Epsilon Archive for Student Projects

Abstract

Milk is produced in the mammary gland in small structures called alveoli. The lumen in the center of these alveoli is surrounded by alveolar epithelial cells and milk production takes place in these cells. HC11 is a murine mammary epithelial cell line deriving from non-tumorigenic mammary epithelial cells that showed normal morphology and function, such as casein induction, in vitro after several sub-cultures. The overall aim of the project is to develop an in vitro model for investigating the transport of toxic compounds into milk by using HC11 cells. In the first part of the project the optimal time for differentiation of the HC11 cells into a milk protein synthesizing phenotype was determined. In the second part transport studies were performed across the HC11 cells to examine the function of breast cancer resistance protein (BCRP). The optimal time for maximum differentiation was determined by measuring the expression of the milk proteins, whey acidic protein (WAP) and β-casein, through Real-Time PCR , after having cultured HC11 cells in differentiating medium. Immunohistochemistry was also performed to examine the morphology and localization of β-casein in the differentiated cells. The transport study was performed in transwells using the BCRP substrate mitoxantrone, measuring both transport across, as well as uptake into the HC11 cells. The results from quantifying the expression of WAP and β-casein, indicate that the optimal time for maximum differentiation of the HC11 cells is 72h. Immunohistochemistry also showed that at this differentiation period the HC11 cells start to form structures resembling alveoli. β-casein expression was predominantly detected in the differentiated HC11 cells closest to the alveolar lumen. The transport experiments gave no useful results concerning mitoxantrone transport across the cells but the results obtained in the uptake experiment indicate that BCRP is upregulated and active in differentiated HC11 cells. In conclusion, the results obtained in this project demonstrate that HC11 cells is not suitable for studies of transport of toxic compounds across the alveolar epithelium but seems to be a promising model for functional studies of transport proteins such as BCRP.