benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and 2-6-dichloro-4-nitrophenol

Previous suggestions of CpG-specific apoptotic commitment implied critical epigenetic modulation of housekeeping genes which have canonical CpG islands at 5' promoter regions. Differential housekeeping gene activity however has not been shown. Using a focussed microarray (genechip) of 22 housekeeping genes we show this in apoptosis induced in human Chang liver cells by DCNP (2,6-dichloro-4-nitrophenol), a non-genotoxic inhibitor of sulfate detoxification. 3-7 folds downregulation of 9 genes in glycolysis, tricarboxylic acid cycle and the respiratory electron transport chain suggested gene-directed energy depletion which was correlated with observed ATP depletion. 4 folds downregulation of the pyruvate dehydrogenease gene suggested gene-directed metabolic acidosis which was correlated with observed cell acidification. Other differential housekeeping gene activity, including 4 folds upregulation of microtubular alpha-tubulin gene, and 2 folds upregulation of ubiquitin, also had a bearing on apoptosis. Broadspectrum zVAD-fmk caspase inhibition abolished 200 bp DNA ladder fragmentations but not the CpG-specific megabase fragmentations and other hallmarks of cell destruction, suggesting a caspase-independent cell death. Death appeared committed at gene-level.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Energy Metabolism; Enzyme Activation; Flow Cytometry; Gene Expression Regulation; Hepatocytes; Humans; Hydrogen-Ion Concentration; Mitochondria; Nitrophenols; Oligonucleotide Array Sequence Analysis