Biomedical subjects

Cem Elbi

Publications and source records attributed to Cem Elbi.

16 recordsLinked to original sources

PubMed2006

Ligand-specific dynamics of the androgen receptor at its response element in living cells.

Androgens have key roles in normal physiology and in male sexual differentiation as well as in pathological conditions such as prostate cancer. Androgens act through the androgen receptor (AR), which is a ligand-modulated transcription factor. Antiandrogens block AR function and are widely used in disease states, but little is known about their mechanism of action in vivo. Here, we describe a rapid differential interaction of AR with target genomic sites in living cells in the presence of agonists which coincides with the recruitment of BRM ATPase complex and chromatin remodeling, resulting in transcriptional activation. In contrast, the interaction of antagonist-bound or mutant AR with its target was found to be kinetically different: it was dramatically faster, occurred without chromatin remodeling, and resulted in the lack of transcriptional inhibition. Fluorescent resonance energy transfer analysis of wild-type AR and a transcriptionally compromised mutant at the hormone response element showed that intramolecular interactions between the N and C termini of AR play a key functional role in vivo compared to intermolecular interactions between two neighboring ARs. These data provide a kinetic and mechanistic basis for regulation of gene expression by androgens and antiandrogens in living cells.

Adenocarcinoma↗

PubMed2006

Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus.

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.

Apoptosis↗

PubMed2006

Chromatin dynamics and the evolution of alternate promoter states.

Eucaryotic gene transcriptional switches utilize changes both in the activity and composition of soluble transcription factor complexes, and epigenetic modifications to the chromatin template. Until recently, alternate states of promoter activity have been associated with the assembly of relatively stable multiprotein complexes on target genes, with transitions in the composition of these complexes occurring on the time scale of minutes or hours. The development of living cell techniques to characterize transcription factor function in real time has led to an alternate view of highly dynamic protein/template interactions. In addition, emerging evidence suggests that energy-dependent processes contribute significantly to the rapid movement of proteins in living cells, and to the exchange of sequence-specific DNA-binding proteins with regulatory elements. Potential mechanisms involved in the unexpectedly rapid flux of factor/template interactions are discussed in the context of a "return-to-template" model for transcription factor function.

Animals↗

PubMed2005

Molecular cloning and characterization of STAMP2, an androgen-regulated six transmembrane protein that is overexpressed in prostate cancer.

We have identified a novel gene, six transmembrane protein of prostate 2 (STAMP2), named for its high sequence similarity to the recently identified STAMP1 gene. STAMP2 displays a tissue-restricted expression with highest expression levels in placenta, lung, heart, and prostate and is predicted to code for a 459-amino acid six transmembrane protein. Using a form of STAMP2 labeled with green flourescent protein (GFP) in quantitative time-lapse and immunofluorescence confocal microscopy, we show that STAMP2 is primarily localized to the Golgi complex, trans-Golgi network, and the plasma membrane. STAMP2 also localizes to vesicular-tubular structures in the cytosol and colocalizes with the Early Endosome Antigen1 (EEA1) suggesting that it may be involved in the secretory/endocytic pathways. STAMP2 expression is exquisitely androgen regulated in the androgen-sensitive, androgen receptor-positive prostate cancer cell line LNCaP, but not in androgen receptor-negative prostate cancer cell lines PC-3 and DU145. Analysis of STAMP2 expression in matched normal and tumor samples microdissected from prostate cancer specimens indicates that STAMP2 is overexpressed in prostate cancer cells compared with normal prostate epithelial cells. Furthermore, ectopic expression of STAMP2 in prostate cancer cells significantly increases cell growth and colony formation suggesting that STAMP2 may have a role in cell proliferation. Taken together, these data suggest that STAMP2 may contribute to the normal biology of the prostate cell, as well as prostate cancer progression.

Adenocarcinoma↗

PubMed2005

Ligand-specific dynamics of the progesterone receptor in living cells and during chromatin remodeling in vitro.

Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

Cell Nucleus↗

PubMed2004

A novel in situ assay for the identification and characterization of soluble nuclear mobility factors.

The development of green fluorescent protein (GFP) technology combined with live cell microscopy techniques have revealed the dynamic properties of GFP-tagged proteins in the nucleus. The mobility of a GFP-tagged protein can be assessed using a quantitative photobleaching technique, fluorescence recovery after photobleaching (FRAP) analysis. FRAP experiments demonstrate that many nuclear proteins are highly mobile within the nucleus. However, the factors within the nucleus that regulate this mobility are not known. This is partly due to an absence of protocols that can be used to identify such nuclear mobility factors. We developed a novel in situ assay that combines a biochemical permeabilization and extraction procedure with a quantitative FRAP technique, a method we used to uncover a new functional role for molecular chaperones in the nuclear mobility of steroid receptors. This assay can readily be adapted to identify and characterize other nuclear mobility factors.

Active Transport, Cell Nucleus↗

PubMed2004

Independent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiproliferative effects of indirubins.

Indirubin, a bis-indole obtained from various natural sources, is responsible for the reported antileukemia activity of a Chinese Medicinal recipe, Danggui Longhui Wan. However, its molecular mechanism of action is still not well understood. In addition to inhibition of cyclin-dependent kinases and glycogen synthase kinase-3, indirubins have been reported to activate the aryl hydrocarbon receptor (AhR), a cotranscriptional factor. Here, we confirm the interaction of AhR and indirubin using a series of indirubin derivatives and show that their binding modes to AhR and to protein kinases are unrelated. As reported for other AhR ligands, binding of indirubins to AhR leads to its nuclear translocation. Furthermore, the apparent survival of AhR-/- and +/+ cells, as measured by the MTT assay, is equally sensitive to the kinase-inhibiting indirubins. Thus, the cytotoxic effects of indirubins are AhR-independent and more likely to be linked to protein kinase inhibition. In contrast, a dramatic cytostatic effect, as measured by actual cell counts and associated with a sharp G1 phase arrest, is induced by 1-methyl-indirubins, a subfamily of AhR-active but kinase-inactive indirubins. As shown for TCDD (dioxin), this effect appears to be mediated through the AhR-dependent expression of p27(KIP1). Altogether these results suggest that AhR activation, rather than kinase inhibition, is responsible for the cytostatic effects of some indirubins. In contrast, kinase inhibition, rather than AhR activation, represents the main mechanism underlying the cytotoxic properties of this class of promising antitumor molecules.

Animals↗

PubMed2004

Kallikrein 4 is a predominantly nuclear protein and is overexpressed in prostate cancer.

Kallikreins (KLKs) are highly conserved serine proteases that play key roles in a variety of physiological and pathological processes. KLKs are secreted proteins that have extracellular substrates and function. For example, prostate-specific antigen (or KLK3) is a secreted protein that is widely used as a diagnostic marker for prostate cancer. KLK4 is a recently identified member of the kallikrein family that is regulated by androgens and is highly specific to prostate for expression. Here, we show that the gene product of KLK4, hK4, is the first member of the KLK family that is intracellularly localized. We provide strong evidence that the previously assigned first exon that was predicted to code for a signal peptide that would target hK4 for secretion is not part of the physiologically relevant form of KLK4 mRNA. In addition to detailed mapping of the KLK4 mRNA 5' end by RT-PCR, this conclusion is supported by predominantly nuclear localization of the hK4 protein in the cell, documented by both immunofluorescence and cell fractionation experiments. Furthermore, in addition to androgens, hK4 expression is regulated by estrogen and progesterone in prostate cancer cells. Finally, in situ hybridization on normal and hyperplastic prostate samples in tissue microarrays indicate that KLK4 is predominantly expressed in the basal cells of the normal prostate gland and overexpressed in prostate cancer. These data suggest that KLK4 has a unique structure and function compared with other members of the KLK family and may have a role in the biology and characterization of prostate cancer.

Animals↗

PubMed2004

Molecular chaperones function as steroid receptor nuclear mobility factors.

Live cell imaging has revealed the rapid mobility of steroid hormone receptors within nuclei and their dynamic exchange at transcriptionally active target sites. Although a number of other proteins have been shown to be highly mobile within nuclei, the identity of soluble factors responsible for orchestrating nuclear trafficking remains unknown. We have developed a previously undescribed in situ subnuclear trafficking assay that generates transcriptionally active nuclei, which are depleted of soluble factors required for the nuclear mobility of glucocorticoid (GR) and progesterone receptors (PR). Using this system and a fluorescence recovery after photobleaching technique, we demonstrate that nuclear mobility of GR recovered on incubation with reticulocyte lysate was inhibited by geldanamycin, a drug that blocks the chaperone activity of heat-shock protein 90. Direct proof of molecular chaperone involvement in steroid receptor subnuclear trafficking was provided by the ATP-dependent recovery of nuclear mobility of GR and PR on incubation with various combinations of purified chaperone and/or cochaperone proteins. Additionally, for both receptors, the inclusion of hormone during the recovery period leads to a retardation of nuclear mobility. Thus, our results provide a description of soluble nuclear mobility factors and furthermore demonstrate a previously unrecognized role for molecular chaperones in the regulation of steroid receptor function within the nucleus.

Active Transport, Cell Nucleus↗

PubMed2004

Phosphorylation of p53 at serine 37 is important for transcriptional activity and regulation in response to DNA damage.

The p53 tumor suppressor protein plays a critical role in mediating cellular response to stress. Upon DNA damage, post-translational modifications stabilize and activate this nuclear phosphoprotein. To determine the effect of phosphorylation site mutants in the context of the whole p53 protein, we performed reporter assays in p53 and MDM2 knockout mouse embryonic fibroblasts transfected with full-length p53 constructs. We show that mutation of S37 causes a decrease in p53 transcriptional activity compared to wild-type p53. Our data further suggest that the dephosphorylation of p53 at S37 is a regulated event involving protein phosphatase 2A (PP2A). Coimmunoprecipitation and immunofluorescence microscopy studies demonstrate that PP2A and p53 associate with one another in vivo following gamma-irradiation. Consistent with these observations, phosphorylated S37 accumulates in cell extracts prepared from gamma-irradiated Molt-4 cells in the presence of okadaic acid. Furthermore, in vitro phosphatase assays show that PP2A dephosphorylates p53 at S37. These results suggest that dephosphorylation of p53 at S37 plays a role in the transcriptional regulation of the p53 protein in response to DNA damage.

Catalytic Domain↗

PubMed2004

Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins.

Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic biophysical properties, such as off rate constants, residence time, and bound fraction, of a wide range of chromatin proteins of diverse functions in vivo. We demonstrate that most chromatin proteins have a high turnover on chromatin with a residence time on the order of seconds, that the major fraction of each protein is bound to chromatin at steady state, and that transient binding is a common property of chromatin-associated proteins. Our results indicate that chromatin-binding proteins find their binding sites by three-dimensional scanning of the genome space and our data are consistent with a model in which chromatin-associated proteins form dynamic interaction networks in vivo. We suggest that these properties are crucial for generating high plasticity in genome expression.

Animals↗

PubMed2004

Subnuclear trafficking and gene targeting by steroid receptors.

Through the use of novel imaging techniques, we have observed direct steroid receptor binding to a tandem array of a hormone-responsive promoter in living cells. We found that the glucocorticoid receptor (GR) exchanges rapidly with regulatory elements in the continued presence of ligand. We have also reconstituted a GR-dependent nucleoprotein transition with chromatin assembled on promoter DNA, and we discovered that GR is actively displaced from the chromatin template during the chromatin remodeling process. Using high-intensity UV laser crosslinking, we have observed highly periodic interactions of GR with promoter chromatin. These periodic binding events are dependent on GR-directed hSWI/SNF remodeling of the template and require the presence of ATP. Both the in vitro and in vivo results are consistent with a dynamic model ("hit-and-run") in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is simultaneously lost from the template. We also find that receptor mobility in the nucleoplasm is strongly enhanced by molecular chaperones. These observations indicate that multiple mechanisms are involved in transient receptor interactions with nucleoplasmic targets.

Animals↗

PubMed2003

Glucocorticoid-like effects of antihepatocarcinogen Rotenone are mediated via enhanced serum corticosterone levels: Molecular Fitting and Receptor Activation Studies.

BACKGROUND: Recent studies suggest that rotenone alters cell signal transduction pathways in a manner similar to glucocorticoids. Histological and biochemical markers of glucocorticoid effects in vivo, evaluated in our laboratories, provide further evidence for similarities in the activity of glucocorticoids and rotenone. The purpose of this study was to investigate the mechanism by which rotenone produces glucocorticoid-like effects. METHODS: Male B6C3F1 mice were treated for 7 days with rotenone (600 ppm in diet), the glucocorticoid antagonist RU486 (2 mg/kg/day, ip), corticosterone (2 mg/kg/day, ip), or both rotenone and RU 486. Control mice received drug-free diet and the vehicle (corn oil, ip). Following preservation in 10% neutral buffered formalin, tissues were embedded in paraffin. Sections were stained with hematoxylin, eosin, and were examined by light microscopy. Tissue sections were processed for in situ enzymatic end labeling of 3'-hydroxy-DNA strand breaks, a measure of apoptosis. Corticosterone was quantified in sera, using a solid phase radioimmunoassay kit. Cells (cell line 1470.2 derived from C127 mouse mammary adenocarcinoma cells) were transiently transfected with 5 &mgr;g of pLTRLuc and 1 &mgr;g of beta-Galactosidase expression vectors using a BTX square-wave pulser at 155 V, 4 pulses (40 ms each). Cells were then treated with dexamethasone, rotenone, or a mixture of both for 6 hr, harvested and assayed for luciferase and beta-Galactosidase activity. Using Root Mean Square (RMS) fit analysis (Alchemy trade mark, Tripose, Inc., St Louis, MO), we assessed possible structural similarities between rotenone and corticosterone, dehydrocorticosterone, glucocorticoid antagonists ZK 98.299, and RU 486. RMS fit was calculated by selecting three atoms in each of the molecules, followed by calculating the distance between these atoms. An RMS value of zero between two molecules indicates identical molecular characteristics. A positive value suggests diminished similarity with a value of 1 or higher excluding any such similarities. RESULTS: Although the stimulatory effect exerted by rotenone on hepatocellular apoptosis was in the opposite direction of that produced by the glucocorticoid antagonist RU 486, data suggested that rotenone does not directly activate the glucocorticoid receptor. Molecular fitting of rotenone to glucocorticoid receptor agonists and antagonists as well as examination of the transcriptional activation of a glucocorticoid-responsive reporter gene (Mouse MammaryTumorVirus) in response to rotenone indicated that it is highly unlikely that rotenone interacts directly with the glucocorticoid receptor. However, feeding male B6C3F1 mice a diet containing rotenone (600 ppm for 7 days) resulted in a 3-fold increase in serum levels of corticosterone relative to control animals. Corticosterone is the major glucocorticoid in rodents. CONCLUSION: Rotenone does not interact directly with the glucocorticoid receptor. Elevation of serum corticosterone levels in response to rotenone may explain the glucocorticoid-like effects of this compound, and may play a role in its anti-hepatocarcinogenic effect.

Journal Article↗

PubMed2002

Molecular cloning and characterization of STAMP1, a highly prostate-specific six transmembrane protein that is overexpressed in prostate cancer.

We have identified a novel gene, six transmembrane protein of prostate 1 (STAMP1), which is largely specific to prostate for expression and is predicted to code for a 490-amino acid six transmembrane protein. Using a form of STAMP1 labeled with green fluorescent protein in quantitative time-lapse and immunofluorescence confocal microscopy, we show that STAMP1 is localized to the Golgi complex, predominantly to the trans-Golgi network, and to the plasma membrane. STAMP1 also localizes to vesicular tubular structures in the cytosol and colocalizes with the early endosome antigen 1 (EEA1), suggesting that it may be involved in the secretory/endocytic pathways. STAMP1 is highly expressed in the androgen-sensitive, androgen receptor-positive prostate cancer cell line LNCaP, but not in androgen receptor-negative prostate cancer cell lines PC-3 and DU145. Furthermore, STAMP1 expression is significantly lower in the androgen-dependent human prostate xenograft CWR22 compared with the relapsed derivative CWR22R, suggesting that its expression may be deregulated during prostate cancer progression. Consistent with this notion, in situ analysis of human prostate cancer specimens indicated that STAMP1 is expressed exclusively in the epithelial cells of the prostate and its expression is significantly increased in prostate tumors compared with normal glands. Taken together, these data suggest that STAMP1 may have an important role in the normal prostate cell as well as in prostate cancer progression.

Amino Acid Sequence↗

PubMed2002

Protein dynamics in the nuclear compartment.

The classic view of a transcriptional initiation complex is that of an assembly of factors with many protein-protein contacts, leading to a multi-component complex whose existence is a result of the stabilizing influence of the many intermolecular interactions. Recent findings from protein mobility experiments in living cells indicate that many kinds of nuclear factors move rapidly and exchange quickly with multiple targets. Two countervailing views of factor/regulatory site interactions emerge from the current literature.

Animals↗

PubMed2002

Recruitment of dioxin receptor to active transcription sites.

The aryl hydrocarbon receptor (AhR or dioxin receptor) is a ligand-activated transcription factor that heterodimerizes with the AhR nuclear translocator (ARNT/HIF-1beta) to form an AhR/ARNT transcription factor complex. This complex binds to specific DNA sites in the regulatory domains of numerous target genes and mediates the biological effects of exogenous ligands. Herein, we have investigated the subcellular distribution of the AhR/ARNT complex in response to ligand stimulation, by using live-cell confocal and high-resolution deconvolution microscopy. We found that unliganded AhR shows a predominantly cytoplasmic diffuse distribution in mouse hepatoma cells. On addition of ligand, AhR rapidly translocates to the nucleus and accumulates in multiple bright foci. Inhibition of transcription prevented the formation of AhR foci. Dual- and triple-immunolabeling experiments, combined with labeling of nascent RNA, showed that the foci are transcription sites, indicating that upon ligand stimulation, AhR is recruited to active transcription sites. The interaction of AhR with ARNT was both necessary and sufficient for the recruitment of AhR to transcription sites. These results indicate that AhR/ARNT complexes are recruited to specific subnuclear compartments in a ligand-dependent manner and that these foci represent the sites of AhR target genes.

Amanitins↗