5-LOX mRNA Research Articles

The 12-lipoxygenase gene-transfected MCF-7 human breast cancer cell line exhibits estrogen-independent, but estrogen and omega-6 fatty acid-stimulated proliferation in vitro, and enhanced growth in athymic nude mice.

The estrogen-dependent, linoleic acid (LA)-unresponsive, MCF-7 breast cancer cell line was transfected with 12-lipoxygenase (12-LOX) cDNA (MCF-7/12-LOX cells). The transfectant stably expressed high levels of 12-LOX mRNA and protein, and secreted large quantities of 12-hydroxyeicosatetraenoic acid when cultured with arachidonate. The transfectant grew in vitro in the absence of estrogen, and its growth was stimulated by LA. The MCF-7/12-LOX cells formed small solid tumors when injected into the mammary fat pads of ovariectomized nude mice. Despite this estrogen independence, MCF-7/12-LOX cell growth was stimulated further by estradiol both in vitro and in vivo, and to a greater extent than parental MCF-7 cells.

Lipoxygenase products and expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human cultured synovial cells

5-Lipoxygenase products are pro-inflammatory mediators. Their roles and cellular origin in chronic inflammatory rheumatisms such as rheumatoid arthritis (RA) are poorly understood. The expression of arachidonate 5-lipoxygenase (5-LOX, arachidonate: oxygen 5-oxydoreductase; EC 1.13.11.34) and the 5-lipoxygenase activating protein (FLAP) genes in osteoarthritis and RA synoviocytes was studied at the transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) methodology. Arachidonic acid metabolism was analyzed by reverse-phase high pressure liquid chromatography. 5-LOX and FLAP mRNA were detectable using RT-PCR in all sources of synoviocytes tested. The expression of 5-LOX and FLAP mRNA led to the synthesis of 5-LOX metabolites. 12- and 15-LOX activities were also present. These LOX products can participate in inflammatory processes leading to joint destruction in RA.

Elevated 12-lipoxygenase mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer

Objectives Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in males in the United States. The mortality is due mainly to distant metastasis. Therefore, predicting the prognosis of prostate cancer patients is an important clinical problem. Previously, we demonstrated that a 12-lipoxygenase (12-LOX) metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid, enhances the invasiveness of prostate cancer cells and that a 12-LOX-selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide) reduces experimental metastasis in animal model systems. In this study, we investigated the potential of 12-LOX as a predictor for the aggressiveness of prostate cancer. Methods The mRNA expression levels of 12-LOX in 122 matching prostate normal and cancerous tissues were measured by quantitative reverse transcription-polymerase chain reaction. Possible association between 12-LOX expression and histologic grade, pathologic and clinical stage, margin positivity, age, and race was analyzed. Results 12-LOX mRNA levels were elevated in cancer cells and the expression associated with poor differentiation and invasiveness of prostate cancer. Overall, 46 (38%) of 122 evaluable patients showed elevated levels of 12-LOX mRNA in prostate cancer tissues compared with the matching normal tissues. A statistically significantly greater number of cases were found to have an elevated level of 12-LOX among T3, high grade, and surgical margin-positive than T2, intermediate, and low grade, and surgical margin-negative prostatic adenocarcinomas. Conclusions Our data suggest that elevation of 12-LOX mRNA expression occurs more frequently in advanced stage, high-grade prostate cancer and that 12-LOX may serve as an indicator for progression and prognosis of prostate cancer. This enzyme also may be a novel target for the development of anti-invasive and antimetastatic agents.

Granulocyte-Macrophage Colony-Stimulating Factor Increases 5-Lipoxygenase Gene Transcription and Protein Expression in Human Neutrophils

The complex process of neutrophil activation and accumulation is orchestrated by a cascade of cytokines and bioactive lipids produced at the site of inflammation. Neutrophils are a rich source of the potent inflammatory lipid leukotriene B4(LTB4). Granulocyte-macrophage colony-stimulating factor (GM-CSF) can activate neutrophils for an exponential increase in LTB4 production in response to a number of subsequent stimuli. In this report, we examined the temporal regulation, by GM-CSF, of the gene and protein expression of 5-lipoxygenase (5-LOX), a key enzyme of the LTB4 production pathway. Human neutrophils were exposed to 10 ng/mL of GM-CSF for various periods of time and 5-LOX mRNA was measured by Northern blot analysis. We observed no change in 5-LOX mRNA at early time points (0.25 to 3 hours); however, by 18 hours we observed a significant augmentation of 5-LOX-specific message (4.3 +/- 1.7-fold increase; n = 6). Nuclear transcription assays indicated that the rate of 5-LOX gene transcription was augmented threefold in neutrophils incubated with GM-CSF, whereas the half-life of the message was not markedly changed. Parallel experiments indicated that the levels of 5-LOX protein were also increased by GM-CSF. The augmentation was observed within 30 minutes after stimulation and was maximal (5.23 +/- 2.6; n = 4) at 18 hours. Incubation of GM-CSF-stimulated neutrophils with protein synthesis inhibitors resulted in a time-dependent impairment of their ability to produce LTB4, with no inhibition seen during the first hours, a 75% decrease seen by 12 hours, and greater than 95% inhibition seen at 18 hours. Collectively, our data imply that GM-CSF can regulate LTB4 production by two distinct mechanisms: a short-term increase that is not related to increased 5-LOX mRNA expression and is independent of protein synthesis, and a sustained increase in LTB4 production that is associated with the transcriptional activation of the 5-LOX gene, increase in 5-LOX mRNA levels, and dependence on protein synthesis. Such transcriptional modulation of 5-LOX enzyme expression may provide new approaches for therapeutic intervention in protracted inflammatory conditions.

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°C. However at 24°C and 10°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 μM free Ca 2+ and subsequent separate analyses of cytosol and membrane fractions indicated that, as in platelets, an increase in intracellular free Ca 2+ 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.

Expression of 5-lipoxygenase in lymphoblastoid B and T cells

The expression of arachidonate 5-lipoxygenase (5-LOX, arachidonate : oxygen 5-oxidoreductase; EC 1.13.11.34) and the 5-lipoxygenase activating protein (FLAP) genes in lymphoblastoid B and T cell lines was studied at the transcriptional level by reverse transcription-PCR analysis. Two B cell lines, CESS and SKW 6.4, were found to express both 5-LOX and FLAP mRNA. One T cell line, MOLT 4, expressed only FLAP mRNA. Upon stimulation of intact cells by calcium ionophore, B lymphoblastoid cells produced 5, 12 and 15 hydroxyeicosatetraenoic acids as well as leukotriene B 4.