In vitro data from our laboratory demonstrate that vitamin B6 (B6) supplementation of estrogen receptor - positive and - negative breast cancer cells is growth inhibitory. Others have reported that dietary B6 supplementation resulted in increased fibrosarcoma pyridoxal phosphate (PLP) concentrations and a significant inverse relationship between tumor PLP concentration and tumor volume in mice. This suggests that, in contrast to data reported for normal cells, tumor cells are capable of accumulating supplemental B6. In the current study, we investigated the effects of dietary B6 supplementation on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in rats. Specifically, we aimed to identify the effect of pyridoxine (PN) supplementation on tumor growth and vitamin uptake by tumor cells. To accomplish this, 50 d old female Sprague Dawley rats were gavaged with 15 mg DMBA and fed a diet containing either 7, 350, or 1050 mg PN-HCl/kg diet, which is the equivalent of 1, 50, or 150x the National Research Council's B6 requirement for rats, respectively. These levels of PN have previously been shown to produce no overt signs of toxicity in rats. Throughout the experiment, the percent of rats with tumors and the average number of tumors per rat remained similar between groups. Mammary tumor growth rates were significantly increased in response to dietary B6 supplementation (P < 0.05). Liver PLP and pyridoxal (PL) concentrations did not differ between dietary treatment groups. Plasma PL and PLP concentrations were significantly higher in the group fed the 150x diet compared with the 1x diet (P < 0.001, P < 0.05). Mammary tissue PL concentrations of the 150x group were significantly higher (P < 0.05) than the 1x group, but no differences were observed in mammary PLP concentrations. Similarly to mammary tissue, no differences between groups were observed in tumor PLP concentration. However, tumor PL concentrations in both the 50x and 150x dietary treatment groups were significantly higher than those from the rats fed the 1x diet (P < 0.002). These data demonstrate that previously reported inhibitory effects of supplemental B6 on breast cancer growth in vitro do not occur in response to dietary supplementation at 50 or 150 times the B6 requirement in vivo. In fact, dietary B6 at 150x the requirement may actually promote mammary tumor growth. In light of these results, investigation of the effects of supplemental B6 on cancer growth in humans is warranted. Supported by American Cancer Society Grant # IRG-99-225-01. / Master of Science

Pre-pubertal bovine mammary development involves ductal and stromal tissue changes. In mice, this process is impacted by presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. We studied the presence, location and changes in numbers of eosinophils, mast cells and macrophages in pre-pubertal bovine mammary tissue. Chemical stains and immunofluorescence were used to identify the cells in formalin fixed, paraffin embedded mammary tissue. The first set (ONT) included samples (n=4/week) from birth to 6 weeks of age. Another set (OVX) determined the influence of ovaries, 19 animals were intact or ovariectomized 30 days before sampling. They were 90, 120 or 150 days old at examination. The third set (EST) allowed examination of the potential influence of exogenous estrogen on innate immune cells in the mammary gland. Samples were from calves given estrogen implants (n=6) or placebo (n=4) at 56 days old, and sampled at 70 days old. We examined 20 images each of NEAR and FAR stroma from every animal. More eosinophils were observed in NEAR versus FAR in the ONT and OVX , more mast cells observed in NEAR versus FAR in ONT. More macrophages were observed in NEAR versus FAR in ONT and EST. We show, for the first time, that innate immune cells are present in prepubertal bovine mammary tissue and that abundance is related to the epithelial structure. We suggest a possible role for these cells in control of bovine mammary development. / Master of Science

Rapid rearing of dairy heifers during late prepuberty has been linked to impairments in mammary development and reductions in milk yield. Our objective was to determine how varying energy intakes between 2 and 14 wk of age affect mammary parenchymal development. At 2 wk of age, Holstein calves were assigned to 1 of 4 treatments (HH, HL, LH, and LL) with 2 levels of energy intake (High or Low) and 2 periods of growth (2 to 8 and 8 to 14 wk of age). At 14 wk, parenchyma at the stromal interface, mid-gland, and above the cistern were collected, fixed, and embedded in paraffin. Digital images of stained sections were used to determine tissue composition (% epithelium, lumen, and stroma). Immunochemistry revealed estrogen receptor (ER) and Ki67 (nuclear proliferation antigen) positive cells, type IV collagen, fibronectin (FN), and laminin. Images representing 4 increasing grades were used to quantify ECM protein deposition. Lumenal and stromal areas were 3.5 ± 1.4% higher (p<0.01) and 4.0 ± 1.7% lower (p<0.01), respectively, in HH and HL heifers. Ki67 labeling in terminal ductular units and subtending ducts was 2.1 ± 0.8% (p<0.01) and 1.4 ± 0.7% (p<0.05) lower for the same feeding level combination. FN deposition was also increased (p<0.05) in HH and HL heifers. High rates of gain between 2 and 14 wk of age resulted in greater lumenal area and reduced cell proliferation in mammary parenchyma at 14 wk of age. Changes in FN deposition could have mediated growth differences. / Master of Science

The majority of bovine mammary gland research focuses on the main cell types - mammary epithelial cells and fibroblasts. However, the extracellular matrix (ECM) within the mammary gland is also of importance for its ability to regulate cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in immortalized bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. However, the experiment was unable to confirm of reject this hypothesis. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary epithelial cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands. / Ph. D. / The mammary gland is a heterogeneous tissue with two main cell types - mammary epithelial cells and fibroblasts. These two cells types produce the majority of the extracellular matrix (ECM) within the mammary gland. The ECM is important for regulation of cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands.

Le développement de l’épithélium mammaire dépend des cellules souches qui, en proliférant et se différenciant, donnent naissance aux cellules du lignage épithélial. Les cellules souches sont ensuite sollicitées à chaque gestation pour régénérer une partie de l’épithélium. L’objectif de cette thèse est d’identifier et de caractériser en profondeur les différents types de cellules du lignage épithélial mammaire bovin par des approches de cytométrie en flux et de tri cellulaire. Nous avons défini et étudié des (sous)populations épithéliales engagées dans le développement mammaire à la puberté. A ce stade, l’épithélium mammaire contient de rares cellules souches mammaires, des cellules progénitrices à typage mixte luminal/basal et des cellules luminales et basales. Ces (sous)populations diffèrent soit en proportion soit en caractéristiques, voire les deux, aux stades physiologiques majeurssuivants (lactation et tarissement). Les cellules basales diminuent en lactation et au tarissement. Elles possèdent une signature moléculaire propre à chaque stade. La population luminale, composée de plusieurs sous-populations, est la plus variable. Elle arbore une réceptivité hormonale différente à chaque stade. Des (sous)populations épithéliales n’apparaissent qu’à un stade précis (puberté ou lactation) et disparaissent aux autres, comme certaines cellules prolifératives définies comme cellules progénitrices. Enfin, la fraction de rares cellules souches putatives diminue graduellement, de la puberté aux stades suivants, tout en conservant des caractéristiques moléculaires similaires. Ces / The development of the mammary epithelium relies on the mammary stem cells which, by proliferating and differentiating, give rise to the luminal, basal and progenitor cells of the epithelial lineage. The stem cells are then solicited at each gestation to regenerate a part of the epithelium. In this thesis work, we aim at identifying and characterizing in depth the different types of cell constitutive of the bovine mammary epithelial cell lineage by flow cytometry and cell sorting approaches. We defined and studied epithelial (sub)populations committed to mammary development at puberty. At this stage, the mammary epithelium contains rare mammary stem cells, mixed luminal/ basal progenitors, as well as luminal and basal cells.These (sub)populations were found to differ in proportion and/or characteristics at thesubsequent major physiological stages (lactation and dry periods). Basal cells decrease at lactation and dry off. They harbor a specific molecular signature at each stage. The luminal population, composed of several subpopulations, is the most variable. The hormonal receptivity of these cells changes at each physiological stage. Interestingly, some epithelial populations only appear at specific stages (puberty or lactation) and disappear at others, as one subpopulation of proliferative cells that we defined as progenitor cells. At last, the pool of rare putative stem cells gradually decreases from puberty to the next stages while maintaining a similar molecular signature. These are novel insights that show that the epithelial lineage evolves substantially through the

While a great deal is known about the changing hormonal environment and the structural development of the mammary gland from pregnancy to lactation, very little is known about the molecular mechanisms governing differentiation of the mammary epithelium into a milk-secreting phenotype. It is important to acknowledge the diversity among the mammary glands of different species in order to better understand applications in human health and the dairy industry. In this study, we examined global protein expression during two states of differentiation in mammary epithelial cells from two species: in vitro proliferating and differentiated MAC-T cells (a bovine immortal cell-line), and primary mammary epithelial cells isolated from pregnant and lactating mice. When comparing the lists of proteins that differed in abundance in the two experiments, we observed many similarities in proteins related to structural dynamics and mRNA processing within these two mammary epithelial cell types. Intriguingly, we observed several differences in the regulation of metabolic proteins, highlighting the distinct pathways by which different species probably metabolize energy and synthesize milk components.

The Met receptor tyrosine kinase (RTK) is expressed in the mammary gland under both normal and neoplastic conditions. Overexpression of the Met receptor is found in 15--20% of human breast cancers and is correlated with shortened disease-free interval and overall survival. In order to explore the role of dysregulated Met receptor signaling on the development of mammary tumors I have characterized a transgenic mouse model that expresses either wild type or a dysregulated Met receptor in the mammary epithelium under the control of the mouse mammary tumor virus promoter/enhancer (MMTV-Met). The Met receptor variants contained a mutation that results in decreased receptor ubiquitination and prolonged receptor signaling (Y1003F) or an activating mutation that was originally observed in patients with papillary renal carcinoma (M1250T) or both mutations (YF/MT). In vitro and in vivo transformation assays demonstrated that each mutation singly is weakly transforming, however, there was an additive effect on transformation when both mutations were present. This additive effect was observed in the transgenic mice where multiparous MMTV-Met-YF/MT mice developed tumors earlier and with much greater penetrance than did mice expressing either of the single mutants. This provides the first in vivo model that demonstrates a role for ubiquitination in suppression of transforming activity of an RTK. MMTV-Met-YF/MT tumors displayed a range of histological phenotypes but were mainly comprised of luminal lineage cells. Notably, MMTV-Met-M1250T tumors contained cells from both the basal and luminal populations, suggesting transformation of a progenitor cell. Progenitor cell transformation in RTK transgenic mouse models is uncommon and highlights distinct signaling differences and potentially lineage specificity of the two Met mutants. / Through assays of overexpression in vivo and inhibition in vitro, Met receptor signaling has been correlated with the development of the mammary gland. To examine the effects of loss of Met receptor signaling on mammary gland development I have utilized the Cre/LoxP1 recombination system to knock-out the Met receptor from the mammary epithelium. Mammary-specific Cre recombinase efficiently excised floxed DNA as visualized by activation of a beta-galactosidase reporter In Met+/+ glands, however, few beta-galactosidase positive cells are retained In the Mefl/fl glands and an intermediate number are retained in the Met fl/+ glands. This indicates that Met-null cells are selected against and supports a role for Met in the development of the mammary gland.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references.

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