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Cystic fibrosis (CF) is a monogenic disease, characterized by massive chronic lung inflammation. The observed variability in clinical phenotypes in monozygotic CF twins is likely associated with the extent of inflammation. This study sought to investigate inflammation-related aberrant DNA methylation in CF twins and to determine to what extent acquired methylation changes may be associated with lung cancer.Blood-based genome-wide DNA methylation analysis was performed to compare the DNA methylomes of monozygotic twins, from the European CF Twin and Sibling Study with various degrees of disease severity. Putatively inflammation-related and differentially methylated positions were selected from a large lung cancer case-control study and investigated in blood by targeted bisulphite next-generation-sequencing. An inflammation-related locus located in the Plakophilin-3 (PKP3) gene was functionally analysed regarding promoter and enhancer activity in presence and absence of methylation using luciferase reporter assays.We confirmed in a unique cohort that monozygotic twins, even if clinically discordant, have only minor differences in global DNA methylation patterns and blood cell composition. Further, we determined the most differentially methylated positions, a high proportion of which are blood cell-type-specific, whereas others may be acquired and thus have potential relevance in the context of inflammation as lung cancer risk factors. We identified a sequence in the gene body of PKP3 which is hypermethylated in blood from CF twins with severe phenotype and highly variably methylated in lung cancer patients and controls, independent of known clinical parameters, and showed that this region exhibits methylation-dependent promoter activity in lung epithelial cells.

Cystic fibrosis (CF) is a monogenic disease, characterized by massive chronic lung inflammation. The observed variability in clinical phenotypes in monozygotic CF twins is likely associated with the extent of inflammation. This study sought to investigate inflammation-related aberrant DNA methylation in CF twins and to determine to what extent acquired methylation changes may be associated with lung cancer.Blood-based genome-wide DNA methylation analysis was performed to compare the DNA methylomes of monozygotic twins, from the European CF Twin and Sibling Study with various degrees of disease severity. Putatively inflammation-related and differentially methylated positions were selected from a large lung cancer case-control study and investigated in blood by targeted bisulphite next-generation-sequencing. An inflammation-related locus located in the

For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status (p = 0.020) and occurrence of brain metastases (p = 0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients (p = 0.022) and also for poor overall survival in metastatic lung cancer patients (p = 0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer.

Background: Pancreatic cancer is characterized by high resistance to chemotherapy. Such chemoresistance can be mediated by multidrug resistance proteins (MRPs), breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein. However, the contribution of individual MRP isoforms to chemoresistance in pancreatic carcinoma is unclear. We studied ATP-binding cassette (ABC) transporter expression in human pancreatic carcinoma cell lines as compared to primary pancreatic duct cells, and analyzed the MRP expression profile in 5-fluorouracil-resistant cells. Methods: Transporter expression was analyzed by quantitative and qualitative RT-PCR, by immunoblot, and chemoresistance by cytotoxicity assay. Results: Primary pancreatic duct cells expressed MRP1, MRP3, MRP4, and MRP5, but not MRP2 mRNA. The established carcinoma cell lines expressed MRP1, MRP4, and MRP5, most of them also MRP2, MRP3, MRP7, and BCRP, but none contained detectable amounts of MRP6, MRP8, or MRP9 mRNA. Immunoblot analyses demonstrated presence of MRP1, MRP4, and MRP5 protein in all, but MRP3 and BCRP protein only in some of these cells. Compared to parental Capan-1 cells, Capan-1 cells with acquired chemoresistance towards 5-fluorouracil showed an upregulated mRNA and protein expression of MRP3, MRP4, and MRP5. In addition, silencing of MRP5 by RNA interference resulted in enhanced sensitivity of parental Capan-1 cells towards 5-fluorouracil cytotoxicity. Conclusion: MRP3, MRP4, and MRP5 are upregulated in 5-fluorouracil-resistant cells, and MRP5 contributes to 5-FU resistance in pancreatic carcinoma cells.

For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status (p = 0.020) and occurrence of brain metastases (p = 0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients (p = 0.022) and also for poor overall survival in metastatic lung cancer patients (p = 0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer.

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.

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.

Mast cells are known to secrete endogenously synthesized leukotriene C4 (LTC4), but the identity of the responsible export pump in human mast cells was unknown. The multidrug resistance proteins MRP1 and MRP2 have been identified as primary-active ATP-dependent export pumps for various amphiphilic anions including the glutathione conjugate LTC4. We therefore studied the expression at the RNAand protein levels of both MRP1 and MRP2 as well as the ATP-dependent LTC4 transport in the human mast cell line HMC-1. Upon stimulation by 1 microM ionomycin, intact HMC-1 cells generated 26 pmol LTC4/10(8) cells within 20 min. Transport experiments using inside-out HMC-1 membrane vesicles demonstrated an ATP-dependent LTC4 transport amounting to 1.4 pmol x (mg protein)(-1) x min(-1). Reverse transcription PCR indicated that HMC-1 cells express mRNA of MRP1, but not of MRP2 or MRP3. Cloning and sequencing of the amplified PCR fragment confirmed its identity with the human MRP1 sequence. Immunoblots using antibodies against MRP1 and MRP2 demonstrated that HMC-1 cells contain the MRP1 but not the MRP2 protein. Our results indicate that the 190 kDa integral membrane glycoprotein MRP1 mediates the ATP-dependent export of LTC4 from human mast cells to the extracellular space.

Tumor cell proliferation and metastasis proceed via a network of interdependent molecular events with a vast array of molecular players and signal transduction mechanisms differing in various types of human tumors. In the sequence of events necessary for carcinogenesis, arachidonate metabolites have been documented to play a significant role at several steps. Arachidonate metabolism in human cells occurs via several enzymatic pathways, including enzymes such as cyclo-oxygenases and lipoxygenases. This review pays particular attention to one member of the lipoxygenase family of enzymes, namely 12-lipoxygenase, since an arachidonate metabolite generated via 12-lipoxygenase action, 12(S)-HETE, has been shown to elicit various prometastatic effects of tumor cells in vivo and in vitro. We focus especially on mechanisms of activation and modulation of 12-lipoxygenase expression in human tumor cells, since various tumor cells express 12-lipoxygenase or are responsive to metabolites derived from 12-lipoxygenase action, thus offering a potential for successful therapeutic intervention against such tumors

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 μM. After 24-hr incubation with 10 μM 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.

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.

Epigenetic changes occur in all types of cancers and include alterations in deoxyribonucleic acid (DNA) methylation, histone modification, or expression changes of non-coding ribonucleic acid (RNA). There is now evidence that epigenetic alterations influence cancer risk and could be developed into useful biomarkers. While it is clear that epigenetic mechanisms can play a key role in mediating environmental influences including dietary folate intake, smoking, other environmental stressors, and age, many of the mechanisms involved remain obscure. There are numerous mechanisms by which genetic and epigenetic factors interact and thus the field of epigenetic epidemiology of cancer faces considerable challenges in tearing apart gene-epigene-environment interactions and in determining how they modulate individual cancer risk. An important question for epigenetic epidemiology of cancer is whether epigenetic changes can be identified in surrogate tissues; e.g., DNA isolated from peripheral lymphocytes, plasma samples, urine, or buccal swabs. The current state of the art of cancer epigenetic epidemiology studies as relating to epigenetic cancer risk factors and markers of diagnostic and prognostic significance is reviewed. In the past, epigenetic work has been carried out in comparative studies of primary human normal/tumor tissue pairs, but recent advances in technologies have made large scale analyses on surrogate tissues within defined cohorts possible. Interindividual variations exist in epigenetic markers, but their origin and relevance needs to be determined. In future studies it will be important to characterize the interaction between environmental, dietary, genetic, and epigenetic factors. Echoing the development in the field of genetics, candidate gene approaches employed thus far will be replaced by genome-wide epigenome analyses in the future. Approaches integrating genetic, epigenetic, and well-documented epidemiological and clinical data are most promising and may improve the currently available risk models for cancer in the future.

The ability of livers to produce endogenous leukotrienes after immunological stimulation was tested with organs from rats and guinea pigs. Passive sensitization of rats in vivo with monoclonal murine antidinitrophenol-IgE before antigen challenge in the isolated perfused liver system elicited a rapid hepatic production and biliary excretion of leukotrienes as judged by radioimmunoassay after separation of individual leukotrienes by high-performance liquid chromatography. Within 10 min after antigen infusion, mainly leukotriene C4, but also leukotriene D4 and N-acetyl-leukotriene E4, appeared in the bile. The biliary excretion rate of antigen-induced cysteinyl leukotrienes rose from

Pancreatic cancer is characterized by high resistance to chemotherapy. Such chemoresistance can be mediated by multidrug resistance proteins (MRPs), breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein. However, the contribution of individual MRP isoforms to chemoresistance in pancreatic carcinoma is unclear. We studied ATP-binding cassette (ABC) transporter expression in human pancreatic carcinoma cell lines as compared to primary pancreatic duct cells, and analyzed the MRP expression profile in 5-fluorouracil-resistant cells.Transporter expression was analyzed by quantitative and qualitative RT-PCR, by immunoblot, and chemoresistance by cytotoxicity assay.Primary pancreatic duct cells expressed MRP1, MRP3, MRP4, and MRP5, but not MRP2 mRNA. The established carcinoma cell lines expressed MRP1, MRP4, and MRP5, most of them also MRP2, MRP3, MRP7, and BCRP, but none contained detectable amounts of MRP6, MRP8, or MRP9 mRNA. Immunoblot analyses demonstrated presence of MRP1, MRP4, and MRP5 protein in all, but MRP3 and BCRP protein only in some of these cells. Compared to parental Capan-1 cells, Capan-1 cells with acquired chemoresistance towards 5-fluorouracil showed an upregulated mRNA and protein expression of MRP3, MRP4, and MRP5. In addition, silencing of MRP5 by RNA interference resulted in enhanced sensitivity of parental Capan-1 cells towards 5-fluorouracil cytotoxicity.MRP3, MRP4, and MRP5 are upregulated in 5-fluorouracil-resistant cells, and MRP5 contributes to 5-FU resistance in pancreatic carcinoma cells.

Metabolism of leukotrienes via omega-oxidation represents a major degradative and inactivating pathway of these biologically active icosanoids. Isonicotinic acid hydrazide (isoniazid) inhibited this process in rats in vivo, in the isolated perfused rat liver, and in hepatic microsomes. The in vivo catabolism of leukotriene E4 via N-acetyl-leukotriene E4 to its omega-oxidized metabolites was inhibited by 50% or 71% using single intravenous isoniazid doses of 0.6 mmol or 1.0 mmol/kg body mass, respectively. Isoniazid interfered with leukotriene catabolism at the initial omega-oxidation step, resulting in an accumulation of N-acetyl-leukotriene E4. Analogous although weaker inhibition of leukotriene omega-oxidation in vivo was observed by pretreatment with isonicotinic acid 2-isopropylhydrazide and monoacetyl hydrazine. In the isolated perfused liver, isoniazid at concentrations varying over 0.2-10 mM decreased the omega-oxidation of cysteinyl leukotrienes dose-dependently by up to 94%. omega-Oxidation of both leukotriene E4 and leukotriene B4 by rat liver microsomes was inhibited by isoniazid, isonicotinic acid 2-isopropylhydrazide, and monoacetyl hydrazine with half-maximal concentrations in the range of 5-15 mM. Our measurements indicate that the impairment of leukotriene omega-oxidation by isoniazid involves both cytochrome-P450-dependent enzyme systems responsible for omega-oxidation of leukotriene E4 and leukotriene B4. In effect, under isoniazid treatment one can expect a prolongation of the proinflammatory actions of endogenously produced leukotrienes.

As previous studies suggested the expression of a 12-LOX enzyme in murine and human melanoma cell lines, the primary aim of this project was to genetically identify the 12-LOX enzyme (platelet-, leukocyte- or epithelial form). By using reverse transcriptase-polymerase chain reaction, sequencing and various immunological techniques we have demonstrated conclusively the expression of the platelet-type 12-LOX in human melanoma cells of different origin, in their transplanted xenografts and in fresh human skin tumors. Furthermore, we found that p12-LOX is able to provide a survival signal for melanoma cells since inhibition of the enzyme by general LOX or selective 12-LOX inhibitors induced apoptosis in vitro. p12-LOX of human melanoma has been shown to be involved in the control of the metastatic phenotype, since we have detected the upregulation of the 12-LOX protein expression in spontaneously metastasizing xenografts and in thick human skin tumors (3.0 mm) characterized by high risk for the development of metastasis. Co-expression of two megakaryocytic genes, p12-LOX and alphaIIb integrin chains, was found to be a frequent phenomenon in human melanoma (approximately 70%) suggesting a common regulatory defect in this tumor.

Background/Aims : Expression and localization of human hepatocellular transporters and of radixin, cross-linking actin with some membrane transporters, may change in cholestatic liver diseases. Methods : We investigated the uptake transporters OATP2 (SLC21A6), OATP8 (SLC21A8), and NTCP (SLC10A1), the export pumps MRP2 (ABCC2), MRP3 (ABCC3), MRP6 (ABCC6), and P-glycoproteins (ABCB1, ABCB4, ABCB11), and radixin, in non-icteric primary biliary cirrhosis (PBC stages I–III) and control human liver needle-biopsies using immunofluorescence microscopy and semi-quantitative RT-PCR. Results : Expression and localization of all transporters were unchanged in PBC I-II. Immunostaining intensities of uptake transporters decreased in PBC III with a concomitant decrease in mRNA levels. Immunostaining intensities and mRNA levels of export pumps were similar in controls and PBC I–III, however, irregular MRP2 immunostaining suggested redistribution of MRP2 into intracellular structures in PBC III. Areas of irregular MRP2 immunostaining showed largely reduced radixin immunostaining, whereas normal hepatocytes had MRP2 and radixin confined to the canalicular membrane. Disrupted localization of radixin and MRP2 supports the concept that radixin contributes to the canalicular localization of MRP2. Conclusions : Down-regulation of uptake transporters may contribute to the impaired hepatobiliary elimination in advanced PBC, and partially altered localization of MRP2 may reflect the onset of changes leading to icteric PBC.

The apical multidrug resistance protein MRP2 (symbol ABCC2) is an ATP-dependent export pump for anionic conjugates in polarized cells. MRP2 has only 48% amino acid identity with the paralog MRP1 (ABCC1). In this study we show that purified recombinant MRP2 reconstituted in proteoliposomes is functionally active in substrate transport. The Km values for ATP and LTC4 in the transport by MRP2 in proteoliposomes were 560 microM and 450 nM, respectively. This transport function of MRP2 in proteoliposomes was dependent on the amount of MRP2 protein present and was determined to 2.7 pmol x min(-1) x mg MRP2(-1) at 100 nM LTC4. Transport was competitively inhibited by the quinoline derivative MK571 with 50% inhibition at about 12 microM. Our data document the first reconstitution of transport-active purified recombinant MRP2. Binding and immunoprecipitation experiments indicated that MRP2 preferentially associates with the chaperone calnexin, but co-reconstitution studies using purified MRP2 and purified calnexin in proteoliposomes suggested that the LTC4 transport function of MRP2 is not dependent on calnexin. The purified, transport-active MRP2 may serve to identify additional interacting proteins in the apical membrane of polarized cells.

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, Ml, 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.

Various human tumor cells including the epidermoid carcinoma A431 cells express the platelet-type isoform of 12-lipoxygenase (12-LOX)1–3. Tumor cell 12-LOX and the arachidonate metabolite generated via 12-LOX activity, 12(S)-HETE, have been shown to contribute to the metastatic potential of tumor cells in vivo and in vitro 4. Most recently, elevated expression of 12-LOX mRNA in prostate epithelial cells was demonstrated to correlate with poor prognosis in human prostate cancer5.

Tumor cell proliferation and metastasis proceed via a network of interdependent molecular events with a vast array of molecular players and signal transduction mechanisms differing in various types of human tumors. In the sequence of events necessary for carcinogenesis, arachidonate metabolites have been documented to play a significant role at several steps. Arachidonate metabolism in human cells occurs via several enzymatic pathways, including enzymes such as cyclo-oxygenases and lipoxygenases. This review pays particular attention to one member of the lipoxygenase family of enzymes, namely 12-lipoxygenase, since an arachidonate metabolite generated via 12-lipoxygenase action, 12(S)-HETE, has been shown to elicit various prometastatic effects of tumor cells in vivo and in vitro. We focus especially on mechanisms of activation and modulation of 12-lipoxygenase expression in human tumor cells, since various tumor cells express 12-lipoxygenase or are responsive to metabolites derived from 12-lipoxygenase action, thus offering a potential for successful therapeutic intervention against such tumors.

The 12-lipoxygenase enzyme (12-LOX) has been found in platelets (1) and several other cell types including leukocytes, epidermal and epithelial cells (2–4), as well as in human (5,6) and rodent tumor cells (7,8). In tumor cells, the major metabolite, 12(S)-HETE, regulates their metastatic potential (9–11) through a protein kinase C-dependent mechanism (7). The intracellular distribution of 12-LOX activity varies between different cells ranging from a predominantly cytosolic (2,4,12) to an exclusive or preferred localization in membranes (5,13,14). This distribution of 12-LOX activity can change upon stimulation of cells suggesting a translocation of the enzyme (15). However, these earlier studies on 12-LOX activity did not investigate in parallel the respective subcellular distribution of the 12-LOX protein itself. In order to obtain a more complete concept of the intracellular regulation and distribution of 12-LOX, we therefore analyzed the distribution of both 12-LOX activity and of 12-LOX protein in murine Lewis lung carcinoma cells (3LL cells) and compared their 12-LOX activity with homologous 12-LOX activity in platelets. Further we asked whether activation of the 12-LOX enzyme in 3LL cells encompasses a Ca2+-dependent translocation to membranes as is the case for 12-LOX in platelets or other enzymes of arachidonate metabolism such as phospholipase A2 and 5-LOX (16,17).

Eicosanoids are a group of oxygenated arachidonic acid (AA) metabolites including prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), lipoxins (LXs), and various hydroperoxy and hydroxy fatty acids (1–3). AA is released from phospholipids mainly by the action of phospholipase A2 and is the substrate for lipoxygenases (LOXs), cyclooxygenase (COX) and cytochrome P-450 monooxygenase (also called epoxygenase). AA is metabolized to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) by 5-lipoxygenase (5-LOX), to 12-HPETE by 12-lipoxygenase (12-LOX; EC 1.13.11.31) and to 15-HPETE by 15-lipoxygenase (15-LOX). Then, 5-HPETE can be converted to 5-hydroxyeicosatetraenoic acid (5-HETE) and LTs, 12-HPETE to 12-HETE and hepoxilins (HXs), 15-HPETE to 15-HETE and LXs. AA can also be metabolized to PGs, prostacyclin and TXs by COX, or to epoxides and diols by cytochrome P-450. Both the LOX and COX pathways have been implicated in several aspects of cancer from carcinogenesis to metastasis (4–11).

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.

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.

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.

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+.

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.

Mercapturic acid formation, an established pathway in the detoxication of xenobiotics, is demonstrated for cysteinyl leukotrienes generated in rats in vivo after endotoxin treatment. The mercapturate N-acetyl-leukotriene E 4 (N-acetyl-LTE 4 ) represented a major metabolite eliminated into bile after injection of [ 3 H]LTC 4 as shown by cochromatography with synthetic N-acetyl-LTE 4 in four different HPLC solvent systems. The identity of endogenoud N-acetyl-LTE 4 elicited by endotoxin in vivo was additionally verified by enzymatic deacetylation followed by chemical N-acetylation. The deacetylation was catalyzed by penicillin amidase. Endogenous cysteinyl leukotrienes were quantified by radioimmunoassay after HPLC separation. A N-acetyl-LTE 4 concentration of 80 nmol/l was determined in bile collected between 30 and 60 min after endotoxin injection. Under this condition, other cysteinyl leukotrienes detected in bile by radioimmunoassay amounted to less than 5% of N-acetyl-LTE 4 . The mercapturic acid pathway, leading from the glutathione conjugate LTC 4 to N-acetyl-LTE 4 , thus plays an important role in the deactivation and elimination of these potent endogenous mediators.

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [γ- 32 P]ATP and [γ- 32 P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa eells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32 P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.

Endogenous protein kinases and acceptor proteins in the focal adhesion plaques of monolayer cells were studied using [γ-32P]ATP as exogenous substrate. Two major phosphoproteins, p135 and pp105, besides some minor phosphoproteins, were phosphorylated in the substrate-attached material after dislodgement of cells. Evidence was obtained, that suspension cells leave the same components on the wall of the culture vessel, resulting from cell collisions with the wall and not from passive adsorption of secreted components or disintegrated cellular material.

N-Acetyl-leukotriene E4, the end product of leukotriene C4 metabolism in the mercapturic acid pathway, was rapidly eliminated from the blood circulation into the bile of rats. Part of the N-acetyl-leukotriene E4 secreted from bile into the intestine underwent enterohepatic circulation. Leukotriene absorption occurred from the small intestine and from the colon. Biliary and urinary excretion within 5.5 h amounted to 15 and 2%, respectively, of the intraduodenally administered N-acetyl- 3H leukotriene E4 in animals anesthetized with ketamine. HPLC analyses indicated that 35% of the biliary radioactivity corresponded to unchanged N-acetyl-3H leukotriene E4, while 65% in bile and 100% in urine were polar metabolites. Enterohepatic circulation extends the biological half-life of N-acetyl-leukotriene E4.

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 mgkg) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase-HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC4 and LTD4 in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC4 and LTD4 which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin-induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.

A significant increase in the production of cysteinyl leukotrienes was observed after mechanical or thermal trauma in the anesthesized rat. The amount of biliary N-acetyl-leukotriene E4, which represents a suitable indicator for blood plasma leukotrienes, was used as a measure of leukotriene generation. Cysteinyl leukotrienes were rapidly eliminated from blood plasma into bile where N-acetyl-leukotriene E4 was the major metabolite. Leukotrienes were at a much lower concentration in blood plasma than in bile and differed in the pattern of metabolites. The detected amounts of leukotrienes were sufficient to induce known phenomena associated with trauma, such as tissue edema and circulatory and respiratory dysfunction. Increased leukotriene generation appears to play an important role in the pathophysiology of tissue trauma.

Rat livers were perfused in a non-recirculating mode at constant pressure via the portal vein with media containing 5 mM glucose, 2 mM lactate, and 0.2 mM pyruvate. [3H]LTC4 was infused for a period of 5 min to a final concentration of 20 nM; it increased glucose and lactate output and reduced perfusion flow. 1) Leukotriene radioactivity was recovered 10 min after the onset of [3H]LTC4 infusion to about 40% in the effluent, to 20% in the bile, and to 40% in the liver. 2) Radioactivity in the effluent increased to a maximum 4-5 min after the onset and decreased again to essentially zero 3 min after completion of [3H]LTC4 infusion. [3H]LTC4 and [3H]LTD4 were the major labeled components in the effluent accounting for 45% and 38%, respectively, of the effluent radioactivity. 3) [3H]LTC4 and [3H]LTD4 were also the major components in bile; they accounted for 50% and 30%, respectively, of the radioactivity excreted, while more polar [3H]leukotriene metabolites accounted for the remainder. 4) In the liver, [3H]LTC4 and [3H]LTD4 were the major and [3H]LTE4, N-acetyl-[3H]LTE4 as well as omega-hydroxy-N-acetyl-[3H]LTE4 and omega-carboxy-N-acetyl-[3H]LTE4 were minor components detected 5 min after completion of [3H]LTC4 infusion. It is concluded from the present findings that during a 5 min infusion period about one third each of the infused LTC4 remained unchanged, was converted to LTD4, and was further degraded to LTE4 and polar metabolites including omega-oxidation products of N-acetyl-LTE4.(ABSTRACT TRUNCATED AT 250 WORDS)

The cysteinyl leukotrienes LTC4, LTD4, and LTE4 are mediators of in flammatory and anaphylactic reactions (Samuelson 1983, Hammarstrom 1983). Injection of cysteinyl leukotrienes into sensitive species causes shock-like reactions, myocardial depression (Lefer 1986, Feuerstein 1984), and widespread extravasation of plasma (Hua et al.1985). Many of these pathophysiologic signs are also observed after endotoxin administration, and direct evidence for a key role of cysteinyl leukotrienes as mediators in endotoxin shock has been presented recently (Hagmann et al. 1984,1985,1986). An involvement of leukotrienes as mediators in tissue trauma is not only indicated by pharmacological antagonism but also by demonstration of systemic leukotriene production after surgical or thermal trauma in the anesthetized rat (Denzlinger et al. 1985). A key to the understanding of the role of cysteinyl leukotrienes in pathophysiological and physiological processes is the elucidation of their metabolism in vivo (Hammarstrom 1983, Hagmann et al. 1984,1985,1986, Keppler et al. 1985,1987, Denzlinger et al. 1985, 1986, Huber et al. 1987), and the determination of their concentrations under pathophysiological conditions in vivo (Hagmann et al. 1984,1985,1986, Denzlinger et al. 1985,1986, Keppler et ai. 1985,1987).

The role of leukotrienes was investigated in frog virus 3-induced hepatitis in rats. Frog virus 3 elicited an enhanced generation of cysteinyl leukotrienes in vivo as monitored by measurement of N-acetyl-leukotriene E4 as the major endogenous metabolite of cysteinyl leukotrienes secreted into rat bile. N-Acetyl-leukotriene E4 concentrations were elevated for more than 4 hr after frog virus 3 injection. In vitro experiments using cultured rat liver Kupffer cells of high purity indicated that these cells can produce and metabolize leukotrienes and are thus a possible source of leukotrienes elicited in vivo by frog virus 3. The selective 5-lipoxygenase inhibitor AA 861 and the dual inhibitor of arachidonate lipoxygenase and cyclooxygenase, BW 755C, reduced the hepatocellular injury after a high dose of frog virus 3 by about 50 and 80%, respectively, as judged from plasma activities of ALT and sorbitol dehydrogenase at 24 hr after frog virus 3 administration. Our in vivo and in vitro studies argue in favor of an important role of leukotrienes as mediators in frog virus 3 hepatitis in rats.

Actions of endotoxin (1ipopolysaccharide, LPS) in vivo have long been suggested to be mediated by arachidonate metabolites (1,2). The important role of arachidonate-derived metabolites including leukotrienes, prostaglandins and thromboxane in experimental LPS shock has been deduced from the LPS resistance of essential fatty acid-deficient rats (3,4), from the altered arachidonate metabolism in LPS-tolerant rats (5), as well as from pharmacological evidence (1,2,6,7). Recent studies showed that the LPS-resistant C3H/HeJ mouse strain, whose macrophages are defective in prostaglandin (8,9) and leukotriene synthesis (10), can be made highly LPS-sensitive by transfer of pure macrophages from LPS-sensitive C3H/HeN mice (11). These data stress the target cell role of macrophages in the in vivo action of LPS (8,11).

The cysteinyl leukotrienes LTC4, LTD4, and LTE4 play an important role within the network of mediators of endotoxin shock since they are capable of eliciting the generation of other lipid mediators of shock. Three lines of evidence support the conclusion that the cysteinyl leukotrienes are involved in the lethal shock-inducing action of endotoxin. (1) Endotoxin elicits the systemic production of cysteinyl leukotrienes in vivo. (2) Injection of these leukotrienes into sensitive species causes shock-like reactions, myocardial depression, and extravasation of plasma. (3) Cysteinyl leukotriene receptor antagonists and inhibitors of leukotriene synthesis prevent experimental endotoxin shock. Moreover, endotoxin induces a strong inhibition of the hepatobiliary elimination of cysteinyl leukotrienes, which normally represents the major pathway for deactivation of these mediators. This dual action of endotoxin, comprising enhanced synthesis and simultaneously impaired deactivation of potent mediators, points to a novel type of toxin action. The role of cysteinyl leukotrienes in endotoxin action does not exclude the involvement of additional shock-inducing factors released by endotoxin and their possible contribution to the formation of lipid mediators, including leukotrienes.

Enterohepatic circulation is an important mechanism responsible for extension of the biological half-lives of many endogenous compounds and xenobiotics (1). Cysteinyl leukotrienes play an important role in lethal endotoxin action (2) and in tissue trauma (3). In the rat,cysteinyl leukotrienes are eliminated from blood preferentially into bile (2) and subsequently into feces (4). In humans,elimination into urine predominates over elimination into feces (4) , raisin., the question of intestinal absorption and enterohepatic circulation of cysteinylleukotrienes.