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

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 μM 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 μM 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.