Search PubMed⌕ Search

Biomedical subjects

G J Nabel

Publications and source records attributed to G J Nabel.

At least 55 records · Page 3Linked to original sources
PubMed1998

Modulation of cytokine-induced HIV gene expression by competitive binding of transcription factors to the coactivator p300.

The host response to viral infection involves the secretion of multiple cytokines which alter immune function and viral replication. These proteins activate several signal transduction pathways in infected cells which must be integrated to regulate cellular and viral gene expression. In this report, we demonstrate that specific transcription factors induced by distinct cytokines regulate HIV transcription by competitive binding to the p300 coactivator. Interferon-alpha (IFN-alpha) was found to inhibit NF-kappaB-dependent HIV gene expression stimulated by tumor necrosis factor-alpha (TNF-alpha). This inhibition was mediated by binding of the IFN-alpha signal transducer and activator of transcription 2, Stat2, to a specific domain of p300 which also binds to the RelA (p65) subunit of NF-kappaB. p300 was found to be limiting with respect to RelA (p65) and Stat2, and this effect was reversed by overexpression of p300. Inhibition by Stat2 was specific for NF-kappaB and was not mediated by Stat1, which is also induced by IFN-alpha. Gene activation induced by the Stat2 transcription domain was also inhibited by expression of RelA. These results demonstrate that HIV transcription can be regulated in the nucleus by competitive binding of specific cytokine-induced transcription factors to a discrete domain of a transcriptional coactivator.

Binding, Competitive↗
PubMed1998

HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300 co-activator.

The accessory protein, Vpr, is a virion-associated protein that is required for HIV-1 replication in macrophages and regulates viral gene expression in T cells. Vpr causes arrest of cell cycle progression at G2/M, presumably through its effect on cyclin B1.Cdc2 activity. Here, we show that the ability of Vpr to activate HIV transcription correlates with its ability to induce G2/M growth arrest, and this effect is mediated by the p300 transcriptional co-activator, which promotes cooperative interactions between the Rel A subunit of NF-kappaB and cyclin B1.Cdc2. Vpr cooperates with p300, which regulates NF-kappaB and the basal transcriptional machinery, to increase HIV gene expression. Similar effects are seen in the absence of Vpr with a kinase-deficient Cdc2, and overexpression of p300 increases levels of HIV Vpr+ replication. Taken together, these data suggest that p300, through its interactions with NF-kappaB, basal transcriptional components, and Cdks, is modulated by Vpr and regulates HIV replication. The regulation of p300 by Vpr provides a mechanism to enhance viral replication in proliferating cells after growth arrest by increasing viral transcription.

CDC2 Protein Kinase↗
PubMed1998

Expression of cyclin-dependent kinase inhibitors in vascular disease.

Arterial lesions in cardiovascular diseases are characterized by proliferation and migration of smooth muscle cells as well as deposition of connective tissue matrix. Factors that stimulate vascular smooth muscle cell (VSMC) proliferation are well described; however, the role of proteins that limit intimal hyperplasia is not well understood. To examine the function of Kip/Cip and INK cyclin-dependent kinase inhibitors (CKIs) in vascular diseases, the expression of p27Kip1 and p16INK was examined in VSMCs in vitro and in porcine arteries and human atherosclerosis in vivo. Western blot and fluorescence activated cell-sorting analysis demonstrated that levels of p27Kip1, but not p16INK, increased during serum deprivation of primary VSMC cultures and caused G1 arrest. p27Kip1 inhibited Cdk2 activity, suggesting that Kip CKIs promote G1 arrest in VSMCs by binding cyclin E/Cdk2. In porcine arteries, p27Kip1, but not p16INK, was constitutively expressed at low levels. Immediately after balloon injury, cell proliferation increased as p27Kip1 levels declined. Three weeks after injury, p27Kip1 was strongly expressed in intimal VSMCs when VSMC proliferation was < 2%, suggesting that p27Kip1 functions as an inhibitor of cell proliferation in injured arteries. In contrast, p16INK expression was detected only transiently early after injury. CKI expression was examined in 35 human coronary arteries, ranging from normal to advanced atherosclerosis. p27Kip1 expression was abundant in nonproliferating VSMCs and macrophages within normal (7 of 8) and atherosclerotic (25 of 27) arteries. p21Cip1 levels were undetectable in normal arteries but were elevated in atherosclerotic (19 of 27) arteries. p16INK could not be detected in normal or atherosclerotic arteries (0 of 35). Thus, the Kip/Cip and INK CKIs have different temporal patterns of expression in VSMCs in vitro and in injured arteries and atherosclerotic lesions in vivo. In contrast to p16INK, p27Kip1 likely contributes to the remodeling process in vascular diseases by the arrest of VSMCs in the G1 phase of the cell cycle.

Adult↗
PubMed1998

Distinct cellular interactions of secreted and transmembrane Ebola virus glycoproteins.

The mechanisms by which Ebola virus evades detection and infects cells to cause hemorrhagic fever have not been defined, though its glycoprotein, synthesized in either a secreted or transmembrane form, is likely involved. Here the secreted glycoprotein was found to interact with neutrophils through CD16b, the neutrophil-specific form of the Fc gamma receptor III, whereas the transmembrane glycoprotein was found to interact with endothelial cells but not neutrophils. A murine retroviral vector pseudotyped with the transmembrane glycoprotein preferentially infected endothelial cells. Thus, the secreted glycoprotein inhibits early neutrophil activation, which likely affects the host response to infection, whereas binding of the transmembrane glycoprotein to endothelial cells may contribute to the hemorrhagic symptoms of this disease.

Cell Line↗
PubMed1998

Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals.

Intracellular expression of gene products that inhibit viral replication have the potential to complement current antiviral approaches to the treatment of AIDS. We previously have shown that a mutant inhibitory form of an essential viral protein, Rev M10, prolongs the survival of T cells transduced with a nonviral vector in HIV-infected individuals. Because these gene-modified cells were not observed in patients beyond 8 weeks, efforts were made to improve the duration of engraftment. In this study, we used retroviral vector delivery of Rev M10 to CD4(+) cells and analyzed relevant immune responses in a pilot study of three HIV-seropositive patients. DNA and RNA PCR analyses revealed that cells transduced with Rev M10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in all three patients. Immune responses were not detected either to Rev M10 or to Moloney murine leukemia virus gp70 envelope protein. Rev M10-transduced cells were detected for an average of 6 months after retroviral gene transfer compared with approximately 3 weeks for the previously reported nonviral vector delivery. These findings suggest that retroviral delivery of an antiviral gene may potentially contribute to immune reconstitution in AIDS and could provide a more effective vector to prolong survival of CD4(+) cells in HIV infection.

Animals↗
PubMed1998

Immunization for Ebola virus infection.

Infection by Ebola virus causes rapidly progressive, often fatal, symptoms of fever, hemorrhage and hypotension. Previous attempts to elicit protective immunity for this disease have not met with success. We report here that protection against the lethal effects of Ebola virus can be achieved in an animal model by immunizing with plasmids encoding viral proteins. We analyzed immune responses to the viral nucleoprotein (NP) and the secreted or transmembrane forms of the glycoprotein (sGP or GP) and their ability to protect against infection in a guinea pig infection model analogous to the human disease. Protection was achieved and correlated with antibody titer and antigen-specific T-cell responses to sGP or GP. Immunity to Ebola virus can therefore be developed through genetic vaccination and may facilitate efforts to limit the spread of this disease.

Animals↗
PubMed1998

Gene transfer into human umbilical cord blood-derived CD34+ cells by particle-mediated gene transfer.

Delivery of genes into hematopoietic progenitor cells offers an attractive means for the introduction of corrective or protective genes into cells of both the myeloid and lymphoid lineage. Previously, investigators have often used murine retroviral vectors for gene delivery which require cells to be cycling for efficient delivery. We describe a nonviral method of gene delivery using particle-mediated gene transfer to obviate many disadvantages of viral vectors related to safety, production costs and the need for cell cycle proliferation. Using a CMV-CAT reporter plasmid, we show transfection of highly purified CD34+ cells isolated from umbilical cord blood. Effective gene transfer was shown in unstimulated and in growth-stimulated cells. Following transfection with a neomycin resistance gene, differentiation into cells of the myeloid lineage was observed, assayed by CFU-GM in the presence of G-418. Both unstimulated and stimulated cells gave rise to CFU-GM in the presence of G-418, indicating that stable expression of the neomycin resistance gene was maintained in early progenitors. These results demonstrate that particle-mediated gene transfer into human hematopoietic cells from umbilical cord blood can be achieved without affecting their CFU-GM differentiation potential. This gene transfer method offers an alternative approach to gene therapy studies involving human hematopoietic progenitor cells.

Anti-Bacterial Agents↗
PubMed1998

Interaction of human immunodeficiency virus type 1 Tat with the transcriptional coactivators p300 and CREB binding protein.

Human immunodeficiency virus type 1 (HIV-1) encodes the transactivator protein Tat, which is essential for viral replication and progression to disease. Here we demonstrate that transcriptional activation by HIV-1 Tat involves p300 or the related cellular transcriptional coactivator CREB binding protein (CBP). Tat transactivation was inhibited by the 12S form of the adenovirus E1A gene product, which inhibits p300 function, and this inhibition was independent of its effect on NF-kappaB transcription. A biochemical interaction of p300 with Tat was demonstrated in vitro and in vivo by coimmunoprecipitation. The carboxy-terminal region of p300, which binds to E1A, was shown to bind specifically to the highly conserved basic domain of Tat, which also mediates binding to the Tat-responsive region RNA stem-loop structure. The ability of Tat to interact physically and functionally with this coactivator provides a mechanism to assemble a basal transcription complex which may subsequently respond to the effect of Tat on transcriptional elongation and represents a novel interaction between an RNA binding protein and a transcriptional coactivator.

CREB-Binding Protein↗
PubMed1998

Combination gene delivery of the cell cycle inhibitor p27 with thymidine kinase enhances prodrug cytotoxicity.

Cytoxicity induced by the herpesvirus thymidine kinase (TK) gene in combination with prodrugs is dependent on cell growth and leads to the elimination of genetically modified cells, thus limiting the duration of expression and efficacy of this treatment in vivo. Here, an effort was made to enhance TK/prodrug efficacy by coexpression of a cyclin-dependent kinase inhibitor (CKI), p27, to render cells resistant to TK/prodrug by inhibiting DNA synthesis. Expression of p27 by transfection substantially reduced cell cycle progression, and its activity was enhanced by mutations designed to stabilize the protein. Coexpression of p27 and TK or a p27/TK fusion protein led to greater prodrug cytotoxicity than that produced by TK alone in the Renca cell line, which is sensitive to bystander killing. Combination gene transfer of this CKI with TK therefore sustained the synthesis of TK by genetically modified cells to enhance the susceptibility of bystander cells to prodrug cytotoxicity and increased the efficacy of this gene transfer approach.

Base Sequence↗
PubMed1998

Distinct biology of Kaposi's sarcoma-associated herpesvirus from primary lesions and body cavity lymphomas.

The DNA sequence for Kaposi's sarcoma-associated herpesvirus was originally detected in Kaposi's sarcoma biopsy specimens. Since its discovery, it has been possible to detect virus in cell lines established from AIDS-associated body cavity-based B-cell lymphoma and to propagate virus from primary Kaposi's sarcoma lesions in a human renal embryonic cell line, 293. In this study, we analyzed the infectivity of Kaposi's sarcoma-associated herpesvirus produced from these two sources. Viral isolates from cultured cutaneous primary KS cells was transmitted to an Epstein-Barr virus-negative Burkitt's B-lymphoma cell line, Louckes, and compared to virus induced from a body cavity-based B-cell lymphoma cell line. While propagation of body cavity-based B-cell lymphoma-derived virus was not observed in 293 cell cultures, infection with viral isolates obtained from primary Kaposi's sarcoma lesions induced injury in 293 cells typical of herpesvirus infection and was associated with apoptotic cell death. Interestingly, transient overexpression of the Kaposi's sarcoma-associated herpesvirus v-Bcl-2 homolog delayed the process of apoptosis and prolonged the survival of infected 293 cells. In contrast, the broad-spectrum caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk failed to protect infected cell cultures, suggesting that Kaposi's sarcoma-associated herpesvirus-induced apoptosis occurs through a Bcl-2-dependent pathway. Kaposi's sarcoma-associated herpesvirus isolates from primary Kaposi's sarcoma lesions and body cavity-based lymphomas therefore may differ and are likely to have distinct contributions to the pathophysiology of Kaposi's sarcoma.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Biochemical analysis of the secreted and virion glycoproteins of Ebola virus.

The glycoproteins expressed by a Zaire species of Ebola virus were analyzed for cleavage, oligomerization, and other structural properties to better define their functions. The 50- to 70-kDa secreted and 150-kDa virion/structural glycoproteins (SGP and GP, respectively), which share the 295 N-terminal residues, are cleaved near the N terminus by signalase. A second cleavage event, occurring in GP at a multibasic site (RRTRR downward arrow) that is likely mediated by furin, results in two glycoproteins (GP1 and GP2) linked by disulfide bonding. This furin cleavage site is present in the same position in the GPs of all Ebola viruses (R[R/K]X[R/K]R downward arrow), and one is predicted for Marburg viruses (R[R/K]KR downward arrow), although in a different location. Based on the results of cross-linking studies, we were able to determine that Ebola virion peplomers are composed of trimers of GP1-GP2 heterodimers and that aspects of their structure are similar to those of retroviruses, paramyxoviruses, and influenza viruses. We also determined that SGP is secreted from infected cells almost exclusively in the form of a homodimer that is joined by disulfide bonding.

Animals↗
PubMed1998

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a 442 amino acid polypeptide-designated viral interferon regulatory factor (vIRF) that displays homology to members of the interferon regulatory factor (IRF) family that bind to consensus interferon sequences and transactivate cellular genes that can modulate growth inhibition. Studies were conducted to determine whether vIRF affects the growth suppression mediated by interferon-alpha (IFN-alpha) in a human B lymphocyte cell line. MATERIALS AND METHODS: The human B lymphocyte cell line Daudi, which is sensitive to the antiproliferative effects of IFN-alpha, was stably transfected to express vIRF, and the proliferative response of vIRF expressing cells to IFN-alpha was compared with controls. The effect of vIRF on IRF- 1 transactivation was analyzed by co-transfection of an IFN-alpha-responsive chloramphenicol acetyltransferase reporter and expression plasmids encoding IRF-1 and vIRF. Electrophoretic mobility shift assays were conducted to determine whether vIRF interferes with the DNA binding activity of IRF-1. RESULTS: Daudi human B lymphocyte cells expressing vIRF were resistant to the antiproliferative effects of IFN-alpha, whereas wild-type Daudi or Daudi cells transformed with vector DNA were growth inhibited by IFN-alpha. The activation of an interferon-responsive reporter by IFN-alpha or IRF-1 was repressed by expression of vIRF. IRF-1 DNA binding activity was unaffected by vIRF, and vIRF alone did not bind to the interferon consensus sequence. CONCLUSIONS: These studies revealed that vIRF functions to inhibit interferon-mediated growth control of a human B lymphocyte cell line by targeting IRF-1 transactivation of interferon-inducible genes. Since KSHV is a B lymphotropic herpesvirus associated with two forms of B lymphocyte neoplasms, these effects of vIRF likely contribute to B cell oncogenesis associated with KSHV infection.

3T3 Cells↗
PubMed1998

Requirements for enhanced transgene expression by untranslated sequences from the human cytomegalovirus immediate-early gene.

BACKGROUND: The cytomegalovirus immediate early (CMV IE) promoter has been widely used for heterologous expression. Further enhancements of gene expression from this potent promoter may allow for the development of improved gene transfer strategies. We aimed to determine whether inclusion of the first exon (5' untranslated) and first intron of the CMV IE gene would increase heterologous transgene expression in primary target cells and to determine the sequences required for any observed increases. MATERIALS AND METHODS: Comparisons of reporter gene expression were made following transient transfection of vascular smooth muscle cells (VSMCs) with plasmids containing the first exon and intron from the CMV IE gene or deletional mutations. Comparisons were also made using a heterologous promoter (RSV). RESULTS: Gene expression from the CMV IE promoter was increased 5.7-fold in VSMC with the inclusion of the first exon and intron. Similar increases were seen with other target cells and from the heterologous RSV promoter. This increase was associated with an increase in steady-state mRNA. Deletion analyses demonstrated that the enhancement was dependent on the presence of the 5' portion of the first exon while deletion of large segments within the intron was associated with similar levels of expression compared with the parental plasmid. CONCLUSIONS: Inclusion of the first exon and intron from the CMV IE gene increases expression from the CMV IE promoter. This enhancement is seen with the heterologous RSV promoter and is associated with an increase in steady-state mRNA. Deletion analyses suggest that this enhancement is associated with inclusion of sequences within the 5' portion of the first exon and inclusion of an intron.

Animals↗
PubMed1997

Gene transfer of Fas ligand induces tumor regression in vivo.

The Fas-Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growth in vivo. Although such inhibition is expected in Fas+ tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas+ Renca tumor by inducing cell death, whereas the elimination of Fas- CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas+ cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.

Adenoviridae↗