Glutathione: A novel control switch that regulates the expression of erythropoietin gene under normoxic condition in human hepatocellular carcinoma cells
School of Medical Technology and Graduate Institute of Medical Biotechnology, Chang Gung University, Kwei-Shan, Taoyuan, Taiwan
We present herein several lines of evidence that intracellular glutathione (GSH) contents can play a pivotal role in the control and regulation of the expression of erythropoietin (EPO) gene under normoxic condition. First, using a group of five human hepatocellular carcinoma (HCC) lines with varying degrees of differentiation as the experimental model, we demonstrated that the endogenous GSH contents of these HCC cells were differentially upregulated depending on the degrees of differentiation with an order of abundance being HepG2 > Hep3B > J5 > Mahlavu > SK-Hep-1. Coincidently, we also found that γ-glutamylcysteine synthetase (γGCS) heavy subunit activities as well as its mRNA contents of these cells correlated precisely with an identical order with that of the GSH contents in these cells. However, among these, only two well-differentiated sublines, HepG2 and Hep3B expressed EPO gene under normoxic condition implying that the latter process was dependent on GSH contents and suggested a notion that a threshold level might be required for its optimal activation. Second, to further obtain the evidence to substantiate this possible role of GSH, we then supplemented to the cell culture media with an excessive quantity of nonlethal N-acetylcysteine (10 mM) for the purpose of reinforcing the endogenous GSH biosynthesis. Interestingly, we found that this manipulation could revert the activation of EPO gene in cell lines, such as J5, Mahlavu, and SK-Hep-1, in which their EPO gene expressions were otherwise repressed under a normoxic condition. Finally, we were able to demonstrate, using duplex RT-PCR and Western blotting techniques, that the expression of EPO gene could be reverted in GCS30, a SK-Hep-1 subline that was permanently transfected withγ-GCSh and is capable of overexpressing endogenous GSH contents can act as a positive regulatory switch for the expression of EPO gene under normoxic condition in human HCC cell lines possibly via a GSH regulated H2O2-mediated oxidative stress pathway.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 892 (Susceptibility genes).