12-Lipoxygenase isoenzymes in mouse skin tumor development.
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.
Krieg
P
P
Research Program on Tumor Cell Regulation, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Kinzig
A
A
Ress-Löschke
M
M
Vogel
S
S
Vanlandingham
B
B
Stephan
M
M
Lehmann
W D
WD
Marks
F
F
Fürstenberger
G
G
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D015127:9,10-Dimethyl-1,2-benzanthracene; D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004817:Epidermis / Q000201:enzymology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006893:Hydroxyeicosatetraenoic Acids / Q000737:chemistry; D007527:Isoenzymes / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D012334:RNA, Neoplasm / Q000235:genetics; D012878:Skin Neoplasms / Q000201:enzymology*; D013237:Stereoisomerism; D013755:Tetradecanoylphorbol Acetate
D018076:DNA, Complementary; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D012334:RNA, Neoplasm; D015127:9,10-Dimethyl-1,2-benzanthracene; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase; D013755:Tetradecanoylphorbol Acetate