12‐lipoxygenase isoenzymes in mouse skin tumor development

Abstract

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.

Murine epidermal lipoxygenase (Aloxe) encodes a 12‐lipoxygenase isoform1

“…Construction of cDNA library from pooled mouse skin papillomas and screening with a 1.23 kb 3'-EcoRI fragment of human platelettype 12-lipoxygenase as 32 P-labeled probe was done as described earlier [11]. By screening of 8X10 5 recombinants (0.5XSSC, 0.5% SDS, 60°C, 20 min) one cDNA clone (C12014) was isolated which was different from previously isolated murine 12-lipoxygenase cDNA clones.…”

“…Expression of 5-lipoxygenase has been reported in human keratinocytes [5] and 8-and 15-lipoxygenase activities were found in tumor promoter-treated mouse epidermis [6] and in human and rat skin [7,8], respectively. Among the 12-lipoxygenases the platelet-type was found to be the predominant isozyme expressed in human and murine skin epidermis [9][10][11].…”

Platelet-type Arachidonate 12-Lipoxygenase in Mouse Gastrointestinal Tract.

“…In addition to mouse enteroendocrine cells, 12-lipoxygenase of platelet-type was found in mouse epidermal cells [2,9] and platelets [2,12]. These three cell types have membrane-bounded granules.…”

“…However, the tissue distribution of the platelet-type enzyme has not yet been investigated in detail. In addition to platelets of various animal species, the enzyme has so far been found only in human and mouse keratinocytes [1,2,7,9,13,18]. Recently we prepared an antiserum against recombinant 12-lipoxygenase of human platelet, and immunostained immature and mature murine megakaryocytes [12] and keratinocytes [13].…”

Formação de adutos exocíclicos com bases de DNA: implicações em mutagênese e carcinogênese

“…Paralelamente, os níveis de edAdo e edCyd aumentaram, respectivamente, em 12 e 9 vezes 16 . Alguns estudos mostraram que tumores de pele induzidos experimentalmente acumulam altos níveis de prostaglandinas 291 e ácidos 8-HETE e 12-HETE (precursores dos leucotrienos) [292][293][294] e têm expressão aumentada das ciclooxigenases e lipoxigenases envolvidas na síntese dessas substâncias. A inibição dessas enzimas inibe significativamente o desenvolvimento de tumor em pele de camundongos 295 .…”