TRANSFORMATION OF EPITHELIAL-CELLS STABLY TRANSFECTED WITH H2O2-GENERATING PEROXISOMAL URATE OXIDASE

Peroxisome proliferators, a group of structurally diverse nongenotoxic agents, induce predictable pleiotropic responses in liver, including the development of liver tumors in rats and mice. These agents transcr iptionally activate the three genes of the peroxisomal beta oxidation enzyme system by interacting with the peroxisome proliferatol-activate d receptor(s), It has been proposed that H2O2 generated by the pgroxis omal beta oxidation system leads to DNA damage and neoplastic transfor mation. Consistent with this hypothesis is that cells stably transfect ed with H2O2-generating peroxisomal fatty acyl-CoA oxidase cDNA, which encodes the first and rate-limiting enzyme of the beta oxidation syst em, undergo transformation in the presence of a fatty acid substrate. To test whether H2O2 generated by other peroxisomal oxidases can also lead to transformation, a full-length cDNA encoding rat urate oxidase (UOX), which oxidizes uric acid to allantoin and in the process genera tes H2O2, was introduced into African green monkey kidney cells (CV-1 cells) under the control of constitutively active human peroxisomal fa tty acyl-CoA oxidase gene promoter, Five stably transfected CV-1 cell lines expressing recombinant rat UOX were isolated in which the recomb inant protein was targeted to peroxisomes and formed crystalloid struc tures or cores similar to those present in rat liver peroxisomes. Incr eased levels of H2O2 were found when cells stably expressing UOX were exposed to the substrate uric acid. These five clones, designated A-U1 to A-U5, exhibited anchorage-independent growth, as demonstrated by t he formation of transformed colonies in soft agar in proportion to the duration of exposure to uric acid. These transformants exhibited clon al growth under serum-deprived conditions, One of these transformed ce ll lines, the A-U3 cell line, was evaluated for tumorigenicity by s,c. injection in nude mice. All five mice injected with transformed A-U3 cells developed adenocarcinomas, but no tumors developed in mite injec ted with control CV-1 cells or cells stably expressing UOX that were n ot exposed to uric acid. These results provide further evidence indica ting that sustained overexpression of a peroxisomal H2O2-generating ox idase causes cell transformation.