Glutathione depletion modulates gene expression in HepG2 cells via activation of protein kinase C alpha.

Abstract Buthionine sulphoximine (BSO; 1 mM) resulted in the depletion of glutathione (GSH) in HepG2 cells to 17 ± 1.5% within 24 h. This was not associated with apoptotic or necrotic cell death over this time period. Use of a human (Phase 1 ® ) cDNA custom toxicology-array and a larger scale (>10,000 gene) Affymetrix U95Av2 array identified a total of 48 and 104 genes, respectively, with a statistically significant (and >1.5-fold) change in expression. A total of 64 differentially expressed genes (6 of which were confirmed by real-time polymerase chain reaction) were suggestive of protein kinase C (PKC) activation. Activation of PKC-α (but not βI or δ) was demonstrated at 24 h through activity measurements and through Western blot analysis of membrane-associated PKC-α protein. Activation did not occur in the presence of additional γ-glutamylcysteine to prevent GSH depletion. Activation of PKC-α by GSH-depletion may, at least in part, be mediated by thiol oxidation and may contribute to a survival signal. If sustained, the activation may be important in non-genotoxic carcinogenesis.