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P J Vickers

Publications and source records attributed to P J Vickers.

16 recordsLinked to original sources
PubMed1992

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).

5-Lipoxygenase-Activating Proteins↗
PubMed1992

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.

5-Lipoxygenase-Activating Proteins↗
PubMed1992

5-Lipoxygenase-activating protein is the target of a novel hybrid of two classes of leukotriene biosynthesis inhibitors.

An 18-kDa leukocyte membrane protein, termed 5-lipoxygenase-activating protein (FLAP), has recently been shown to be the target of two structurally distinct classes of leukotriene biosynthesis inhibitors. These classes of inhibitors are based on indole and quinoline structures and are represented by MK-886 and L-674,573, respectively. A novel class of hybrid structure based on the indole and quinoline classes of inhibitors, termed quindoles, has recently been developed. These compounds, exemplified by L-689,037, are potent inhibitors of leukotriene biosynthesis, both in vitro and in vivo. In the present study, we have developed and characterized a potent radioiodinated photoaffinity analogue of L-689,037, termed [125I]L-691,678. This compound was used in immunoprecipitation studies with FLAP antisera to show that the quindole series of leukotriene biosynthesis inhibitors interact directly with FLAP. In addition, we show that MK-886, L-674,573, and L-689,037 specifically compete, in a concentration-dependent manner, with both [125I]L-691,678 and [125I]L-669,083, a photoaffinity analogue of MK-886, for binding to FLAP. These results suggest that these three classes of leukotriene biosynthesis inhibitors share a common binding site on FLAP, providing further evidence that FLAP represents a suitable target for structurally diverse classes of leukotriene biosynthesis inhibitors.

5-Lipoxygenase-Activating Proteins↗
PubMed1992

Characterization of a 5-lipoxygenase-activating protein binding assay: correlation of affinity for 5-lipoxygenase-activating protein with leukotriene synthesis inhibition.

A binding assay has been developed to measure the affinity of leukotriene synthesis inhibitors for 5-lipoxygenase-activating protein (FLAP), using human leukocyte membranes as the source of FLAP and a radioiodinated leukotriene synthesis inhibitor, 125I-L-691,831, as ligand. Linearity of specific binding of radiolabeled ligand was demonstrated with increasing protein and ligand concentrations. Saturation analysis of radioligand binding showed a Kd of 6 nM and a Bmax that, depending on the membrane preparation, varied between 8 and 53 pmol/mg of protein. An excellent correlation was shown between affinity for FLAP in the binding assay and inhibition of leukotriene synthesis in human polymorphonuclear leukocytes for compounds from two structurally distinct classes, namely indoles and quinolines. A large number of membrane-active compounds did not compete with 125I-L-691,831 binding to FLAP. In addition, direct 5-lipoxygenase inhibitors and a selection of eicosanoids were unable to compete for FLAP binding. This study validates a selective binding assay for leukotriene synthesis inhibitors whose protein target is FLAP.

5-Lipoxygenase-Activating Proteins↗
PubMed1992

Identification of amino acid residues of 5-lipoxygenase-activating protein essential for the binding of leukotriene biosynthesis inhibitors.

5-Lipoxygenase-activating protein (FLAP) is specifically labeled by [125I]L-669,083 and [125I]L-691,678, photoaffinity analogues of two classes of potent leukotriene biosynthesis inhibitors. Because human FLAP contains only a single tryptophan residue at position 72 and two internal methionine residues at positions 89 and 125, we have used reagents that specifically cleave at these residues, in conjunction with antipeptide antisera, to localize the site of attachment of the photoaffinity ligands. Immunoprecipitation of specifically labeled peptide fragments after digestion of photoaffinity-labeled FLAP by iodosobenzoic acid at 72Trp demonstrates that the inhibitors bind to FLAP amino-terminal to this residue. This finding is consistent with similar immunoprecipitation studies after digestion at methionine residues using cyanogen bromide. These findings localize the site of attachment of the inhibitors to a region of FLAP that includes the hydrophilic loop between the proposed first and second transmembrane regions. Based on these findings, site-directed mutagenesis of human FLAP was performed to define key amino acids involved in inhibitor binding. Using a radioligand binding assay, analysis of mutants of human FLAP expressed in COS-7 cells demonstrates that a number of residues in the amino-terminal half of the first hydrophilic loop of the protein can be deleted without significantly affecting inhibitor binding. In contrast, no inhibitor binding was detectable with mutants in which amino acid residues in the carboxyl-terminal half of this loop were deleted. Furthermore, a point mutation of 62Asp to asparagine results in a mutant with dramatically reduced affinity for inhibitors. This loss of affinity was not displayed by a mutant in which 62Asp was mutated to a glutamate residue, suggesting that a negative charge associated with residue 62 may be critical for inhibitor binding. The roles that amino acid residues in the carboxyl-terminal half of the first hydrophilic loop of FLAP may play in the binding of leukotriene biosynthesis inhibitors are currently under investigation.

5-Lipoxygenase-Activating Proteins↗
PubMed1990

Correlation between expression of 5-lipoxygenase-activating protein, 5-lipoxygenase, and cellular leukotriene synthesis.

Previous studies involving transfection of cDNAs for 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase into osteosarcoma cells have shown that both these proteins are essential for leukotriene synthesis (Dixon, R. A. F., Diehl, R. E., Opas, E., Rands, E., Vickers, P. J., Evans, J. F., Gillard, J. W., and Miller, D. K. (1990) Nature 343, 282-284). In the present study we show that FLAP is present in a variety of cells known to produce leukotrienes, but is absent from a number of cells which do not synthesize leukotrienes. Furthermore, differentiation of the human promyelocytic HL-60 cell line towards granulocytic cells following exposure to dimethylsulfoxide is associated with the concurrent induction of both FLAP and 5-lipoxygenase and an increased capacity to synthesize leukotrienes. Cellular leukotriene synthesis in this system is functionally dependent on FLAP as shown by its inhibition by the leukotriene biosynthesis inhibitor MK-886, a compound which specifically binds to FLAP.

5-Lipoxygenase-Activating Proteins↗
PubMed1990

Identification and isolation of a membrane protein necessary for leukotriene production.

Several inflammatory diseases, including asthma, arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages. The initial enzymatic step in the formation of leukotrienes is the oxidation of arachidonic acid by 5-lipoxygenase (5-LO) to leukotriene A4. Osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations, but no leukotriene metabolites are produced by these cells when stimulated with the calcium ionophore A23187, indicating that an additional component is necessary for cellular 5-LO activity. A new class of indole leukotriene inhibitor has been described that inhibits the formation of cellular leukotrienes but has no direct inhibitory effect on soluble 5-LO activity. We have now used these potent agents to identify and isolate a novel membrane protein of relative molecular mass 18,000 which is necessary for cellular leukotriene synthesis.

Affinity Labels↗
PubMed1990

Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis.

Leukotrienes, the biologically active metabolites of arachidonic acid, have been implicated in a variety of inflammatory responses, including asthma, arthritis and psoriasis. Recently a compound, MK-886, has been described that blocks the synthesis of leukotrienes in intact activated leukocytes, but has little or no effect on enzymes involved in leukotriene synthesis, including 5-lipoxygenase, in cell-free systems. A membrane protein with a high affinity for MK-886 and possibly representing the cellular target for MK-886 has been isolated from rat and human leukocytes. Here, we report the isolation of a complementary DNA clone encoding the MK-886-binding protein. We also demonstrate that the expression of both the MK-886-binding protein and 5-lipoxygenase is necessary for leukotriene synthesis in intact cells. Because the MK-886-binding protein seems to play a part in activating this enzyme in cells, it is termed the five-lipoxygenase activating protein (FLAP).

5-Lipoxygenase-Activating Proteins↗
PubMed1989

Relation between cytochrome P450IA1 expression and estrogen receptor content of human breast cancer cells.

Multidrug resistance (MDR) in an MCF-7 human breast cancer cell line (MCF7/Adr) is associated with decreased drug accumulation and overexpression of P-glycoprotein as well as alterations in the levels of specific drug-metabolizing enzymes, including decreased activity of the phase I drug-metabolizing enzyme aryl hydrocarbon hydroxylase (AHH) and increased expression of the anionic form of the phase II drug-metabolizing enzyme glutathione S-transferase. Since the development of MDR in this MCF-7 cell line is also associated with a loss of estrogen receptors (ER), we have examined the expression of cytochrome P450IA 1, the gene encoding AHH activity, in other breast cancer cell lines not selected for drug resistance but expressing various levels of ER. These studies show that a relationship exists between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AHH activity and the ER content in a series of breast cancer cell lines. In these cell lines expression of AHH activity is regulated, at least in part, at the level of P450IA 1 RNA. While TCDD-specific binding proteins (Ah receptors) were found in each of the breast cancer cell lines, there was no apparent relation between the level of nuclear TCDD-binding proteins and the level of TCDD-inducible P450IA 1 expression. Previous studies from our laboratory have described an inverse relationship between levels of the anionic form of glutathione S-transferase and ER in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Aryl Hydrocarbon Hydroxylases↗
PubMed1988

Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer.

The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). We have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (AdrR MCF7), the anionic isozyme of glutathione S-transferase (GST pi). Hybridization with this GST pi cDNA, GST pi-1, demonstrated that increased GST pi activity in AdrR MCF7 cells is associated with overexpression but not with amplification of the gene. We mapped the GST pi gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GST pi overexpression are associated with the loss of ERs in AdrR MCF7 cells, we examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines we found an inverse association between GST pi expression and ER content. We also examined RNA from 21 primary breast cancers and found a similar association between GST pi expression and ER content in vivo. GST pi mRNA content in 11 ER-positive tumors (less than or equal to 10 fmol/mg of protein) was significantly different from the GST pi content of 10 ER-negative tumors (P = 0.002; Mann-Whitney Wilcoxon test for two independent samples). The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GST pi than ER-positive tumors.

Breast Neoplasms↗
PubMed1988

A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo.

MCF-7 human breast cancer cells provide a useful in vitro model system to study hormone-responsive breast cancer as they contain receptors for estrogen and progesterone, and estrogen both induces the synthesis of specific proteins in these cells and increases their rate of proliferation. An MCF-7 cell line which was selected for resistance to adriamycin (MCF-7/AdrR) exhibits the phenotype of multidrug resistance (MDR), and displays multiple biochemical changes. MDR in MCF-7/AdrR is also associated with a loss of mitogenic response to estrogen and the development of cross-resistance to the antiestrogen 4-hydroxytamoxifen. In addition, while the parental MCF-7 cell line responds to estrogen with increased levels of progesterone receptors and the secretion of specific proteins, these estrogen responses are lost in MCF-7/AdrR. Furthermore, while the formation of tumors in nude mice by wild-type MCF-7 cells is dependent upon the presence of estrogen, MCF-7/AdrR cells form tumors in the absence of exogenous estrogen administration. These changes in hormonal sensitivity and estrogen-independent tumorigenicity of the multidrug-resistant MCF-7 cell line are associated with a loss of the estrogen receptor and a concomitant increase in the level of receptors for epidermal growth factor. Thus, in MCF-7/AdrR cells, the development of MDR is associated with alterations in the expression of both cytosolic and membrane receptors, resulting in resistance to hormonal agents and the expression of hormone-independent tumor formation.

Animals↗
PubMed1985

Activation of folypolyglutamate synthetase by pteroic acid.

The glutamylation of methotrexate catalyzed by beef liver folypolyglutamate synthetase (FPGS) is activated by addition of pteroic acid. Pteroic acid causes greater stimulation of FPGS, including glutamylation of tetrahydrofolate, at neutral pH values (i.e., below the pH optimum of 8.4). We have attributed this activation to a conformational change of FPGS induced by pteroic acid.

Animals↗
PubMed1985

Folate analogues as substrates of mammalian folylpolyglutamate synthetase.

The antifolate drugs methotrexate (MTX) and aminopterin (AM) have been tested as substrates for folylpolyglutamate synthetase (FPGS) partially purified from beef liver. The Km for MTX is 100 microM, and that for AM is 25 microM. These values are considerably higher than those for either tetrahydrofolate or folinic acid. Based on their ratios of Vmax to Km, AM is a better substrate than is MTX for the beef liver FPGS. Both are poorer substrates than tetrahydrofolate. The 7-hydroxy metabolites of MTX and AM also are substrates for FPGS. The reactivity of 7-hydroxymethotrexate is similar to that of MTX, but 7-hydroxyaminopterin is a poorer substrate than AM. Folinic acid, often used as the rescue agent in high-dose MTX therapy, has a low Km with mammalian FPGS (7 microM). Its activity is comparable to that of the best substrate, tetrahydrofolate. Low concentrations of folinic acid prevent the formation of polyglutamates of MTX. This inhibition is competitive, presumably because folinic acid and MTX are competing substrates for FPGS. The activities of folate and antifolate substrates also have been determined with rat liver FPGS. With near-saturating concentrations of AM, MTX, or 7-hydroxymethotrexate, the reaction velocity exceeds that with an optimal concentration of tetrahydrofolate. However, the Km values of the folate analogues all are greater than those of the tetrahydrofolate coenzymes. In contrast to the formation of long-chain polyglutamates observed when tetrahydrofolate or folinic acid was the substrate, beef liver FPGS, under our reaction conditions, cannot catalyze the formation from MTX monoglutamate of polyglutamates longer than the triglutamate. MTX di- and triglutamates are poorer substrates than is MTX itself. Longer polyglutamates of MTX, while having no activity as substrates, must bind to the enzyme, because they are inhibitors. Our observations using MTX and AM with the enzymatic FPGS system help to rationalize the therapeutic use of antifolates.

Aminopterin↗
PubMed1984

Folypolyglutamate synthetase from beef liver: assay, stabilization, and characterization.

A reliable assay for folylpolyglutamate synthetase has been devised and tested. Conditions have been established for the complete separation of [3H]glutamate and the tritium-labelled products on columns of DEAE-cellulose. The availability of this assay has aided us in partially purifying and characterizing the synthetase from extracts of beef liver. Suitable conditions have been found for the stabilization of the activity of both crude and partially purified folylpolyglutamate synthetase. The apparent Km values for L-glutamate (0.82 mM), dl-L-tetrahydrofolate (9 microM), ATP (25 microM with 10 mM MgCl2), KCl (3 mM), and 2-mercaptoethanol (5 mM) have been estimated. Several oxidized pteridine substrates have been tested. Of the antifolates tested, aminopterin is the more active substrate. The chain lengths of folate polyglutamates have been measured by chromatography on columns of DEAE-cellulose, with elution by a gradient of sodium acetate. Conjugates as long as hexaglutamates have been detected. The identities of the polyglutamates of tetrahydrofolate have been verified by hydrolysis in the presence of conjugase and by double-labelling experiments.

Animals↗
PubMed1973

N.B.T. test.

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Bacterial Infections↗