Biomedical subjects

J R Wands

Publications and source records attributed to J R Wands.

At least 19 recordsLinked to original sources

PubMed1999

A short region in the genome of hepatitis B virus is critical for maintenance of high transcript levels.

The majority of hepatitis B virus (HBV) transcripts are not normally spliced during the viral life cycle, but several splice donor and acceptor sites are conserved on HBV transcripts. In particular, the genome region between nt 450 and 500 of the HBV genome appears to be rich in such sequences. In this study we deleted a short 30-nt sequence between a conserved splice donor site at HBV genome position 462 and a splice acceptor site at position 491, thus deleting the surface/polymerase open reading frames by 10 amino acid residues. At the transcriptional level, this deletion led to >99% reduction of the 2.1-kb class of subgenomic transcripts in transfected cells. Nuclear run-on experiments revealed that the transcription rate of the deleted 2.1-kb transcript is unchanged when compared with the wildtype, suggesting a posttranscriptional mechanism for the downregulation of the deleted transcript. In addition, experiments with a replication-competent HBV mutant containing the 30-nt deletion showed that the corresponding 10-amino-acid sequence within the reverse transcriptase domain of the polymerase protein appeared to be nonessential.

Amino Acid Sequence↗

PubMed1999

Targeted gene transfer to hepatocellular carcinoma cells in vitro using a novel monoclonal antibody-based gene delivery system.

Gene therapy approaches for the treatment of malignant tumors will require high-level expression of therapeutic genes in tumors compared with normal tissues. This may be achieved either by targeted gene delivery to tumor cells or by the use of tumor-specific promoters. Here, we describe the use of a novel conjugate consisting of a tumor-reactive monoclonal antibody (mAb), designated AF-20, coupled to a DNA-binding cationic amphiphile, cholesteryl-spermine, for gene delivery to hepatocellular carcinoma (HCC) cells. The high-affinity mAb, AF-20, recognizes a rapidly internalized 180-kd cell-surface glycoprotein that is abundantly expressed on HCC and other human tumors. The AF-20 mAb and an isotype-matched control antibody (C7-57) were covalently coupled to cholesteryl-spermine. Binding and internalization of AF-20-cholesteryl-spermine was confirmed by fluorescence microscopy using fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG antibody. Following transfection of FITC-labeled oligonucleotides and ethidium monoazide-labeled plasmid DNA, cellular uptake and intracellular localization of nucleic acids were examined by laser scanning confocal microscopy. Transfection of luciferase or beta-galactosidase reporter genes complexed to AF-20-cholesteryl-spermine resulted in high levels of gene expression in AF-20 antigen-positive tumor cells. Very low levels of gene expression were observed using the control compound (C7-57-cholesteryl-spermine), which does not recognize the AF-20 tumor antigen or when AF-20 antigen-negative NIH 3T3 cells were transfected with AF-20-cholesteryl-spermine. Thus, covalent coupling of the AF-20 mAb to cholesteryl-spermine generated a highly specific and efficient nonviral vector system for targeted gene delivery to AF-20 antigen-positive HCC cells.

Animals↗

PubMed1999

Biologic significance of angiopoietin-2 expression in human hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is generally a highly vascular tumor, but the mechanisms of neovascularization that permit rapid growth have not been defined. Angiopoietins (Ang) recently have been identified as ligands for vascular endothelial-specific Tie2 receptor tyrosine kinase and may be important growth factors in the generation of new blood vessels. We investigated Ang expression in 23 samples of HCC and paired adjacent uninvolved liver samples to determine if these genes have a potential role in the growth and spread of this disease. The full coding sequence of a variant angiopoietin-2 (Ang2) cDNA was obtained from HCC specimens, and the biologic consequences of overexpression on tumor formation and hemorrhage were determined in an animal model system. Angiopoietin-1 (Ang1) was equally expressed in HCC and adjacent noncarcinomatous liver tissue. Surprisingly, Ang2 was found to be highly expressed only in tumor tissue. In addition, Ang2 was expressed in 10 of 12 hypervascular HCC, but only in 2 of 11 hypovascular HCC. Ectopic expression of Ang2 in nonexpressing HCC cells promotes the rapid development of human hepatomas and produces hemorrhage within tumors in nude mice. These results suggest a role for Ang2 in the neovascularization of HCC. This enhanced gene expression may contribute to the clinical hypervascular phenotype, as well as tumor formation and progression.

Amino Acid Sequence↗

PubMed1998

Genetic immunization generates cellular and humoral immune responses against the nonstructural proteins of the hepatitis C virus in a murine model.

Exposure to hepatitis C virus (HCV) is associated with a high prevalence of persistent viral infection and the development of chronic liver disease and hepatocellular carcinoma. Recovery from acute infection may depend upon the generation of broad-based cellular immune responses to viral structural and nonstructural proteins. We used the DNA-based immunization approach in BALB/c mice to determine whether the HCV nonstructural proteins NS3, NS4, and NS5 will induce Ab responses, CD4+ Th cell proliferation, and cytokine release in response to stimulation by recombinant proteins as well as generate CD8+ CTL activity both in vitro and in vivo. We found that the nonstructural proteins were particularly good immunogens and produced cellular immune responses when administered as a DNA construct. Indeed, a tumor model was established following inoculation of syngenic SP2/0 cells stably transfected with NS5. We observed protection against tumor formation and growth only in mice immunized with the NS5-encoding DNA construct, establishing the generation of significant CTL activity in vivo by this technique. The results indicate that genetic immunization may define the cellular immune response of the host to HCV nonstructural proteins and is a promising approach for vaccine development.

Animals↗

PubMed1998

Cloning, bacterial synthesis, and characterization of immunoglobulin variable regions of a monoclonal antibody specific for the hepatitis B virus X protein.

The nucleotide (nt) sequences encoding the variable regions of the heavy (H) and light (L) chains were determined for a murine monoclonal antibody, 12/231/93, which is specific for a linear epitope located between amino acids 90 and 102 of the hepatitis B virus (HBV) X protein (HBx). The variable (V) regions of the H and L chains were shown to belong to the mouse H chain subgroup II (C) and kappa L chain group III, respectively. The cloned variable region sequences were used for the production of a Fab fragment in Escherichia coli, which had binding activity for membrane immobilized recombinant HBx. These gene sequences may be useful for the study of HBx function in cells that will support HBV replication.

Amino Acid Sequence↗

PubMed1998

A novel frizzled gene identified in human esophageal carcinoma mediates APC/beta-catenin signals.

A novel member of the human frizzled (Fz) gene family was cloned and found to be specifically expressed in 3 of 13 well differentiated (23%), 13 of 20 moderately differentiated (62%), and 12 of 14 poorly differentiated (86%) squamous cell esophageal carcinomas compared with the adjacent uninvolved normal mucosa. The FzE3 cDNA encodes a protein of 574 amino acids and shares high sequence homology with the human FzD2 gene particularly in the putative ligand binding region of the cysteine-rich extracellular domain. Functional analysis revealed that transfection and expression of the FzE3 cDNA in esophageal carcinoma cells stimulates complex formation between adenomatous polyposis coli (APC) and beta-catenin followed by nuclear translocation of beta-catenin. Furthermore, cotransfection of a mutant construct encoding a FzE3 protein with a C-terminal truncation completely inhibited the interaction of APC with beta-catenin in cells. Finally, coexpression of FzE3 with Lef-1 transcription factor enhanced beta-catenin translocation to the nucleus. These observations suggest that FzE3 gene expression may down-regulate APC function and enhance beta-catenin mediated signals in poorly differentiated human esophageal carcinomas.

Adenomatous Polyposis Coli Protein↗

PubMed1998

A novel variant of human Grb7 is associated with invasive esophageal carcinoma.

The cDNAs of a putative growth factor-bound (Grb) 7 signal transduction molecule and Grb7V novel splice variant were isolated from an invasive human esophageal carcinoma. Although both Grb7 isoforms share homology with the Mig-10 cell migration gene, the Grb7V isoform lacks 88 base pairs in the C terminus; the resultant frame shift leads to substitution of an SH2 domain with a short hydrophobic sequence. The wild-type Grb7 protein, but not the Grb7V isoform, is rapidly tyrosyl phosphorylated in response to EGF stimulation in esophageal carcinoma cells. Analysis of human esophageal tumor tissues and regional lymph nodes with metastases revealed that Grb7V was expressed in 40% of Grb7-positive esophageal carcinomas. More importantly, Grb7V expression was enhanced after metastatic spread to lymph nodes as compared to the original tumor tissues. Finally, transfection of an antisense Grb7 RNA expression construct lowered endogenous Grb7 protein levels and suppressed the invasive phenotype exhibited by esophageal carcinoma cells. These findings suggest that Grb7 isoforms are involved in cell invasion and metastatic progression of human esophageal carcinomas.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Tightly regulated expression of the entire hepatitis C virus structural region in continuous human cell lines.

Investigation of the hepatitis C virus (HCV) life cycle is limited by the lack of an efficient cell culture system. Employing a tetracycline-regulated gene expression system we generated a panel of continuous human cell lines allowing the inducible expression and faithful processing of HCV structural proteins as well as of a functional NS2-3 autoprotease. HCV proteins were found in the cytoplasm in a pattern characteristic for the endoplasmic reticulum. High-level expression of HCV proteins was found to be cytotoxic. These cell lines represent a unique in vitro system in which to further investigate the structural proteins of HCV and to evaluate novel antiviral strategies against hepatitis C in a well-defined and reproducible cellular context.

Cell Compartmentation↗

PubMed1998

Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective.

Hepatitis B virus (HBV) variant strains may develop during therapy for chronic infection with the nucleoside analog 2',3'-dideoxy-3'-thiacytidine (3TC). HBV mutants result from isoleucine (I) or valine (V) substitutions in the methionine (M) of the YMDD motif in the viral reverse-transcriptase catalytic domain. In addition, other mutations in the reverse-transcriptase "B domain" involving either a phenylalanine (F)-to-leucine (L) at amino acid 501 (F501L) or an L-to-M substitution at amino acid 515 (L515M) have been observed during 3TC and Famciclovir therapy as well. To determine the biologic consequences of these mutations on viral replication, variant viral genomes were constructed and transiently transfected into hepatocellular carcinoma (HCC) and HEK 293 human embryo kidney-derived cell lines. In transiently transfected HCC cells, the viruses bearing the YI/VDD or F501L mutations had greatly impaired replication as compared to wild-type virus, whereas the virus carrying the L515M substitution showed the least defect. Double mutants with the L515M substitution showed intermediate defect between the YI/VDD or F501L and the L515M single-mutant strains. In contrast, when transfected into HEK 293 cells, the viruses bearing the YI/VDD or L515M mutation replicated as wild-type. However, under conditions of deoxynucleotide depletion produced by hydroxyurea treatment of HEK 293 cells, all mutants but not the wild-type virus exhibited a reduced replication phenotype similar to that observed in HCC cells. In both HCC and HEK 293 cells, the mutant viruses carrying the F501L substitution showed a decreased pregenomic RNA encapsidation level, suggesting that the defect in HBV DNA synthesis occurs at the RNA packaging level. These findings show that 3TC and Famciclovir selected mutations alter the properties of the HBV reverse transcriptase, resulting in impaired viral replication within the cell.

2-Aminopurine↗

PubMed1998

Cytokine and hepatitis B virus DNA co-immunizations enhance cellular and humoral immune responses to the middle but not to the large hepatitis B virus surface antigen in mice.

Genetic immunization is a potentially useful strategy to prevent or treat hepatitis B virus (HBV) infection. We have previously shown that HBV envelope proteins are highly immunogenic using this technique. The large envelope protein (LHBs), however, induced significantly weaker humoral and cellular immune responses when compared with the middle envelope protein (MHBs). We studied the effect of co-immunizations with cytokine DNA expression constructs encoding for interleukin (IL)-2 and (GM-CSF) on the immunogenicity of LHBs at the B-and T-cell level. Co-immunizations of mice with plasmids encoding for MHBs and IL-2 or GM-CSF increased anti-HBs responses, helper T-cell proliferative activity, and cytotoxic T lymphocyte (CTL) killing. In contrast, co-immunizations of plasmids encoding for LHBs and IL-2 or GM-CSF had no effect on humoral and cellular immune responses. LHBs did not inhibit the production or secretion of IL-2 and GM-CSF. In addition, IL-2, tumor necrosis factor alfa (TNF-alpha), and interferon gamma (IFN-gamma) had no suppressive effect on HBV envelope protein expression in vitro. Based on these data, MHBs, but not LHBs, genetic immunization can be augmented by IL-2 or GM-CSF cytokines.

Animals↗

PubMed1998

Ethanol inhibits hepatocyte proliferation in insulin receptor substrate 1 transgenic mice.

BACKGROUND & AIMS: Long-term ethanol consumption is known to impair the ability of the liver to regenerate, but the molecular mechanisms are poorly understood. Multiple growth factors promote hepatocyte proliferation, some of which involve the insulin receptor substrate 1 (IRS-1)-mediated signal transduction pathway. To explore effects of ethanol on the IRS-1 signal liver growth in vivo, studies in transgenic mice overexpressing IRS-1 in the liver were performed because these mice show constitutive activation of the downstream signal transduction pathways leading to enhanced hepatocyte proliferation. METHODS: Tyrosyl phosphorylation of IRS-1 and subsequent protein-protein interactions were examined in liver lysates from animals fed ethanol or control diet. Activity of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) was assessed by specific enzymatic assays. Hepatocyte proliferation was measured by incorporation of [3H]thymidine into liver DNA. RESULTS: Tyrosyl phosphorylation of IRS-1, association of IRS-1 with PI3K, and activation of downstream PI3K and MAPK pathways were greatly reduced as a result of long-term ethanol consumption. Ethanol virtually abolished the enhanced hepatocyte DNA synthesis induced by expression of the IRS-1 transgene. CONCLUSIONS: Altered transmission of growth signals through the IRS-1-mediated signal transduction cascade may represent a molecular mechanism of how ethanol inhibits hepatocyte proliferation.

Animals↗

PubMed1998

Antisense RNA complementary to hepatitis B virus specifically inhibits viral replication.

BACKGROUND & AIMS: Chronic infection with the hepatitis B virus (HBV) is a major public health problem, and currently available therapies have limited efficacy. Gene therapy strategies for HBV infection are under active investigation. We evaluated the potential of antisense RNA transcribed from antisense genes to interfere with HBV replication. METHODS: Subgenomic fragments of the HBV genome were studied with respect to the property of inhibiting HBV replication when intracellularly expressed in the antisense orientation. RESULTS: Antisense RNAs derived from the HBV genome specifically inhibited HBV replication and antigen expression in human hepatocellular carcinoma cells by 60%-75%. DNA sequences corresponding to the identified RNAs had no effect on HBV replication, indicating that inhibitory effects are mediated by RNA. Transcripts corresponding to the inhibitory subgenomic fragments were present at high levels. One antisense RNA was found to reduce the amount of pregenomic RNA encapsidated into core particles as a molecular mechanism of antiviral effects. CONCLUSIONS: Certain antisense RNA molecules will have substantial antiviral effects against HBV. Antisense RNAs derived from the HBV genome are promising candidates as antiviral agents and may serve as novel tools to identify functionally important regions of HBV transcripts.

Base Sequence↗

PubMed1998

Total chemical synthesis of the 3' untranslated region of the hepatitis C virus with long oligodeoxynucleotides.

Hepatitis C Virus (HCV) is the major causative agent of chronic hepatitis. Since it has been difficult to obtain full-length cDNA clones of HCV including the 3' untranslated region (UTR) that give rise to replication competent virus, we generated the 3'UTR by a modified protocol of total chemical synthesis (TCS) with overlap-extension-PCR using four long oligodeoxynucleotides. A synthetic cDNA fragment of about 340 nucleotides (nt) in length was generated, subcloned and sequenced. This approach represents a rapid and easy alternative to RT-PCR from infectious serum and may be a highly valuable method to generate partial cDNA clones of HCV and other viruses including defined variants.

3' Untranslated Regions↗

PubMed1998

Differential cellular and humoral immune responses to HCV core and HBV envelope proteins after genetic immunizations using chimeric constructs.

Development of a broad based cellular and humoral immune response to hepatitis C virus (HCV) structural proteins may be important for eradication of viral infection. In previous studies in mice we demonstrated that facilitated DNA-based immunization with an HCV core DNA-expression construct stimulated the generation of weak cytotoxic T lymphocyte (CTL), helper T cell (Th), and humoral immune responses against HCV core related epitopes. To enhance the immunogenicity of this non-secreted viral structural protein at both the B- and T-cell level, several chimeric HBV-HCV constructs were prepared which were designed to express and secrete HCV core protein along with various regions of the hepatitis B envelope protein. No secretion of the chimeric proteins into the culture supernatant was detected using sensitive radioimmunoassays. However, such chimeric proteins were capable of generating CD4+ inflammatory T cell and CD8+ CTL activity against both HBV and HCV components of the fusion proteins. It was determined that the proliferative activity of T cells as well as the humoral immune responses to HCV core protein were substantially enhanced by some chimeric fusion proteins as compared to the HCV core protein alone. The strength of the immune responses appeared directly related to the level of Th1 cytokines produced by CD4+ T cells obtained from immunized animals. Further characterization of the immune responses stimulated by these DNA constructs studied helped to define some of the most immunogenic regions of the chimeric proteins that they encode.

Animals↗

PubMed1998

Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication.

The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.

Adaptor Proteins, Signal Transducing↗

PubMed1998

The native form and maturation process of hepatitis C virus core protein.

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles.

Animals↗

PubMed1998

P53- and CD95-associated apoptosis in neurodegenerative diseases.

Apoptosis is likely to be an important mechanism of cell loss in neurodegenerative diseases, but the signaling cascades activated before DNA fragmentation have not yet been determined. p53 or CD95 gene up-regulation precedes apoptosis in many cell types, and a potential role for these molecules in apoptosis of neurons and glial cells has already been demonstrated in Alzheimer's disease (AD). To determine whether apoptosis in other neurodegenerative diseases is mediated by similar mechanisms, p53 and CD95 expression were examined in postmortem central nervous system tissues from patients with diffuse Lewy body disease (DLBD), Pick's disease (PkD), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Down's syndrome plus Alzheimer's disease (DN+AD). Quantitative immunoblot analysis demonstrated higher temporal lobe levels of p53 and CD95 proteins in DLBD, PkD, and DN+AD, and higher temporal lobe levels of CD95 only in MSA and PSP relative to PD and aged controls (for all, p < 0.01). In histologic sections, increased p53 immunoreactivity was localized in neuronal and glial cell nuclei, neuronal perikarya, and dystrophic neuritic and glial cell processes in the frontal (Area 1 1) and temporal (Area 21) lobes in DLBD, PkD, and DN+AD, the motor cortex and spinal ventral horns in ALS, and the striatum and midbrain in DLBD, MSA, PD, and PSP. Increased CD95 expression and nuclear DNA fragmentation were present in the same cell types and structures that manifested increased nuclear p53 immunoreactivity. The results suggest that p53- or CD95-associated apoptosis may be a common mechanism of cell loss in several important neurodegenerative diseases. In addition, the presence of abundant p53-immunoreactive neurites and glial cell processes appears to be a novel feature of neurodegeneration shared by these distinct diseases.

Adult↗

PubMed1998

Ethanol inhibition of insulin signaling in hepatocellular carcinoma cells.

Chronic ethanol toxicity impairs liver regeneration, inhibits DNA synthesis, and mutes cellular responses to growth factor stimulation. Previous studies demonstrated that the adverse effects of ethanol are mediated by inhibition of tyrosyl phosphorylation of the insulin receptor and the insulin receptor substrate-type 1 (IRS-1). However, overexpression of IRS-1 leads to increased DNA synthesis and cellular transformation due to constitutive activation of mitogen-activated protein (MAP) kinase. The present study examines the effects of ethanol on insulin signaling through IRS-1 in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, to determine whether such cells were resistant to the inhibitory effects of ethanol. The results demonstrated that ethanol treatment (100 mM) caused 30 to 50% reductions in the levels of insulin-stimulated tyrosyl phosphorylation of the insulin receptor beta-subunit, tyrosyl phosphorylation of IRS-1, phosphorylation of Erk2, association of phosphatidylinositol-3 kinase with tyrosyl-phosphorylated IRS-1, and MAP kinase and phosphatidylinositol-3 kinase activities. In contrast, ethanol treatment had no effect on epidermal growth factor-stimulated tyrosyl phosphorylation of Shc. Corresponding with the pronounced inhibition of MAP kinase, ethanol treatment resulted in 30 to 50% reductions in the expression levels of two important insulin-responsive genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA). The findings suggest that, in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, ethanol treatment substantially inhibits IRS-1 and MAP kinase signaling and growth-associated gene expression, but has no effect on Shc phosphorylation, which activates p21ras through an IRS-1 independent pathway.

Carcinoma, Hepatocellular↗