Biomedical subjects

G J Nabel

Publications and source records attributed to G J Nabel.

At least 145 records · Page 8Linked to original sources

PubMed1993

Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3).

Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.

Animals↗

PubMed1993

Recombinant platelet-derived growth factor B gene expression in porcine arteries induce intimal hyperplasia in vivo.

Platelet-derived growth factor (PDGF) B chain induces cell proliferation in vitro and is associated with arterial lesions that cause cardiovascular disease. However, it has been difficult to document the biological response to PDGF B gene expression in arteries in vivo. To determine the biologic effects of this growth factor in vivo, we have introduced an eukaryotic expression vector plasmid encoding recombinant PDGF B by direct gene transfer into porcine iliofemoral arteries using DNA liposome complexes. The presence of PDGF B plasmid DNA and expression of recombinant mRNA were confirmed by polymerase chain reaction analysis, and recombinant PDGF protein was demonstrated by immunohistochemistry. Intimal thickening was observed in porcine arteries 21 days following transfection with the recombinant PDGF B gene compared with arteries transduced with a control gene, E. coli beta-galactosidase. An eightfold increase in intimal to medial ratio was present in PDGF B gene transfected arteries compared with control transfected arteries (P = 0.001). This study suggests that expression of a recombinant PDGF B gene in vivo can play a role in the induction of intimal hyperplasia, which can lead to cardiovascular diseases.

Animals↗

PubMed1992

Isolation of a cDNA clone encoding a zinc finger protein highly expressed in T-leukemia lines.

A cDNA clone encoding a novel zinc finger protein expressed in lymphoid cells has been isolated. This protein contains 5 repeats of the C2H2 motif previously described in the Drosophila gap gene, Krüppel, which is involved in embryo segmentation. Northern blot analysis showed that the messenger RNA (mRNA) encoding this protein is expressed at high levels in a variety of T-leukemia cell lines, at lower levels in some B cells, but is not observed in nonlymphoid cells. Within the T lineage, the mRNA is found at high levels in both alpha beta and gamma delta T cells. These data suggest that this cDNA, designated Hkr-T1, represents a gene that may contribute to the determination of the differentiation and the specificity within lymphoid cells.

Amino Acid Sequence↗

PubMed1992

Cellular distribution of nuclear factor kappa B binding activity in rat liver.

The cellular localization of nuclear factor kappa B (NF-kappa B) binding activity in rat liver has been investigated using electrophoretic mobility shift assay on extracts of highly purified hepatocytes and Kupffer cells obtained from liver perfused in vivo with collagenase. Constitutive NF-kappa B binding activity was demonstrated in nuclear extracts of control Kupffer cells, and this was not apparently influenced by injection of lipopolysaccharide (LPS) into rats 24 h before perfusion. In contrast, little nuclear NF-kappa B binding activity was present in hepatocytes from control animals, although there was detectable inactive, inhibitor-bound, NF-kappa B in the cytoplasm. However, nuclear NF-kappa B binding activity was increased in hepatocytes from LPS-treated animals and after in vitro culture of control rat hepatocytes. Thus NF-kappa B binding activity has been demonstrated in highly purified hepatocytes and appears to be inducible both in vivo and in vitro. These findings support a role for NF-kappa B in hepatocyte gene regulation which may be important in the modulation of the hepatic acute phase response.

Animals↗

PubMed1992

Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication.

The human immunodeficiency virus (HIV) Rev protein is essential for viral structural protein expression (Gag, Pol, and Env) and, hence, for viral replication. In transient transfection assays, mutant forms of Rev have been identified that inhibit wild-type Rev activity and therefore suppress viral replication. To determine whether such transdominant Rev proteins could provide long-term protection against HIV infection without affecting T cell function, T leukemia cell lines were stably transduced with a retroviral vector encoding a transdominant mutant of the Rev protein, M10. While all the M10-expressing cell lines remained infectable by HIV-1, these same cells failed to support a productive replication cycle when infected with a cloned isolate of HIV-1. In addition, two out of three M10-expressing CEM clones were also resistant to highly productive infection by a heterogeneous HIV-1 pool. Expression of M10 did not affect induction of HIV transcription mediated by the kappa B regulatory element or Tat. Importantly, constitutive expression of Rev M10 did not alter the secretion of interleukin 2 in response to mitogen stimulation of EL-4 and Jurkat cells. The inhibition of HIV infection in cells stably expressing a transdominant Rev protein, in the absence of any deleterious effect on T cell function, suggests that such a strategy could provide a therapeutic effect in the T lymphocytes of acquired immunodeficiency syndrome patients.

Clone Cells↗

PubMed1992

Transduction of a foreign histocompatibility gene into the arterial wall induces vasculitis.

Autoimmune vasculitis represents a disease characterized by focal inflammation within arteries at multiple sites in the vasculature. Therapeutic interventions in this disease are empirical and often unsuccessful, and the mechanisms of immune injury are not well-defined. The direct transfer of recombinant genes and their expression in the arterial wall provides an opportunity to explore the pathogenesis and treatment of vascular disease. In this report, an animal model for vasculitis has been developed. Inflammation has been elicited by direct gene transfer of a foreign class I major histocompatibility complex gene, HLA-B7, to specific sites in porcine arteries. Transfer and expression of this recombinant gene was confirmed by a polymerase chain reaction and immunohistochemistry, and cytolytic T cells specific for HLA-B7 were detected. These findings demonstrate that expression of a recombinant gene in the vessel wall can induce a focal immune response and suggest that vessel damage induced by cell-mediated immune injury can initiate vasculitis.

Animals↗

PubMed1992

Distinct combinations of NF-kappa B subunits determine the specificity of transcriptional activation.

The nuclear factor that binds to the kappa light-chain enhancer of B cells (NF-kappa B) is a transcription factor that regulates the expression of a variety of cellular and viral genes. NF-kappa B is composed of distinct subunits, and at least four independent genes (p105, p100, p65, and c-rel) have been isolated that encode related proteins that bind kappa B sites. Because it is possible that specific interactions of different subunits can allow selective gene activation, we have characterized the specificity of transcriptional activation by various combinations of these subunits. When tested alone, an approximately 49-kDa form (p49) of the p100 protein bound weakly to kappa B, but p49 associated with p65 to bind efficiently to this site. Furthermore, p49 acted in combination with either p65 or a Rel/VP16 fusion protein to activate kappa B-dependent transcription in Jurkat T leukemia cells. The p49/p65 or p49/Rel combination stimulated transcription mediated by the canonical kappa B site but did not stimulate reporter genes containing interleukin 2 receptor alpha or major histocompatibility complex kappa B elements, despite its ability to bind to these sites. Transactivation mediated by the p49/p100 and p65 NF-kappa B proteins is therefore sensitive to minor changes in the sequence of the kappa B site. Specificity determined by the association of NF-kappa B subunits provides a mechanism to selectively regulate variant kappa B sites associated with different cellular and viral genes.

Base Sequence↗

PubMed1992

Related subunits of NF-kappa B map to two distinct loci associated with translocations in leukemia, NFKB1 and NFKB2.

The chromosomal locations of the human genes NFKB1 and NFKB2, which encode two alternative DNA binding subunits of the NF-kappa B complex, p105 and p49/p100, respectively, have been determined. p105 was assigned to 4q21.1-q24 and p49/p100 to chromosome 10 by Southern blot analysis of panels of human/Chinese hamster cell hybrids. The locations were confirmed by fluorescence in situ hybridization and mapped with greater resolution to 4q23 and 10q24, respectively. These results demonstrate that these members of the NF-kappa B family are unlinked. Interestingly, p49/p100 as well as p105 maps to regions associated with certain types of acute lymphoblastic leukemia.

Animals↗

PubMed1992

Localization of the gene encoding R kappa B (NFRKB), a tissue-specific DNA binding protein, to chromosome 11q24-q25.

Although NF (nuclear factor)-kappa B binds in vitro to several of the kappa B regulatory elements found in cellular and viral genes, another DNA binding protein, R kappa B, also binds to a related variant of the kappa B site that regulates interleukin-2 receptor alpha-chain gene expression, a critical event in T cell activation. Southern blot analysis of a human-mouse somatic cell hybrid panel and in situ hybridization using a fluorescent genomic R kappa B probe have allowed assignment of the R kappa B gene (NFRKB) to 11q24-q25. The NFRKB locus is in close proximity to the chromosomal breakpoint implicated in Ewing sarcoma, but it does not appear to span this region. Nonetheless, NFRKB may be particularly useful as the most telomeric marker thus far assigned to 11q.

Animals↗

PubMed1992

Gene transfer in vivo with DNA-liposome complexes: safety and acute toxicity in mice.

DNA can be introduced into a variety of cell types after formation of liposomal complexes with cationic lipids. In this report, conditions have been established to optimize the production of DNA-liposome complexes that efficiently transfect cells. The safety and toxicity of this method of gene delivery have been assessed after in vivo administration, either by intravenous or direct intratumor injection. Nine to eleven days after intravenous injection, DNA was found primarily in heart and lung tissue by PCR analysis. No abnormalities were evident from histologic examination of tissue, examination of tissue-specific serum enzymes, routine biochemical parameters, or electrocardiographic monitoring. DNA-liposome complexes can therefore be used for the delivery of recombinant genes in vivo with minimal toxicity.

Animals↗

PubMed1992

Gene transfer in vivo with DNA-liposome complexes: lack of autoimmunity and gonadal localization.

Direct gene transfer into localized arterial segments can be performed in vivo by transfection with DNA-liposome complexes. This technique holds promise for the treatment of human diseases, including malignancy and cardiovascular disorders. We have previously characterized the potential toxicity of this form of treatment in mice in vivo (Stewart et al., 1992). In this report, we examined two issues relevant to long-term expression of foreign recombinant genes: (i) the potential for autoimmune damage to major organs and (ii) DNA localization in gonadal tissue. Autoimmunity and toxicity of allogeneic major histocompatibility (MHC) gene transfer was assessed in mice after induction of an immune response to a recombinant murine class I MHC gene by direct gene transfer in vivo. Histological examination of brain, heart, lung, liver, kidney, spleen, and skeletal muscle revealed no clinically significant immunopathology or organ damage. The toxicity of gene delivery by DNA liposomes was also analyzed in pigs and rabbits in vivo. No histopathology was observed following the introduction of plasmids encoding several different gene products, and analysis of serum following DNA liposome delivery revealed no abnormalities of serum biochemical parameters. The potential for transfer of recombinant DNA into testes and ovary in animals was evaluated by the polymerase chain reaction. Although evidence of recombinant plasmid was consistently observed in transfected, but not untransfected, arterial sites and occasionally in lung, kidney, spleen, and liver, no plasmid DNA was detected in testes or ovary. These studies suggest that uptake of recombinant DNA following direct gene transfer by liposomal transfection in major organs is not associated with autoimmunity, toxicity, or gonadal localization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A novel Ets-related transcription factor, Elf-1, binds to human immunodeficiency virus type 2 regulatory elements that are required for inducible trans activation in T cells.

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are structurally related retroviruses which both cause AIDS in humans. Although both viruses establish latency in quiescent human-peripheral-blood T cells, the asymptomatic phase of HIV-2 infection may be more prolonged than that of HIV-1. The latent phases of both HIV-1 and HIV-2 infection have been shown to be disrupted by T-cell activation, a process that requires host cell transcription factors. In the case of HIV-1, the transcription factor NF-kappa B is sufficient for inducible transcriptional activation. In contrast, factors in addition to NF-kappa B are required to activate HIV-2 transcription in infected T cells. In this report, we demonstrate that a novel Ets-related transcription factor, Elf-1, binds specifically to two purine-rich motifs in the HIV-2 enhancer. Mutagenesis experiments demonstrated that these Elf-1 binding sites are required for induction of HIV-2 transcription following T-cell-receptor-mediated T-cell activation. Moreover, Elf-1 is the only factor present in activated T-cell nuclear extracts that binds to these sites in electrophoretic mobility shift assays. Thus, Elf-1 is a novel transcription factor that appears to be required for the T-cell-receptor-mediated trans activation of HIV-2 gene expression. These results may explain differences in the clinical spectra of diseases caused by HIV-1 and HIV-2 and may also have implications for the design of therapeutic approaches to HIV-2 infection.

Adult↗

PubMed1992

Specific NF-kappa B subunits act in concert with Tat to stimulate human immunodeficiency virus type 1 transcription.

NF-kappa B is a protein complex which functions in concert with the tat-I gene product to stimulate human immunodeficiency virus (HIV) transcription. To determine whether specific members of the NF-kappa B family contribute to this effect, we have examined the abilities of different NF-kappa B subunits to act with Tat-I to stimulate transcription of HIV in Jurkat T-leukemia cells. We have found that the p49(100) DNA binding subunit, together with p65, can act in concert with Tat-I to stimulate the expression of HIV-CAT plasmid. Little effect was observed with 50-kDa forms of p105 NF-kappa B or rel, in combination with p65 or full-length c-rel, which do not stimulate the HIV enhancer in these cells. These findings suggest that the combination of p49(100) and p65 NF-kappa B can act in concert with the tat-I gene product to stimulate the synthesis of HIV RNA.

Cells, Cultured↗

PubMed1992

Activation of the human immunodeficiency virus type 1 enhancer is not dependent on NFAT-1.

The function of a putative NFAT-1 site in the human immunodeficiency virus type 1 enhancer has been analyzed. Activation by the T-cell antigen receptor is minimal in Jurkat cells and is mediated by the kappa B sites. The putative NFAT-1 region is not required for the response to anti-CD3 or to mitogens in T-cell, B-cell, or monocyte/macrophage leukemia lines, nor is it a cis-acting negative regulatory element.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1992

Activation of the human immunodeficiency virus type 2 enhancer is dependent on purine box and kappa B regulatory elements.

Human immunodeficiency virus type 2 (HIV-2) displays several features which distinguish it from HIV-1. Among the differences in these two viruses are the responses of their enhancer regions to T-cell activation. For example, stimulation of HIV-1 transcription is largely dependent on two kappa B regulatory elements. In contrast, the HIV-2 enhancer has a single kappa B site and contains additional cis-acting sequences responsive to induction. One of these sites, previously termed CD3R, is a purine-rich site, also called PuB1, which is responsive to stimulation of the CD3 component of the T-cell receptor complex and binds Elf-1, a member of the ets proto-oncogene family. In this report, we examine the interaction of the PuB1 site with other sites in the HIV-2 enhancer. We demonstrate that the PuB1 site confers responsiveness to T-cell activators only in cooperation with additional enhancer elements. Induction of the HIV-2 enhancer is dependent on at least two other cis-acting regulatory elements in addition to PuB1 and kappa B. One of these elements is another purine-rich site (PuB2), which also binds recombinant Elf-1. An adjacent region, proximal to the PuB2 ets (pets) site, shows protection in DNase footprinting experiments with extracts from Jurkat T cells. Mutation of either the kappa B, PuB1, PuB2, or pets site significantly reduces the response of the HIV-2 enhancer to T-cell stimulation, an effect which is mediated at the RNA level. Therefore, activation of the HIV-2 enhancer is dependent on at least four cis-acting elements, only one of which is found in HIV-1, which act in synergy with one another. Despite their sequence similarity, the organization and function of the HIV-2 enhancer have diverged considerably from those of HIV-1.

Base Sequence↗

PubMed1992

cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, Elf-1.

The recent definition of a consensus DNA binding sequence for the Ets family of transcription factors has allowed the identification of potential Ets binding sites in the promoters and enhancers of many inducible T-cell genes. In the studies described in this report, we have identified two potential Ets binding sites, EBS1 and EBS2, which are conserved in both the human and murine interleukin-2 enhancers. Within the human enhancer, these two sites are located within the previously defined DNase I footprints, NFAT-1 and NFIL-2B, respectively. Electrophoretic mobility shift and methylation interference analyses demonstrated that EBS1 and EBS2 are essential for the formation of the NFAT-1 and NFIL-2B nuclear protein complexes. Furthermore, in vitro mutagenesis experiments demonstrated that inducible interleukin-2 enhancer function requires the presence of either EBS1 or EBS2. Two well-characterized Ets family members, Ets-1 and Ets-2, are reciprocally expressed during T-cell activation. Surprisingly, however, neither of these proteins bound in vitro to EBS1 or EBS2. We therefore screened a T-cell cDNA library under low-stringency conditions with a probe from the DNA binding domain of Ets-1 and isolated a novel Ets family member, Elf-1. Elf-1 contains a DNA binding domain that is nearly identical to that of E74, the ecdysone-inducible Drosophila transcription factor required for metamorphosis (hence the name Elf-1, for E74-like factor 1). Elf-1 bound specifically to both EBS1 and EBS2 in electrophoretic mobility shift assays. It also bound to the purine-rich CD3R element from the human immunodeficiency virus type 2 long terminal repeat, which is required for inducible virus expression in response to signalling through the T-cell receptor. Taken together, these results demonstrate that multiple Ets family members with apparently distinct DNA binding specificities regulate differential gene expression in resting and activated T cells.

Adult↗

PubMed1991

Cloning of an NF-kappa B subunit which stimulates HIV transcription in synergy with p65.

The transcription factor NF-kappa B is a protein complex which comprises a DNA-binding subunit and an associated transactivation protein (of relative molecular masses 50,000 (50K) and 65K, respectively). Both the 50K and 65K subunits have similarity with the rel oncogene and the Drosophila maternal effect gene dorsal. The 50K DNA-binding subunit was previously thought to be a unique protein, derived from the 105K gene product (p105). We now report the isolation of a complementary DNA that encodes an alternative DNA-binding subunit of NF-kappa B. It is more similar to p105 NF-kappa B than other family members and defines a new subset of rel-related genes. It is synthesized as approximately 100K protein (p100) that is expressed in different cell types, contains cell cycle motifs and, like p105, must be processed to generate a 50K form. A 49K product (p49) can be generated independently from an alternatively spliced transcript; it has specific kappa B DNA-binding activity and can form heterodimers with other rel proteins. In contrast to the approximately 50K protein derived from p105, p49 acts in synergy with p65 to stimulate the human immunodeficiency virus (HIV) enhancer in transiently transfected Jurkat cells. p49/p100 NF-kappa B could therefore be important in the regulation of HIV and other kappa B-containing genes.

Amino Acid Sequence↗

PubMed1991

Lymphoid expression and TATAA binding of a human protein containing an Antennapedia homeodomain.

In an effort to identify human proteins that bind to the TATAA box, a lambda gt-11 expression library was screened with a radiolabeled DNA probe containing 12 copies of the TATAA sequence. A cDNA encoding a specific TATAA binding protein was isolated and found to contain a homeobox domain identical at 59 of 60 residues to the Drosophila Antennapedia (Antp) homeodomain, as well as another conserved motif found in homeotic genes, the homeo-specific pentapeptide. Although this and other Antp-like homeobox proteins have been described previously in neuronal cells and fibroblasts, we report the expression of this gene in lymphoid cells. This cDNA, isolated from a B-cell library, hybridizes to a 1.6-kb messenger RNA in several T- and B-cell lines, and the expected protein was identified in Jurkat T-lymphoid cells by Western blot analysis. The DNA binding specificity of this human Antp clone was analyzed using single-base mutations of the TATAA sequence. The first thymidine, as well as the last three bases (TAA), were important for homeobox binding. Finally, the function of the highly conserved homeospecific pentapeptide protein region was investigated in both the human and Drosophila Antp proteins. The homeospecific pentapeptide region was not required for DNA binding, and Drosophila Antp proteins mutated in the pentapeptide region were able to transactivate the Ubx promoter in Schneider L2 cells, in contrast to a homeodomain mutation, suggesting an alternative function for the homeospecific pentapeptide in homeotic genes. Because the human Antp TATAA binding protein is expressed in both lymphoid and non-lymphoid cells, we suggest that this homeobox gene has evolved a more general transcriptional regulatory function in higher eukaryotic cells.

Amino Acid Sequence↗