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Transformation of Epithelial Cells Stably Transfected with H₂O₂-generating Peroxisomal Urate Oxidase¹

Department of Pathology Northwestern University Medical School, Chicago, Illinois 60611

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 transcriptionally activate the three genes of the peroxisomal ß oxidation enzyme system by interacting with the peroxisome proliferator-activated receptor(s). It has been proposed that H₂O₂ generated by the peroxisomal ß oxidation system leads to DNA damage and neoplastic transformation. Consistent with this hypothesis is that cells stably transfected with H₂O₂-generating peroxisomal fatty acyl-CoA oxidase cDNA, which encodes the first and rate-limiting enzyme of the ß oxidation system, 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 generates H₂O₂, was introduced into African green monkey kidney cells (CV-1 cells) under the control of constitutively active human peroxisomal fatty acyl-CoA oxidase gene 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 crystalloid structures or cores similar to those present in rat liver peroxisomes. Increased 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 the formation of transformed colonies 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-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 mice injected with control CV-1 cells or cells stably expressing UOX that were not exposed to uric acid. These results provide further evidence indicating that sustained overexpression of a peroxisomal H₂O₂-generating oxidase causes cell transformation.

¹ This research was supported by Veterans Affairs Merit Review Research Support (to A. V. Y.), NIH Grant GM23750 (to J. K. R.), and the Joseph L. Mayberry, Sr., Research Endowment Fund.

² To whom requests for reprints should be addressed, at Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. Phone: (312) 503-7948; Fax: (312) 503-8249.

Received 8/27/96. Accepted 9/18/96.

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