Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis.

The effect of overexpression of 15-lipoxygenase-1 (15-LO-1) was studied in the human prostate cancer cell line, PC-3. Stable PC-3 cell lines were generated by transfection with 15-LO-1-sense (15-LOS), 15-LO-1-antisense (15-LOAS) or vector (Zeo) and selection with Zeocin. After characterization by RT-PCR, western and HPLC, a PC3-15LOS clone was selected that possessed 10-fold 15-LO-1 enzyme activity compared with parental PC-3 cells. The PC3-15LOAS clone displayed little or no 15-LO-1 activity. These PC-3 cell lines were characterized for properties of tumorigenesis. The proliferation rates of the cell lines were as follows: PC3-15LOS > PC-3 = PC3-Zeo > PC3-15LOAS. Addition of a specific 15-LO-1 inhibitor, PD146176, caused a dose-dependent inhibition of proliferation in vitro. Overexpression of 15-LO-1 also caused [(3)H]thymidine incorporation to increase by 4.0-fold (P < 0.01). Compared with parental and PC-3-Zeo cells, PC3-15LOS enhanced whereas PC3-15LOAS reduced the ability of PC-3 cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. These data suggested a pro-tumorigenic role for 15-LO-1 in PC-3 cells in vitro. Therefore, to clarify the role of 15-LO-1 in vivo, the effect of 15-LO-1 expression on the growth of tumors in nude mice was investigated. The PC-3 cell lines were inoculated subcutaneously into athymic nude mice. The frequency of tumor formation was increased and the sizes of the tumors formed were much larger in the PC3-15LOS compared with PC3-15LOAS, parental PC-3 and PC-3-Zeo cells. Immunohistochemistry for 15-LO-1 confirmed expression throughout the duration of the experiment. The expression of factor VIII, an angiogenesis marker, in tumor sections was increased in tumors derived from PC3-15LOS cells and decreased in those from PC3-15LOAS cells compared with tumors from parental or Zeo cells. These data further supported the evaluation by ELISA of vascular endothelial growth factor (VEGF) secretion by PC-3 cells in culture. Secretion of this angiogenic factor was elevated in PC3-15LOS cells compared with the other cell lines. These results support a role for 15-LO-1 in a novel growth-promoting pathway in the prostate.

F(2)-isoprostanes mediate high glucose-induced TGF-beta synthesis and glomerular proteinuria in experimental type I diabetes.

BACKGROUND: The recently discovered arachidonic acid derivatives, isoprostanes, are increased in pathological conditions associated with oxidative stress, such as diabetes. No role has yet been described for isoprostanes during the development of diabetic nephropathy. Cell culture in high ambient glucose has been used as a model in elucidating cellular mechanisms underlying diabetic nephropathy. Among the growth factors involved in the effect of high glucose, transforming growth factor-beta (TGF-beta) has been described as playing a key role in the development of nephropathy. METHODS: Streptozotocin-induced diabetic rats were supplemented in their diet with the antioxidant vitamin E (1000 U/kg diet). Blood and urine samples were taken to determine renal function and isoprostane concentration, as determined by gas chromatography/mass spectrometry. Glomerular mesangial and endothelial cells were cultured in high ambient glucose to determine the synthesis of isoprostanes and the role of isoprostanes in high glucose-induced synthesis of TGF-beta. RESULTS: Streptozotocin-induced diabetic rats had marked increases in plasma levels and urinary excretion rates of F(2)-isoprostanes. Dietary supplementation with vitamin E normalized (plasma) and reduced (urine) isoprostane levels and, surprisingly, improved proteinuria and blood urea nitrogen (BUN) levels. High ambient glucose increased F(2)-isoprostane synthesis in glomerular endothelial and mesangial cells in culture. Incubation of glomerular cells with F(2)-isoprostanes stimulated the production of TGF-beta. CONCLUSIONS: Increased F(2)-isoprostane synthesis during diabetes appears to be responsible in part for the increase in renal TGF-beta, a well-known mediator of diabetic nephropathy.

Concordant induction of 15-lipoxygenase-1 and mutant p53 expression in human prostate adenocarcinoma: correlation with Gleason staging.

We recently reported that the mutant form of the tumor-suppressor gene p53 up-regulates 15-LO-1 gene expression in a murine cell line. Here, we examine the expression of 15-lipoxygenase (LO)-1 and mutant p53 (mtp53) in human prostatic tissues and 15-LO-1 in the human prostate adenocarcinoma cell line PC-3. Reverse transcription-PCR and western analyses conclusively demonstrated expression of 15-LO-1 in PC-3 cells. Western blotting for 15-LO-1 in freshly resected 'normal' and prostate adenocarcinoma specimens showed 15-LO-1 expression in normal tissue, but significantly higher levels were detected in prostate adenocarcinomas. Prostate adenocarcinoma tissues generated chirally pure 13-S-hydroxyoctadecadienoic acid from exogenous linoleic acid, a preferred substrate of 15-LO-1. To study the correlation of 15-LO-1 expression with mtp53 in prostate cancer, we immunostained 48 prostatectomy specimens obtained by transurethral resection of the prostate and needle biopsy (median age 68 years, range 52-93) of different Gleason grades (n = 48), using antibodies specific for 15-LO-1, mtp53 and MIB-1 (a proliferation marker). We compared staining in cancerous foci with adjacent normal appearing prostate tissues. In only 5 of 48 patients did 'normal' tissue adjacent to cancerous foci display staining for 15-LO-1. However, no staining for mtp53 was observed in any of the normal tissues. In cancer foci, robust staining was observed for both 15-LO-1 (36 of 48, 75%) and mtp53 (19 of 48, 39%). Furthermore, the intensities of expression of 15-LO-1 and mtp53 correlated positively with each other (P < 0.001) and with the degree of malignancy, as assessed by Gleason grading (P < 0.01). By immunohistochemistry, 15-LO-1 was located in secretory cells of peripheral zone glands, prostatic ducts and seminal vesicles, but not in the basal cell layer or stroma. Based on these and other studies, we propose a model describing a possible role for 15-LO-1 expression in influencing the malignant potential and pathobiological behavior of adenocarcinomas.

Reciprocal regulation of LTA(4) hydrolase expression in human monocytes by gamma-interferon and interleukins 4 and 13: potential relevance to leukotriene regulation in glomerular disease.

BACKGROUND/AIMS: Leukotriene A(4) (LTA(4)) hydrolase catalyzes the final step in the synthesis of leukotriene B(4) (LTB(4)). TH-1- and TH-2-derived cytokines may regulate LTB(4) synthesis by monocytes through their actions on the expression of LTA(4) hydrolase. METHODS: Freshly isolated monocytes were incubated with pro- and anti-inflammatory cytokines for 36 h. mRNA expression was determined by Northern blot, protein expression was determined by Western blot and LTB(4) synthesis was determined by ELISA. RESULTS: Interferon-gamma (a TH-2-derived cytokine) increased significantly LTA(4) hydrolase mRNA expression, whereas interleukin (IL)-4 and IL-13 (both TH-2-derived cytokines) decreased LTA(4) hydrolase mRNA expression in these cells. The same effects were seen on the expression of immunoreactive LTA(4) hydrolase after incubating the monocytes with either TH-1- or TH-2-derived cytokines. The monocyte-derived cytokine IL-1 beta did not show any significant effect on LTA(4) hydrolase mRNA expression. When LTB(4) release was measured, both IL-1 beta and interferon-gamma significantly increased LTB(4) production by monocytes, while TH-2 cytokines (IL-4 and IL-13) decreased it. CONCLUSION: The opposing effects of TH-1- and TH-2-derived cytokines on the expression of LTA(4) hydrolase mRNA may regulate LTB(4) synthesis by monocytes during inflammation.

15-Lipoxygenase in glomerular inflammation.

Glomerulonephritis is a significant factor fueling the rapid increase in the population of patients with end-stage renal disease. Novel therapeutic strategies targeting specific mechanisms of glomerular destruction are the most reasonable approaches to arrest ongoing injury. In this review, we summarize some of our results obtained in our effort to characterize the role of 15-lipoxygenase activation as one of the mechansisms operative during the early, prefibrotic stage of glomerular immune injury. We also summarize the effects of cytokines released during these processes, as well as the activation by aspirin of the synthesis of 15-R-HETE (see text). Finally, we will propose a clinical approach to this group of disorders, based on emerging concepts of the pathophysiology of glomerulonephritis from our work and that of several other investigators.

Ku autoantigen (DNA helicase) is required for interleukins-13/-4-induction of 15-lipoxygenase-1 gene expression in human epithelial cells.

As reported previously in human monocytes, a human lung epithelial cell line, A549, showed de novo induction of 15-Lipoxygenase-1 (15-LO-1) in response to interleukins-13 (IL-13) and -4 (IL-4). In this cell line, 15-LO-1 expression, by RT-PCR and western blotting, was observed following 6 and 24 h of exposure to human IL-13 (ED50 5 ng/ml) and IL-4 (ED50 0.2 ng/ml). We have previously shown that no cis-acting regulatory elements exist within the 15-LO-1 promoter region. To define IL-13 and IL-4 responsive trans-acting elements, we identified a region (DP2: -353 to -304 bp site) within the 15-LO-1 promoter (by footprinting experiments) to which IL-13-responsive elements (or factors) bind specifically (Kelavkar et al, 1998, Mol Biol Rep 25, 173-182). To further delineate this region, we constructed (by site-directed mutagenesis) several deletion mutants in the 'LOPB5' region containing the 29 bp within the -353 to -304 bp of the DP2 core element. These were: DP3 (site totally deleted), DP4 (5 bp deleted at the center of the site), DP5 (8 bp at the 5'-end of the site) and DP6 (13 bp at the 3'-end of the site). Cotransfection of these deletion constructs (driving luciferase reporter genes) was associated with 90% (DP4, DP5 and DP6) or 100% (DP3) abrogation of promoter activity at 24 h. Purification of nuclear protein extracts from IL-13 and IL-4-stimulated A549 cells, using a DP2 core containing affinity column, identified a 150 kDa protein under non-denaturing conditions, and two, 70 and 85 kDa proteins under denaturing conditions. These were not detectable by Coomassie blue staining in control nuclear protein extracts. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of the tryptic digests of these proteins, identified one as the 86 kDA Lupus KU autoantigen protein P86 and the second as the 70 kDa Lupus KU autoantigen protein P70. Gel shift and supershift experiments using monoclonal antibodies toward Ku antigen and its individual subunits, and utilizing DP2 and other mutant oligonucleotides with purified nuclear protein extracts from control and cytokine-treated A549 cells, confirmed our findings. Furthermore, electroporation of neutralizing anti-Ku70, Ku 80 and Ku70/80 antibodies into A549 cells totally suppressed IL-13 and IL-4-stimulated 15-LO-1 induction in these cells. Further, immunoprecipitation experiments data suggests that IL-4 and IL-13 activate Ku antigens and 15-LO-1 expression through distinct signaling events. In summary, in A549 cells, Ku antigen is induced in response to the cytokines, IL-13 and -4, and a 29 bp region within the -353 to -304 bp region of the 15-LO-1 promoter is required for its binding and subsequent induction of 15-LO-1 gene expression. The findings may provide an important link between the established dysregulated function of Ku antigen in auto-immune diseases, such as systemic lupus erythematosus and thyroiditis, and the increasingly recognized 'anti-inflammatory' role of 15-LO-1.

Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis.

The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene B(4) biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.

Free-radical-generated F2-isoprostane stimulates cell proliferation and endothelin-1 expression on endothelial cells.

BACKGROUND: Free-radical-generated F2-isoprostane stimulates DNA synthesis and endothelin-1 (ET-1) expression on endothelial cells. 8-Iso-prostaglandin F2alpha (8-iso-PGF2alpha) is a member of the recently discovered family of prostanoids, the F2-isoprostanes, produced in vivo by cyclooxygenase-independent, free-radical-catalyzed lipid peroxidation. The goal of our study is to establish the effect of isoprostane on ET-1 production by endothelial cells, as well to determine the receptors responsible for these effects. METHODS: The proliferative effect of isoprostanes was measured as an increase of viable cell number and [3H]-thymidine uptake. ET-1 gene expression and protein synthesis were determined by Northern blot and radioimmunoassay, respectively. We also determined inositol 1,4,5-trisphosphate synthesis. Thromboxane A2 (TXA2) receptor antagonist SQ29,548 was used to establish the role of TXA2 receptor in isoprostane effect, as well as to determine the type of receptors involved in these effects. RESULTS: Our results show that physiological concentrations of 8-iso-PGF2alpha stimulated cell proliferation, DNA synthesis, and ET-1 mRNA and protein expression in bovine aortic endothelial cells (BAECs). The proliferative effect was partially abolished by treatment with anti-endothelin antibody. 8-Iso-PGF2alpha also increased inositol 1, 4,5-trisphosphate formation in these cells. These effects were partially inhibited by SQ29,548. In competitive binding assays, two binding sites were recognized on BAECs with dissociation constants (Kd) and binding site densities at equilibrium similar to those previously described in smooth muscle cells and likely represent [3H]-8-iso-PGF2alpha binding to its own receptor (high-affinity binding site) and cross-recognition of the TXA2 receptor (low-affinity binding site). CONCLUSION: These studies expand the potential scope of the pathophysiologic significance of F2-isoprostanes, released during oxidant injury, to include alteration of endothelial cell biology.

MK-591 acutely restores glomerular size selectivity and reduces proteinuria in human glomerulonephritis.

BACKGROUND: Leukotrienes are 5-lipoxygenated (5-LO) metabolites of arachidonic acid that mediate some of the glomerular hemodynamic and structural changes in experimental and human glomerulonephritis. METHODS: We conducted an open-label, pilot study of the short-term effects of leukotriene biosynthesis inhibition using an orally active 5-LO activating protein (FLAP) antagonist (MK-591) on glomerular function in patients with glomerulonephritis. Eleven adult patients (seven women, median age 38 years) with glomerulonephritis (5 lupus nephritis, 2 IgA nephropathy, 1 membranoproliferative, 1 membranous, 1 C1q-deficiency, and 1 idiopathic crescentic) and moderate renal insufficiency [glomerular filtration rate (GFR) 62 +/- 9 ml/min/1.73 m2] were given MK-591 at a dose of 100 mg orally twice a day for four days. RESULTS: MK-591 reduced proteinuria (albumin and IgG excretion rates) from 3233 +/- 1074 to 1702 +/- 555 microg/min and from 196 +/- 78 to 148 +/- 55 microg/min for albumin and IgG, respectively (P < 0.05 for both). This was not accompanied by a reduction in systemic arterial pressure, GFR, or renal plasma flow. By analysis of the fractional clearance of polydisperse dextrans, baseline proteinuria resulted from a loss of size selectivity with enhanced passage of large (>52 A) dextrans as compared with healthy controls. Treatment with MK-591 caused a selective improvement in the enhanced passage of large (>58 A) dextrans without affecting the handling of smaller dextrans, indicating an improvement in glomerular size selectivity. MK-591 was well tolerated, and no adverse effects were observed. CONCLUSIONS: Short-term therapy with MK-591 reduces proteinuria by restoring glomerular size selectivity and thus reduces transglomerular protein trafficking. These benefits may result from glomerular leukotriene biosynthesis inhibition, but other MK-591-specific actions cannot be excluded.

Effects of mutant p53 expression on human 15-lipoxygenase-promoter activity and murine 12/15-lipoxygenase gene expression: evidence that 15-lipoxygenase is a mutator gene.

Human 15-lipoxygenase (h15-LO) is present on chromosome 17p13.3 in close proximity to the tumor-suppressor gene, p53. 15-LO is implicated in antiinflammation, membrane remodeling, and cancer development/metastasis. The murine BALB/c embryo fibroblast cell line, (10)1val, expresses p53 in mutant (mt) conformation when grown at 39 degrees C and in wild-type conformation when grown at 32 degrees C. Transfection of h15-LO promoter constructs (driving luciferase reporter) into (10)1val cells and into p53-deficient (10)1 cells resulted in a marked increase in h15-LO promoter activity in (10)1val cells at 39 degrees C, but not at 32 degrees C, or as compared with (10)1 cells. Transfection of h15-LO promoter deletion constructs, however, resulted in total loss of activity in both cell types at 32 degrees C and 39 degrees C. Cotransfection of (10)1 cells with h15-LO promoter (driving luciferase reporter) along with increasing levels of a mt p53 expression vector demonstrated dose-dependent capacity of mt p53 to induce 15-LO promoter activity. No effect was observed with wild-type p53. In contrast to h15-LO promoter activity, (10)1val cells had significantly lower levels of endogenous (murine) 12/15-LO (mouse analog of h15-LO) mRNA and protein when grown at 39 degrees C compared with cells grown at 32 degrees C. Our data support the hypothesis that loss of a tumor-suppressor gene (p53), or "gain-of-function activities" resulting from the expression of its mutant forms, regulates 15-LO promoter activity in man and in mouse, albeit in directionally opposite manners. The studies define a direct link between 15-LO activity and an established tumor-suppressor gene located in close chromosomal proximity.

Novel approaches to treatment of glomerulonephritis.

Our improved understanding of the mechanisms of glomerular injury has allowed the design of novel therapeutic strategies to treat glomerulonephritis. While elimination of the etiologic factors continues to be a difficult task, modulation of the inflammatory response seems more promising at present. Several proinflammatory mediators have been identified as therapeutic targets and their inhibition has ameliorated glomerular injury in experimental animals. The role of anti-inflammatory molecules and the phenomenon of immunotolerance have gained particular attention and may prove to be useful therapeutically. Mediators of tissue repair have been found to contribute to glomerular destruction, and their inhibition was protective in a variety of experiments. In this review we discuss some of these novel approaches which may be targeted against human glomerulonephritis in the near future.

Expression of recombinant rat interleukin-13 (IL-13) and generation of a neutralizing rat IL-13 antiserum.

Using baculoviral and bacterial systems, we expressed biologically active recombinant rat IL-13 and generated neutralizing rat IL-13 antiserum. Recombinant rat IL-13 produced by baculovirus-infected insect cells stimulated proliferation of TF-1 premyeloid cell line and induced expression of 15-lipoxygenase mRNA in human peripheral blood monocytes. Antiserum generated by immunizing a rabbit with recombinant bacterial rat IL-13 specifically inhibited TF-1 proliferation induced by baculoviral rat IL-13 but did not neutralize human IL-13 mitogenic activity. Western blotting with anti-rat IL-13 serum revealed a approximately 12 kD protein band in supernatants of insect cells infected with recombinant baculovirus carrying the rat IL-13 cDNA. The availability of recombinant rat IL-13 and rat IL-13 antibodies should facilitate studying the role of IL-13 in rat models of human inflammatory disorders.

Contrasting effects of proinflammatory and T-helper lymphocyte subset-2 cytokines on the 5-lipoxygenase pathway in monocytes.

Human peripheral blood monocytes (HPBMs) express 5-lipoxygenase (5-LO) and 5-LO activating protein (FLAP), and hence have an ability to synthesize proinflammatory leukotrienes (LTs). Regulation of 5-LO and FLAP expression is a major determinant of cellular LT synthesis. We examined the effects of proinflammatory [interleukin (IL)-1 and interferon (IFN)-gamma] and T helper lymphocyte subset 2 (TH-2; IL-4 and IL-13) cytokines on (1) LTB4 production, and (2) 5-LO and FLAP expression in HPBMs. We show that IL-1 and IFN-gamma stimulate, whereas IL-4 and IL-13 inhibit ionophore-activated LTB4 release. The stimulatory effects of IL-1 and IFN-gamma were apparent at 16 to 36 hours of incubation. IL-1 modestly increased FLAP mRNA and significantly increased 5-LO mRNA steady state levels at 24 and 36 hours of incubation, respectively. IFN-gamma did not change the mRNA or protein expression of either 5-LO or FLAP. The inhibitory effects of IL-4 and IL-13 were associated with decreased FLAP mRNA and protein steady state levels. These results demonstrate that regulation of monocyte LTB4 biosynthesis by different cytokines proceeds via different pathways that partly involve modulation of the expression of the key proteins, 5-LO and FLAP. In addition, the contrasting effects of proinflammatory and TH-2-derived cytokines on monocyte LTB4 production demonstrate mechanisms by which cytokine subpopulations may modulate monocyte function in inflammation.

Evidence for the distinct nature of F2-isoprostane receptors from those of thromboxane A2.

In rat glomeruli and mesangial cells, the thromboxane A2 (TxA2) mimetic, U-46,619, but not 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), reduced glomerular inulin space and increased inositol 1,4,5-trisphosphate production, effects abolished by SQ-29,548. In competitive binding studies using 8-iso-[3H]PGF2alpha or [3H]SQ-29,548, mesangial cells displayed TxA2 binding sites but not ones for 8-iso-PGF2alpha. In contrast, rat aortic smooth muscle cells possessed specific binding sites for both TxA2 and 8-iso-PGF2alpha and displayed functional responses to both agonists, such as time- and dose-dependent activation of mitogen-activated protein kinases. In these cells, the mean dissociation constant value for the isoprostane receptor was 31.8 +/- 5.7 nM. When human TxA2 receptor cDNA was expressed in Xenopus oocytes injected with the Ca2+-specific photoprotein, aequorin, 8-iso-PGF2alpha gave much weaker responses than U-46,619. These studies provide the first radioligand binding characteristics of the F2-isoprostane receptor and demonstrate its specific and heterologous cellular localization. These studies support the distinct nature and biological significance of isoprostane receptors and provide a tool for their further molecular characterization.

Mechanism of induction of prostaglandin E2 production by endothelin 1 in cultured rat mesangial cells.

The phospholipase A2 inhibitors mepacrine, ONO-RS-082, and AACOCF3 completely inhibited prostaglandin E2 production induced by endothelin 1 in cultured rat mesangial cells, suggesting that phospholipase A2 is a critical enzyme in this process. TMB-8, an inhibitor of calcium mobilization from intracellular stores, abolished its production, while neither nicardipine nor chelation of extracellular calcium by EGTA did. The protein kinase C inhibitors, H-7 and staurosporine, and downregulation of protein kinase C could not inhibit prostaglandin E2 production, while W-7, a calmodulin inhibitor, abolished it. Pertussis toxin never influenced its production. Thus, endothelin 1 evokes prostaglandin E2 production in mesangial cells mainly through the activation of phospholipase A2, dependent on intracellular calcium and calmodulin and independent of extracellular calcium, protein kinase C, and pertussis toxin sensitive guanosine 5'-triphosphate binding proteins.