Personal dosimetry for positron emitters, and occupational exposures from clinical use of gallium-68.

The current status and issues regarding positron dosimetry in nuclear medicine are summarized. The suitability of the United Kingdom Health Security Agency extremity and eye beta-gamma personal thermoluminescence dosemeters are then considered. Monte Carlo modelling is performed to determine their responses and derive sets of calibration factors, along withHp(0.07) andHp(3) conversion coefficients, for carbon-11, nitrogen-13, oxygen-15, fluorine-18 and gallium-68 sources, which are commonly used in positron emission tomography (PET) computed tomography; data for these isotopes is assumed extrapolatable to other positron sources. It is found that the dosemeters are adequate for assessing exposures to PET radionuclides, even if their routine calibrations to caesium-137 were maintained. An idealized set of measurements representing gallium-68 exposure scenarios is then described, including reproducible mock-ups of individuals manipulating vials and syringes. Finally, a short case-study is presented that explores occupational doses during routine clinical use of gallium-68. The extremity dosemeter results demonstrated significant variations dependent upon the exposure conditions, with some seen to be comparatively large; whole-body and eye dose rates per activity were found to be lower. The importance of routine dose monitoring of workers is emphasized, with the need for a longer-termed follow-up study demonstrated.

A new, highly selective murine peroxisome proliferator-activated receptor δ agonist increases responsiveness to thermogenic stimuli and glucose uptake in skeletal muscle in obese mice.

AIM: We investigated how GW800644, the first pharmacologically selective murine peroxisome proliferator-activated receptor δ (PPARδ) agonist, affects energy balance, glucose homeostasis and fuel utilization by muscle in obese mice. METHODS: Potencies were determined in transactivation assays. Oral glucose tolerance was determined after 14 and 22 days' administration (10 mg/kg body weight, twice daily) to Lep(ob)/Lep(ob) mice. Food intake and energy expenditure were measured during a 26-day experiment, and plasma metabolites and 2-deoxyglucose uptake in vivo at termination. Palmitate oxidation and 2-deoxyglucose uptake by isolated soleus muscles were measured after 14 (in lean and obese mice) and 26 days. RESULTS: GW800644 activated murine PPARδ (EC(50) 2 nM), but caused little to no activation of PPARα or PPARγ up to 10 µM. It did not increase liver weight. GW800644 reduced food intake and body weight in obese mice after 8 days. It did not affect resting energy expenditure, but, compared to pair-fed mice, it increased the response to a ß(3)-adrenoceptor agonist. It improved glucose tolerance. GW800644, but not pair-feeding, reduced plasma glucose, insulin and triglyceride concentrations. It increased 2-deoxyglucose uptake in vivo in adipose tissue, soleus muscle, heart, brain and liver, and doubled 2-deoxyglucose uptake and palmitate oxidation in isolated soleus muscle from obese but not lean mice. CONCLUSIONS: PPARδ agonism reduced food intake and independently elicited metabolic effects that included increased responsiveness to ß(3)-adrenoceptor stimulation, increased glucose utilization and fat oxidation in soleus muscle of Lep(ob)/Lep(ob) but not lean mice and increased glucose utilization in vivo in Lep(ob)/Lep(ob) mice.

Orphan nuclear receptors: shifting endocrinology into reverse.

Steroid and thyroid hormones and vitamin A metabolites (retinoids) regulate the expression of complex gene programs by binding to members of the nuclear receptor family of ligand-activated transcription factors. The nuclear receptor family also includes many "orphan" members that currently lack known ligands but that represent candidate receptors for new hormones. Recently, natural and synthetic ligands have been identified for several orphan receptors and used to dissect their biological roles. This "reverse endocrinology" strategy has resulted in the discovery of unanticipated nuclear signaling pathways for retinoids, fatty acids, eicosanoids, and steroids with important physiological and pharmacological ramifications.

Bile acids: natural ligands for an orphan nuclear receptor.

Bile acids regulate the transcription of genes that control cholesterol homeostasis through molecular mechanisms that are poorly understood. Physiological concentrations of free and conjugated chenodeoxycholic acid, lithocholic acid, and deoxycholic acid activated the farnesoid X receptor (FXR; NR1H4), an orphan nuclear receptor. As ligands, these bile acids and their conjugates modulated interaction of FXR with a peptide derived from steroid receptor coactivator 1. These results provide evidence for a nuclear bile acid signaling pathway that may regulate cholesterol homeostasis.

The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity.

The human nuclear pregnane X receptor (hPXR) activates cytochrome P450-3A expression in response to a wide variety of xenobiotics and plays a critical role in mediating dangerous drug-drug interactions. We present the crystal structures of the ligand-binding domain of hPXR both alone and in complex with the cholesterol-lowering drug SR12813 at resolutions of 2.5 and 2.75 angstroms, respectively. The hydrophobic ligand-binding cavity of hPXR contains a small number of polar residues, permitting SR12813 to bind in three distinct orientations. The position and nature of these polar residues were found to be critical for establishing the precise pharmacologic activation profile of PXR. Our findings provide important insights into how hPXR detects xenobiotics and may prove useful in predicting and avoiding drug-drug interactions.

The nuclear receptor PPARgamma - bigger than fat.

Work reported over the past year has provided insights into the mechanisms whereby ligand activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) regulates systemic glucose and lipid homeostasis. PPARgamma has also been implicated recently in the biology of monocytes and in cell-cycle regulation and cancer. Polyunsaturated fatty acids and eicosanoids bind and activate PPARgamma, suggesting that these lipids may serve as hormonal regulators of a variety of biological processes.

PPARbeta/delta selectively induces differentiation and inhibits cell proliferation.

Peroxisome proliferator-activated receptor (PPAR) beta-null mice exhibit exacerbated epithelial cell proliferation and enhanced sensitivity to skin carcinogenesis, suggesting that ligand activation of PPARbeta will inhibit keratinocyte proliferation. By using of a highly specific ligand (GW0742) and the PPARbeta-null mouse model, activation of PPARbeta was found to selectively induce keratinocyte terminal differentiation and inhibit keratinocyte proliferation. Additionally, GW0742 was found to be anti-inflammatory due to inhibition of myeloperoxidase activity, independent of PPARbeta. These data suggest that ligand activation of PPARbeta could be a novel approach to selectively induce differentiation and inhibit cell proliferation, thus representing a new molecular target for the treatment of skin disorders resulting from altered cell proliferation such as psoriasis and cancer.

Suckling defect in mice lacking the soluble haemopoietin receptor NR6.

Cytokines control a variety of cellular responses including proliferation, differentiation, survival and functional activation, via binding to specific receptors expressed on the surface of target cells [1]. The cytokine receptors of the haemopoietin family are defined by the presence of a conserved 200 amino acid extracellular domain known as the haemopoietin domain [2]. We report here the isolation of NR6, a haemopoietin receptor that, like the p40 subunit of interleukin-12 (IL-12) [3] and the EBI3 gene induced by Epstein-Barr virus infection in lymphocytes [4], contains a typical haemopoietin domain but lacks transmembrane and cytoplasmic domains. Although in situ hybridisation revealed NR6 expression at multiple sites in the developing embryo, mice lacking NR6 did not display obvious abnormalities and were born in the expected numbers. Neonatal NR6(-/-) mice failed to suckle, however, and died within 24 hours of birth, suggesting that NR6 is necessary for the recognition or processing of pheromonal signals or for the mechanics of suckling itself. In addition, NR6(-/-) mice had reduced numbers of haemopoietic progenitor cells, suggesting a potential role in the regulation of primitive haemopoiesis.

Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARgamma is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARgamma-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor-deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-alpha and gelatinase B, indicating both systemic and local actions of PPARgamma. These findings suggest that PPARgamma agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARgamma ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities.

The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions.

The cytochrome P-450 monooxygenase 3A4 (CYP3A4) is responsible for the oxidative metabolism of a wide variety of xenobiotics including an estimated 60% of all clinically used drugs. Although expression of the CYP3A4 gene is known to be induced in response to a variety of compounds, the mechanism underlying this induction, which represents a basis for drug interactions in patients, has remained unclear. We report the identification of a human (h) orphan nuclear receptor, termed the pregnane X receptor (PXR), that binds to a response element in the CYP3A4 promoter and is activated by a range of drugs known to induce CYP3A4 expression. Comparison of hPXR with the recently cloned mouse PXR reveals marked differences in their activation by certain drugs, which may account in part for the species-specific effects of compounds on CYP3A gene expression. These findings provide a molecular explanation for the ability of disparate chemicals to induce CYP3A4 levels and, furthermore, provide a basis for developing in vitro assays to aid in predicting whether drugs will interact in humans.

Probing the structure and function of the estrogen receptor ligand binding domain by analysis of mutants with altered transactivation characteristics.

We have developed a genetic screen for the yeast Saccharomyces cerevisiae to isolate estrogen receptor (ER) mutants with altered transactivation characteristics. Use of a "reverse" ER, in which the mutagenized ligand binding domain was placed at the N terminus of the receptor, eliminated the isolation of truncated constitutively active mutants. A library was screened with a low-affinity estrogen, 2-methoxyestrone (2ME), at concentrations 50-fold lower than those required for activation of the unmutagenized ER. Several mutants displaying enhanced sensitivity to 2ME were isolated. We further characterized a mutant carrying the substitution L536P, which was located immediately N terminal to the AF-2-activating domain of the receptor. Amino acid 536 corresponds to a ligand contact residue in retinoic acid receptor gamma, suggesting that key contact points are conserved among receptors. Introduction of L536P into the original ER cDNA isolate HE0, which contains the substitution G400V, rendered the receptor more sensitive to a variety of agonists. When introduced into the wild-type ER HEG0, L536P also rendered the receptor more sensitive to agonists, and, in addition, induced high levels of constitutive activity that could be inhibited by antiestrogens. Estrogens containing a keto substitution in the steroid D ring, but not those containing a hydroxyl group, were full agonists of L536P-HEG0. Limited proteolytic analysis suggested that the L536P substitution, which is located immediately N terminal to the AF-2 domain, induces a conformational change in the ER that partially mimics binding by hormone. Both HEG0 and L536P-HEG0 formed complexes with hsp90 in vitro, indicating a lack of correlation between interaction with hsp90 in vitro and hormonal regulation of ER transactivation in vivo. This supports the idea that a factor(s) acting downstream of hsp90 is important for controlling activity of the hormone-free receptor.

A new ligand for the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), GW7845, inhibits rat mammary carcinogenesis.

We have tested a new ligand for peroxisome proliferator-activated receptor-gamma, GW7845, as an inhibitor of experimental mammary carcinogenesis, using the classic rat model with nitrosomethylurea as carcinogen. Rats were first treated with a single dose of nitrosomethylurea (50 mg/kg body weight, i.p.). Starting 1 week later, they were fed GW7845, at either 60 or 30 mg/kg of diet, for 2 months. This agent significantly reduced tumor incidence, tumor number, and tumor weight at both doses. This is the first report of the use of a ligand for peroxisome proliferator-activated receptor-gamma to prevent experimental breast cancer.

Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor.

Tamoxifen inhibits estrogen receptor (ER) transcriptional activity by competitively inhibiting estradiol binding and inducing conformational changes in the receptor that may prevent its interaction with coactivators. In bone, the cardiovascular system, and some breast tumors, however, tamoxifen exhibits agonist activity, suggesting that the tamoxifen-ER complex is not recognized identically in all cells. We used phage display to demonstrate that the antiestrogen GW5638 induces a unique structural change in the ER. The biological significance of this conformational change was revealed in studies that demonstrated that tamoxifen-resistant breast tumor explants are not cross-resistant to GW5638. Because of these properties, this drug is currently being developed as a potential therapeutic for tamoxifen-resistant breast cancers.

Expression of wild-type estrogen receptor beta and variant isoforms in human breast cancer.

It has been shown in previous studies that a variety of estrogen receptor (ER) beta mRNA transcripts are expressed in human breast cancer cell lines and tumors. To complement the RNA expression studies, we have developed ER-beta-specific antibodies to characterize ER-beta protein expression in breast cancer cell lines and tumors. Monoclonal antibodies were made against a peptide representing the first 18 amino acids of the longest ER-beta open reading frame reported to date, and polyclonal antibodies were made against a peptide within the ER-beta B domain. By Western blot analysis, we show that ER-beta protein is expressed in all cancer cell lines tested and in three of five breast tumor samples. The breast cancer cell lines showed variation in the size of the expressed ER-beta protein. The longest form detected was consistent with the 530-amino acid, full-length ER-beta sequence. Shorter ER-beta isoforms were detected in the ER-alpha-negative MDA-MB-231 and MDA-MB-435 breast cancer cell lines, likely corresponding to previously described COOH-terminal RNA variant isoforms.

The human IL-11 receptor requires gp130 for signalling: demonstration by molecular cloning of the receptor.

We describe the molecular cloning of a cDNA for the alpha chain of the human IL-11 receptor (IL-11R alpha) and demonstrate the requirement of either the human or mouse gp130 molecule for signalling. cDNA clones encoding IL-11R alpha were isolated from a bone marrow cDNA library using a fragment from the murine IL-11R alpha as a probe. The human receptor was predicted to consist of 422 amino acids and was found to share 84% identity with the murine protein. In the extra-cellular region it exhibited a single hemopoietin domain with conserved cysteine residues and WSTWS motif. The transmembrane region was followed by a short cytoplasmic tail which did not contain a tyrosine kinase domain. Interaction of the human IL-11R alpha with murine gp130 was demonstrated: expression of the human IL-11R alpha in murine M1 cells which constitutively express murine gp130 (and murine LIF receptor), resulted in the generation of specific high-affinity binding sites for IL-11 (Kd = 250 pM). In addition, expression of the human IL-11R alpha in these cells permitted the induction of macrophage differentiation in response to IL-11. These results suggested that the human IL-11R alpha chain was able to form a functional receptor complex in association with murine gp130. The requirement of gp130 for signalling was confirmed by expression of the human IL-11R alpha in Ba/F3 cells. BaF3 cells that expressed the human IL-11R alpha alone showed binding of radiolabelled IL-11 but no proliferative response. Introduction of human gp130 into these cells resulted in high-affinity IL-11 binding sites and IL-11 dependent cellular proliferation. Thus these results demonstrated the absolute requirement of gp130 for signalling.

A novel N-aryl tyrosine activator of peroxisome proliferator-activated receptor-gamma reverses the diabetic phenotype of the Zucker diabetic fatty rat.

The discovery that peroxisome proliferator-activated receptor (PPAR)-gamma was the molecular target of the thiazolidinedione class of antidiabetic agents suggested a key role for PPAR-gamma in the regulation of carbohydrate and lipid metabolism. Through the use of high-throughput biochemical assays, GW1929, a novel N-aryl tyrosine activator of human PPAR-gamma, was identified. Chronic oral administration of GW1929 or troglitazone to Zucker diabetic fatty (ZDF) rats resulted in dose-dependent decreases in daily glucose, free fatty acid, and triglyceride exposure compared with pretreatment values, as well as significant decreases in glycosylated hemoglobin. Whole body insulin sensitivity, as determined by the euglycemic-hyperinsulinemic clamp technique, was significantly increased in treated animals. Comparison of the magnitude of glucose lowering as a function of serum drug concentrations showed that GW1929 was 2 orders of magnitude more potent than troglitazone in vivo. These data were consistent with the relative in vitro potencies of GW1929 and troglitazone. Isolated perfused pancreas studies performed at the end of the study confirmed that pancreata from vehicle-treated rats showed no increase in insulin secretion in response to a step change in glucose from 3 to 10 mmol/l. In contrast, pancreata from animals treated with GW1929 showed a first- and second-phase insulin secretion pattern. Consistent with the functional data from the perfusion experiments, animals treated with the PPAR-gamma agonist had more normal islet architecture with preserved insulin staining compared with vehicle-treated ZDF rats. This is the first demonstration of in vivo efficacy of a novel nonthiazolidinedione identified as a high-affinity ligand for human PPAR-gamma. The increased potency of GW1929 compared with troglitazone both in vitro and in vivo may translate into improved clinical efficacy when used as monotherapy in type 2 diabetic patients. In addition, the significant improvement in daily meal tolerance may impact cardiovascular risk factor management in these patients.

Peroxisome proliferator-activated receptor agonists.

The peroxisome proliferator-activated receptors are a family of three ligand-activated transcription factors. Fibrate antihyperlipidemic drugs and thiazolidinedione antihyperglycemic drugs were recently identified as synthetic ligands for these receptors. In addition, certain unsaturated fatty acids and eicosanoids were shown to bind the receptors, and thus represent naturally occurring PPAR ligands. The synthetic and natural ligands have proven to be powerful tools in dissecting the biology of these orphan receptors.

Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library.

BACKGROUND: The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown. RESULTS: A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS: The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.

The regulation of hematopoiesis in max 41 transgenic mice with sustained excess granulopoiesis.

max 41 transgenic mice consistently exhibit elevated numbers of mature granulocytes and monocytes in the peripheral blood and of immature and mature cells of these lineages in the marrow, spleen, lymph nodes and liver. The immature populations are not autonomous and exhibit a normal quantitative responsiveness to proliferative stimulation by the four colony-stimulating factors. The present studies examined three other candidate regulators of granulocyte formation and showed that max 41 cells exhibit normal quantitative responsiveness to stem cell factor, slightly enhanced responsiveness to IL-6 but reduced responsiveness to Flk-ligand. Serum levels of growth factors were not unusually elevated in max 41 mice before or after the injection of endotoxin nor were excessive levels of the four CSFs or IL-6 produced in cultures of max 41 organs. Responses to injected G-CSF were not unusually high in terms of fold-elevations in max 41 mice. Levels of mRNA for various growth factors were not abnormal in max 41 marrow populations although, in crowded cultures, max 41 marrow cells exhibited a higher level of endogenously stimulated colony formation than control cells. max 41 cells also exhibited elevated responsiveness to stimulation by mixtures of growth factors, particularly those in organ-conditioned media. The present observations suggest some possible mechanisms by which a max 41 mouse might achieve a sustained elevation of granulocyte and monocyte production but the data seem insufficient to provide a complete explanation and indicate persisting deficiencies in knowledge of how granulocyte and monocyte production is regulated.

Structure and expression of the GM-CSF receptor alpha and beta chain genes in human leukemia.

In this paper we report on the structure and expression of the genes encoding the alpha and beta chains of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in human leukemia. The alpha chain gene is highly polymorphic in normal individuals and no evidence for rearrangement within this locus was found in 47 hemopoietic, nine non-hemopoietic malignancies and five human cell lines. Using the polymerase chain reaction the gene for the alpha chain was shown to be expressed in 18/18 primary myeloid as well as 8/8 primary lymphoid leukemias analysed. To investigate the integrity of the mRNA, polymerase chain reactions (PCR) using a combination of oligonucleotides spanning the entire coding region of the alpha chain were performed. Normal sized fragments were generated with all combinations of oligonucleotides from all but one leukemia. One chronic lymphoid leukemia displayed an apparent alteration at the 3' end of the 3' untranslated region of the alpha chain mRNA. No polymorphisms were detected in the beta chain gene which was also not rearranged in any of the samples analysed. The beta chain mRNA was expressed in 17/18 primary myeloid and 7/8 primary lymphoid leukemias and in those leukemias there was no evidence for any lesions in the mRNA, as judged by PCR fragment size. Thus gross structural lesions in the genes encoding the GM-CSF receptor alpha and beta chains appear to be infrequent in hemopoietic neoplasms.