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.
White
Angela T
AT
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Spence
Fiona J
FJ
Chipman
James K
JK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D020869:Gene Expression Profiling / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000172:deficiency* / Q000378:metabolism*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008113:Liver Neoplasms / Q000201:enzymology / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors
D020033:Protein Isoforms; D019328:Buthionine Sulfoximine; D007770:L-Lactate Dehydrogenase; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D005978:Glutathione