Recently, several studies have focused on Zn's role in preventing apoptosis. The objective of this study was to develop a cell culture model to examine the role of Zn in both cell proliferation and apoptosis. To this end. depleted Zn media was obtained by dialyzing fetal calf serum (FCS) against the chelator DPTA, followed by removal of the Zn-DPTA complex by dialysis against 20 mM Hepes. Media (DMEM + 7% FCS) were used as is (0.5 uM) or supplemented with Zn to final concentrations of 5,15,50, and 100 uM Zn. Control media (DMEM + 7% non-dialyzed FCS) was supplemented with Zn (5,15,50,100 uM) or not supplemented (2.5 uM). [Cu] and [Fe] were fixed at 1 uM and 5 uM. respectively. HL60 cells were sampled at 24,48,72, & 96 h. for cell number/viability/morphology, and apoptosis markers (DNA laddering and/or binding of flourescein-conjugated annexin V). Culturing cells in 0.5 uM Zn resulted in extensive apoptosis by 72 h., and by 96 h. only 17% of the cell population was viable. The onset/severity of apoptosis was not prevented by supplementing the media with 50 ng/ml IGF-I. Culturing in 5 uM Zn resulted in a balance of cell proliferation and apoptosis after 96 h. Maximal proliferation of HL-60 cells plateaued in dialyzed media at 50 uM Zn and in non-dialyzed media at 15 uM Zn. Surprisingly, no inhibition of growth occurred in dialyzed or non-dialyzed media at 100 uM Zn. Flow cytometry of cells cultured in 0.5 uM Zn media and subsequently loaded with Zinquin indicated that the rate of apoptosis could be correlated with the depletion of free intracellular Zn.
|Original language||English (US)|
|State||Published - 1997|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology