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1.
Clin Pharmacol Ther ; 88(2): 204-13, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20164833

ABSTRACT

Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor in humans. Systemic immunity against gene therapy vectors has been shown to hamper therapeutic efficacy; however, helper-dependent high-capacity adenovirus (HC-Ad) vectors elicit sustained transgene expression, even in the presence of systemic anti-adenoviral immunity. We engineered HC-Ads encoding the conditional cytotoxic herpes simplex type 1 thymidine kinase (TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Flt3L). Flt3L expression is under the control of the regulatable Tet-ON system. In anticipation of a phase I clinical trial for GBM, we assessed the therapeutic efficacy, biodistribution, and clinical and neurotoxicity with escalating doses of HC-Ad-TetOn-Flt3L + HC-Ad-TK in rats. Intratumoral administration of these therapeutic HC-Ads in rats bearing large intracranial GBMs led to long-term survival in approximately 70% of the animals and development of antiglioma immunological memory without signs of neuropathology or systemic toxicity. Systemic anti-adenoviral immunity did not affect therapeutic efficacy. These data support the idea that it would be useful to develop HC-Ad vectors further as a therapeutic gene-delivery platform to implement GBM phase I clinical trials.


Subject(s)
Adenoviridae/genetics , Brain Neoplasms/therapy , Genetic Vectors/pharmacokinetics , Genetic Vectors/therapeutic use , Glioblastoma/therapy , Adenoviridae/immunology , Adjuvants, Immunologic/therapeutic use , Animals , Behavior, Animal , Brain Neoplasms/psychology , Clinical Trials, Phase I as Topic , Dose-Response Relationship, Immunologic , Gene Dosage , Genetic Therapy , Genetic Vectors/adverse effects , Glioblastoma/psychology , Humans , Immunohistochemistry , Injections , Neoplasm Transplantation , Rats , Survival Analysis , Tissue Distribution , Transgenes/genetics
2.
J Virol ; 83(4): 2004-10, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19073729

ABSTRACT

Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3' end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences.


Subject(s)
Adenoviridae/genetics , Genetic Vectors , Herpesvirus 1, Human/enzymology , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism , Gene Expression , Hepatitis B Virus, Woodchuck/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
3.
Hum Gene Ther ; 17(5): 531-44, 2006 May.
Article in English | MEDLINE | ID: mdl-16716110

ABSTRACT

First-generation adenoviral (Ad) and high-capacity adenoviral (HC-Ad) vectors are efficient delivery vehicles for transferring therapeutic transgenes in vivo into tissues/organs. The initial successes reported with adenoviral vectors in preclinical trials have been limited by immune-related adverse side effects. This has been, in part, attributed to the use of poorly characterized preparations of adenoviral vectors and also to the untoward immune adverse side effects elicited when high doses of these vectors were used. HC-Ads have several advantages over Ads, including the lack of viral coding sequences, which after infection and uncoating, makes them invisible to the host's immune system. Another advantage is their large cloning capacity (up to approximately 35 kb). However, accurate characterization of HC-Ad vectors, and of contaminating replication-competent adenovirus (RCA) or helper virus, is necessary before these preparations can be used safely in clinical trials. Consequently, the development of accurate, simple, and reproducible methods to standardize and validate adenoviral preparations for the presence of contaminant genomes is required. By using a molecular method that allows accurate, reproducible, and simultaneous determination of HC-Ad, contaminating helper virus, and RCA genome copy numbers based on real-time quantitative PCR, we demonstrate accurate detection of these three genomic entities, within CsCl-purified vector stocks, total DNA isolated from cells transduced in vitro, and from brain tissue infected in vivo. This approach will allow accurate assessment of the levels and biodistribution of HC-Ad and improve the safety and efficacy of clinical trials.


Subject(s)
Adenoviridae/genetics , Genetic Vectors/standards , Genome, Viral , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/standards , Adenoviridae/isolation & purification , Animals , Base Sequence , Biological Assay , Brain/virology , Cell Line , DNA, Viral/metabolism , Female , Genetic Vectors/adverse effects , Helper Viruses/genetics , Helper Viruses/isolation & purification , Mice
4.
J Biol Chem ; 276(21): 18066-74, 2001 May 25.
Article in English | MEDLINE | ID: mdl-11278484

ABSTRACT

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a beta-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize beta-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not beta-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via beta-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a beta-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta-arrestin-dependence was observed. Visualization of beta-arrestin/GFP redistribution confirmed a loss or gain of beta-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for beta-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by beta-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the beta-arrestin-dependent pathway.


Subject(s)
Arrestins/metabolism , Receptors, LHRH/metabolism , Receptors, Thyrotropin-Releasing Hormone/metabolism , Amino Acid Sequence , Animals , COS Cells , Casein Kinase II , Molecular Sequence Data , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptors, LHRH/genetics , Receptors, Thyrotropin-Releasing Hormone/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , beta-Arrestins
5.
J Biol Chem ; 276(16): 12736-43, 2001 Apr 20.
Article in English | MEDLINE | ID: mdl-11278883

ABSTRACT

The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the thyrotropin-releasing hormone receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing hormone (GnRH) or beta(2)-adrenergic receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time- and dose-dependent increase in the amount of energy transfer. Inhibition of receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, beta-arrestin, with TRHR and the absence of an interaction of beta-arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes.


Subject(s)
Receptors, Thyrotropin-Releasing Hormone/chemistry , Receptors, Thyrotropin-Releasing Hormone/physiology , Triptorelin Pamoate/analogs & derivatives , Amino Acid Substitution , Animals , Arrestins/metabolism , Bacterial Proteins/analysis , Bacterial Proteins/genetics , COS Cells , Cell Line , Cell Membrane/physiology , Chlorocebus aethiops , Coated Pits, Cell-Membrane/physiology , Energy Transfer , Humans , Iodine Radioisotopes , Luciferases/analysis , Luciferases/genetics , Luminescent Measurements , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Macromolecular Substances , Mutagenesis, Site-Directed , Rats , Receptors, LHRH/agonists , Receptors, LHRH/chemistry , Receptors, LHRH/physiology , Receptors, Thyrotropin-Releasing Hormone/agonists , Recombinant Fusion Proteins/agonists , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Transfection , Triptorelin Pamoate/pharmacokinetics , beta-Arrestins
6.
J Biol Chem ; 275(24): 18432-40, 2000 Jun 16.
Article in English | MEDLINE | ID: mdl-10748079

ABSTRACT

Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the -106 to -88 bp region of the TNF-alpha promoter.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Glucocorticoids/pharmacology , Monocytes/metabolism , NF-kappa B/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Tumor Necrosis Factor-alpha/biosynthesis , Activating Transcription Factor 2 , Animals , Base Sequence , Binding Sites , Cell Line , Humans , Lipopolysaccharides/pharmacology , Luciferases/metabolism , Mice , Molecular Sequence Data , Transcriptional Activation/drug effects , Transfection , Tumor Necrosis Factor-alpha/genetics
7.
Cytokine ; 12(2): 110-9, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10671295

ABSTRACT

The authors have previously demonstrated that the tumour necrosis factor (TNF) -308 G/A polymorphism affects the binding of transcription factors. In transient transfection assays in PMA stimulated U937 monocytes and Jurkat T cells, the A-containing TNF2 promoter has a 2-3-fold greater transcriptional activity than the TNF1 promoter in the presence of the TNF 3'UTR. In this study it was found that a difference in TNF1 and TNF2 promoter activities was only observed in U937 and Jurkat cells, and not in Raji (B cell line), HeLa (epithelial carcinoma cell line), HepG2 (hepatoma cell line) or THP-1 (monocyte), suggesting cell-type specific transcription factors or modifications may be involved in the formation of the -308 protein/DNA complex. Physiological stimulators, TNF and interferon gamma (IFN-gamma) did not cause differential promoter activity between TNF1 and TNF2, but LPS did with only the TNF2 promoter/3'UTR construct being significantly responsive to lipopolysaccharide (LPS) in U937 cells. In U937 cells, the -308 polymorphism affected transcription following differentiation by phorbol myristate acetate (PMA), retinoic acid, PMA plus LPS and PMA plus retinoic acid with an increase in nuclear factor binding to both TNF1 and TNF2 in the -323 to -285 region being observed. The greatest difference between TNF2 and TNF1 promoter activities (5-fold) was observed following PMA plus retinoic acid treatment of transfected U937 cells for 48h. During this time, U937 differentiated into cells with a macrophage-like morphology. An understanding of the cell type and stimuli specific requirements for differential expression of the -308 polymorphism may help elucidate the role the TNF -308 polymorphism plays in diseases where elevated TNF levels are thought to be important.


Subject(s)
Polymorphism, Genetic , Promoter Regions, Genetic , Tumor Necrosis Factor-alpha/genetics , 3' Untranslated Regions , Base Sequence , Binding Sites/genetics , Cell Differentiation , Cell Line , DNA/genetics , DNA/metabolism , Gene Expression , HeLa Cells , Humans , Jurkat Cells , Nuclear Proteins/metabolism , Transfection , U937 Cells
8.
J Leukoc Biol ; 66(4): 562-6, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10534109

ABSTRACT

A biallelic G (TNF1 allele) to A (TNF2 allele) polymorphism 308 nucleotides upstream from the transcription initiation site in the tumor necrosis factor (TNF) promoter is associated with elevated TNF levels and disease susceptibilities observed in human subjects. The TNF2 allele is strongly associated with the high-TNF-producing autoimmune MHC haplotype HLA-A1, B8, DR3, with elevated serum TNF levels and a more severe outcome in infectious diseases, such as cerebral malaria. A number of groups have set out to determine whether the -308 polymorphism could affect transcription factor binding and hence influence TNF transcription and expression levels. Although some studies have failed to show any functional difference between the two allelic forms, others have shown that the -308 polymorphism effected transcription factor binding to the region encompassing -308, with the region in the TNF2 allele showing altered binding characteristics. The -308 polymorphism also has been found by some groups to be functionally significant in reporter gene assays in Raji B cells, Jurkat T cells, and U937 pre-monocytic cells. Up to fivefold differences can be measured between TNF1 and TNF2 allelic constructs when the TNF 3'UTR is present, indicating a role in the expression of the polymorphism. Although controversial, the majority of the data support a direct role for the TNF2 -308 allele in the elevated TNF levels observed in TNF2 homozygotes and HLA-A1, B8, DR3 individuals. Elevated TNF levels due to the -308 polymorphism may alter the immune response such that it confers susceptibility to certain autoimmune and infectious diseases.


Subject(s)
Gene Expression Regulation , Polymorphism, Genetic , Promoter Regions, Genetic , Tumor Necrosis Factor-alpha/genetics , Animals , Humans , Immunity , Transcription Factors/metabolism , Transcription, Genetic , Tumor Necrosis Factor-alpha/immunology
10.
Mol Immunol ; 34(5): 391-9, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9293772

ABSTRACT

Since the tumor necrosis factor alpha (TNF-alpha) gene was found to be located in the central major histocompatibility complex (MHC) there has been much speculation concerning a genetic association between particular TNF alleles and disease susceptibility. A relationship between the MHC haplotype A1, B8, DR3, TNF-alpha expression levels and susceptibility to autoimmune disease has been suggested by several groups. The identification of the -308 polymorphism and its association with the HLA A1, B8, DR3 haplotype have led to speculation that the polymorphism may play a role in the altered expression of TNF-alpha. We have demonstrated that the region (-323 to -285) encompassing -308 in the TNF2 allele binds nuclear factors differently to the same region in the promoter of the more common TNF1 allele. The G/A -308 polymorphism affected the affinity of factor binding and resulted in a factor binding to TNF2 but not TNF1. The observed differential binding was shown to be functional, with the 38bp region from TNF2 causing a two-fold greater activity of a heterologous promoter over that due to the same region in TNF1. To further substantiate the functional consequences of the TNF-alpha -308 polymorphism, we analysed both allelic forms of the TNF-alpha promoter region (-993 to +110) in a transient transfection assay, using luciferase as a reporter gene. The results showed that when present with the 3'UTR the -308A allelic form gave a two-fold greater level of transcription than the 308G form in PMA-stimulated Jurkat and U937 cells. This suggests that the -308 G/A polymorphism may play a role in the altered TNF-alpha gene expression observed in individuals with the HLA A1, B8, DR3 haplotype.


Subject(s)
Polymorphism, Genetic , Promoter Regions, Genetic/immunology , Transcription, Genetic/immunology , Tumor Necrosis Factor-alpha/genetics , Base Sequence , Carcinoma, Hepatocellular , Cell Line, Transformed , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Jurkat Cells , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolism
11.
Biochem Mol Biol Int ; 40(1): 43-51, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8886268

ABSTRACT

We have identified a region of the human TNF-alpha promoter between nucleotides -323 and -285, capable of influencing transcriptional activity. This region encompasses the -308 polymorphism and contains a 10 bp sequence homologous to the consensus binding site of activator protein-2 (AP-2). Protein complexes derived from U937 and Jurkat cells were found to bind to this element. Competitive EMSA using a consensus AP-2 oligonucleotide indicated that AP-2 may be involved. Functional assays demonstrate that this region can repress activity of a heterologous promoter in the Jurkat T-cell line, but act as an inducible enhancer of transcription in U937 cells.


Subject(s)
DNA-Binding Proteins/chemistry , Transcription Factors/chemistry , Tumor Necrosis Factor-alpha/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation , Humans , Jurkat Cells , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factor AP-2 , Transfection
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