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

• Published in: Toxicology (Amsterdam) Vol. 216; no. 2; pp. 168 - 180
• Main Authors: White, Angela T., Spence, Fiona J., Chipman, James K.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 15.12.2005; Amsterdam Elsevier Science
• Subjects: Animals; Apoptosis - drug effects; Biological and medical sciences; Blotting, Western; Buthionine Sulfoximine - pharmacology; Carcinoma, Hepatocellular - enzymology; Carcinoma, Hepatocellular - metabolism; Cell Line, Tumor; Cell signalling; Cell Survival - drug effects; Enzyme Activation - drug effects; Gene Expression Profiling - methods; Gene Expression Regulation, Enzymologic; Glutathione; Glutathione - deficiency; Glutathione - metabolism; Humans; L-Lactate Dehydrogenase - metabolism; Liver Neoplasms - enzymology; Liver Neoplasms - metabolism; Medical sciences; Oligonucleotide Array Sequence Analysis; Oxidative stress; Phosphorylation - drug effects; Protein Isoforms; Protein kinase C; Protein Kinase C - metabolism; Protein Kinase C-alpha - drug effects; Protein Kinase C-alpha - metabolism; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction - methods; Time Factors; Toxicogenomics; Toxicology; Cell signalling; Oxidative stress; RAR; Protein kinase C; PKC; SSH; DAG; γ-GCS; Toxicogenomics; LDH; 4-HNE; RT-PCR; ROS; GSH; BSO; ODS; Glutathione; Depletion; Signaling pathway; Toxicogenomics: Oxidative stress; Enzyme; Transferases; Gene expression; In vitro; Toxicogenomic
• ISSN: 0300-483X; 1879-3185
• DOI: 10.1016/j.tox.2005.08.001

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.