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W Hagmann

Publications and source records attributed to W Hagmann.

At least 19 recordsLinked to original sources
PubMed2000

Expression, subcellular localization and putative function of platelet-type 12-lipoxygenase in human prostate cancer cell lines of different metastatic potential.

The involvement of 12-lipoxygenase (12-LOX) expression and function in tumor metastasis has been demonstrated in several murine tumor cell lines. In addition, 12-LOX expression was detected in human prostatic tumors and correlated to the clinical stage of disease. Here we provide data that human prostate cancer cell lines express the platelet-type isoform of 12-LOX at both the mRNA and protein levels, and immunohistochemistry revealed 12-LOX expression in human prostate tumors. The enzyme was localized to the plasma membrane, cytoplasmic organelles and nucleus in non-metastatic cells (PC-3 nm) and to the cytoskeleton and nucleus in metastatic cells (DU-145). After orthotopic/intraprostatic injection of tumor cells into SCID mice, the metastatic prostate carcinoma cells (DU-145) expressed 12-LOX at a significantly higher level compared with the non-metastatic counterparts, PC-3nm. The functional involvement of 12-LOX in the metastatic process was demonstrated when DU-145 cells were pretreated in vitro with the 12-LOX inhibitors N-benzyl-N-hydroxy-5-phenylpentamide (BHPP) or baicalein, the use of which significantly inhibited lung colonization. These data suggest a potential involvement of 12-LOX in the progression of human prostate cancer.

Animals↗
PubMed1999

Purification of the human apical conjugate export pump MRP2 reconstitution and functional characterization as substrate-stimulated ATPase.

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP-binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C-terminal (His)6-tag. Immunoblot and immunofluorescence analyses detected the MRP2-(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2-(His)6-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C4 and were photoaffinity-labelled with 8-azido-[alpha-32P]ATP. MRP2-(His)6 was solubilized from membranes of MRP2-(His)6-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2-(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein-dependent manner with a Km for ATP of 2.1 mM and a Vmax of 25 nmol ADP x mg MRP2-1 x min-1. This ATPase activity was substrate-stimulated by oxidized and reduced glutathione and by S-decyl-glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

12-lipoxygenase expression in human melanoma cell lines.

12-lipoxygenase (12-LOX) expression and function in the regulation of the metastatic phenotype was demonstrated in several murine melanoma lines before. Here we have provided novel evidences that, though at a low level (in max. 15% of the cell population), human melanoma lines (HT168, M1, HT199, HT18 and WM35) express the platelet-type isoform of 12-LOX both at mRNA and protein levels. 12-LOX expression was demonstrated in cultured tumor cells and in skin tumor xenografts. Comparison of the expression of 12-LOX in skin primary tumors and its lung metastases indicated a stable expression. The low level of 12-LOX expression in human melanoma cell lines suggests that other lipoxygenase(s) could also be responsible for the metabolism of arachidonic acid to 12-HETE breakdown products.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1997

Requirement for epidermal growth factor receptor tyrosine kinase and for 12-lipoxygenase activity in the expression of 12-lipoxygenase in human epidermoid carcinoma cells.

We studied the dependency of basal 12-lipoxygenase (12-LOX; arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) expression and activity on functional protein tyrosine kinase of the epidermal growth factor receptor (EGF-R) and on 12-LOX activity in human A431 epidermoid carcinoma cells. Treatment of cells with inhibitors of high specificity for EGF-R tyrosine kinase, namely PD 153035 and 4,5-dianilinophthalimide (DAPH1), decreased cellular 12-LOX at mRNA, protein, and activity levels in a time- and dose-dependent manner, with PD 153035 being effective at concentrations below 1 microM. After 24-hr incubation with 10 microM PD 153035 or DAPH1, 12-LOX activity dropped to 14% (39%), and 12-LOX protein to 25% (24%) of control level. Inhibition of 12-LOX activity by the compound N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP) also resulted in a substantial decrease in 12-LOX protein expression. 12-LOX mRNA levels were diminished or undetectable by reverse transcription-polymerase chain reaction after cell treatment with these inhibitors. Our results suggest that basal 12-LOX expression in A431 tumor cells largely depends on functional EGF-R tyrosine kinase, and that 12-LOX activity is required in the EGF-elicited intracellular signaling maintaining the expression of 12-LOX.

Arachidonate 12-Lipoxygenase↗
PubMed1996

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.

Arachidonate 12-Lipoxygenase↗
PubMed1996

Differential secretion of cathepsins B and L from normal and tumor human lung cells stimulated by 12(S)-hydroxy-eicosatetraenoic acid.

Cathepsins B and L play roles in intracellular and extracellular proteolysis in normal and malignant processes. A directed extracellular proteolysis by regulated secretion could facilitate the process of invasion. We have therefore investigated the effect of the physiological signal mediator 12(S)-hydroxy-eicosatetraenoic acid on the release of cathepsins B and L in normal and malignant human lung cells. Quantitative determinations of cathepsin activities were done by flow cytometry and spectrofluorometry using synthetic dipeptidyl substrates coupled to fluorogens. Most interestingly, a difference in the secretion of cathepsins B and L was found: only release of active cathepsin B was detected. The effect was specific for 12(S)-hydroxy-eicosatetraenoic acid, 12(R)-hydroxy-eicosatetraenoic acid, and 5(S)-hydroxy-eicosatetraenoic acid were ineffective. The response was immediate but a substantial amount of nonreleasable activity remained cell bound. Alveolar macrophages, Wi-38 fibroblasts, and tumor cells derived from large cell carcinomas and adenocarcinomas were sensitive to 12(S)-hydroxy-eicosatetraenoic acid, but cells from undifferentiated squamous cell carcinomas were not. Sensitivity did not parallel malignancy but more likely the degree of differentiation of cells. The investigated tumor cell lines showed no detectable endogenous 12-lipoxy-genase activity to synthesize 12(S)-hydroxy-eicosatetraenoic acid from arachidonate; therefore, we assume a paracrine mechanism for 12(S)-hydroxy-eicosatetraenoic acid action. Protein kinase C alpha, a key enzyme involved in 12(S)-hydroxy-eicosatetraenoic acid-elicited responses, was expressed in all sensitive tumor cells, but insignificantly in a sensitive normal cell line and an insensitive tumor cell line. From our experiments we propose two separate intracellular pools of active cathepsin B: an unreleasable, lysosomal fraction and a fraction available for regulated secretion. Different processing and sorting mechanisms may be responsible for the generation of these cathepsin B-fractions in these pools.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

OBJECTIVE: To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo. METHODS: The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay. RESULTS: Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor. CONCLUSION: These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.

Aggrecans↗
PubMed1995

In vivo effects of stromelysin on the composition and physical properties of rabbit articular cartilage in the presence and absence of a synthetic inhibitor.

OBJECTIVE: To characterize the effects of intraarticular injection of recombinant human stromelysin (SLN) on the matrix composition and physical properties of cartilage from lapine stifle joints and the modulation of these effects by the systemic administration of an N-carboxyalkyl synthetic matrix metalloproteinase inhibitor, L-696,418. METHODS: Female 6-8-week-old New Zealand white rabbits received an intraarticular injection of 100 micrograms activated SLN in 1 stifle joint and buffer in the contralateral control knee; these animals were killed after 1 hour. A separate group of animals received an intravenous injection of either 30 mg/kg L-696,418 or buffer prior to intraarticular injection of SLN. Joints were dissected and analyzed for proteoglycan (PG) loss into joint fluid, tissue biochemical composition, and histology by toluidine blue or anti-VDIPEN antibody staining, or were frozen for physical property analysis. Disks of femoropatellar groove cartilage were harvested from the stifle joint and tested in uniaxially confined compression for determination of electromechanical and mechanical properties. RESULTS: Lapine stifle joints that received injection of SLN without systemic administration of L-696,418 showed a 13-fold increase in loss of PG into synovial fluid. Cartilage from these joints showed significant decreases in streaming potential at 1 Hz and electrokinetic coupling coefficient, but no change in equilibrium modulus, dynamic stiffness, or hydraulic permeability. Systemic treatment with L-696,418 resulted in a significant decrease in loss of PG into joint fluid and elimination of changes in cartilage high-frequency streaming potential and coupling coefficient in joints that were injected with SLN. CONCLUSION: The 1-hour exposure to SLN in vivo resulted in loss of PG and exposure of the VDIPEN epitope of the aggrecan core protein in the superficial region of the tissue near the articular surface. This highly localized degradation resulted in electromechanical behavior changes, but little or no change occurred in mechanical properties. Systemic administration of L-696,418 significantly decreased loss of PG from cartilage and prevented the highly localized tissue degradation and the resultant changes in electromechanical behavior caused by intraarticular SLN injection.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Cell proliferation status, cytokine action and protein tyrosine phosphorylation modulate leukotriene biosynthesis in a basophil leukaemia and a mastocytoma cell line.

Mast cells, mastocytoma cells and basophil leukaemia cells are well-established producers of leukotrienes when grown and stimulated appropriately. I report that the cells' ability to produce leukotrienes is dependent on the cells' proliferative status or their provision with growth factors. Proliferating MC/9 and subconfluent RBL2H3 cells respond maximally to stimulation by 1 microM ionomycin with the production of 56 and 32 pmol of cysteinyl-leukotrienes/10(6) cells respectively. In contrast, confluent RBL2H3 or growth-arrested MC/9 cells lose their ability to generate leukotrienes in response to ionomycin treatment. This rapid down-regulation of leukotriene synthesis is also observed when proliferating RBL2H3 cells are transferred to growth-factor-free medium, wherein cellular leukotriene-synthesis capacity has an apparent half-lifetime of 60 min. Transfer back into growth medium results in the regeneration of leukotriene synthesis capacity within 6 h. In growth-arrested MC/9 cells, leukotriene production ability can at least partially be restored by priming the cells with interleukin 3, but not with interleukin 4. In RBL2H3 cells, pretreatment with protein tyrosine kinase inhibitors such as genistein (5 min, 37 microM), herbimycin A (6 h, 3 microM) or tyrphostin 25 (16 h, 100 microM) completely inhibits leukotriene generation, whereas okadaic acid (15 min, 0.5 microM) has no effect. Under these conditions, both genistein and herbimycin A strongly impair ionomycin-induced protein tyrosine phosphorylation. Our study indicates that leukotriene generation in these tumour cells is tightly regulated by their proliferation status and supply with growth factors, and cell stimulation towards leukotriene synthesis appears to involve protein tyrosine kinase activity.

Animals↗
PubMed1994

Endogenous 12(S)-HETE production by tumor cells and its role in metastasis.

12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] is the 12-lipoxygenase metabolite of arachidonic acid. Previously, we have demonstrated that exogenous 12(S)-HETE can activate protein kinase C, increase cell surface expression of integrins, enhance adhesion, induce endothelial cell retraction, and increase experimental metastasis of tumor cells. Because of these prominent effects of exogenous 12(S)-HETE on tumor cell metastatic potential, it is important to determine whether there is endogenous 12(S)-HETE production by tumor cells. In the present study, mRNAs from human, rat, and mouse platelets as well as human colon carcinoma (Clone A), rat Walker carcinoma (W256), and mouse melanoma (B16a) and lung carcinoma (3LL) were reverse transcribed and amplified by polymerase chain reaction with platelet 12-lipoxygenase specific primers. Identity of the polymerase chain reaction fragments was confirmed by sequencing. 12-Lipoxygenase protein was detected by Western blotting. Tumor cell-derived 12-HETE was determined by reverse phase-high performance liquid chromatography analysis. In addition, the effect of endogenous 12(S)-HETE on tumor cells was studied by using a platelet-type 12-lipoxygenase selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide). Our results suggest that some tumor cells express platelet-type 12-lipoxygenase mRNA, protein and metabolize arachidonic acid to 12(S)-HETE and that endogenous 12(S)-HETE, like the exogenous 12(S)-HETE, may play an important role in tumor cell adhesion to matrix in vitro and lung colonization in vivo.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

12-lipoxygenases and 12(S)-HETE: role in cancer metastasis.

Arachidonic acid metabolites have been implicated in multiple steps of carcinogenesis. Their role in tumor cell metastasis, the ultimate challenge for the treatment of cancer patients, are however not well-documented. Arachidonic acid is primarily metabolized through three pathways, i.e., cyclooxygenase, lipoxygenase, and P450-dependent monooxygenase. In this review we focus our attention on one specific lipoxygenase, i.e., 12-lipoxygenase, and its potential role in modulating the metastatic process. In mammalian cells there exist three types of 12-lipoxygenases which differ in tissue distribution, preferential substrates, and profile of their metabolites. Most of these 12-lipoxygenases have been cloned and sequenced, and the molecular and biochemical determinants responsible for catalysis of specific substrates characterized. Solid tumor cells express 12-lipoxygenase mRNA, possess 12-lipoxygenase protein, and biosynthesize 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid], as revealed by numerous experimental approaches. The ability of tumor cells to generate 12(S)-HETE is positively correlated to their metastatic potential. A large collection of experimental data suggest that 12(S)-HETE is a crucial intracellular signaling molecule that activates protein kinase C and mediates the biological functions of many growth factors and cytokines such as bFGF, PDGF, EGF, and AMF. 12(S)-HETE plays a pivotal role in multiple steps of the metastatic 'cascade' encompassing tumor cell-vasculature interactions, tumor cell motility, proteolysis, invasion, and angiogenesis. The fact that 12-lipoxygenase is expressed in a wide diversity of tumor cell lines and 12(S)-HETE is a key modulatory molecule in metastasis provides the rationale for targeting these molecules in anti-cancer and anti-metastasis therapeutic protocols.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1993

Activity and protein distribution of 12-lipoxygenase in HEL cells: induction of membrane-association by phorbol ester TPA, modulation of activity by glutathione and 13-HPODE, and Ca(2+)-dependent translocation to membranes.

The understanding of the intracellular regulation of 12-lipoxygenase requires a knowledge of the distribution of both enzyme protein and its activity. In human erythroleukemia cells, the membrane fraction contains about 90% of the total cellular 12-lipoxygenase activity, whereas only approximately 10% of 12-lipoxygenase activity resides in the cytosol. However, the majority of the cellular 12-lipoxygenase protein is found in the cytosol. Pretreatment of cells for 0-3 days with 160 nM TPA caused a marked, time-dependent increase in membrane-bound 12-lipoxygenase activity and protein, respectively. In contrast, the cytosolic amount of 12-lipoxygenase protein and activity, respectively, were minimally altered by this TPA treatment. Recombining the active membrane fraction with cytosol resulted in no significant inhibition of its 12-lipoxygenase activity, but the addition of GSH to the membrane fraction inhibited 12-lipoxygenase activity in a dose-dependent manner. On the other hand, the cytosolic enzyme can be rendered active in the presence of 1 microM 13-hydroperoxyoctadecadienoic acid. In HEL cell homogenates, a partial translocation of the cytosolic enzyme to the membrane takes place in a Ca(2+)-dependent manner, resulting in an increase in membrane-associated 12-lipoxygenase activity and a concomitant decrease in cytosolic 12-lipoxygenase activity above 0.1 microM Ca2+.

Arachidonate 12-Lipoxygenase↗
PubMed1992

Resident mast cells are the main initiators of anaphylactic leukotriene production in the liver.

During anaphylaxis the sensitized liver can have substantial capacity for leukotriene production. However, the intrahepatic cellular source for these potent eicosanoid mediators has been unclear so far. We therefore analyzed the appropriate role of resident liver cells in organ-specific generation of leukotrienes by immunohistochemical localization of 5-lipoxygenase, by measurement of cysteinyl leukotriene production in animals or isolated livers and by histochemical monitoring of mast cells in rat, guinea pig and mouse livers, respectively. During anaphylaxis in vivo, these species all generated large amounts of leukotrienes. Immunohistochemistry with rat liver demonstrated resident mast cells as the predominant cell type in liver containing 5-lipoxygenase. Rat and guinea pig livers contained numerous mast cells and produced substantial amounts of leukotrienes on antigen challenge; in contrast, mouse livers neither showed detectable mast cells nor generated leukotrienes when stimulated analogously. Infusion of histamine or serotonin (1 mmol/L each) or of the degranulating substance P (8 mumol/L) did not elicit leukotriene generation in rat livers. Furthermore, substantial degranulation of liver mast cells by compound 48/80 (0.5 mg/kg body mass) was paralleled by only modest leukotriene formation (63 +/- 10 pmol in bile/kg body mass/30 min). These results indicate that during anaphylaxis mast cells are the main intrahepatic cells initiating leukotriene production and that such leukotriene generation is likely to be independent of mast cell degranulation or the release of histamine or serotonin.

Anaphylaxis↗