Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to understand the structural features that might lead to an estrogen receptor (ER) based breast tumor imaging agent with improved uptake characteristics, we have synthesized several new analogs of 16 beta-fluoroestradiol (beta FES) and studied their tissue distribution in immature rats. The compounds we prepared were 11 beta-methoxy-beta FES (7a), 11 beta-ethyl-beta FES (7b), 17 alpha-ethynyl-beta FES (8c), 17 alpha-ethynyl-11 beta-methoxy-beta FES (8a), and 11 beta-ethyl-17 alpha-ethynyl-beta FES (8b). All of the analogs exhibit good affinity for ER, ranging at 25 degrees C from 10 to 460, with estradiol equal to 100. Measurement of their octanol/water partition coefficients by an HPLC method allowed us to estimate their level of nonspecific binding and thereby to predict their binding selectivity indices (BSI, i.e., the ratio of their ER-specific to nonspecific binding); the BSI values of three fluorine-substituted analogs exceed that of estradiol. These ligands have been labeled in the 16 beta position with fluorine-18 by the nucleophilic displacement of an alpha-disposed trifluoromethanesulfonate by [18F]fluoride ion. Reduction with lithium aluminum hydride produced the estradiol series ([18F]-7a-c), while treatment with lithium trimethylsilylacetylide afforded the ethynylated series ([18F]-8a-c). The synthesis time was 85 min for [18F]-7a-c and 120 min for [18F]-8a-c, with radiochemical yields ranging from 16 to 43%, and effective specific activities being 90-2900 Ci/mmol (3.3-107 TBq/mmol). In tissue distribution studies in immature female rats, all of the labeled analogs demonstrated ER-selective uptake in the principal target tissues, the uterus and the ovaries, and also in organs with lower titers of ER, the secondary target sites kidney, thymus, fat, and muscle. Although factors other than specific and nonspecific binding obviously affect the tissue distribution of these 16 beta-fluoroestrogens, we find that their ER-specific uptake by both the principal and the secondary target tissues correlates with their BSI values at a high level of statistical significance in most cases. The ethynylated-11 beta-methoxy analog [18F]-8a had high selectivity (uterus to blood ratio) after 3 h and exhibited the highest uterine uptake (percent injected dose/gram) of any fluorine-substituted estradiol ligand we have studied to date. This compound has been chosen for more detailed studies (to be described elsewhere), including clinical trials in human patients diagnosed with primary breast cancer.
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PMID:16 beta-([18F]fluoro)estrogens: systematic investigation of a new series of fluorine-18-labeled estrogens as potential imaging agents for estrogen-receptor-positive breast tumors. 768 51

Activated forms of the nuclear and cytoplasmic tyrosine kinase c-Abl are completely cytoplasmic and oncogenic. The overexpression of c-Abl, and in certain fibroblast cell lines even of v-Abl, leads to a cell cycle arrest revealing an alternative Abl function. To facilitate the analysis of this growth inhibitory function we have taken advantage of regulable Abl-estrogen receptor (ABL:ER) fusion proteins. Oncogenic in the presence of estrogen, they are reversibly switched to inhibit cell proliferation upon removal of hormone. Using this system, we demonstrate that inhibition is effected by Abl derivatives which we have previously shown to be hypo-phosphorylated and to have low kinase activity. Since an almost exclusively cytoplasmic ABL:ER protein is fully growth inhibitory, relevant interactions may occur in the cytoplasm. We identify the cell cycle arrest as an early G1 or G0-like block. Interestingly, growth inhibition correlates with an altered expression pattern of early serum response genes; c-Jun mRNA and c-Fos protein levels are elevated in Abl-blocked cells. In view of the two functional modes of overexpressed Abl proteins, one can speculate that normal c-Abl may be involved in relaying growth regulatory signals from the membrane to the nucleus.
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PMID:Cell cycle arrest by tyrosine kinase Abl involves altered early mitogenic response. 773 83

Estrogen receptors are expressed in several brain areas of various animal species, and steroid hormones exert physiologic and biochemical effects on the central nervous system. The aim of the present study was to evaluate in female adult rats, the suitability of 16 alpha [18F]fluoro-17 beta-estradiol ([18F]FES), a selective estrogen receptor ligand, for the in vivo assessment of brain estrogen receptors. This was considered to be a preliminary step in evaluating the potential usefulness of [18F]FES for studies of cerebral estrogen receptors with positron emission tomography (PET) in nonhuman primates and human subjects. We evaluated (a) the time course of the metabolic degradation of [18F]FES in blood; (b) the time course of distribution of the tracer in discrete cerebral areas; (c) the inhibitory effect of increasing doses of cold estradiol on cerebral [18F]FES uptake; and (d) the possibility of in vivo quantification of estrogen receptor binding parameters using both equilibrium and dynamic kinetic analyses. We quantified [18F]FES binding to estrogen receptors using both equilibrium and dynamic kinetic analyses. The results of this study indicate that [18F]FES is a suitable tracer for the measurement of estrogen receptors in the pituitary and hypothalamus, using either the equilibrium or the kinetic analysis. However, [18F]FES is inadequate for the in vivo investigation of estrogen binding sites in brain areas with low receptor density, such as the hippocampus.
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PMID:Systemic and cerebral kinetics of 16 alpha [18F]fluoro-17 beta-estradiol: a ligand for the in vivo assessment of estrogen receptor binding parameters. 786 Jun 63

We have measured in vivo the uptake of 16 alpha-[18F]estradiol (FES) by target tissues in the immature rat at increasing dose levels (obtained by dilution of [18F]FES with unlabeled estradiol). This was done to examine the binding capacity of target tissues in vivo and to determine whether the uptake in receptor-rich tissues was flow limited, as this has implications concerning the appropriateness of using receptor-rich tissues in experimental animals as models for FES uptake by receptor-poor breast tumors in humans. We also wanted to establish the dose level of the anti-estrogen tamoxifen required to block target tissue uptake of FES. We found that in untreated rats, specific uptake in the uterus saturated at c. 180 pmol/g, in the ovary at c. 54 pmol/g and in the muscle at c. 2 pmol/g. At an intermediate dose of tamoxifen (570 micrograms/kg), uptake saturated at somewhat lower levels, and at a high tamoxifen dose (1710 micrograms/kg), yet lower specific uptake was evident. In the FES titrations at low dose levels of FES, both the uterus and the ovaries, but not the muscle, showed characteristics of flow-limited uptake, i.e. the uptake-to-dose ratio reached a maximum level. This flow limitation suggests that only when receptor levels are sufficiently low will the FES uptake be related to receptor concentration. While receptor-rich tissues such as the rat uterus will show this flow limitation, the receptor concentration in most primary and metastatic human breast tumors is sufficiently low, so that the uptake should parallel receptor content. In in vivo distribution studies, target tissues (or tumors) with low receptor content will be more fully saturated and ligand more readily displaced. Also, uptake by secondary target tissues (i.e. those with a lower content of estrogen receptor, such as muscle, thymus and kidney) may be better models for assessing the effectiveness of new breast tumor imaging agents than uptake by receptor-rich tissues.
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PMID:Titration of the in vivo uptake of 16 alpha-[18F]fluoroestradiol by target tissues in the rat: competition by tamoxifen, and implications for quantitating estrogen receptors in vivo and the use of animal models in receptor-binding radiopharmaceutical development. 840 74

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-beta (ER-beta) and the orphan nuclear receptor nur 77. No receptors for ER-alpha, progesterone, LH, or PRL could be detected. This cell line also expressed 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20alpha-HSD mRNA expression. In addition, estradiol enhanced ER-beta expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.
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PMID:Establishment and characterization of a simian virus 40-transformed temperature-sensitive rat luteal cell line. 952 80

Progesterone receptor (PR) is an estrogen-stimulated gene which has a CpG island that is heavily methylated in a significant fraction of estrogen receptor (ER)-negative/PR-negative human breast cancers and cell lines, including MDA-MB-231 cells. Treatment of MDA-MB-231 cells with the demethylating agent, 5-aza-2'-deoxycytidine (deoxyC) led to demethylation and expression of ER and PR. However, simultaneous treatment with antiestrogen prevented PR transcription, suggesting that demethylation of PR alone is not sufficient to reactivate the PR gene. To examine the effects of ER on the methylation status of the PR CpG island, we stably transfected MDA-MB-231 cells with an inducible expression vector for ER. Surprisingly, in two cell clones, we found that induction of PR gene expression by ligand-bound ER does not require demethylation of the PR CpG island. In contrast, induction of PR transcription was inhibited by blocking the interaction of ER with SRC-1A, a coactivator of ER function. For the first time, we show that a transcription factor with the potential to remodel heterochromatin can activate gene expression without altering the methylation status of the CpG island. These results raise the possibility that demethylation and histone acetylation are distinct but complementary mechanisms for destabilizing heterochromatin and activating transcription.
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PMID:Demethylation of the progesterone receptor CpG island is not required for progesterone receptor gene expression. 970 23

Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3') in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARgamma activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.
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PMID:Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. 980 23

MCF-7 human breast carcinoma cultures grown in the presence of 17-beta estradiol form solid, multicellular nodules, a process that reflects changes in cell-substrate adhesions and loss of growth inhibition. We examined the effects of estradiol on the status of tyrosine phosphorylation in focal adhesion kinase (FAK) and the association of FAK with paxillin using immunoprecipitations and then probing western blots for FAK, phosphotyrosine, and paxillin. Culture of MCF-7 cells for seven days in the presence of 1 nM E2 resulted in decreased tyrosine phosphorylation of FAK compared to controls. The estradiol-induced effect was blocked by 100 nM of the estrogen receptor antagonist 4-hydroxytamoxifen, indicating dephosphorylation of FAK is an estrogen receptor-mediated event. FAK immunoprecipitated from either estradiol or DMSO-treated cells phosphorylated the exogenous substrate poly(Glu,Tyr), suggesting that the potential kinase activity of FAK was not changed by estradiol. Estradiol treatment also resulted in a reduced association between FAK and paxillin. The decreased phosphorylation levels and reduced association between FAK and paxillin may be important steps leading to the loss of stable focal contacts and loss of growth inhibition during MCF-7 nodulation.
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PMID:Decreased tyrosine phosphorylation of focal adhesion kinase after estradiol treatment of MCF-7 human breast carcinoma cells. 987 83

16beta-[18F]Fluoromoxestrol ([18]betaFMOX) is an analog of 16alpha-[18F]fluoroestradiol-17beta ([18F]FES), a radiopharmaceutical known to be an effective positron emission tomography (PET) imaging agent for estrogen receptor-positive (ER+) human breast tumors. Based on comparisons of target tissue uptake efficiency and selectivity in a rat model, [18F]betaFMOX was predicted to be as effective an imaging agent as [18F]FES. However, in a preliminary PET imaging study with [18F]FMOX of 12 patients, 3 of whom had ER+ breast cancer, no tumor localization of [18F]betaFMOX was observed. In search for an explanation for the unsuccessful [18F]betaFMOX clinical trial, we have examined the rate of metabolism of [18F]FMOX and [18F]FES in isolated rat, baboon, and human hepatocytes. We have also studied the effect of the serum protein sex hormone-binding globulin (SHBG), which binds [18F]FES better than [18F]betaFMOX, on these rates of metabolism. Immature rat hepatocytes were found to metabolize [18F]FES 31 times faster than [18F]betaFMOX, whereas mature rat cells metabolized [18F]FES only 3 times faster, and baboon and human hepatocytes only 2 times faster than [18F]betaFMOX. In the presence of SHBG, the metabolic consumption rate for [18F]FES in mature rat hepatocytes decreased by 26%. Thus, the very favorable target tissue uptake characteristics of [18F]betaFMOX determined in the rat probably result from its comparative resistance to metabolism (vis-a-vis [18F]FES) in this species, an advantage that is strongly reflected in comparative metabolism rates in rat hepatocytes. In the baboon and human, [18F]FES is extensively protein bound and protected from metabolism, an effect that may be reflected to a degree as a decrease in the rate of metabolism of this compound in baboon and human hepatocytes relative to [18F]betaFMOX. Thus in primates, SHBG may potentiate the ER-mediated uptake of [18F]FES in ER+ tumors by selectively protecting this ligand from metabolism and ensuring its delivery to receptor-containing cells. In addition to current screening methods for 18F-estrogens that involve evaluating in vivo ER-mediated uptake in the immature female rat, studies comparing the metabolism of the new receptor ligands in isolated hepatocytes, especially those from primates or humans, may assist in predicting the potential of these ligands for human PET imaging.
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PMID:Comparative breast tumor imaging and comparative in vitro metabolism of 16alpha-[18F]fluoroestradiol-17beta and 16beta-[18F]fluoromoxestrol in isolated hepatocytes. 1009 12

Dendritic cells are professional antigen presenting cells that capture antigens and migrate to lymphoid tissues to elicit specific T cell responses. Here we used an in vitro differentiation system for generating highly motile dendritic cells from chicken bone marrow progenitors by employing the conditional v-Rel estrogen receptor (ER) fusion protein v-RelER. Molecular mechanisms of dendritic cell motility were investigated. Differentiation of v-relER progenitors into dendritic cells is associated with a reduction in cell-cell and cell-extracellular matrix interactions as cells acquire motility. We demonstrate that v-relER progenitors and dendritic cells express several adhesion receptors and components of adhesion complexes. Differentiation of v-relER cells was accompanied by downregulation of focal adhesion kinase (FAK), a key molecule of adhesion complexes, but ectopic FAK expression did not affect cell adhesion and motility. Interestingly, v-relER dendritic cells exhibit a polarised expression pattern of actin and vimentin, with actin being highly concentrated at the leading edge of the cells where lamellipodia are formed. FAK, paxillin and tyrosine phosphorylated proteins are found at both poles of the cell and colocalise with actin at the leading edge, while surface beta1 integrin is confined to the uropod at the rear. CD34(+ )stem cell-derived human dendritic cells also exhibited an elongated bipolar morphology, mode of migration and a polarised pattern of actin-vimentin expression similar to v-relER dendritic cells.
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PMID:Polarised expression pattern of focal contact proteins in highly motile antigen presenting dendritic cells. 1031 61


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