Regulatory mechanisms in the stabilization of p53 tumor suppressor gene in zinc depleted hepatoblastoma cells

Abstract

The influence of zinc status on the expression of p53, the human tumor suppressor gene, as well as other proteins that may be involved in p53 stability were examined in hepatoblastoma cells (HepG2). Cells were cultured in the zinc-depleted and supplemented media. Chelex 100 resin, a divalent ion-chelating resin, was used to deplete zinc from fetal bovine serum FBS. The Dulbecco's Modified Eagle Medium (DMEM) with 10% chelexed FBS, containing 0.2 μmol/L and 0.4 μmol/L zinc added were termed the zinc deficient ZD0.2 and ZD0.4 media, respectively. The other media consisted of the zinc-normal (ZN), zinc-adequate (ZA), and zinc supplement (ZS) groups, contained 4, 16, and 32 μmol/L zinc, respectively. Cells growth was depressed only in ZD0.2 cells to 78% of ZN cells. As compared to ZN cells, cellular zinc levels were reduced 67% and 56% in ZD0.2 and ZD0.4, respectively, but increased 84% and 127% in ZA and ZS cells, respectively. Nuclear p53 levels were almost 100% and 40% higher in ZD0.2 and ZD0.4 cells, respectively, than ZN cells. In contrast, p53 mRNA abundance was increased 40% in ZD0.4 cells and depressed 60% in ZD0.2 cells as compared with ZN cells. No differences in nuclear p53 protein and p53 mRNA levels were observed among ZN, ZA, and ZS cells. Total cellular and nuclear p21 protein, a major downstream p53 target, as well as p21 mRNA levels were markedly reduced in ZD0.2 and ZD0.4 cells, but were not altered in ZA and ZS cells, when compared to ZN cells. Mdm2 protein, which modulates p53 nuclear export and degradation, was more than twofold higher in the nuclear of ZD0.2 and ZD0.4 cells as compared to ZN cells. In contrast, Mdmx, known to bind Mdm2 and interfere with Mdm2-dependent p53 nuclear export, was depressed in the cytoplasm but not in the nucleus of ZD0.2 and ZD0.4 cells as compared with ZN cells. Moreover, c-Abl, capable of binding mdm2 and enhancing its nuclear accumulation in a p53-independent manner, was not alter in the total but lower in the nucleus of ZD0.2 and ZD0.4 cells than in ZN cells. However, the amount of Mdm2 bound to p53 was depressed and that bound to Mdmx and c-Abl were increased in ZD0.2 and ZD0.4 cells. In zinc deficiency, the reduced binding of Mdm2 to p53 may have resulted from the observed enhanced phosphorylation of p53 at serine 15 and 392, and Mdm2 tyrosine phosphorylation. Most importantly, the accumulation of nuclear p53 in ZD0.2 and ZD0.4 may have resulted from the marked reduction in the nuclear p300, a platform for bringing Mdm2, p53, and other factors together, for p53 nuclear export and degradation.