrexate carrier (Mtx) 1; divalent metal transporter (Dmt) 1; and the milk protein ?-casein. Transporter expression patterns in MEO differed from whole tissue for ?-actin, Mdr1a, Mdr1b, Oct1, Octn3, Ent3, Cnt1, Cnt3, Ncbt1, Pept2, Mtx1, and ?-casein. This brings into question whether whole mammary gland tissue is truly appropriate for an understanding of transporter expression in the mammary epithelium. Nevertheless, four general transporter expression patterns emerged in isolated MEO: decline throughout lactation (Mdr1a, Mdr1b, Mrp1 & Dmt1), increase throughout lactation (Cnt1 & Octn3), increase in early lactation (Oct1, Octn2, Ent1, Cnt2, Cnt3, Pept2 & Mtx1) and constant expression throughout lactation (Octn1, Ent2, Ent3, Ncbt1, Ncbt2 & Pept1). These expression patterns will provide insight into the critical windows of nutrient delivery to the breast milk to provide adequate nutritional stimuli to the suckling infant. Furthermore, MEO cultured in an extracellular matrix-rich environment maintained transporter expression at the mRNA level, which underscores the potential of the primary MEO in vitro model system as a screening tool for xenobiotic-transporter interactions at the mammary gland. Transporter expression patterns in MEO were unique for each transporter evaluated. This information accompanied by an in vitro screening tool may allow for predictions of xenobiotic interference with breast milk composition to help safeguard infant health.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

Content removed due to copyright restriction: Appendix 6 Cooper, C. V., Stelwagen, K., Singh, K., Farr, V. C., Prosser, C. G., and Davis, S. R. (2004): Expression of the tight junction protein zonula occludens-1 during mammary engorgement. Proceedings of the New Zealand Society of Animal Production 64,43-47. Singh, K., Dobson, J., Phyn, C. V. C., Davis, S. R., Farr, V. C., Molenaar, A. J., and Stelwagen, K. (2005): Milk accumulation decreases expression of genes involved in cell-extracellular matrix communication and is associated with induction of apoptosis in the bovine mammary gland. Livestock Production Science 98,67-78. Appendix 7 McMahon, C.D., Farr, V.C., Singh, K., Wheeler, T.T. and Davis, S.R. (2004). Decreased expression of ß1-integrin and focal adhesion kinase in epithelial cells may initiate involution of mammary glands. Journal of Cellular Physiology 200, 318-325 / Extended periods of milk accumulation result in loss of secretory activity, increased apoptosis and eventually, involution of mammary glands. This process is associated with increased permeability of the tight junction (TJ) complexes between adjacent mammary epithelial cells (MECs). The change in cell shape during mammary engorgement from a cuboidal to a flattened morphology may initiate changes in protein and gene expression (mechanotransduction) that trigger these processes. Therefore, this study examined the regulation of the major TJ protein components during mammary engorgement, and in particular the role of physical distension of the mammary epithelium in the regulatory process. Expression of the integral transmembrane TJ proteins, occludin and claudin-1, and the cytoplasmic TJ protein, ZO-1, were down-regulated in both bovine and rat mammary glands during the early stages of mammary apoptosis and involution following the abrupt cessation of milk removal. In the rat, these responses were locally regulated as they occurred only in teat-sealed glands in a hemi-suckled model. Furthermore, the down-regulation of TJ proteins is consistent with a loss of TJ integrity during mammary engorgement. Induced physical distension of rat mammary glands in vivo transiently up-regulated the expression levels of occludin protein and mRNA, and ZO-1 mRNA, followed by an accelerated decrease in expression compared with the effects of milk accumulation alone. This was associated with the initiation of apoptosis, the up-regulation of the pro-apoptotic factor pSTAT3, and the down-regulation of the cell-ECM survival factor βl-integrin. An in vitro model was also developed to stretch MECs, mimicking the flattening in cell shape during mammary engorgement in vivo. While stretching MECs in vitro did not conclusively alter TJ protein expression, the overall results of this project support further investigation into the role of the TJ complex in mechanotransduction pathways. In addition, the results point to crosstalk between cell-ECM survival signalling and STAT3 death signalling as a candidate for regulation by physical distension of the mammary epithelium. In conclusion, this study supports the hypothesis that physical distension during engorgement of the mammary glands with milk is a primary trigger initiating apoptosis of MECs through changes in the regulation of gene pathways controlling cell survival and death, and the disruption of TJ function.

Le cancer du sein est une pathologie hétérogène au plan clinique et au moins 5 sous-types moléculaires ont pu être définis sur la base de différences d’expression ARNm. Ces sous-types présentent des différences de profils d’anomalies génomiques et de méthylation des cytosines. Ces différences génétiques et épigénétiques s’expliqueraient par des types cellulaires d’origines distincts au sein de l’épithélium mammaire, toutefois, ceci n’a pas été confirmé clairement à ce jour. Alternativement, il a été proposé que l’activation de voies oncogéniques différentes pouvait avoir un impact significatif sur les modifications génétiques ou épigénétiques. Dans ce travail nous avons voulu vérifier cette hypothèse en l’appliquant à un modèle de cellules épithéliales mammaires normales primaires humaines, que nous avons isolé des à partir de glandes mammaires. Ces cellules ont été transformées en deux étapes par transduction avec (i) un shARN ciblant TP53, (ii) un oncogène. Nous avons sélectionné 3 oncogènes qui activent des voies de signalisations distinctes CCNE1, HRAS-v12 et WNT1. Nous avons établi un modèle de transformation tumorale en trois étapes, cellules normales, immortalisées et transformées, permettant de suivre les modifications moléculaires associées à chaque étape et de vérifier si l’activation de voies oncogéniques distinctes produisait des profils d’anomalies différents. Les différents modèles ont été analysés par CGH-array, RRBS, transcriptome et miRNA à des temps de culture définis.Nos résultats montrent que l’activation de la voie RAS aboutit à des profils d’anomalies génétiques et de méthylation des CpG radicalement différents de ceux obtenus après surexpression des gènes CCNE1 et WNT1. Ces différences apparaissent très rapidement après transduction des oncogènes alors que les profils des cellules CCNE1 et WNT1 divergent plus tardivement. Enfin, l’inactivation de p53 n’induit pas par elle-même une instabilité élevée, mais produit un contexte de plasticité favorable aux modifications génétiques et épigénétique.Par ailleurs, nous avons noté des différences phénotypiques entre les HMEC RAS (mésenchymateuses) et les HMEC CCNE1 et les HMEC WNT1 (épithéliales). Dans ce travail, je montre que les HMEC shp53 immortalisées présentent une plasticité phénotypique, une partie des cellules entrant en EMT spontanément, l’autre restant épithéliales. J’ai montré que la transduction RAS sélectionnait les cellules ayant effectué une EMT, alors que la transduction de CCNE1 ou WNT1 sélectionnait les cellules épithéliales. J’ai cherché à identifier les déterminants de ces changements phénotypiques et mes résultats suggèrent qu’ils résultent d’une balance entre une signalisation TGFB1/BMP1, qui favorise l’EMT, et BMP4/WNT7 qui favorise la MET. / Breast cancer is a heterogeneous pathology. Based on the differences of mRNA expression, at least five molecular subtypes have been defined. These subtypes show differences in profiles of genomic abnormalities and CpG methylation. These molecular subtypes are thought to originate from different cell lineages in the mammary gland. However, this has not yet been clearly demonstrated. Alternatively, it has been proposed that the activation of different oncogenic pathways could have a significant impact on genetic or epigenetic changes.We wanted to verify this hypothesis by applying it to a normal primary human mammary epithelial cells (HMEC) model, which we isolated from normal mammary explants. These cells were transformed in two step process by sequential transduction of (i) a shRNA targeting TP53, (ii) an oncogene. We selected 3 oncogenes that activate distinct signaling pathways CCNE1, HRAS-v12 and WNT1. Our tumor transformation model was established in three-step, normal, immortalized and transformed cells, allowing us to monitor the molecular changes associated with each step and to verify whether the activation of distinct oncogenic pathways produced different profiles of genetic and epigenetic modifications. The different models were analyzed at defined culture times by CGH-array, RRBS, transcriptome and miRNA. Our results show that genetic abnormalities and CpG methylation profiles are different between cells where the RAS pathway was activated and cells overexpressing WNT1 or CCNE1. These differences appear rapidly after oncogene transduction, whereas the profiles of the CCNE1 and WNT1 cells diverged later. Finally, inactivation of p53 by itself does not induce high instability, but produces a context of plasticity favorable to genetic and epigenetic changes.In addition, we noted phenotypic differences between HMEC RAS (mesenchymal) and HMEC CCNE1 and HMEC WNT1 (epithelial). In this work, I show that the immortalized HMEC shp53 exhibit a phenotypic plasticity, where some cells enter a spontaneous EMT and the others remain epithelial. I showed that RAS transduction selected cells that are undergoing an EMT, whereas transduction with CCNE1 or WNT1 selected the epithelial cells. I have sought to identify the determinants of these phenotypic changes and my results suggest that a balance exists between TGFβ1 / BMP1 signaling, which promotes EMT, and BMP4 / WNT7a which promotes TEM.

La leptine est une hormone peptidique ayant une action sur de nombreux tissus. Une dérégulation de la sécrétion de cette hormone est observée au cours de l’obésité. L’obésité est fréquemment associée à des troubles de santé dont les principaux sont le diabète de type II, l’hypertension artérielle et les maladies cardiovasculaires. Elle est également un facteur de risque du cancer du sein, particulièrement en post-ménopause favorisant la récidive et augmentant la mortalité. Ces perturbations, associées à un état de stress oxydant défini par un excès de production des espèces réactives de l’oxygène (ERO) par rapport aux systèmes de défense antioxydants, pourraient avoir un impact majeur dans le risque de carcinogenèse chez le sujet obèse. Il est clairement établi aujourd’hui que le statut oxydatif des cellules est directement corrélé aux capacités de prolifération mais aussi de survie des cellules dans leur environnement. A ce jour, très peu de données existent concernant le rôle de la leptine dans la modulation du statut oxydatif des cellules épithéliales mammaires saines et tumorales. L’objectif de cette thèse était d’étudier d'abord les mécanismes d’action et les effets de la leptine sur le statut oxydatif et inflammatoire des cellules épithéliales mammaires saines et néoplasiques ; puis dans un deuxième temps, une étude expérimentale a été conduite pour caractériser in vivo l’impact de l’obésité associée ou non à l’activité physique sur la croissance tumorale et le statut oxydatif et inflammatoire des tumeurs. Le projet avait également pour but de mettre en œuvre une nouvelle technique d’analyse basée sur la détection de fluorescence native induite par excitation laser à 224 nm afin d’évaluer la production de composés bio-actifs de la famille des éicosanoïdes, dont les isoprostanes, impliqués dans le processus inflammatoire. Nous avons exploré in vitro l’impact de la leptine sur le statut oxydatif des cellules épithéliales mammaires. Cette étude nous a permis d’établir que la réponse au signal leptinique varie en fonction du statut néoplasique de la lignée considérée, en fonction du temps de contact et non de la dose testée. Ensuite, nous avons étudié l’impact de l’obésité associée ou non à l’activité physique sur la croissance tumorale et sur le statut oxydatif et inflammatoire des tumeurs à l’aide d’un modèle de souris âgées C57BL/6 nourries avec un régime hyper-lipidique (HL) pendant 14 semaines, et hébergées soit dans un environnement enrichi (EE) pour favoriser l’activité physique et les interactions sociales, soit dans un environnement standard pendant 8 semaines, après quoi des cellules syngéniques de tumeur mammaire EO771 ont été implantées dans les quatrièmes coussinets adipeux mammaires. In vitro, la leptine a stimulé la production de ROS de façon indépendante de la dose et cette augmentation était dépendante de la production d'O2 cytosolique. Les résultats montrent une augmentation significative du poids dans les groupes recevant le régime HL à prise alimentaire journalière identique. La composition corporelle à 8 semaines montre une prise de masse grasse significative sous régime HL, majorée par l’ovariectomie et partiellement limitée par l’activité physique. Après implantation des tumeurs, le régime HL favorise la croissance tumorale et la perte de l’activité locomotrice. Par contre, l’EE prévient la perte d’activité physique des animaux. L’ensemble de ces travaux montre que la leptine contribue à l’apparition d’un stress oxydant en lien avec le statut tumoral des cellules épithéliales mammaires. Ceci peut expliquer en partie l’augmentation du risque de cancer mammaire associée à l’obésité en post-ménopause. Ces résultats permettront d'objectiver le bénéfice d'une intervention nutritionnelle ciblée afin de moduler la réponse des cellules aux stimulations des adipokines. A terme, cette étude doit contribuer à mieux comprendre l’intégration des signaux issus de l’environnement cellulaire. / Obesity is now considered, as a risk factor for developing breast cancer in postmenopausal women and for mortality in response to this pathology. Obesity, which is frequently associated with hyperleptinemia, induces cellular signalling pathways, some of which involving reactive oxygen species (ROS) as intracellular messengers. High levels of ROS contribute to oxidative stress, cellular damages and pathogenesis. Therefore, ROS production associated to obesity could be a major risk factor for mammary carcinogenesis. Furthermore, increased oxidative stress and inflammation characterised by infiltration of immune cells into adipocytes are described. This is associated with a lipid peroxidation and the production of bio-active compounds including isoprostanes.The aim of this study was to determine the impact of leptin in modulating the oxidative and inflammatory status of epithelial mammary cells and in tumor mammary tissue. Moreover, the purpose of this work was to develop a new analysis technique based on native fluorescence detection induced by laser excitation at 224 nm to evaluate the production of bio-active compounds from the family of eicosanoids, involved in the inflammatory process, including isoprostanes.Initially we identified in vitro the leptin effects on ROS production in 3 human epithelial mammary cell models which present different neoplastic status (healthy primary (HMEC) cells, MCF-7 and MDA-MB-231) in presence of two leptin concentrations (10 ng/ml close to physiological values, 100 ng/ml as obesity level). To better understand the potential involvement of adipocyte tumor microenvironment in mammary carcinogenesis, we secondly explored in vivo the impact of high fat diet (HFD) and of enriched environment (EE) on mammary tumor development. Female C57BL/6 mice were fed with a HFD versus a normo-caloric diet (NC) for 14 weeks. After 8 weeks mammary tumor syngeneic cells EO771 were implanted into the fourth mammary fat pads. Before injection, mice were housed in EE or in standard environment (ES) for 8 weeks. In vitro, leptin stimulated ROS production in dose-independent manner and this increase was dependent of cytosolic O2•- production. This ROS production contributed to a different antioxidative response depending of the neoplastic cell status. Leptin induced the antioxidative enzymes expression and activities such as heme-oxygenase or glutathione peroxidase only in HMEC cells. In neoplastic cells, these enzyme activities did not change whatever the leptin concentration used.Thus, high fat diet promoted mammary tumor development associated with a decrease in body fat and an increase in volume and weight of tumors that was not limited by physical activity. This diet induced a decrease of adiponectin and an increase of leptin plasma level compared to NC diet however, leptinemia was not influenced by EE.The native fluorescence isoprostanes determination method, turned out not to be quite sensitive. Therefore, the native fluorescence of these compounds is too low to allow their detection in biological media used. In contrast, the native fluorescence appears to be a potential cellular exploration tool.Through this work, we have shown that leptin contributes to the onset of oxidative stress linked to the status of mammary epithelial tumor cells. This may partly explained the increase of risk of breast cancer recurrence observed in situations of obesity. The results obtained in vivo eventually will support the benefit of a nutrition intervention to modulate cell response to adipokines stimulation. Ultimately, this study contributes to better understand the integration of signals from the cell environment.

Bovine mastitis, or inflammation of the mammary gland, has substantial economic and animal welfare implications. A genetic basis for mastitis resistance traits is recognized and can be used to guide selective breeding programs. The discovery of regions of the genome associated with mastitis resistance, and knowledge of the underlying molecular mechanisms responsible, can facilitate development of efficient mastitis control and therapeutic strategies. The objectives of this dissertation research were to identify sites of genetic variation associated with mastitis resistance, and to define the contributions of the milk-secreting epithelial cells to mammary gland immune responses and mastitis resistance. Twenty seven regions of the bovine genome potentially involved in mastitis resistance were identified in Holstein dairy cattle. Additionally, this research demonstrates a role of bovine mammary epithelial cells in mastitis resistance, and provides guidance for the use of an in vitro model for mastitis studies. Primary bovine mammary epithelial cells from mastitis-resistant cows have differential expression of 42 inflammatory genes compared with cells from mastitis-susceptible cows, highlighting the importance of epithelial cells in mastitis resistance. Bovine mammary epithelial cells display both similarities and differences in pro-inflammatory gene expression compared to fibroblasts, and their expression of inflammatory genes is influenced by administration of the enzyme phospholipase A2. The growth potential of milk-derived bovine mammary epithelial cells in vitro can be extended, facilitating their use in mastitis studies, by transfection with a viral protein. Collectively, this research contributes to current knowledge on bovine mastitis resistance and in vitro models.

Mammary gland (MG) is a dynamic organ that is essential for the production and secretion of breast milk. During pregnancy the MG undergoes a critical phase of remodeling, which is accompanied by the differentiation of mammary epithelial cells (MECs). During lactation, the MG requires high level of energy for proper folding of proteins in the secretory pathway, which takes place in the endoplasmic reticulum (ER). Limited evidence has been reported on the impact of cannabis or its components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on the differentiation of MECs. However, both THC and CBD have been reported to induce ER stress in various cell types, resulting in impacting cellular function. Furthermore, consequences of cannabis use in the perinatal period on breast milk composition have not been reported. Using the HC11 cell line, we investigated whether THC and CBD evoke ER stress in MECs resulting in the impaired cellular function. We also reported on the effects of cannabis use during pregnancy and lactation on the levels of macronutrients and bioactive factors in the breast milk of cannabis users. Relative to control, 10μM THC and 10μM CBD reduced mRNA levels of milk proteins (CSN2 and WAP) and lipid synthesizing enzymes (FASN, FABP4, PLIN2 and LPL), as well as whey acidic protein and lipid levels. In addition, 10μM THC, 10μM CBD, and the combination of 10μM THC + 10μM CBD significantly induced the expression of ER stress genes in HC11 cells. Furthermore, in the milk of women who used cannabis during pregnancy and lactation, the levels of secretory immunoglobulin A (SIgA) were decreased, relative to non-users. Altogether, our findings indicate that cannabis use in the perinatal period may have implications on breast milk composition and infant health. / Thesis / Doctor of Philosophy (Medical Science) / Breast milk is crucial to the nutrition and immunity of the newborn. It is produced by the mammary gland (MG), which is composed of mammary epithelial cells (MEC) that play a key role in producing and secreting proteins and factors into the milk. During pregnancy, the MG undergoes remodeling which is accompanied by differentiation of the MECs (change from unspecialized to a specialized stage that allows the MECs to produce milk proteins and other factors). The disruption in MEC differentiation can result in altered milk production and composition. Cannabis is used by women during pregnancy and breastfeeding. In this study, we investigated the impact of cannabinoids on the MEC differentiation and examined the effect of cannabis use in the perinatal period on the breast milk composition in humans. We reported that cannabinoids reduced the differentiation of MECs and maternal cannabis use during breastfeeding reduced levels of an essential immune factor.

The mammary alveolar cell-T (MAC-T) cell line is able to uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin and prolactin and is extensively used to study bovine mammary epithelial cell function. Growth hormone (GH) has been shown to increase milk protein synthesis both in vivo and in mammary cell models, and induce cytoskeletal rearrangement in 3T3 fibroblast cell line and a Chinese hamster ovary (CHO) cell line. Few studies have focused on identifying the mechanisms involved in differentiated MAC-T cells’ response to GH. We tested the hypothesis that MAC-T cells would respond directly to GH and that the response would include alterations in milk protein gene expression, leading to a more appropriate model for mammary cell function than treatment with dexamethasone, insulin and prolactin alone. To identify mechanisms that are involved in MAC-T cells’ response to GH, global protein was assessed through two-dimensional gel electrophoresis and differentially expressed proteins were identified through mass spectrometry. Differentiated cells expressed GH receptor mRNA, and addition of GH to the differentiation medium increased production of α-S1 casein and α-lactalbumin mRNA. Proteins that were differentially expressed are related to metabolism, the cytoskeleton, protein folding, RNA and DNA processing, detoxifying and calcium metabolism. These results indicate that GH is an important factor in inducing a lactogenic phenotype in the MAC-T cell line, and supports GHs involvement in differentiation, while altering cell metabolism in preparation for synthesis and secretion of milk components.

No description available.