Transformation of epithelial cells stably transfected with HO-generating peroxisomal urate oxidase

Abstract

Peroxisome proliferators, a group of structurally diverse nongenotoxic agents, induce predictable pleiotropic responses in liver, including the developmentofliver tumorsin rats and mice. These agentstranscription ally activate the three genes ofthe peroxisomal fi oxidation enzymesystem by interacting with the peroxisome proliferator-activated receptor(s). It has been proposedthat H202 generatedby the peroxisomal(I oxidation system leads to DNA damage and neoplastictransformation.Consistent with this hypothesis is that cells stably transfected with H202-generating peroxisomal fatty acyl-CoA oxidase eDNA, which encodes the first and rate-limiting enzymeofthe fi oxidation system, undergo transformation in the presenceof a fatty acid substrate.To test whether 11202generatedby other peroxisomal oxidases can also lead to transformation, a full-length cDNA encoding rat urate oxidase (UOX), which oxidizes uric acid to allantoinand in the processgeneratesH202, was introducedinto African green monkey kidney cells (CV-1 cells) under the control of constitutively active human peroxisomalfatty acyl.CoAoxidasegene promoter. Five stably transfected CV-1 cell lines expressing recombinant rat UOX were isolated in which the recombinant protein was targeted to peroxisomes and formed crystalloidstructuresor cores similar to those present in rat liver peroxisomes.Increasedlevels of H202 were found when cells stably expressing UOX were exposed to the substrate uric acid. These five dones, designated A-Ui to A-U5, exhibited anchorage-independent growth, as demonstratedby the formation of transformedcolonies in soft agar in proportion to the duration of exposure to uric acid. These transformants exhibited clonal growth under serum-deprived conditions. One of these transformed cell lines, the A-U3cell line, was evaluated for tumorigenicity by s.c. injection in nude mice. All five mice injected with transformed A-U3 cellsdevelopedadenocarcinomas,but no tumors developedin mice injected with control CV-i cells or cells stably expressing UOX that were not exposed to uric acid. These results provide further evidence indicating that sustained overexpressionof a peroxisomalH202-generatingoxidase causes cell transformation.