Induction of c-fos, and cytochrome c oxidase subunits I and II by gossypol acetic acid in rat liver cells.

ABSTRACT

The hypothesis that oxidative stress induced by acute, sublethal, gossypol treatment induces transcription of stress genes in a rodent liver cell line was tested. In northern blot analysis of gossypol-treated cells, there was a dose-dependent increase in c-fos, a component of the redox-regulated transcription factor activator protein-1 (AP-1). Induction of c-fos was biphasic. A rapid 3.33+/-1.37 fold induction in c-fos was detected after treatment with 5 micromol/L gossypol for 30 min. Additionally, treatment with 5 micromol/L gossypol for 12 h caused a 2.66+/-0.67 fold increase in c-fos expression. PCR-based subtractive hybridization was used to generate a subtracted complementary DNA (cDNA) pool representing mRNA species increased in response to gossypol exposure. Several thousand clones were grown from bacteria transformed with the subtracted cDNA pool. After screening and confirmation by northern blotting, five clones were confirmed to be induced by gossypol. Sequence analysis confirmed that four of these clones contained DNA sequences from cytochrome-c oxidase subunit II (COX II), and one contained a DNA sequence from cytochrome-c oxidase subunit I (COX I). A five-fold induction (5.13+/-1.39 fold) in COX II occurred after 1 h of gossypol exposure, and a three-fold induction (3.24 fold) in COX I occurred after 3 h of gossypol exposure. These studies provide further evidence that mitochondria are a major site of the cytotoxic action of gossypol based upon an adaptive response to gossypol involving the upregulation of genes from the mitochondrial genome.