Lipoxygenase (LOX) metabolites of arachidonic acid (AA) and linoleic acid (LA) namely 12(S)-hydroxyeicosatetraenoic acid [l2(S)-HETE] and 13(S)- hydroxyoctadecadienoic acid [13(S)-HODE] have been shown to modulate in a positive and negative aspect respectively numerous parameters which determine the metastatic phenotype. Therefore the regulation of production of these two important fatty acids during the metastatic process suggests that inhibitors of 12-LOX or analogs of 13-HODE may be developed as antimetastatic agents. Therefore we ask three questions about tumor cell LOXs. (1) Which LOX-like enzyme(s) is responsible for 12-HETE, 13-HODE generation? (2) What is the substrate specificity and metabolite profiles? and (3) What is the role of LOX-like enzymes in metastasis? We will identify the LOX-like enzyme(s) responsible for 12-HETE and 13-HODE production by screening a murine tumor cell cDNA library with probes against various known LOXs. Thus far no LOX enzyme has been identified in cell lines established from solid tumors. Positive clones will be sequenced and a full-length cDNA clone constructed. The tumor LOX cDNA will be expressed in a baculovirus expression system. The profile of AA and LA metabolism, substrate specificity and effect of inhibitors will be determined. We will generate polyclonal antibody(s) against tumor cell LOX specific peptide(s) designed based on the cDNA sequence and characterize the antibody(s). High metastatic (HM) and low metastatic (LM) cells form higher amounts of 12(S)-HETE. Tumor cell LOX mRNA will be analyzed by Northern blot to determine if higher level of message is expressed in high metastatic cells. Nuclear run off assays will be performed and RNA stability will be analyzed by the chase labeling assay. The amount of LOX protein will be assessed in high and low metastatic cell lines by Western blotting and immunoprecipitation using peptide generated polyclonal antibodies. Finally we will down and up regulate the tumor cell LOX expression to assess its affect on the metastatic phenotype. Antisense constructs will be generated and transfected into HM tumor cells. Downregulation will be confirmed at the mRNA and AA/LA metabolism levels. Transfectants will be assayed for adhesion to endothelium, ability to spread on fibronectin, motility, and lung colonization. Eukaryotic expression constructs will be generated and transfected into LM tumor cells. Positive clones will be selected and confirmed at mRNA, protein and AA/LA metabolism levels and the transfectants used in the above mentioned assays. This study is expected to yield important information on the enzymes responsible for the generation of key fatty acids which play an important role in cell signaling and which may be targets for the development of antimetastatic therapies based on the manipulation of cell signaling mechanisms.
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