Study on the effect of ethanol on peripubertal mice mammary glands: Through gene expression studies and in vitro analysis of ethanol treated mammary epithelial cells
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One in every eight women in the United States will develop breast cancer in her lifetime, and intake of as little as one drink per day increases this risk by 10%. The specific mechanisms whereby ethanol increases breast cancer susceptibility have been proposed to work through several pathways, including folate depletion, DNA damage from ethanol-derived acetaldehyde accumulation, changes in cell adhesion, and increased estrogen levels. Previous studies have demonstrated that folate depletion does not mimic or exacerbate the effects of ethanol on the peripubertal developing mouse mammary gland. In fact, folate replete status resulted in an ethanol-induced increase in ductal branching and epithelial multi-layering, a sign of alterations in cell adhesion-dependent survival. The first Aim of this project was to examine the changes in gene expression in the peripubertal mouse mammary gland to define the pathways that are altered by ethanol exposure. This was brought about using a gene array plate to check for differential expression of genes involved in the pathways of mouse breast cancer. Pik3R1, Cdk2 and Myc were significantly down regulated by 70, 42 & 34 fold, respectively. Validations of twelve such targets were carried out through qRT PCR. RT validation did not however show any significant fold regulation true to the regulation shown in the mouse cancer pathway plate except for Ifnb1 which was upregulated in all four ethanol treated experimental mice as compared to the control. Taking only the RT validation results into consideration, a p53 related pathway has been proposed which can be verified by further analysis on the genes involved. Immunohistochemical analysis could give a better understanding on the effects of ethanol on the epithelial cells in the mammary glands. The second Aim of this project was to develop a three-dimensional tissue culture model to determine if ethanol similarly allows multi-layering to persist during the maturation of spheroids formed by a mammary epithelial cell line, TM12. TM12 cells grown on plastic or Matrigel were exposed 0%, 0.5%, 1% and 2% ethanol for 3 days. Mitochondrial activity as a measure of viable cell number was quantified by MTT assay, and analysis of spheroid number, mean diameter and percentage of multi-layering were determined by histological analysis of H&E stained paraffin sections. The MTT assay showed significant decrease in mitochondrial activity only at 2% ethanol for the cells grown on plastic; when cells were grown on Matrigel, ethanol as low as 0.5% decreased mitochondrial activity, suggesting that Matrigel altered the sensitivity of the cells to ethanol. In contrast, evaluation of histological changes in the spheroids did not show any significant change in spheroid number, mean diameter or percentage of multilayering. TM12 cells show incomplete cavitation (lumen formation) in all treatment groups by day 3. A different mammary epithelial cell line, MCF10A, may be more appropriate to confirm these studies.