Modulation of p21 Human Tumor Suppressor Gene Expression By Zinc Status In Human Hepatoblastoma And Normal Human Bronchial Epithelial Cells

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WONG, Hon-Kit
Lei, David K.Y.
The transcriptional regulation of p21 gene in both human hepatoblastoma (HepG2) cells and normal human bronchial epithelial (NHBE) cells in response to different zinc status has been examined. In both zinc deficient (ZD) and zinc marginal deficient (ZD0.4) HepG2 cells, the p21 mRNA and nuclear protein levels as well as p21 promoter activity were repressed as compared to that of zinc normal (ZN) control cells. However, they were not altered in zinc adequate (ZA) and zinc supplement (ZS) cells as compared to ZN cells. Moreover, transfection of a construct consisted of a constitutive promoter fused with a full length p21 coding sequence in ZD cells, normalized p21 protein expression to that of the ZN cells, but failed to correct cell growth reduction. Similar transfection in ZN cells overexpressed p21 and repressed cell growth. Thus, the present data indicate that in zinc-deficient HepG2 cells, the p21 transcriptional process is depressed. However, depressed p21 transcriptional process is not responsible for repressed cell growth in zinc-deficient HepG2 cells. In NHBE cells, the nuclear p21 protein level and mRNA abundance as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost 2-fold when compared to ZN control cells. G2/M blockage in ZS cells was coupled with enhanced p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage. A similar gene knock-down approach was used to establish if the p21 upregulation in ZS cells was p53 dependent. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53-dependent. Furthermore, the normalization of p53 protein by siRNA in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status induced G2/M blockage and growth depression. Thus, high zinc status in NHBE cells upregulated p53 expression which in turn elevated p21 that eventually induced G2/M blockage.