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Results for “Targeted expression”

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PubMed2005

Isogenic human cell lines for drug discovery: regulation of target gene expression by engineered zinc-finger protein transcription factors.

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF-generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

Amino Acid Sequence↗

PubMed2002

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced STAT5 activation and target-gene expression during human monocyte/macrophage differentiation.

GM-CSF signals through JAK2 and STAT5 and stimulates the expression of STAT5 target genes, such as pim-1 and CIS. Analyzed by EMSA, GM-CSF stimulation led to much stronger STAT5 DNA-binding to pim-1 or CIS GAS elements in primary human monocytes compared with mature macrophages. Similarly, GM-CSF-induced expression of pim-1 and CIS mRNAs was much stronger in monocytes. These differencies were not a result of downregulation of the GM-CSF receptor system or STAT5 expression, because monocytes and macrophages readily expressed GM-CSF receptor, JAK2, STAT5A, and STAT5B mRNAs and proteins. Monocytes expressed significant amounts of truncated STAT5 forms that took part in STAT5-DNA complex formation in GM-CSF-stimulated monocytes. This resulted in faster moving STAT5 complexes compared with macrophages in EMSA. Our results demonstrate that STAT5 isoform expression, GM-CSF-induced STAT5 activation, and STAT5 target-gene expression are altered significantly during monocyte/macrophage differentiation.

Cell Differentiation↗

PubMed2004

Neuroblastoma and pre-B lymphoma cells share expression of key transcription factors but display tissue restricted target gene expression.

BACKGROUND: Transcription factors are frequently involved in the process of cellular transformation, and many malignancies are characterized by a distinct genetic event affecting a specific transcription factor. This probably reflects a tissue specific ability of transcription factors to contribute to the generation of cancer but very little is known about the precise mechanisms that governs these restricted effects. METHODS: To investigate this selectivity in target gene activation we compared the overall gene expression patterns by micro-array analysis and expression of target genes for the transcription factor EBF in lymphoma and neuroblastoma cells by RT-PCR. The presence of transcription factors in the different model cell lines was further investigated by EMSA analysis. RESULTS: In pre-B cells mb-1 and CD19 are regulate by EBF-1 in collaboration with Pax-5 and E-proteins. We here show that neuroblastoma cells express these three, for B cell development crucial transcription factors, but nevertheless fail to express detectable levels of their known target genes. Expression of mb-1 could, however, be induced in neuroblastoma cells after disruption of the chromatin structure by treatment with 5-azacytidine and Trichostatin A. CONCLUSION: These data suggest that transcription factors are able to selectively activate target genes in different tissues and that chromatin structure plays a key role in the regulation of this activity.

Antigens, CD↗

PubMed2001

Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumors after systemic application.

Systemic application of positively charged polycation/DNA complexes has been shown to result in predominant gene expression in the lungs. Targeting gene expression to other sites, eg distant tumors, is hampered by nonspecific interactions largely due to the positive surface charge of transfection complexes. In the present study we show that the positive surface charge of PEI (25 kDa branched or 22 kDa linear)/DNA complexes can be efficiently shielded by covalently incorporating transferrin at sufficiently high densities in the complex, resulting in a dramatic decrease in nonspecific interactions, eg with erythrocytes, and decreased gene expression in the lung. Systemic application of transferrin-shielded PEI/DNA complexes into A/J mice bearing subcutaneously growing Neuro2a tumors via the tail vein resulted in preferential (100- to 500-fold higher) luciferase reporter gene expression in distant tumors as compared with the major organs including the lungs. Tumor targeting is also demonstrated by DNA uptake and beta-galactosidase gene expression in tumor cells. Assessing DNA distribution following systemic application significant amounts of DNA were found in the liver and tumor. However, in the liver, DNA was mainly taken up by Kupffer cells and degraded without significant transgene expression. In the tumor, DNA was associated mainly with tumor cells and frequently found near structures which resemble primitive blood vessels.

Animals↗

PubMed2000

Rapid activation of NF-kappaB and AP-1 and target gene expression in postischemic rat intestine.

BACKGROUND & AIMS: The molecular mechanisms underlying intestinal mucosal damage-repair processes induced by ischemia-reperfusion (IR) remain unknown. We determined nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1) activities and the expression of potential target genes relevant to damage-repair events. METHODS: Rat jejunal segment was subjected to ischemia for 30 minutes followed by reperfusion for defined times. NF-kappaB and AP-1 activities; mucosal p105, p50, and inhibitor kappaB-alpha (IkappaB-alpha) levels; and c-fos, neurotensin, and ferritin H expression were determined by electrophoretic mobility shift assay and Western and Northern analyses, respectively. RESULTS: NF-kappaB and AP-1 activities were significantly elevated from 1 to 12 hours after reperfusion. The activated NF-kappaB in the nuclear extract consisted of solely p50 homodimers. Activation of p50 was associated with a decrease of p105, generation of p50, and increased phosphorylation and degradation of IkappaB-alpha. The activated AP-1 contained c-fos but not c-jun, fosB, and Fra-1. Reperfusion induced a transient elevation of c-fos, prolonged increase of neurotensin, and early reduction followed by recovery of ferritin H messenger RNA. CONCLUSIONS: The intestine shows organ-specific responses to IR, characterized by prolonged NF-kappaB and AP-1 activation involving NF-kappaB p50 dimers and excluding AP-1 c-jun protein. Degradation of the IkappaB-gamma component of p105 and partial reduction IkappaB-alpha selectively activate p50/p50 dimers. Temporal patterns of target gene expression reflect functional relevance to mucosal damage-repair processes after IR.

Animals↗

PubMed2002

Multiplexed, targeted gene expression profiling and genetic analysis on electronic microarrays.

BACKGROUND: Electronic microarrays comprise independent microelectrode test sites that can be electronically biased positive or negative, or left neutral, to move and concentrate charged molecules such as DNA and RNA to one or more test sites. We developed a protocol for multiplexed gene expression profiling of mRNA targets that uses electronic field-facilitated hybridization on electronic microarrays. METHODS: A multiplexed, T7 RNA polymerase-mediated amplification method was used for expression profiling of target mRNAs from total cellular RNA; targets were detected by hybridization to sequence-specific capture oligonucleotides on electronic microarrays. Activation of individual test sites on the electronic microarray was used to target hybridization to designated subsets of sites and allow comparisons of target concentrations in different samples. We used multiplexed amplification and electronic field-facilitated hybridization to analyze expression of a model set of 10 target genes in the U937 cell line during lipopolysaccharide-mediated differentiation. Performance of multiple genetic analyses (single-nucleotide polymorphism detection, gene expression profiling, and splicing isoform detection) on a single electronic microarray was demonstrated using the ApoE and ApoER2 genes as a model system. RESULTS: Targets were detected after a 2-min hybridization reaction. With noncomplementary capture probes, no signal was detectable. Twofold changes in target concentration were detectable throughout the ( approximately 64-fold) range of concentrations tested. Levels of 10 targets were analyzed side by side across seven time points. By confining electronic activation to subsets of test sites, polymorphism detection, expression profiling, and splicing isoform analysis were performed on a single electronic microarray. CONCLUSIONS: Microelectronic array technology provides specific target detection and quantification with advantages over currently available methodologies for targeted gene expression profiling and combinatorial genomics testing.

Alternative Splicing↗

PubMed2000

Targeting gene expression to tumor cells with loss of wild-type p53 function.

The tumor suppressor protein p53 is a transcription factor that can positively regulate the expression of critical target genes involved in negative control of cell growth or induction of apoptosis; p53 is also able to suppress the transcription of other genes by virtue of its ability to bind components of the basal transcription machinery. Over 50% of human tumors are characterized by p53 mutations that result in a loss of wild-type p53 (wtp53) function in the transcriptional control of these target genes. We have exploited this loss of p53 function in the regulation of gene transcription to develop a novel gene therapy strategy that maximizes expression of the potential therapeutic gene in tumors while simultaneously down-regulating the same gene in normal cells. In one construct (unit I), the potential therapeutic gene (in this case represented by a luciferase reporter) is placed under the control of a promoter such as the heat shock protein 70 gene promoter, which is repressed by wtp53 but overexpressed in many tumor cells with defective p53 function. Residual expression of the reporter in normal cells is repressed by cotransfection of another construct (unit II) consisting of a repressor of unit I under the control of a promoter that is activated by wtp53 expression. Unit II contains a promoter with a consensus wtp53 binding site driving a transcriptional repressor or an antisense construct for the gene in unit I. Our results suggest that this dual control approach may represent a strategy with wide applications in the field of cancer gene therapy.

Feasibility Studies↗

PubMed2005

B cell antigen receptor signaling enhances IFN-gamma-induced Stat1 target gene expression through calcium mobilization and activation of multiple serine kinase pathways.

The signal transducers and activators of transcription 1 (Stat1) are essential for the majority of interferon-gamma (IFN-gamma)-regulated gene expression. Phosphorylation of serine 727 in the transcription activation domain of Stat1 is induced in response to IFN-gamma for maximal transcription activity. In this report, we show that crosslinking of B cell antigen receptor (BCR) or T cell antigen receptor (TCR) can enhance S727 phosphorylation in Stat1 and result in increased expression of Stat1 target genes. We further demonstrate that this enhancement by BCR cross-linking involves the widely used secondary messenger Ca2+ and simultaneous activation of multiple serine kinase pathways. When cells are exposed to both IFN-gamma and a Ca2+ fluxing reagent, the level of S727 phosphorylation is enhanced, resulting in increased transcription activation of Stat1 target genes. We directly demonstrate that the biochemical function of phospho-Ser-727 is to enhance the recruitment of transcription coactivator CBP/p300 to the promoters of Stat1 target genes. Furthermore, we show that both the p38 mitogen-activated protein kinase (MAPK) and the Ca(2+)/calmodulin-dependent kinase (CaMKII) are activated in response to BCR signaling to converge on Stat1 S727 for maximal gene expression. These studies demonstrate that a wide variety of noncytokine signaling pathways can modulate cytokine signaling through modulation of Stat1 serine phosphorylation.

Animals↗

PubMed2001

[Using Hsp70 promoter to regulate target gene expression in tumor].

OBJECTIVE: To regulate gene expression in the desired tumor cells both in vitro and in vivo. METHODS: Heat inducible green fluorescent protein (GFP) expression plasmid and adenovirus were built by using different sizes of 5' end regulating sequence from human heat shock protein (Hsp) gene as promoter and GFP cDNA as report gene. GFP expression turned on by heating was observed in the cultured cells and the tumor grown in the dorsal skin window chamber. RESULTS: A 400 bp of Hsp gene 5' end regulating sequence can be activated by heating and it can drive report gene expression effectively both in vitro and in vivo. CONCLUSIONS: Heating can selectively induce gene expression in the targeted tumor. It provides an useful tool for cancer gene therapy because it possibly maximizes tumor cell killing and minimizes normal tissue damage.

Adenoviridae↗

PubMed2005

[Construction and expression of siRNA expression constructs targeting epidermal growth factor receptor].

OBJECTIVE: To construct the small interfering RNA (siRNA) expression constructs targeting epidermal growth factor receptor(EGFR) and express them in TJ905 human malignant cells. METHODS: Two target sequences from Receptor L domain and catalytic domain were selected to create two expression constructs using psiRNA-NeoG2. Furthermore, the siRNA constructs were transfected into TJ905 cells as mediated by Lipofectamin. Meanwhile, an antisense EGFR construct p-anti-hEGFR was set as control. Immunofluorescence and Western blot were performed to detect EGFR expression. RESULTS: With the successful construction of the two siRNA expression plasmids and the stable transfection to TJ905 cells, the expression of EGFR was down-regulated to 90% and 92% respectively, but to 82% in the anti-sense EGFR group. CONCLUSION: The siRNA expression constructs targeting EGFR could specifically inhibit EGFR expression, and should be a new strategy in glioma gene therapy targeting EGFR.

Blotting, Western↗

PubMed2000

Target-specific expression of pre- and postsynaptic mechanisms.

Target-specific expression of pre- and postsynaptic mechanisms of synaptic transmission has been shown in a variety of central neurons by a number of laboratories. These data have demonstrated that synaptic transmission between single axons diverging onto distinct target neurons can behave independently, differentially influencing activity in the target neuron. Similarly, single neurons are capable of manufacturing molecularly distinct ligand-gated receptors and targeting them to synapses innervated by distinct converging afferent projections. A picture is emerging consistent with a role for both pre- and postsynaptic mechanisms in influencing the target-specific nature of transmission at numerous diverse synapses throughout the mammalian CNS. This target specificity adds another level of complexity in unravelling the roles played by individual neurons within a computational network. To begin to understand the coordinated activity of large ensembles of neurons it is becoming clear that the nature of transmission between individual pre- and postsynaptic elements within a circuit must first be understood for each and every neural element involved.

Animals↗

PubMed2003

Evidence for lack of enhanced hedgehog target gene expression in common extracutaneous tumors.

Abnormal hedgehog signaling, most commonly caused by loss of PTCH1 inhibitor activity,drives tumorigenesis of basal cell carcinomas (BCCs). To assess whether other tumors also have abnormal hedgehog signaling, we have assayed RNA from common cancers at nine different sites for levels of expression of hedgehog target genes that are up-regulated uniformly in BCCs. We report here that such dysregulation appears not to be common in the types of non-BCC cancers studied, indicating that the molecular pathogenesis of BCCs, like their frequency and behavior, differs markedly from that of most other cancers.

Adolescent↗

PubMedDate not supplied

Molecular imaging in drug discovery and development: potential and limitations of nonnuclear methods.

Noninvasive conventional imaging methods are established technologies in modern drug discovery and development providing valuable morphological, physiological, and metabolic information to characterize disease phenotypes, to evaluate the efficacy of therapy and to identify and develop potential biomarkers for clinical drug evaluation. The development of target-specific or molecular imaging has added a new dimension: molecular events such as the target expression, the drug-target interaction, or the activation of signal transduction pathways can be studied in the intact organism with high spatial and temporal resolution. Molecular imaging is inherently a multimodality approach. In this article, we review the role of molecular imaging for drug discovery and development focusing on nonnuclear imaging methods, i.e., magnetic resonance imaging (MRI) and optical imaging techniques based on fluorescence and bioluminescence readouts. Examples discussed are direct visualization of target expression using target-specific ligands or reporter genes, pathway imaging, and cell-trafficking studies.

Animals↗

PubMed2001

Targeting transgene expression for cystic fibrosis gene therapy.

We have developed an expression cassette for cystic fibrosis (CF) gene therapy using control elements from the human cytokeratin 18 gene (KRT18, also known as K18). KRT18 is naturally expressed in a spatial pattern similar to that of CFTR, the gene mutated in CF. We delivered a KRT18-driven lacZ plasmid complexed with cationic liposomes intravenously to mice and examined expression in various tissues. We found expression in nasal and bronchial epithelium, airway submucosal glands, gall bladder, and kidneys. Expression was low in pancreas and gut, and absent from liver and alveolar lung. This is consistent with the expression pattern reported for a K18lacZ transgenic mouse. Following delivery of a cytomegalovirus (CMV) major immediate-early promoter/enhancer-driven lacZ plasmid, we found expression in bronchi, submucosal glands, alveolar cells, liver, and kidney. We did not detect expression in nose, pancreas, gall bladder, or gut. Using fluorescently labeled plasmid delivered by means of liposomes, we identified the liver, alveolar lung, and kidneys as the major plasmid deposition sites. Our data demonstrate that a KRT18-driven expression vector delivered systemically can target gene expression to CF-affected tissues, despite an uneven distribution of plasmid DNA. A KRT18-based vector may be a useful alternative to viral promoter-based vectors in clinical gene therapy trials to treat CF.

Animals↗

PubMed2002

Mks1 in concert with TOR signaling negatively regulates RTG target gene expression in S. cerevisiae.

The target of rapamycin (TOR) signaling pathway allows eukaryotic cells to regulate their growth in response to nutritional cues. In S. cerevisiae, TOR controls the expression of genes involved in several nutrient-responsive biosynthetic pathways. In particular, we have demonstrated that TOR negatively regulates a concise cluster of genes (termed RTG target genes) that encode mitochondrial and peroxisomal enzymes required for de novo amino acid biosynthesis. TOR acts in part by regulating the subcellular localization of the Rtg1/Rtg3 transcription factor complex. Nuclear entry of this complex requires the cytoplasmic protein Rtg2, whose precise function has remained ill defined. Here we establish that the likely role of Rtg2 is to antagonize the activity of another protein, Mks1, which we demonstrate is itself a negative regulator of RTG target gene activation. Results of epistasis analyses suggest that Rtg2 and Mks1 act downstream of TOR and upstream of Rtg1 and Rtg3. Moreover, we find that Mks1 phosphorylation responds to TOR as well as to each of the Rtg1-Rtg3 proteins, indicative of complex regulation within this branch of TOR signaling. In addition to RTG target genes, microarray analysis reveals robust expression of lysine biosynthetic genes in mks1Delta cells, which depends on a functional RTG pathway. This latter result provides a molecular explanation for the previous identification of MKS1 as LYS80, a negative regulator of lysine biosynthesis [8].

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed1993

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Animals↗

PubMed2005

PML interacts with Myc, and Myc target gene expression is altered in PML-null fibroblasts.

c-myc is a well-known proto-oncogene encoding for a transcription factor that needs to be tightly regulated in order to preserve cell homeostasis. The Promyelocytic Leukaemia gene product PML plays an important role in cell growth and survival, and resides in discrete subnuclear structures called Nuclear Bodies (NB). We performed comparative analysis of the expression of 40 Myc target genes and of Myc binding to their regulatory regions both in wild-type and PML knockout cells. We demonstrate that if PML is absent, despite Myc binding to the DNA regulatory sequences is unchanged, the expression profile of several Myc target genes is altered. PML is largely involved in gene regulation, via recruitment of several transcription factors and cofactors to the NB. Consistently, we show that Myc partially localizes to the NB and physically interacts with PML, and that this localization depends on Myc expression levels. As deregulation occurs to both activated and repressed Myc target genes, we propose that PML influences Myc transcriptional activity through a mechanism that involves the control of Myc post-translational modifications.

Animals↗

PubMed1989

Rejection of skin allografts by CD4+ T cells is antigen-specific and requires expression of target alloantigen on Ia- epidermal cells.

The effector mechanism of skin allograft rejection has been characterized as Ag specific, rejecting cells that express the target alloantigen but sparing those that do not. However, the rejection of MHC class II disparate skin grafts, in which very few cells (Langerhans cells) actually express the target Ia Ag could conceivably proceed by either one of two distinct rejection mechanisms. One possibility is that Ia- cells are destroyed by a sequence of events in which CD4+ T cells, activated by Ia+ LC, elaborate soluble factors that are either directly cytolytic or that recruit and activate non-specific effector cells. The alternative possibility is that activated CD4+ T cells elaborate soluble factors which induce Ia expression on Ia- cell populations, and that these Ia+ cells are subsequently destroyed by effector cells specific for the induced Ia alloantigens. We found that rejection of Ia+ LC was not of itself sufficient to cause rejection of skin grafts, indicating that skin allograft rejection is contingent on the destruction not only of LC but of other graft cell populations as well. We then investigated whether CD4+ T cells rejected allogeneic skin grafts in an antigen specific fashion. To do so, we engrafted immunoincompetent H-2b nude mice with trunk skin grafts from B6----A/J allophenic mice because such skin is composed of mutually exclusive cell populations expressing either H-2a or H-2b histocompatibility Ag, but not both. The engrafted mice were subsequently reconstituted with H-2b CD4+ T cells. The CD4+ T cells destroyed keratinocytes of A/J origin but spared keratinocytes of B6 origin, even though neither cell population constitutively expresses target IAk alloantigen. The targeted rejection of A/J keratinocytes but not of B6 keratinocytes indicates that the target Ia alloantigen must have been induced on Ia- A/J keratinocytes, rendering them susceptible to destruction by anti-Iak-specific CD4+ effector cells. These data demonstrate that CD4+ T cell rejection of skin allografts is mediated by Ag-specific CD4+ cytolytic T cells and hence, requires the induction of target Ia alloantigens on epidermal cells within the graft.

Animals↗