Biomedical subjects

O M Andrisani

Publications and source records attributed to O M Andrisani.

At least 19 recordsLinked to original sources

PubMed2001

Hepatitis B virus X protein differentially activates RAS-RAF-MAPK and JNK pathways in X-transforming versus non-transforming AML12 hepatocytes.

The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronic HBV patients by an unknown mechanism. Activities of pX likely relevant to hepatocyte transformation include activation of the mitogenic RAS-RAF-MAPK and JNK pathways. To assess the importance of mitogenic pathway activation by pX in transformation, we employed a cellular model system composed of two tetracycline-regulated, pX-expressing cell lines, constructed in AML12-immortalized hepatocytes. This system includes the differentiated 3pX-1 and the de-differentiated 4pX-1 hepatocytes. Our studies have demonstrated that conditional pX expression transforms only 3pX-1 cells. Here, comparative in vitro kinase assays and various in vivo analyses demonstrate that pX affects an inverse activation of RAS-RAF-MAPK and JNK pathways in 3pX-1 versus 4pX-1 cells. Sustained pX-dependent RAS-RAF-MAPK pathway activation is observed in pX-transforming 3pX-1 cells, whereas sustained pX-dependent JNK pathway activation is observed in pX non-transforming 4pX-1 cells. This differential, pX-dependent mitogenic pathway activation affects differential activation of cAMP-response element-binding protein and c-Jun and determines the proliferative response of 3pX-1 and 4pX-1 cells. Furthermore, tetracycline-regulated, pX-NLS-expressing cell lines demonstrate that expression of the nuclear pX-NLS variant minimally activates the RAS-RAF-MAPK pathway and results in markedly reduced transformation. These results link sustained, pX-mediated activation of RAS-RAF-MAPK pathway to hepatocyte transformation.

Cell Division↗

PubMed2001

The hepatitis B virus HBx protein induces adherens junction disruption in a src-dependent manner.

Chronic hepatitis B virus infection is strongly associated with the development of hepatocellular carcinoma (HCC). Epithelial tumors are frequently characterized by loss of cadherin expression or function. Cadherin-dependent adhesion prevents the acquisition of a migratory and invasive phenotype, and loss of its function is itself enough for the progression from adenoma to carcinoma. The HBx protein of hepatitis B virus is thought to contribute to the development of the carcinoma, however, its role in the oncogenic and metastatic processes is far from being fully understood. We report herein the ability of HBx to disrupt intercellular adhesion in three different cell lines stably transfected with an inducible HBx expression vector. The linkage between the actin cytoskeleton and cadherin complex, which is essential for its function, is disrupted in the presence of HBx, as indicated by detergent solubility and immunoprecipitation experiments. In addition, beta-catenin was tyrosine phosphorylated in HBx-expressing cells. Inhibition of the src family of tyrosine kinases resulted in the prevention of the disruption of adherens junctions. These results suggest that HBx is able to disrupt intercellular adhesion in a src-dependent manner, and provide a novel mechanism by which HBx may contribute to the development of HCC.

Adherens Junctions↗

PubMed2001

BMP-2 stimulates tyrosinase gene expression and melanogenesis in differentiated melanocytes.

Cells of the vertebrate neural crest (crest cells) differentiate in vitro to melanocytes and sympathoadrenal (SA) progenitor cells. We have shown previously, using primary J. quail neural crest cultures, the combinatorial effect of bone morphogenetic protein-2 (BMP-2) and cAMP signaling on SA cell development. Herein, we report that in primary J. quail neural crest cultures, BMP-2 and cAMP signaling similarly exert a combinatorial effect on melanocyte development. We demonstrate that BMP-2 treatment of neural crest cells increases melanogenesis by promoting the synthesis of melanin. This increased melanin synthesis by BMP-2 is effected by the selective increase in the transcription of the tyrosinase gene, encoding the rate-limiting enzyme of the melanin biosynthetic pathway. By contrast, BMP-2 exerts no effect on the expression of the tyrosine-related proteins 1 and 2 (Tyrpl and Dct), also involved in the melanin biosynthetic process, or on the expression of microphalmia (Mitf) gene, supporting the fact that BMP-2 does not affect melanocyte differentiation. Employing transient transfection analysis of tyrosinase-reporter constructs in B16 melanoma cells, we demonstrate that the BMP-2 response-element is localized between 900 and 1,100 bp upstream from the tyrosinase transcriptional start site. These studies support a role for BMP-2 in melanogenesis by selectively targeting the expression of the tyrosinase gene involved in melanin biosynthesis.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Differential expression of sympathoadrenal lineage-determining genes and phenotypic markers in cultured primary neural crest cells.

Bone morphogenetic protein-2 (BMP-2) promotes the development of primary neural crest cells grown in tissue culture to the sympathoadrenal (SA) lineage. Independent studies have characterized the expression patterns of SA-lineage genes in developing chicken embryo; however, studies using cultured primary neural crest cells have characterized only the expression patterns of the catecholaminergic markers, tyrosine hydroxylase (TH) and catecholamines (CAs). To further explore the molecular mechanisms that control SA-cell development using the in vitro model system, it is crucial to define the expression patterns of both the catecholaminergic markers and the genes regulating SA-lineage determination. Accordingly, we defined, in the absence and presence of BMP-2, the temporal expression patterns of TH and CA, the SA lineage-determining genes ASH-1, Phox2a, and Phox2b, the GATA-2 gene, and the pan-neuronal SCG10 gene. Comparison of these data with the reported temporal and spatial patterns of expression in vivo demonstrate that the inductive steps of SA-lineage determination, including the specification of neurotransmitter identity and neuronal fate, are recapitulated in the neural-crest culture system.

Adrenal Glands↗

PubMed2000

Different regions of hepatitis B virus X protein are required for enhancement of bZip-mediated transactivation versus transrepression.

The hepatitis B virus X protein (pX) interacts directly with the bZip transactivator CREB and the bZip repressors ICERIIgamma and ATF3, increasing their DNA-binding affinity in vitro and their transcriptional efficacy in vivo. However, the mechanism of bZip-pX interaction and of the pX-mediated increase in the bZip transcriptional efficacy remains to be understood. In this study with deletion mutants of pX, we delineated a 67-amino-acid region spanning residues 49 to 115 required for direct CREB, ATF3, and ICER IIgamma interaction in vitro and in vivo and increased bZip/CRE binding in vitro. Transient transfections of the pX deletion mutants in AML12 hepatocytes demonstrate that pX(49-115) is as effective as the full-length pX in enhancing the ATF3- and ICERIIgamma-mediated transrepression. However, this pX region is inactive in increasing the transactivation efficacy of CREB; additional amino acid residues present in pX(49-140) are required to mediate the increased transactivation efficacy of CREB in vivo. This requirement for different regions of pX in affecting CREB transactivation suggests that amino acid residues 115 to 140 integrate additional events in effecting pX-mediated transactivation, such as concomitant interactions with select components of the basal transcriptional apparatus.

Basic-Leucine Zipper Transcription Factors↗

PubMed2000

Cyclic AMP signaling functions as a bimodal switch in sympathoadrenal cell development in cultured primary neural crest cells.

Cells of the vertebrate neural crest (crest cells) are an invaluable model system to address cell fate specification. Crest cells are amenable to tissue culture, and they differentiate to a variety of neuronal and nonneuronal cell types. Earlier studies have determined that bone morphogenetic proteins (BMP-2, -4, and -7) and agents that elevate intracellular cyclic AMP (cAMP) stimulate the development of the sympathoadrenal (SA, adrenergic) lineage in neural crest cultures. To investigate whether interactive mechanisms between signaling pathways influence crest cell differentiation, we characterized the combinatorial effects of BMP-2 and cAMP-elevating agents on the development of quail trunk neural crest cells in primary culture. We report that the cAMP signaling pathway modulates both positive and negative signals influencing the development of SA cells. Specifically, we show that moderate activation of cAMP signaling promotes, in synergy with BMP-2, SA cell development and the expression of the SA lineage-determining gene Phox2a. By contrast, robust activation of cAMP signaling opposes, even in the presence of BMP-2, SA cell development and the expression of the SA lineage-determining ASH-1 and Phox2 genes. We conclude that cAMP signaling acts as a bimodal regulator of SA cell development in neural crest cultures.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Differential immediate early gene expression in conditional hepatitis B virus pX-transforming versus nontransforming hepatocyte cell lines.

We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1alpha and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1beta and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation. Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. The pX-transforming 3pX lineage displays early, pX-dependent induction of ATF3 and prolonged induction of c-fos. The nontransforming 4pX cells display an absence of pX-dependent ATF3 induction and transient induction of c-fos. Our results support the direct link of pX expression to oncogenic transformation in 3pX lineage clones and underscore the advantage of this conditional cellular model system for studying mechanisms of pX-mediated oncogenesis.

Activating Transcription Factor 3↗

PubMed1999

The transcriptional function of the hepatitis B virus X protein and its role in hepatocarcinogenesis (Review).

The hepatitis B virus (HBV) encodes a 16.5 kDa multifunctional protein termed pX or HBx, required for transcription of the viral genome and implicated in the development of hepatocellular carcinoma (HCC) in chronic HBV-infected patients. However, the mechanism of pX-mediated hepatocarcinogenesis remains unknown. pX is a multifunctional protein exhibiting a number of activities affecting transcription, cell growth, and apoptotic cell death. Although pX does not directly bind DNA, pX is regarded as a promiscuous transactivator, acting via a dual mechanism: in the cytoplasm, pX activates mitogenic signaling cascades; in the nucleus, pX interacts directly with members of the bZip class of transcription factors and with specific components of the basal transcriptional apparatus. The focus of this review is to describe the transactivation function of pX and its role in hepatocarcinogenesis.

Animals↗

PubMed1999

CREB-mediated transcriptional control.

cAMP-response-element-binding protein, CREB, is a 43-kDa leucine zipper transcription factor identified and cloned via the study of cAMP-regulated genes. In the last decade, numerous studies have contributed much to our understanding of CREB structure, function, and CREB-mediated transcription. CREB binds to the cAMP-response-element (CRE) as a homodimer formed via the leucine zipper motif present at its C-terminus; its transcriptional activity is regulated by phosphorylation at Ser133, located within the N-terminal transactivation domain. Active, Ser133-phosphorylated CREB effects transcription of CRE-dependent genes via interaction with the 265-kDa co-activator protein CREB-binding-protein, CBP, which bridges the CRE/CREB complex to components of the basal transcriptional apparatus. This mechanism of CREB activation is effected by diverse signals, including those regulating the intracellular levels of cAMP and Ca+2, growth factors, and cellular stress. Accordingly, CREB-mediated transcription regulates diverse cellular responses, including intermediary metabolism, neuronal signaling, cell proliferation, and apoptosis. In addition to the regulation of CREB by phosphorylation, the viral oncoproteins HBV pX and HTLVI Tax regulate CREB transcriptional efficacy by an alternative mechanism, by increasing its DNA-binding affinity for viral and/or cellular CRE sites. In this review I describe key experiments that have defined the mechanism of CREB activation, with primary emphasis on emerging evidence linking CREB to cellular growth and development.

Amino Acid Sequence↗

PubMed1998

Multiple regions of the porcine alpha-skeletal actin gene modulate muscle-specific expression in cell culture and directly injected skeletal muscle.

Transcriptional regulation of the porcine alpha-skeletal actin gene was investigated by comparative transient transfection assays in cultured mammalian cells and by direct DNA injection in skeletal muscle. Intron I sequences were necessary to direct high-level, cell-specific porcine alpha-skeletal actin expression in C2C12 myotubes, but they inhibited transcription in skeletal muscle. A 5' distal sequence (-1929 to -550), had enhancer-like activity in C2C12 myotubes and directly injected muscle, and inhibited transcription in Hela cells. In contrast, a central region (-550 to -388) enhanced basal transcription in directly injected muscle, but not in C2C12 myotubes. A distal regulatory element localized to the 3' untranslated region modulated SV40 promoter activity only in cell culture studies. These results suggest that the intragenic and 3' distal regulatory element may be differentially utilized during differentiation and maturation of skeletal muscle. All three regions decreased SV40 promoter activity in Hela cells, suggesting that they play a role in defining tissue-specific expression of porcine alpha-skeletal actin. Furthermore, different regulatory programs of alpha-skeletal actin expression appear to exist in these two experimental systems.

Actins↗

PubMed1997

The hepatitis B virus X protein enhances the DNA binding potential and transcription efficacy of bZip transcription factors.

The hepatitis B virus X protein interacts with the basic-region, leucine zipper protein (bZip) domain of cAMP response element-binding protein increasing its affinity for the cAMP response element site in vitro and its transcriptional efficacy in vivo (Williams, J. S., and Andrisani, O. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3819-3823). Here we examine pX interactions with bZip transcription factors ATF3, gadd153/Chop10, ICER IIgamma, and NF-IL6. We demonstrate direct interactions in vitro between pX and the bZip proteins tested. In contrast MyoD and Gal4(1-147) fail to interact with pX. We also demonstrate by the mammalian two-hybrid assay the direct interaction of pX with cAMP response element- binding protein, ICER IIgamma, ATF3, and NF-IL6 in hepatocytes. In addition, pX increases the DNA binding potential of bZip proteins for their cognate DNA-binding site in vitro. In transient transfections in hepatocytes (AML12 cell line), pX increases the transcriptional efficacy of the bZip transcription factors. NF-IL6-mediated transcriptional activation is enhanced 3-fold by pX. Most interestingly, pX augments the repression mediated by bZip repressors ATF3 and ICER IIgamma, by 6- and 7-fold, respectively, demonstrating for the first time the involvement of pX in gene repression. We conclude that pX is an enhancer of the DNA binding potential of bZip transcription factors, thereby increasing the transactivation or repression efficacy of bZip-responsive genes.

Animals↗

PubMed1995

Cloning of the canine interleukin-2-encoding cDNA.

Here we report the nucleotide sequence of the canine interleukin-2 (IL-2)-encoding cDNA. Cloning of the canine IL-2 cDNA was achieved by the polymerase chain reaction employing, as a template, a cDNA derived from mitogen-stimulated canine splenic lymphocyte mRNA. The deduced amino acid (aa) sequence of canine IL-2 consists of 155 aa and displays 84% sequence similarity to human IL-2.

Amino Acid Sequence↗

PubMed1995

The hepatitis B virus X protein targets the basic region-leucine zipper domain of CREB.

The X gene product encoded by the hepatitis B virus, termed pX, is a promiscuous transactivator of a variety of viral and cellular genes under the control of diverse cis-acting elements. Although pX does not appear to directly bind DNA, pX-responsive elements include the NF-kappa B, AP-1, and CRE (cAMP response element) sites. Direct protein-protein interactions occur between viral pX and the CRE-binding transcription factors CREB and ATF. Here we examine the mechanism of the protein-protein interactions occurring between CREB and pX by using recombinant proteins and in vitro DNA-binding assays. We demonstrate that pX interacts with the basic region-leucine zipper domain of CREB but not with the DNA-binding domain of the yeast transactivator protein Gal4. The interaction between CREB and pX increases the affinity of CREB for the CRE site by an order of magnitude, although pX does not alter the rate of CREB dimerization. Methylation interference footprinting reveals differences between the CREB DNA and CREB-pX DNA complexes. These experiments demonstrate that pX titers the way CREB interacts with the CRE DNA and suggest that the basic, DNA-binding region of CREB is the target of pX. Transfection assays in PC12 cells with the CREB-dependent somatostatin promoter demonstrate a nearly 15-fold transcriptional induction after forskolin stimulation in the presence of pX. These results support the significance of the CREB-pX protein-protein interactions in vivo.

Animals↗

PubMed1994

A secondary phosphorylation of CREB341 at Ser129 is required for the cAMP-mediated control of gene expression. A role for glycogen synthase kinase-3 in the control of gene expression.

The cAMP-dependent protein kinase (PKA) phosphorylates CREB327/341 at a single serine residue, Ser119/133, respectively. Phosphorylation at this site creates the sequence motif SXXXS(P), a consensus site of the glycogen synthase kinase-3 (GSK-3) enzyme (Fiol, C.J., Mahrenholz, A.M., Wang, Y., Roeske, R.W., and Roach, P.J. (1987) J. Biol. Chem. 262, 14042-14048). We examined the phosphorylation of CREB at the SXXXS(P) consensus site and its role in CREB transactivation to cAMP induction. Neither isoform of the GSK-3 enzyme (GSK-3 alpha or beta) utilizes CREB as its substrate unless CREB is already phosphorylated at Ser119/133. A 13-amino acid peptide containing the sequence surrounding Ser119/133 was phosphorylated by GSK-3, at Ser115/129, only after the primary phosphorylation of the peptide by PKA (at Ser119/133), suggesting that Ser115/129 is a GSK-3 phosphoacceptor site. Mutant CREB327/341 proteins containing Ser-->Ala substitutions confirmed Ser115/129 as the only GSK-3 phosphorylation site. Transfection assays of wild type and mutant Gal4-CREB fusion proteins in PC12 cells demonstrated that Ser-->Ala substitution of residue 129 of CREB341 impairs the transcriptional response to cAMP induction. Analogous mutation in CREB327 results in 70% decrease in its transactivation response to cAMP. In undifferentiated F9 cells, which are refractory to cAMP induction, transfected GSK-3 beta kinase induces a 60-fold increase in cyclic AMP response element-dependent transcription, mediated via the endogenous CREB protein. We propose that the hierarchical phosphorylation at the PKA and GSK-3 sites of CREB are essential for cAMP control of CREB.

Amino Acid Sequence↗

PubMed1993

Bacterial expression and characterization of the CREB bZip module: circular dichroism and 2D 1H-NMR studies.

In this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB327. The bZip peptide, CREB259-327, purified to near homogeneity, maintains the sequence-specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259-327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The Kd of the purified native and modified CREB259-327 for the CRE site was determined by gel retardation assays to be on the order of 10(-7) M. We employed CD spectroscopy to study the folding properties of the native and modified CREB259-327. The CD analyses of the native/modified CREB259-327 peptide demonstrated a 20% increase in the alpha-helical content upon binding to the cAMP response-element. Only a 5% increase in the alpha-helical content of CREB259-327 is observed upon binding to the AP-1 site. This observation contrasts with CREB from the GCN4 protein (Weiss, M.A., et al., 1990, Nature 347, 575-578). In addition, the two-dimensional (2D) 1H-NMR studies of the bZip CREB peptide further support the distinct features of the CREB protein, in comparison to GCN4. Analysis by CD and 2D NMR of the dimerization domain of CREB suggests that the distinct DNA binding characteristics of CREB reside in the basic portion of the bZip module.

Amino Acid Sequence↗

PubMed1993

Binding constant determination studies utilizing recombinant delta CREB protein.

In this study, we report the binding constants (Kd) of the cAMP-responsive element binding protein (delta-CREB) for various cAMP-response element (CRE) motifs. We utilized purified recombinant delta CREB protein in binding reactions with natural CRE motifs found in the promoter of two neuropeptide hormone genes and with several variant CRE motifs. The Kd of delta CREB for the perfectly palindromic CRE, TGACG-TCA, found within the somatostatin promoter is estimated to be 5.0 x 10(-9) M. The Kd of delta CREB for the variant CRE motif TG_CGTCA found within the enkephalin promoter is calculated to be in the 3 x 10(-8) M. These studies provide an in vitro quantitative assessment of the binding affinity of delta CREB for various CRE motifs.

Amino Acid Sequence↗

PubMed1991

Involvement of lysine residues 289 and 291 of the cAMP-responsive element-binding protein in the recognition of the cAMP-responsive element.

The molecular interactions resulting in specific binding of trans-acting factors to distinct cis-acting elements is not well understood. Here we report our attempt to understand the involvement of distinct amino acid residues of the basic domain of cAMP-responsive element-binding protein (delta CREB) in the determination of binding toward the cAMP-responsive element (CRE). Using in vitro mutagenesis, we constructed site-directed mutants of distinct amino acid residues within the DNA contact region of delta CREB. The activities of the mutant proteins were analyzed by gel retardation, methylation interference, and CRE competition analyses. We demonstrate that a single lysine to glutamine substitution at positions 289 and 291 of delta CREB alters the methylation interference pattern of the mutant protein for the CRE site. Additional mutants constructed at these positions demonstrate that only identical basic residues at both positions 289 and 291 of delta CREB can restore the wild type methylation interference pattern of the mutant delta CREB protein for the CRE site. These observations point to the importance of the lysine residues at positions 289 and 291 in the process of CRE binding. In addition, this observation suggests that the symmetrical nature of the CRE site is reflected in the DNA contact region of the protein.

Amino Acid Sequence↗

PubMed1990

Somatostatin gene regulation.

Since the cloning of the somatostatin cDNA and gene, the efforts of a number of laboratories have contributed to the understanding of somatostatin gene expression and regulation. A genetic element located approximately 40 nucleotides upstream from the somatostatin mRNA cap site is important in the expression and cAMP-induced transcription of this neuropeptide gene. The identification of this genetic element has enabled the identification, purification, and cloning of a new class of proteins: the somatostatin gene transactivator named CREB. The availability of cloned CREB will permit studies designed to understand not only the mechanism of somatostatin gene expression and regulation, but also the pathway of signal transduction triggered by cAMP-stimulated events.

Animals↗