12-lipoxygenase-catalyzed arachidonic acid metabolism in normal and neoplastic mouse epidermis was assessed by cDNA cloning of the epidermal 12-lipoxygenases and by studying their expression patterns, enzyme activities, and product levels. Papillomas and squamous cell carcinomas induced by the initiation/promotion protocol contained 50- to 60-fold more 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) than normal epidermis. The ratio of S to R enantiomers was 9:1. This indicates that most of this eicosanoid was of enzymatic origin. Accordingly, cell-free preparations of the tumors exhibited about fivefold elevated 12-lipoxygenase activities. A papilloma-derived cDNA library was screened with human platelet-type 12-lipoxygenase cDNA probes. Two cDNA clones encoding the platelet-type and the leukocyte-type isoforms of murine 12-lipoxygenase were isolated, demonstrating the coexpression of the isoenzymes in the same tissue and species. When expressed in COS-7 cells, the recombinant enzymes showed the characteristic substrate selectivity and product profile, with the leukocyte-type enzyme metabolizing linoleic and arachidonic acid to 13-hydroxy-9,11-octadecadienoic acid and to 12- and 15-HETE, respectively, and the platelet-type enzyme oxygenating exclusively arachidonic acid to 12-HETE. In epidermis in vivo and in keratinocytes in culture, only the platelet-type 12-lipoxygenase (mRNA and protein) was detectable. In mouse epidermis both isoenzymes were induced transiently by phorbol esters. Most tumors showed constitutive overexpression of platelet-type mRNA, whereas leukocyte-type specific transcripts were detectable only in a few tumors. These data suggest that the platelet-type enzyme is the 12-lipoxygenase isoform of keratinocytes that is responsible for the generation of most of the 12-HETE found in neoplastic epidermis.

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A cDNA, recently cloned (by Krieg et al. (1998)) from mouse skin, was shown to encode a 12(R)-lipoxygenase. When expressed in HEK cells, the recombinant protein converted methyl arachidonate into the corresponding 12-HETE ester which was shown to be the R-enantiomer by chiral phase chromatography. Neither arachidonic acid nor linoleic acid were substrates for the recombinant protein. The structure of the 12(R)-lipoxygenase gene is unique among all animal lipoxygenases in that it is divided into 15 exons and 14 introns spanning approximately 12.5 kb. By interspecific backcross analysis, the 12(R)-lipoxygenase gene was localized to the central region of mouse chromosome 11.z 1999 Federation of European Biochemical Societies.

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The expression pattern, enzymatic activity, and products of 8-lipoxygenase (LOX) were analyzed in normal and neoplastic skin of NMRI mice. While barely detectable in normal epidermis, 8-LOX was transiently induced by 12-O-tetradecanoylphorbol-13-acetate and constitutively expressed in papillomas but not carcinomas obtained by the initiation-promotion protocol of mouse skin carcinogenesis. The product profile and chirality of both the native and the recombinant protein produced the S enantiomers of 8-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8-HETE) and 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE) as the main arachidonic acid- and linoleic acid-derived metabolites. As compared with normal epidermis, papillomas exhibited 25- and 4-fold elevated levels of 8-HETE and 9-HODE, respectively. However, the varying S to R ratios of 8-HETE and the predominance of 9(R)-HODE indicated that in addition to 8(S)-LOX, other enzymes yet to be defined may be involved in 8-HETE and 9-HODE production. The massive accumulation of both 8-HETE and 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE) point to a critical role of these LOX pathways in epidermal tumor development, in particular in the papilloma stage. Here we showed that 8- and 12-hydroperoxyeicosatetraenoic acids and 8- and 12-HETE induce chromosomal alterations in cycling primary basal keratinocytes.

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