Title

Regulation of zinc homeostasis in cultured cells

Date of Completion

January 2008

Keywords

Biology, Molecular|Biology, Cell|Health Sciences, Nutrition

Degree

Ph.D.

Abstract

Zinc is an essential micronutrient and has a finely regulated homeostatic mechanism in the body. Previous studies in our laboratory have demonstrated that in the presence of extracellular metal chelator diethylene triamine penta acetate (DTPA), GH3 rat anterior pituitary tumor cells prelabled with DTPA retained radioactive zinc whereas rat primary hepatocytes gave out zinc. Thus the main objective of this dissertation is to investigate the differential zinc homeostatic response to DTPA in transformed and primary cells. ^ The differential effects of DTPA on radioactive zinc flux were further investigated in H4IIE rat hepatoma cell line, MCF-7 human breast cancer cell line, rat primary anterior pituitary cells, and Hs578Bst non transformed human mammary cell line. Cells pre-labeled with 65Zn showed an increased retention of radioactive zinc in H4IIE, MCF-7 and Hs578Bst in presence of DTPA whereas pituitary cells gave out more radioactive zinc, illustrating different homeostatic responses of cell lines and primary cells. Time course experiments showed that zinc efflux is shut down rapidly in presence of DTPA in transformed cells whereas primary cells continue to lose zinc. To further understand the mechanism of the differential responses of transformed and primary cells, effect of zinc (100 μM) and DTPA (50 μM) treatment on mRNA (48 hour treatment) expression, protein expression (2 hour and 48 hour treatments) and localization (2 hour treatment) of the plasma membrane zinc efflux transporter ZnT-1 was studied in H4IIE cells and primary hepatocytes. ZnT-1 mRNA or protein expression did not change with either zinc or DTPA treatment. Though ZnT-1 is thought to be localized on the plasma membrane, membrane localization of ZnT-1 was not seen. Under the influence of zinc, the mRNA expression of metallothionein-1 (MT-1) which is considered as a marker of zinc status, was increased whereas DTPA had no effect. ^ These studies demonstrated that the transformed cells and the primary cells from the same tissue of origin respond differently to restricted zinc availability and this differential response is due to the nature of the cells and is independent of the tissue of origin. The differential response of cells could not be explained by the amount or localization of ZnT-1 suggesting either a regulation of its activity or the presence of a yet undiscovered zinc efflux transport system. ^