12-Lipoxygenase in Lewis lung carcinoma cells: molecular identity, intracellular distribution of activity and protein, and Ca(2+)-dependent translocation from cytosol to membranes.
Recently we demonstrated that Lewis lung (3LL) tumor cells express 12-lipoxygenase (12-LOX) mRNA and protein, respectively. In this study we partially sequenced the 12-LOX cDNA after reverse-transcription polymerase chain reaction amplification of 12-LOX mRNA from cultured 3LL cells. Comparison with platelet and leukocyte 12-LOX indicates that 3LL 12-LOX is identical with the platelet-type enzyme at least within the sequenced region. Further, we investigated the intracellular distribution of both 12-LOX enzyme protein and its activity which are prerequisites for understanding 12-LOX regulation. 12-LOX activity was monitored via the production of 12-hyroxyeicosatetraenoic acid from 3LL cells and their subcellular fractions using reverse-phase high performance liquid chromatography. 12-LOX protein was measured by direct slot blot and by Western Blotting. In 3LL cells, both 12-LOX activity and 12-LOX protein were predominantly localized in the cytosol. This 12-LOX activity was optimal at 37 degrees C. However at 24 degrees C and 10 degrees C, it showed 87% and 61% of this activity, respectively, thus differing distinctly from 12-LOX in platelets or rat basophilic leukemia cells. Incubation of 3LL cell homogenates with 0-100 microM free Ca2+ and subsequent separate analyses of cytosol and membrane fractions indicated that, as in platelets, an increase in intracellular free Ca2+ caused a loss of cytosolic 12-LOX activity. However, no significant Ca(2+)-induced increase in membrane-associated 12-LOX activity was observed under these conditions in 3LL cells. In contrast, at the 12-LOX protein level we observed a Ca(2+)-dependent loss in the cytosol and a concomitant increase in the membrane fraction. Thus, we suggest that 12-LOX in 3LL cells undergoes rapid translocation from cytosol to membrane in a Ca(2+)-dependent manner, but is no longer active or becomes inactivated at the membrane site.
Hagmann
W
W
Department of Radiation Oncology, Wayne State University, Detroit, Michigan, USA.
Gao
X
X
Zacharek
A
A
Wojciechowski
L A
LA
Honn
K V
KV
D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000032:analysis* / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport; D001792:Blood Platelets / Q000201:enzymology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D018827:Carcinoma, Lewis Lung / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology* / Q000378:metabolism; D018076:DNA, Complementary; D004789:Enzyme Activation; D007962:Leukocytes / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D014407:Tumor Cells, Cultured / Q000201:enzymology
D018076:DNA, Complementary; D012333:RNA, Messenger; D001092:Arachidonate 12-Lipoxygenase; D002118:Calcium