Regulation of mitochondrial cytochrome b mRNA by copper in cultured human hepatoma cells and rat liver

Copper overload and deficiency are known to cause morphological and functio nal mitochondrial abnormalities. The reverse transcriptase-polymerase chain reaction (RT-PCR)-based method of differential display of mRNA was used to identify genes with altered expression in cultured human hepatoma cells (H ep G2) exposed to increasing concentrations of copper (0-100 mu M, 24 h). C opper regulation of a cloned PCR product, identified as the gene for the mi tochondrially encoded cytochrome b, was confirmed by Northern analysis and in situ hybridization. Copper toxicity increased cytochrome b mRNA abundanc e up to 3.6-fold, and copper chelation reduced it by 50%. Hepatic cytochrom e b mRNA was also increased in rats fed a high-copper diet. Thapsigargin tr eatment resulted in a significant increase in cytochrome b mRNA, suggesting that an increase in intracellular calcium may be involved in the mechanism of copper action. Furthermore, although cyclohexamide (CHX) alone did. not increase cytochrome b mRNA, the addition of CHX and copper resulted in a s ixfold increase. These data suggest a role for cytochrome b in the response to increases or decreases in hepatic copper.