Implication of hydrogen peroxide generation and apoptosis in the neoplastic transformation of mouse fibroblasts overexpressing peroxisomal fatty acyl-CoA oxidase.

Abstract

Receptor-mediated overexpression of H2O2-generating peroxisomal fatty acyl-CoA oxidase (AOX) has been implicated in peroxisome proliferator-induced hepatocarcinogenesis. To investigate the role of rat AOX generated H2O2 in transformation, we overexpressed this enzyme in a non-tumorigenic mouse fibroblast cell line (LM tk-) under control of mouse urinary protein promoter. The clones overexpressing rat peroxisomal AOX, when exposed to a fatty acid substrate (100 microM linoleic acid) for 6 to 96 h, demonstrated > 10-fold increase of intracellular H2O2. This increase in H2O2 concentration was associated with increased apoptosis as evidenced by DNA fragmentation, in situ terminal deoxynucleotide transferase dUTP nick end-labeling (TUNEL). These cell lines stably expressing AOX formed colonies in soft agar in proportion to the duration (1-7 weeks) of exposure to a fatty acid substrate (100 microM linoleic acid, erucic acid or nervonic acid) and these transformants developed into fibrosarcomas when injected in athymic nude mice. These results suggest that H2O2 generated by AOX overexpression in immortalized fibroblasts leads to apoptosis, and the extent and duration of H2O2 and possibly other DNA damaging reactive oxygen species generated by the overexpression of peroxisomal AOX can influence apoptosis and neoplastic transformation.