12-Lipoxygenase in A431 cells: genetic identity, modulation of expression, and intracellular localization.
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.
Hagmann
W
W
Department of Radiation Oncology, Wayne State University, Detroit, Michigan 48202, USA.
Gao
X
X
Timar
J
J
Chen
Y Q
YQ
Strohmaier
A R
AR
Fahrenkopf
C
C
Kagawa
D
D
Lee
M
M
Zacharek
A
A
Honn
K V
KV
D001092:Arachidonate 12-Lipoxygenase / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D016895:Culture Media, Serum-Free; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D018076:DNA, Complementary; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D014158:Transcription, Genetic
D016895:Culture Media, Serum-Free; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D001092:Arachidonate 12-Lipoxygenase; D011505:Protein-Tyrosine Kinases