Biomedical subjects

C A Rosen

Publications and source records attributed to C A Rosen.

At least 19 recordsLinked to original sources

PubMed1994

Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription.

TFIIA is thought to play an important role in transcriptional regulation in higher eukaryotes, but its precise function is unclear. A human cDNA encoding a protein with 45% identity to the small subunit of yeast TFIIA has been isolated. TFIIA activity could be reconstituted by the mixing of recombinant large (alpha beta) and small (gamma) subunits. TFIIA-depleted HeLa nuclear extracts were used to demonstrate that TFIIA is essential for basal and activated transcription by several distinct classes of activators. Recombinant TFIIA functioned in transcriptional activation whether expressed as a dimer (alpha beta+gamma) or as a trimer (alpha+beta+gamma), which closely resembles the native form. Yeast TFIIA also functioned in transcriptional activation, and the human gamma subunit was functionally interchangeable with TOA2, its yeast homolog. Recombinant TFIIA mediated the stimulation of TFIID binding to the TATA region and downstream promoter sequences by the Zta transcriptional activator. Significantly, we found that TFIIA bound directly to Zta in an activation domain-dependent manner. One consequence of the TFIIA-mediated interaction between Zta and TFIID was the formation of a promoter-bound complex resistant to TATA oligonucleotide competition. These results demonstrate that TFIIA is an evolutionarily conserved general factor critical for activator-regulated transcription.

Amino Acid Sequence↗

PubMed1994

Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of man's commonest hereditary diseases. Several studies have implicated a defect in DNA mismatch repair in the pathogenesis of this disease. In particular, hMSH2 and hMLH1 homologues of the bacterial DNA mismatch repair genes mutS and mutL, respectively, were shown to be mutated in a subset of HNPCC cases. Here we report the nucleotide sequence, chromosome localization and mutational analysis of hPMS1 and hPMS2, two additional homologues of the prokaryotic mutL gene. Both hPMS1 and hPMS2 were found to be mutated in the germline of HNPCC patients. This doubles the number of genes implicated in HNPCC and may help explain the relatively high incidence of this disease.

Adenosine Triphosphatases↗

PubMed1994

Synergistic transcriptional activation of the IL-8 gene by NF-kappa B p65 (RelA) and NF-IL-6.

Transcriptional activation of the IL-8 gene by several inflammatory mediators, including the cytokines IL-1 and TNF-alpha, is mediated through sequences located between nucleotide -94 and -71 of the IL-8 promoter. Because adjacent binding sites for the inducible transcription factors NF-kappa B and NF-IL-6 are located within this region, we examined the functional interaction of these two transcription factor families in IL-8 gene regulation. Maximal transcriptional activation by PMA in Jurkat T lymphocytes was shown to require intact binding sites for both NF-kappa B and NF-IL-6. Electrophoretic mobility shift analysis indicates that NF-IL-6, as well as other related members of this family, bind specifically to the NF-IL-6 site in the IL-8 promoter. In addition, NF-kappa B p65 (RelA), but not NF-kappa B p50 (NFKB1), binds specifically to the NF-kappa B site. When incubated together, RelA and NF-IL-6/C/EBP form a ternary complex with this region of the IL-8 promoter; this binding is dependent on intact binding sites for both NF-IL-6 and RelA. Transient cotransfection analyses indicate that the cooperative association of NF-IL-6 and RelA with the IL-8 promoter results in synergistic transcriptional activation. Mutational analyses of RelA demonstrate that the C-terminal transactivation domain and the DNA binding domain are required for synergistic activation with NF-IL-6. In addition, overexpression of the NF-kappa B inhibitor molecule, I kappa B, abolished the RelA- and RelA/NF-IL-6-dependent synergistic activation. These data demonstrate that RelA and members of the C/EBP/NF-IL-6 family can functionally cooperate in transcriptional activation of the IL-8 gene and suggest a common mechanism for inducible regulation of cytokine gene expression.

Base Sequence↗

PubMed1994

Mutation of a mutL homolog in hereditary colon cancer.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site.

Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with c-Rel/p65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV cross-linking studies revealed the presence of c-Rel and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells.

Animals↗

PubMed1993

Antisense inhibition of the p65 subunit of NF-kappa B blocks tumorigenicity and causes tumor regression.

The NF-kappa B transcription factor, composed of two proteins, p50 and p65, is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Various cell adhesion molecules have NF-kappa B binding sites and may play an important role in inflammatory response, tumorigenicity, and metastasis. In an earlier study, we demonstrated that adhesion of diverse transformed cells was blocked by antisense inhibition of the p65 subunit of NF-kappa B. Since cell-substratum interactions play an important role in tumorigenicity, we reasoned that antisense p65 could inhibit tumorigenicity. In diverse transformed cell lines, phosphorothioate antisense oligonucleotides to p65 inhibited in vitro growth, reduced soft-agar colony formation, and eliminated the ability of cells to adhere to an extracellular matrix. Stable transfectants of a fibrosarcoma cell line expressing dexamethasone-inducible antisense RNA to p65 showed inhibition of in vitro growth and in vivo tumor development. In response to inducible expression of antisense RNA, a pronounced tumor regression was seen in nude mice. The administration of antisense but not sense p65 oligonucleotides caused a pronounced inhibition of tumorigenicity in nude mice injected with diverse tumor-derived cell lines. Inhibitors of NF-kappa B function may thus be useful in the treatment of cancer.

Animals↗

PubMed1993

Antisense oligonucleotides to the p65 subunit of NF-kappa B block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes.

NF-kappa B is a pleiotropic regulator of a variety of genes implicated in the cellular response to injury. This function has been attributed to the coordinated binding of subunits of NF-kappa B to distinct regions of the promoter elements of numerous genes, including cytokines, growth factor receptors, and adhesion molecules. Antisense phosphorothioate oligonucleotides to the p50 and p65 subunits of the NF-kappa B complex were used to define the physiologic role of this transcription factor in resting and stimulated granulocytes. A reduction in the expression of p65 was produced by treatment with the phosphorothioate antisense oligodeoxynucleotide. This reduction was accompanied by rapid changes in the cellular adhesion of dimethyl sulfoxide-differentiated HL-60 leukemia cells stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These effects were characterized by a marked reduction in CD11b integrin expression on the surface of treated cells. Furthermore, the p65 antisense oligomer effectively abolished an upregulation of CD11b that was produced by formyl-met-leu-phe and TPA. However, the p65 antisense phosphorothioate oligodeoxynucleotide had no significant effect on the production of reactive oxygen intermediates or on phagocytosis by these cells. These findings indicate that antisense oligomers to p65 can be used to define the role of NF-kappa B in the activation pathways of neutrophils.

Antigens, CD↗

PubMed1993

Cell-type-specific transactivation of the parathyroid hormone-related protein gene promoter by the human T-cell leukemia virus type I (HTLV-I) tax and HTLV-II tax proteins.

The human T-cell leukemia virus type I (HTLV-I) and HTLV-II Tax proteins are potent transactivators of viral and cellular gene expression. Using deletion mutants, the downstream parathyroid hormone-related protein (PTHrP) promoter is shown to be responsive to both HTLV-I and HTLV-II Tax as well as the AP1/c-jun proto-oncogene. Transactivation of PTHrP by Tax was seen in T cells but not in B-cell lines or fibroblasts. A carboxy terminal Tax deletion mutant was deficient in transactivation of both the PTHrP and IL2R alpha promoters but not the HTLV-I long terminal repeat (LTR). Exogenous provision of NFkB rescued IL2R alpha expression but not the PTHrP promoter. Thus, HTLV-I Tax, HTLV-II Tax, and c-jun transactivate PTHrP and may contribute to the pathogenesis of hypercalcemia in adult T-cell leukemia.

Animals↗

PubMed1993

The type 1 human immunodeficiency virus Tat binding protein is a transcriptional activator belonging to an additional family of evolutionarily conserved genes.

The type 1 human immunodeficiency virus Tat protein is a powerful transcriptional activator when bound to an RNA structure (TAR) present at the extreme 5' terminus of viral mRNA. Since transcriptional activation requires binding of Tat to RNA, it has been suggested that Tat enhances initiation or elongation through a direct interaction with cellular transcription factors. Here we show through protein fusion experiments that the previously identified cellular Tat binding protein, TBP-1, although unable to bind DNA, is a strong transcriptional activator when brought into proximity of several promoter elements. Transcriptional activity depends upon the integrity of at least two highly conserved domains: one resembling a nucleotide-binding motif and the other motif common to proteins with helicase activity. Our studies further reveal that TBP-1 represents one member of a large, highly conserved gene family that encodes proteins demonstrating strong amino acid conservation across species. Finally, we identified a second family member that, although 77% similar to TBP-1, does not activate transcription from the promoters examined. This finding, together with the observation that TBP-1 does not activate each promoter examined, suggests that this gene family may encode promoter-specific transcriptional activators.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1993

NF-kappa B subunit-specific regulation of the interleukin-8 promoter.

Interleukin-8 (IL-8), a chemotactic cytokine for T lymphocytes and neutrophils, is induced in several cell types by a variety of stimuli including the inflammatory cytokines IL-1 and tumor necrosis factor alpha TNF-alpha. Several cis elements, including a binding site for the inducible transcription factor NF-kappa B, have been identified in the regulatory region of the IL-8 gene. We have examined the ability of various NF-kappa B subunits to bind to, and activate transcription from, the IL-8 promoter. A nuclear complex was induced in phorbol myristate acetate-treated Jurkat T cells which bound specifically to the kappa B site of the IL-8 promoter and was inhibited by addition of purified I kappa B alpha to the reaction mixture. Only antibody to RelA (p65), but not to NFKB1 (p50), NFKB2 (p50B), c-Rel, or RelB was able to abolish binding, suggesting that RelA is a major component in these kappa B binding complexes. Gel mobility shift analysis with in vitro-translated and purified proteins indicated that whereas the kappa B element in the human immunodeficiency virus type 1 long terminal repeat bound to all members of the kappa B/Rel family examined, the IL-8 kappa B site bound only to RelA and to c-Rel and NFKB2 homodimers, but not to NFKB1 homodimers or heterodimers of NFKB1-RelA. Transient transfection analysis demonstrated a kappa B-dependent expression of the IL-8 promoter in a human fibrosarcoma cell line (8387) and in Jurkat T lymphocytes. Cotransfection with various NF-kappa B subunits indicated that RelA and c-Rel, but neither NFKB1 nor heterodimeric NFKB1-RelA, was able to activate transcription from the IL-8 promoter. Furthermore, cotransfection of NFKB1 and RelA, although able to support activation from the human immunodeficiency virus type 1 long terminal repeat, failed to activate expression from the IL-8 promoter. Antisense oligonucleotides to RelA, but not NFKB1, inhibited phorbol myristate acetate-induced IL-8 production in Jurkat T lymphocytes. These data demonstrate the differential ability of members of the kappa B/Rel family to bind to, and activate transcription from, the IL-8 promoter. Furthermore, while providing a novel example of a kappa B-regulated promoter in which the classical NF-kappa B complex is unable to activate transcription from the kappa B element, these data provide direct evidence for the role of RelA in regulation of IL-8 gene expression.

Base Sequence↗

PubMed1993

Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae.

The NF-kappa B transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo- or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GAL4 and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucin zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-kappa B inhibitory molecule, I kappa B, reduced the transcriptional activity of p65 significantly, suggesting the ability of I kappa B to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-kappa B function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.

Amino Acid Sequence↗

PubMed1993

Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA.

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

Amino Acid Sequence↗

PubMed1993

Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.

Oxidative stress and expression of the vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells are early features in the pathogenesis of atherosclerosis and other inflammatory diseases. Regulation of VCAM-1 gene expression may be coupled to oxidative stress through specific reduction-oxidation (redox) sensitive transcriptional or posttranscriptional regulatory factors. In cultured human umbilical vein endothelial (HUVE) cells, the cytokine interleukin 1 beta (IL-1 beta) activated VCAM-1 gene expression through a mechanism that was repressed approximately 90% by the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC). Furthermore, PDTC selectively inhibited the induction of VCAM-1, but not intercellular adhesion molecule-1 (ICAM-1), mRNA and protein accumulation by the cytokine tumor necrosis factor-alpha (TNF alpha) as well as the noncytokines bacterial endotoxin lipopolysaccharide (LPS) and double-stranded RNA, poly(I:C) (PIC). PDTC also markedly attenuated TNF alpha induction of VCAM-1-mediated cellular adhesion. In a distinct pattern, PDTC partially inhibited E-selectin gene expression in response to TNF alpha but not to LPS, IL-1 beta, or PIC. TNF alpha and LPS-mediated transcriptional activation of the human VCAM-1 promoter through NF-kappa B-like DNA enhancer elements and associated NF-kappa B-like DNA binding proteins was inhibited by PDTC. These studies suggest a molecular linkage between an antioxidant sensitive transcriptional regulatory mechanism and VCAM-1 gene expression that expands on the notion of oxidative stress as an important regulatory signal in the pathogenesis of atherosclerosis.

Antioxidants↗

PubMed1992

Identification of a naturally occurring transforming variant of the p65 subunit of NF-kappa B.

Transcription factor NF-kappa B comprises two proteins, p50 and p65, that have sequence similarity to the v-rel oncogene. In primary hematopoietic cell populations an alternatively spliced form of NF-kappa B p65 mRNA was observed that encoded a protein designated p65 delta. Expression of the p65 delta cDNA in Rat-1 fibroblasts resulted in focus formation, anchorage-independent growth in soft agar, and tumor formation in athymic nude mice, effects not obtained with expression of p65 or a p65 delta mutant that contains a disruption within the transcriptional activation domain. Thus, p65 delta, which associated weakly and interfered with DNA binding by p65, may sequester an essential limiting regulatory factor or factors required for NF-kappa B function.

Animals↗

PubMed1992

HIV regulatory proteins: potential targets for therapeutic intervention.

With the incidence of HIV infection on the rise worldwide, it is obvious that new approaches must be taken to halt the spread of disease. Unfortunately, this is no easy task; of all retroviruses studied to date HIV remains the most complex in terms of genomic organization, regulation of gene expression, and replication. However, as the mechanism of action of the unique viral regulatory proteins is deciphered, new windows of opportunity for attacking the virus like cycle are opened. The essential regulatory function served by both Tat and Rev transacting regulatory proteins makes them attractive targets for prophylactic and therapeutic intervention. This review will focus on our current understanding of Tat and Rev function.

Animals↗

PubMed1992

I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention.

NF-kappa B is an inducible transcription factor comprised of a 50-kD (p50) and a 65-kD (p65) subunit. Induction of NF-kappa B activity, which is a critical event in many signal transduction pathways, involves release from a cytoplasmic inhibitory protein, I kappa B, followed by translocation of the active transcription factor complex into the nucleus. Earlier studies suggested that I kappa B targets the p65 subunit of NF-kappa B. However, we demonstrate by in vitro and in vivo methods that the recently cloned I kappa B/MAD-3 interacts with both the p50 and p65 subunits of NF-kappa B, as well as c-Rel. Furthermore, an alternatively spliced, dimerization-deficient transforming variant of p65 (p65 delta) interacts extremely weakly with I kappa B/MAD-3, suggesting that dimerization is important for interaction. We demonstrate that the conserved nuclear localization sequences (NLSs) of NF-kappa B and c-Rel are the targets for I kappa B/MAD-3 interaction. Indirect immunofluorescence experiments demonstrate that I kappa B/MAD-3 expression retains both p65 and p50 in the cytoplasm. Furthermore, and most important, a p65 that contains an SV40 large T antigen NLS in addition to its own NLS is no longer retained in the cytoplasm in the presence of I kappa B/MAD-3. We propose that I kappa B/MAD-3 masks the NLSs of NF-kappa B and c-Rel and that this constitutes the mechanism for cytoplasmic retention of these proteins.

Alternative Splicing↗

PubMed1992

HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors.

The HIV-1 trans-activator Tat increases the rate of transcription from the HIV-1 LTR promoter through the stem-loop-containing TAR RNA. To analyze the mechanisms of Tat action, a cell-free trans-activation system with no preincubation has been developed. Recombinant Tat specifically increased the level of a long runoff transcript but not a promoter-proximal transcript in a TAR-dependent fashion. These observations and the result of pulse-chase experiments support strongly the hypothesis that Tat enhances the ability of RNA polymerase to elongate over longer distances. Increased levels of the purified cellular factor TFIIF, essential for initiation and also implicated in elongation of transcription, obviated trans-activation by Tat by increasing the basal (Tat-independent) activity. However, another elongation factor, ATN/TFIIS, showed synergistic activation with Tat. An antiserum against a recombinant form of the large subunit of TFIIF (RAP 74) preferentially suppressed the activated level of transcription exerted by Tat. We propose the hypothesis that Tat acts as a processivity factor on RNA polymerase II in an analogous manner to TFIIF.

Amino Acid Sequence↗

PubMed1992

I-Rel: a novel rel-related protein that inhibits NF-kappa B transcriptional activity.

The NF-kappa B transcription factor complex is comprised of two subunits, p50 and p65, that share significant homology to the rel oncogene. We have isolated a cDNA encoding a novel 66-kD rel-related protein, designated I-Rel. Unlike other rel-related proteins, I-Rel does not interact with DNA. I-Rel forms heterodimers with p50, however, and greatly attenuates its DNA-binding activity--an effect probably resulting from the presence of a domain inhibitory to DNA binding present within the 121 amino-terminal residues of I-Rel. In contrast, I-Rel does not associate with p65. Transfection experiments demonstrate that I-Rel suppresses NF-kappa B-induced transcription, probably through its association with p50. Expression of I-Rel mRNA is induced by mitogenic stimulation and accumulates after the appearance of p50 transcripts. Our findings suggest that p50 and I-Rel are components of a feedback pathway where expression of I-Rel may modulate indirectly the expression of genes responsive to the NF-kappa B transcription factor complex.

Amino Acid Sequence↗