12-Lipoxygenase in A431 Cells: Genetic Identity, Modulation of Expression, and Intracellular Localization |
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Authors: | Wolfgang Hagmann Xiang Gao Joszef Timar Yong Q Chen Anja-Rose Strohmaier Chris Fahrenkopf David Kagawa Miu Lee Alex Zacharek Kenneth V Honn |
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Institution: | aDepartment of Radiation Oncology, Wayne State University, Detroit, Michigan, 48202;dDepartment of Pathology, Wayne State University, Detroit, Michigan, 48202;fDepartment of Chemistry, Wayne State University, Detroit, Michigan, 48202;bDivision of Tumor Biochemistry, German Cancer Research Center, D-69120, Heidelberg, Germany;eDivision of Biomedical Structure Research, German Cancer Research Center, D-69120, Heidelberg, Germany;cFirst Institute of Pathology and Experimental Cancer Research, Semmelweis Medical University, Budapest, Hungary |
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Abstract: | Human A431 epidermoid carcinoma cells express 12-lipoxygenase enzymatic activity. However, the isoform identity based on cDNA sequence data is not known. Further, the simultaneous characterization of the intracellular distribution of 12-lipoxygenase protein and activity is lacking. Here we report that the cDNA sequence from RT-PCR-amplified 12-lipoxygenase mRNA is identical with the platelet-type 12-lipoxygenase isoform, and the leukocyte-type isoform of 12-lipoxygenase is not expressed in A431 cells. The predominant amount (78%) of 12-lipoxygenase protein resides in the cytosol. In contrast, the predominant (98%) 12-lipoxygenase activity is localized in the membrane fraction. Western blot and immunofluorescence data demonstrate that epidermal growth factor increases total cellular 12-lipoxygenase protein and enhances the association of 12-lipoxygenase protein with perinuclear or nuclear membrane sites. In addition, epidermal growth factor stimulates 12-lipoxygenase activity resulting in generation of 12(S)-hydroxyeicosatetraenoic acid from cellular arachidonate. In contrast, both 12-lipoxygenase protein and activity decrease approximately 80% within 24 h during serum starvation. The recovery of 12-lipoxygenase expression in serum-deprived cells can be induced by readdition of epidermal growth factor or serum. Further, the basal expression of 12-lipoxygenase depends on signal pathways requiring protein tyrosine kinase activity, since genistein, herbimycin A, and tyrphostin 25 reduce the expression of 12-lipoxygenase protein in A431 cells. |
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