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

Buthionine sulphoximine (BSO; 1mM) resulted in the depletion of glutathione (GSH) in HepG2 cells to 17+/-1.5% within 24h. 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-alpha (but not betaI or delta) was demonstrated at 24 h through activity measurements and through Western blot analysis of membrane-associated PKC-alpha protein. Activation did not occur in the presence of additional gamma-glutamylcysteine to prevent GSH depletion. Activation of PKC-alpha 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.