Phase I, first-in-human study of MSC-1 (AZD0171), a humanized anti-leukemia inhibitory factor monoclonal antibody, for advanced solid tumors.

BACKGROUND: Activation of leukemia inhibitory factor (LIF) is linked to an immunosuppressive tumor microenvironment (TME), with a strong association between LIF expression and tumor-associated macrophages (TAMs). MSC-1 (AZD0171) is a humanized monoclonal antibody that binds with high affinity to LIF, promoting antitumor inflammation through TAM modulation and cancer stem cell inhibition, slowing tumor growth. In this phase I, first-in-human, open-label, dose-escalation study, MSC-1 monotherapy was assessed in patients with advanced, unresectable solid tumors. MATERIALS AND METHODS: Using accelerated-titration dose escalation followed by a 3 + 3 design, MSC-1 doses of 75-1500 mg were administered intravenously every 3 weeks (Q3W) until progression or unmanageable toxicity. Additional patients were enrolled in selected cohorts to further evaluate safety, pharmacokinetics (PK), and pharmacodynamics after escalation to the next dose had been approved. The primary objective was characterizing safety and determining the recommended phase II dose (RP2D). Evaluating antitumor activity and progression-free survival (PFS) by RECIST v1.1, PK and immunogenicity were secondary objectives. Exploratory objectives included pharmacodynamic effects on circulating LIF and TME immune markers. RESULTS: Forty-one patients received treatment. MSC-1 monotherapy was safe and well tolerated at all doses, with no dose-limiting toxicities. The maximum tolerated dose was not reached and the RP2D was determined to be 1500 mg Q3W. Almost half of the patients had treatment-related adverse events (TRAEs), with no apparent trends across doses; no patients withdrew due to TRAEs. There were no objective responses; 23.7% had stable disease for ≥2 consecutive tumor assessments. Median PFS was 5.9 weeks; 23.7% had PFS >16 weeks. On-treatment changes in circulating LIF and TME signal transducers and activators of transcription 3 signaling, M1:M2 macrophage populations, and CD8+ T-cell infiltration were consistent with the hypothesized mechanism of action. CONCLUSIONS: MSC-1 was very well tolerated across doses, with prolonged PFS in some patients. Biomarker and preclinical data suggest potential synergy with checkpoint inhibitors.

Studies of differential gene expression in clinically derived eosinophil populations.

BACKGROUND: Influx of eosinophils into the post-capillary bronchial epithelium and the subsequent release of inflammatory mediators is characteristic of the late phase of asthmatic attacks. The genes that serve to predispose the peripheral blood eosinophils of asthmatics to undergo this process are poorly defined. The aim of this report is to describe the differential gene expression of both the known pro-inflammatory genes 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) and novel cDNA sequences in eosinophils derived from clinical samples. METHODS: Novel cDNA sequences representing genes upregulated in peripheral blood eosinophils of asthmatic as compared with nonasthmatic patients were identified by differential display polymerase chain reaction (DDPCR). The differential expression of these sequences, in addition to known pro-inflammatory genes, were then studied by reverse dot blotting of amplified RNA generated from the eosinophils of nonasthmatic donors, asthmatic donors, asthmatic donors taking steroids, interleukin (IL) -3, IL-5, granulocyte-macrophage colony stimulating factor- (GM-CSF) treated eosinophils from asthmatic donors and the eosinophilic cell line AML14. RESULTS: Four unique DDPCR-generated 3'UTR DNA fragments were identified that showed differing patterns of expression between the eosinophil populations of interest. Expression of each of the novel clones was increased in the peripheral blood eosinophils of asthmatics and downregulated in those donors taking steroids. Expression of 5-lipoxygenase was not found to vary between the different eosinophil populations, whereas FLAP was induced by treatment with the cytokine cocktail in both primary eosinophils and the eosinophilic cell line AML14. CONCLUSION: The differential regulation of the novel cDNA sequences and FLAP in the range of eosinophil populations studied suggest that they may provide clinically relevant therapeutic targets. Moreover, the procedures used in these studies may provide a general approach to the study of differential gene expression in small numbers of cells such as those obtained from clinical samples.

Differential characteristics of human 15-lipoxygenase isozymes and a novel splice variant of 15S-lipoxygenase.

The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5-LO, 12S-LO, 12R-LO, 15-LO and 15S-LO. Using partially purified recombinant 15S-LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15-LO. 15S-LO has a threefold higher Km, similar Vmax and increased specificity of oxygenation for arachidonic acid, and a similar Km but decreased Vmax for linoleic acid in comparison to 15-LO. Unlike 15-LO, 15S-LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S-LO activity is regulated through calcium-dependent association of the enzyme with the membrane fraction of cells. In addition, whilst independently cloning the recently described 15S-LO, we identified a splice variant containing an in-frame 87-bp deletion corresponding to amino acids 401-429 inclusive. Modelling of the 15S-LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete alpha-helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates. Alternative splicing of 15S-LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15-LO isozymes which may reflect differing roles for the proteins in vivo.

Characterization of autocrine inducible prostaglandin H synthase-2 (PGHS-2) in human osteosarcoma cells.

The human osteosarcoma 143.98.2 cell line was found to express high levels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Maximal levels of PGHS-2 induction were attained when the cells were grown beyond confluence. The osteosarcoma cells also secrete IL-1 alpha, IL-1 beta and TNF alpha in the culture medium. PGHS-2 expression was inducible by the exogenous addition of these cytokines as well as conditioned media from auto-induced cultures and inhibitable by treatment with dexamethasone. In contrast, undifferentiated U937 cells selectively express PGHS-1 as analyzed by RT-PCR and Western blotting. The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the cellular PGE2 production mediated by each isoform of human PGHS were determined using osteosarcoma and undifferentiated U937 cells. When cells were preincubated with inhibitors to allow time-dependent inhibition prior to arachidonic acid stimulation, NS-398, CGP 28238, L-745,337, SC-58125 all behaved as potent (IC50 = 1-30 nM) and selective inhibitors of PGHS-2, in contrast to indomethacin, flurbiprofen or diclofenac which are potent inhibitors of enzymes. DuP-697 and sulindac sulfide were also potent inhibitors of PGHS-2 but both compounds inhibited cellular PGHS-1 activity at higher doses (IC50 = 0.2-0.4 microM). Time-dependent inhibition of PGE2 production in osteosarcoma cells was observed for indomethacin, diclofenac and etodolac. The synthesis of PGE2 by U937 cells was strongly dependent on exogenous arachidonic acid (100-fold stimulation) whereas confluent osteosarcoma cells also produced PGE2 without exogenous stimulus (7-fold stimulation by arachidonic acid). Osteosarcoma cells grown beyond confluence released more PGE2 from endogenous substrate than arachidonic acid stimulated undifferentiated U937 cells. These results indicate that osteosarcoma cells selectively express PGHS-2 with an autocrine regulation and effective utilization of endogenous arachidonic acid for PGE2 synthesis.

Altered sensitivity of aspirin-acetylated prostaglandin G/H synthase-2 to inhibition by nonsteroidal anti-inflammatory drugs.

Aspirin (ASA) acetylates Ser516 of prostaglandin G/H synthase-2 (PGHS-2) resulting in a modified enzyme that converts arachidonic acid to 15(R)-hydroxy-eicosatetraeroic acid [15(R)-HETE]. ASA has pharmacological benefits that may not all be limited to inhibition of prostaglandin synthesis, and this study was initiated to further investigate the properties of ASA-acetylated PGHS-2 and of the mutation of Ser516 to methionine, which mimics ASA acetylation. Both the S516M mutant and ASA-acetylated form of PGHS-2 (ASA-PGHS-2) synthesize 15(R)-HETE and have apparent K(m) values for arachidonic acid within 10-fold of the apparent K(m) value for untreated PGHS-2. The time courses of turnover-dependent inactivation were similar for reactions catalyzed by PGHS-2 and ASA-PGHS-2, whereas the PGHS-2(S516M) showed a decrease in both the initial rate of 15-HETE production and rate of enzyme inactivation. The production of 15-HETE by modified PGHS-2 was sensitive to inhibition by most nonsteroidal anti-inflammatory drugs (NSAIDs), including selective PGHS-2 inhibitors. As observed for the cyclooxygenase activity of PGHS-2, the inhibition of 15-HETE production by indomethacin was time-dependent for both ASA-PGHS-2 and PGHS-2(S516M). However, two potent, structurally related NSAIDs, diclofenac and meclofenamic acid, do not inhibit either ASA-PGHS-2 or the PGHS-2(S516M) mutant. These results demonstrate that the sensitivity to inhibition by NSAIDs of the 15-HETE production by ASA-treated PGHS-2 is different than that of prostaglandin production by PGHS-2 and that Ser516 plays an important role in the interaction with fenamate inhibitors. The results also indicate that the conversion of arachidonic acid to 15-HETE by ASA-PGHS-2 is an efficient process providing a unique mechanism among NSAIDs that will not lead to arachidonic acid accumulation or shunting to other biosynthetic pathways.

Arginine 120 of prostaglandin G/H synthase-1 is required for the inhibition by nonsteroidal anti-inflammatory drugs containing a carboxylic acid moiety.

The therapeutic action of nonsteroidal anti-inflammatory drugs (NSAIDs) is exerted through the inhibition of prostaglandin G/H synthase (PGHS), which is expressed as two isoenzymes, termed PGHS-1 and PGHS-2. From the crystal structure of sheep PGHS-1, it has been proposed that the carboxylic acid group of flurbiprofen is located in a favorable position for interacting with the arginine 120 residue of PGHS-1 (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249). Mutation of this Arg120 residue to Glu was performed and expressed in COS-7 cells using a vaccinia virus expression system. Comparison of microsomal enzyme preparations show that the mutation results in a 20-fold reduction in the specific activity of PGHS-1 and in a 100-fold increase in the apparent Km for arachidonic acid. Indomethacin, flurbiprofen, and ketoprofen, inhibitors of PGHS activity containing a free carboxylic acid group, do not exhibit any inhibitory effects against the activity of PGHS-1(Arg120-->Glu). Diclofenac and meclofenamic acid, other NSAIDs containing a free carboxylic acid group, were 50-100-fold less potent inhibitors of the activity of the mutant as compared with the wild type PGHS. In contrast, the nonacid PGHS inhibitors, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a desbromo-sulfonamide analogue of DuP697 (L-746,483), were both more potent inhibitors of PGHS-1(Arg120-->Glu) than of the wild tyupe PGHS-1. Inhibition of PGHS-1(Arg120-->Glu) was time-dependent for diclofenac and time-independent for DuP697, as observed for the wild type enzyme, indicating that the mutation does not alter the basic mechanism of inhibition. Aspirin is an acid NSAID that inhibits PGHS-1 through a unique covalent acetylation of the enzyme and also showed a reduced rate of inactivation of the mutated enzyme. These data provide biochemical evidence of the importance of the Arg120 residue in PGHS-1 for interaction with arachidonic acid and NSAIDs containing a free carboxylic acid moiety.

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.

Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer.

Prostaglandin G/H synthase (PGHS), a key enzyme leading to the formation of prostaglandins, is the target of nonsteroidal antiinflammatory drugs. Two forms of the enzyme have been identified, PGHS-1 and PGHS-2. Epidemiological evidence has suggested that aspirin and other nonsteroidal antiinflammatory drugs may reduce the risk of colorectal cancer. We examined by immunoblot analyses the expression of human PGHS-1 and PGHS-2 protein in 25 matched colon cancer and nontumor tissues, 4 premalignant polyps, 5 control colon tissues from noncancer patients, and 3 matched normal and cancerous breast tissue samples. PGHS-1 was detected in all normal and tumor tissue. In contrast, PGHS-2 was not detected in 23 of 25 normal colon tissues but was detected in 19 of 25 colon tumors. PGHS-2 protein was not observed in four human premalignant polyp samples, control colon from noncancer patients, or matched normal or cancerous breast tissues. These results suggest that the beneficial effects of nonsteroidal antiinflammatory drugs in colon cancer may be mediated by inhibition of PGHS-2.

Structural requirements for the binding of fatty acids to 5-lipoxygenase-activating protein.

5-Lipoxygenase-activating protein is required for cellular leukotriene synthesis and is the target of the leukotriene biosynthesis inhibitors MK-886 (3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H- indol-2-yl]-2,2-dimethylpropanoic acid) and MK-591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)-indol-2-yl] - 2,2-dimethylpropanoic acid). Recent studies demonstrate that 5-lipoxygenase-activating protein binds arachidonic acid and stimulates the utilization of this substrate by 5-lipoxygenase. The present study utilizes a radioligand binding assay to assess the affinity of 5-lipoxygenase-activating protein for arachidonic acid and the specificity of the fatty acid binding site on 5-lipoxygenase-activating protein. Our findings demonstrate that the presence of a free carboxyl group on fatty acids or leukotriene biosynthesis inhibitors which interact with 5-lipoxygenase-activating protein is not required for specific binding to the protein. However, the degree of saturation significantly affects the affinity of fatty acids for 5-lipoxygenase-activating protein.

Mutation of serine-516 in human prostaglandin G/H synthase-2 to methionine or aspirin acetylation of this residue stimulates 15-R-HETE synthesis.

Prostaglandin G/H synthase (PGHS) is a key enzyme in cellular prostaglandin (PG) synthesis and is the target of non-steroidal anti-inflammatory agents. PGHS occurs in two isoforms, termed PGHS-1 and PGHS-2. These isoforms differ in several respects, including their enzymatic activity following acetylation by aspirin. While PG synthesis by both isoforms is inhibited by aspirin, 15-R-hydroxyeicosatetraenoic acid (15-R-HETE) synthesis by PGHS-2, but not PGHS-1, is stimulated by preincubation with aspirin. We have mutated the putative aspirin acetylation site of hPGHS-2, and expressed the mutants in COS-7 cells using recombinant vaccinia virus. Enzyme activity and inhibitor sensitivity studies provide evidence that Ser516 is the aspirin acetylation site of human PGHS-2 and that substitution of a methionine residue at this position can mimic the effects of aspirin acetylation on enzyme activity.

The binding of leukotriene biosynthesis inhibitors to site-directed mutants of human 5-lipoxygenase-activating protein.

Site-directed mutagenesis was used to develop deletion and point mutants of human 5-lipoxygenase-activating protein (FLAP), which were then expressed in COS-7 cells. Membrane preparations from these cells were analyzed in a radioligand binding assay. Binding of leukotriene biosynthesis inhibitors to FLAP mutants containing deletions of 2 to 6 amino acids within the region from residue 48-61 was undetectable. This finding is consistent with previous studies which suggest that residues amino-terminal to the proposed second transmembrane of FLAP are critical for inhibitor binding. The present study also defines residues of FLAP a) amino-terminal to residue 48, b) between the proposed second and third transmembrane regions and c) in the C-terminal region of the protein which are not involved in inhibitor binding.

5-lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes.

The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells. In addition, subcellular fractionation techniques showed > 83% of immunoblot-detectable FLAP protein and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope.

5-Lipoxygenase activity in the human pancreas.

Low levels of 5-lipoxygenase (5-LO), the first committed enzyme in the synthesis of leukotrienes (LTs), have been reported in the porcine pancreas. We have quantitated 5-LO activity in subcellular fractions of pancreas samples from three human donors. 5-LO activity was detectable in all samples, although enzyme activity was lower than in human leukocytes. 5-LO in human pancreas samples displayed highest specific activity in membrane fractions, and did not require arachidonic acid (AA) addition for activity. These unusual characteristics of pancreatic 5-LO appear to be due, at least in part, to the presence of unesterified AA in the pancreas samples. Western blot analysis demonstrated that the human pancreas contains low levels of 5-lipoxygenase-activating protein (FLAP) in addition to 5-LO.

5-lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase.

5-Lipoxygenase (5-LO) and its activating protein (FLAP) are both required for cellular leukotriene (LT) synthesis, with 5-LO catalyzing both the synthesis of (5S)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) from arachidonic acid and the subsequent synthesis of LTA4 from 5-HPETE. We have previously expressed both human 5-LO and human FLAP to high levels in Spodoptera frugiperda (Sf9) insect cells, using recombinant baculoviruses. To study the mechanism by which FLAP activates 5-LO, we compared cellular 5-LO activity in Sf9 cells expressing this enzyme to that in Sf9 cells coexpressing FLAP and 5-LO. In this system, FLAP stimulates the utilization of arachidonic acid by 5-LO as a substrate, and increases the efficiency with which 5-LO converts 5-HPETE to LTA4. LT synthesis in cells coexpressing FLAP and 5-LO is inhibited by 3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H-indol-2-yl]-2,2- dimethyl-propanoic acid (MK-886), an LT biosynthesis inhibitor which specifically binds to FLAP. These studies in Sf9 cells, together with our recent demonstration that FLAP specifically binds arachidonic acid, suggests that FLAP activates 5-LO by acting as an arachidonic acid transfer protein.

Amino acid residues of 5-lipoxygenase-activating protein critical for the binding of leukotriene biosynthesis inhibitors.

5-Lipoxygenase-activating protein (FLAP) plays an essential role in cellular leukotriene (LT) synthesis and represents the target of three classes of LT biosynthesis inhibitors. We have taken three approaches to localize regions of FLAP involved in the binding of these inhibitors. A comparison of the amino acid sequences of FLAP from eight mammalian species identifies regions of the protein which are highly conserved and consequently may be involved in functional and inhibitor binding properties of the protein. Conversely, amino acids not conserved amongst these species are unlikely to play an essential role in inhibitor binding. Immunoprecipitation of peptide fragments of FLAP cross-linked to photoaffinity analogues of LT biosynthesis inhibitors following site-specific peptide cleavage indicates that the inhibitor attachment site is amino-terminal to 72Trp. Taken together, the cross-species analysis and photoaffinity labelling studies suggest a region within the first hydrophilic loop of FLAP which may be important for inhibitor binding. Site-directed mutagenesis of human FLAP followed by the analysis of FLAP mutants in a radioligand binding assay was used to more accurately define critical amino acid residues within this region. Mutagenesis studies reveal that mutants containing deletions of amino acids in regions of FLAP not conserved between species retain the ability to specifically bind inhibitors. Furthermore, mutants containing deletions in a highly conserved region of the protein (residues 42-61) do not bind inhibitors. These studies have therefore localized specific amino acids of FLAP which are essential for inhibitor binding. The roles that these amino acids play in inhibitor binding and may play in 5-LO activation is under investigation.

5-lipoxygenase-activating protein is an arachidonate binding protein.

5-Lipoxygenase-activating protein (FLAP) is an 18-kDa integral membrane protein which is essential for cellular leukotriene (LT) synthesis, and is the target of LT biosynthesis inhibitors. However, the mechanism by which FLAP activates 5-LO has not been determined. We have expressed high levels of human FLAP in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus, and used this system to demonstrate that FLAP specifically binds [125I]L-739,059, a novel photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK-886, an LT biosynthesis inhibitor which specifically interacts with FLAP. These studies suggest that FLAP may activate 5-LO by specifically binding arachidonic acid and transferring this substrate to the enzyme.

Cross-species comparison of 5-lipoxygenase-activating protein.

To identify regions of 5-lipoxygenase-activating protein (FLAP) important for the function of the protein and the binding of leukotriene biosynthesis inhibitors, we performed a cross-species analysis of FLAP. FLAP from all 10 mammalian species analyzed (human, monkey, horse, pig, cow, sheep, rabbit, dog, rat, and mouse) were immunologically cross-reactive and specifically bound leukotriene biosynthesis inhibitors with high affinity. Using the polymerase chain reaction, cDNA clones for FLAP from six species (monkey, horse, pig, sheep, rabbit, and mouse) were isolated and sequenced. The deduced amino acid sequences of FLAP show a high degree of identity to each other and to the published sequences for human and rat FLAP. Two regions of the protein are almost totally conserved among all of the species analyzed. This suggests that these regions have functional significance and may be involved in inhibitor binding.

A23187-induced translocation of 5-lipoxygenase in osteosarcoma cells.

In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation).