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K G Xanthopoulos

Publications and source records attributed to K G Xanthopoulos.

At least 37 records · Page 2Linked to original sources
PubMed1996

A novel human CCAAT/enhancer binding protein gene, C/EBPepsilon, is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to the T-cell receptor alpha/delta locus.

Members of the CsolidusEBP family of transcriptional factors have been implicated in the regulation of genes in a variety of tissues. We report here the isolation and characterization of the human C/EBPepsilon gene (CEBPE). By using low-stringency hybridization conditions and probes derived from the C/EBPalpha and C/EBPdelta genes, we have isolated overlapping genomic clones that cover almost 25 kb of the C/EBPepsilon gene locus and corresponding cDNA clones. DNA sequence analysis reveals that the gene encodes a protein highly homologous to rat CRP1. The gene was assigned to chromosome 14q11.2 by fluorescence in situ hybridization and was physically linked to the genetic marker D14S990. Based on linkage data derived from this marker, we positioned the CEBPE gene between the T-cell receptor alpha/delta locus and a cluster of four serine proteases expressed exclusively in hematopoietic cells. Expression of C/EBPepsilon was detected in Jurkat T-cell and in HL 60 promyelocytic cell lines. From a variety of normal human tissues studied, expression of mRNA was monitored only in peripheral blood mononuclear cells, tissues involved in the immune system, and ovaries. These data demonstrate that the C/EBPepsilon gene shows a restricted pattern of expression, has an intriguing chromosomal location, and suggest a possible role for the regulation of certain genes in cells of myeloid and lymphoid lineages.

Amino Acid Sequence↗
PubMed1995

Molecular cloning, sequence, and expression patterns of the human gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha).

The human gene encoding the transcription factor C/EBP alpha was isolated from an umbilical cord genomic library screened by low stringency hybridization. Two overlapping clones were characterized by restriction enzyme analysis and included 13.2 kb of the C/EBP alpha locus. The entire gene and 471 bp of the promoter were sequenced. The human C/EBP alpha gene is 2783 bp long and encodes a 356 amino acid long protein, which is the same in length as for rat C/EBP alpha. Compared to rat C/EBP alpha, there are two insertions of two amino acids and one deletion of four. The amino acid similarity between the two proteins is over 92%. The human C/EBP alpha gene was found to be expressed at the highest levels in placenta. High expression was also found in liver, lung, skeletal muscle, pancreas, small intestine, colon and in peripheral blood leukocytes. However, the expression was undetectable or very low in brain, kidney, thymus, testis and ovary. These results show that the human C/EBP alpha gene is expressed in a tissue restricted manner.

Amino Acid Sequence↗
PubMed1995

Myc inhibits CCAAT/enhancer-binding protein alpha-gene expression in HIB-1B hibernoma cells through interactions with the core promoter region.

The product of the c-myc proto-oncogene, Myc, has been implicated in the transcriptional regulation of several genes, acting either as an activator or repressor of gene expression. To determine whether Myc is involved in the modulation of the expression of the CCAAT/enhancer-binding protein alpha (C/EBP alpha) gene, we used both stable cell lines overexpressing Myc and transient co-transfection assays. We show that the endogenous C/EBP alpha protein level is repressed in stable cell lines overexpressing Myc. We also show that enforced expression of Myc in mouse hibernoma HIB-1B cells dramatically repressed the expression of C/EBP alpha--promoter-reporter fusion genes. This effect of Myc was mediated through the core promoter region. Mutation of the initiator site could not abolish this affect, indicating that Myc may interact with some component(s) of the basal transcription machinery.

Animals↗
PubMed1995

Expression of the liver-enriched transcription factors C/EBP alpha, C/EBP beta, HNF-1, and HNF-4 in preneoplastic nodules and hepatocellular carcinoma in rat liver.

The expression patterns of the liver-enriched transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta and hepatocyte nuclear factor (HNF)-1 and HNF-4 were studied in liver nodules and hepatocellular carcinomas from male rats treated according to the resistant hepatocyte (RH) model. C/EBP alpha expression was lower at the transcriptional, mRNA, and protein levels in persistent nodules than in the respective surrounding livers. Expression was further decreased in the tumors. Transcriptional downregulation of C/EBP alpha gene expression was observed already in very early nodules, isolated 3 wk after partial hepatectomy in the RH model. However, no detectable changes were observed in preneoplastic nodules in the transcription or in steady-state mRNA levels of C/EBP beta, HNF-1, and HNF-4. A slight decrease in C/EBP beta protein and a more pronounced attenuation of HNF-1 and HNF-4 levels was observed in nodules, being 67%, 37%, and 46% of the levels in the corresponding surrounding livers, respectively. In conclusion, differential regulation of several transcription factors that are associated with the maintenance of the differentiated state of the hepatocytes was observed in preneoplastic and neoplastic liver lesions. This could have an impact on the regulation of a wide array of genes during liver carcinogenesis. Furthermore, the attenuation of C/EBP alpha expression, regarded as a negative growth regulator, could contribute to the proliferative advantage of nodules during liver carcinogenesis.

Animals↗
PubMed1995

Retroviral-mediated gene transfer into human bone marrow stromal cells: studies of efficiency and in vivo survival in SCID mice.

Retroviral-mediated gene transfer into bone marrow cells is extensively used in gene therapy protocols. Cytokines are needed for stimulation, to achieve a high rate of gene transfer. However, the stromal cell compartment of bone marrow is characterized by rapid proliferation even without cytokines. In this study, human bone marrow stromal cells were isolated and subsequently infected with recombinant retrovirus in a cell-free supernatant. The LN retroviral vector used in this study carries the bacterial neomycin phosphotransferase gene (neoR). Transduction efficiency was significantly enhanced by repeated cycles of infection, with a maximum of 91% transduced cells by four rounds of infection. Presence of the neoR-gene was detected by PCR from all stromal cells selected by G418. After culture in vitro for 3 months, cells were still positive for PCR amplification of the neoR-gene. Transduced stromal cells were also injected into SCID mice to study their homing and survival ability in vivo.

Animals↗
PubMed1994

Retroviral-mediated gene transfer into human myeloma cells.

We have studied retroviral-mediated gene transfer into human myeloma cells. Bone marrow cells were obtained from four patients with advanced myeloma, where the marrow was heavily infiltrated with myelomatous plasma cells. Myeloma cells were isolated by immunomagnetic separation, using the high-affinity B-B4 monoclonal antibody. Following separation, cells were transduced with the LN retroviral vector, which carries the gene for neomycin phosphotransferase, by incubation in cell-free supernatant with or without a growth-factor combination of IL-3, IL-6 and SCF. After infection, the cells were cultured for 9 d in RPMI-1640 and 10% FCS, either in the presence or absence of the neomycin analogue G418. Transduction efficiency was 1.5-3.8%, when compared to the number of cells at initiation of the culture, and 5.0-50.0% when compared to the number of surviving infected cells cultured without G418. The gene transfer rate was similar whether or not growth factors were present during the retroviral infection. These preclinical data provide evidence that retroviral-mediated gene transfer into human myeloma cells is feasible, and form part of the basis for current clinical studies of gene marking of bone marrow or peripheral blood progenitor cells before autologous stem cell transplantation in multiple myeloma.

Aged↗
PubMed1994

Basic fibroblast growth factor increases retroviral-mediated gene transfer into human hematopoietic peripheral blood progenitor cells.

Retroviral vector-mediated gene transfer into human hematopoietic stem cells (HSC) may permit gene therapy of certain genetic diseases. Stimulation of HSC with hematopoietic growth factors (GF) has been shown to increase the level of retroviral transduction. We have studied the effect of basic fibroblast growth factor (bFGF), alone and in combination with other GFs, on the efficiency of transfer of the bacterial neomycin phosphotransferase (neoR) gene into human CD34(+)-enriched peripheral blood hematopoietic progenitor cells. The combination of bFGF, interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in a transduction efficiency of 37 and 35% for G418-resistant colony-forming units-granulocyte/macrophage (CFU-GM) and mixed colonies multipotent colony-forming units (CFU-GEMM), respectively, which was significantly higher than the corresponding figures obtained with IL-3, IL-6, and SCF. The optimal concentration of bFGF was between 20 and 200 ng/mL. bFGF alone had no effect on the transduction rate. These results indicated a synergism in the action of bFGF, IL-3, IL-6, and SCF to enhance gene transduction rates into human hematopoietic progenitor cells.

Antigens, CD↗
PubMed1993

Gene regulation in rodent hepatocytes during development, differentiation and disease.

The expression of genes in the liver is mostly controlled at the transcriptional level and depends on the regulatory interactions between cis-acting sequences and trans-acting molecules. Proximal promoters and distant enhancers in combination with a number of hepatocyte-enriched DNA-binding proteins and general transcription factors interact specifically with these elements and control the expression of liver-specific genes. Hepatocyte-enriched regulatory proteins have been isolated from liver nuclear extracts, characterized, and their corresponding genes have been cloned. These include the hepatocyte nuclear factors 1, 3, 4 (HNF-1,3,4), some members of the CAAAT/enhancer binding protein (C/EBP) family, and D site binding protein (DBP). These factors belong to larger families and are able to form heterodimers, perhaps with the exception of the HNF-3 family, with other members of the same family. Interestingly, the majority of the genes encoding such proteins are themselves regulated at the transcriptional level, although both transcriptional and post-transcriptional events modulate their expression during development, hepatocyte differentiation and disease, suggesting that a transcriptional cascade may play a critical role in mammalian liver development and differentiation.

Animals↗
PubMed1993

High level activity of the mouse CCAAT/enhancer binding protein (C/EBP alpha) gene promoter involves autoregulation and several ubiquitous transcription factors.

The promoter region of the mouse CCAAT-Enhancer Binding Protein (C/EBP alpha) gene is capable of directing high levels of expression of reporter constructs in various cell lines, albeit even in cells that do not express their endogenous C/EBP alpha gene. To understand the molecular mechanisms underlying this ubiquitous expression, we have characterized the promoter region of the mouse C/EBP alpha gene by a variety of in vitro and in vivo methods. We show that three sites related in sequence to USF, BTE and C/EBP binding sites and present in promoter region -350/+3, are recognized by proteins from rat liver nuclear extracts. The sequence of the C/EBP alpha promoter that includes the USF binding site is also capable of forming stable complexes with purified Myc+Max heterodimers and mutation of this site drastically reduces transcription of C/EBP alpha promoter luciferase constructs both in liver and non liver cell lines. In addition, we identify three novel protein-binding sites two of which display similarity to NF-1 and a NF kappa B binding sites. The region located between nucleotides -197 and -178 forms several heat-stable complexes with liver nuclear proteins in vitro which are recognized mainly by antibodies specific for C/EBP alpha. Furthermore, transient expression of C/EBP alpha and to a lesser extent C/EBP beta expression vectors, results in transactivation of a cotransfected C/EBP alpha promoter-luciferase reporter construct. These experiments support the notion that the C/EBP alpha gene is regulated by C/EBP alpha but other C/EBP-related proteins may also be involved.

Animals↗
PubMed1993

Differential adrenergic regulation of C/EBP alpha and C/EBP beta in brown adipose tissue.

We investigated the regulation of the expression of two members of the C/EBP family of transcriptional activators, C/EBP alpha and C/EBP beta, in brown adipose tissue in mice. Less than one hour of cold exposure led to dramatic changes in the expression of both genes. C/EBP alpha steady-state mRNA and protein levels were drastically and rapidly reduced whereas C/EBP beta mRNA and protein levels were induced severalfold. Also norepinephrine injection affected the expression of the transcription factors. Preconfluent cells in brown fat primary cultures responded to norepinephrine with a decrease in C/EBP alpha and an increase in C/EBP beta mRNA; in confluent cells the expression of both factors was increased. Thus, C/EBP alpha and C/EBP beta gene expression is under adrenergic control both in vivo and in vitro but the type of response is directed by the degree of differentiation of the cells.

Adipose Tissue, Brown↗
PubMed1993

Differential patterns of expression of three C/EBP isoforms, HNF-1, and HNF-4 after partial hepatectomy in rats.

Regenerating liver provides a system for studying the mechanisms controlling regulated proliferation of differentiated hepatocytes. A set of transcription factors termed hepatocyte nuclear factors (HNF-1, -3, -4) and CCAAT/enhancer binding protein (C/EBP) isoforms are known to regulate several genes predominantly expressed in the liver. To assess whether these factors might be involved in the hepatocyte proliferation program, we have studied the expression of the three C/EBP isoforms C/EBP alpha, C/EBP beta, and C/EBP delta and the two hepatocyte-enriched transcription factors, HNF-1 and HNF-4, in rat liver at various time points after partial hepatectomy and sham operations using transcriptional "run-on" assays and Northern blot and Western blot experiments. We report here that partial hepatectomy in rats leads to dramatic changes in the pattern of expression of some of these genes. The three C/EBP isoforms are differentially regulated in response to partial hepatectomy and are likely to play different roles in determining the proliferation/differentiation state of hepatocytes. In particular, C/EBP alpha expression is rapidly down-regulated, whereas C/EBP delta is induced. C/EBP beta expression is also increased, although an increase is also observed after sham operation. The drastic decrease in C/EBP alpha under these conditions of active DNA synthesis and rapid cell proliferation further supports the concept of a potential incompatibility between high C/EBP alpha protein levels and cell proliferation. The patterns of transcriptional rates of HNF-1 and HNF-4 during the different stages of the regenerative process are similar. However, HNF-1 steady-state mRNA and protein levels are significantly changed while HNF-4 remains virtually unaffected, indicating that post-transcriptional mechanisms are also involved in the regulation of HNF-1 gene expression.

Animals↗
PubMed1992

Transiently and stably introduced CCAAT/enhancer-binding-protein genes are constitutively expressed in cultured cells.

CCAAT/enhancer-binding protein (C/EBP) is expressed in certain cell types including hepatocytes and adipocytes. In order to understand the mechanisms that control the expression of the mouse C/EBP gene in the liver as well as in adipocytes, we have studied both the endogenous gene and transfected C/EBP gene constructs. The initiation site of transcription was identified and a strong liver-specific DNase-I hypersensitive site located at -3 kb, which does not appear to contribute functionally to the regulation of the gene in a variety of either transiently or stably transfected cells with constructs which include sequences up to 6-kb upstream of the transcription start. C/EBP gene expression during the transition from preadipocytes to adipocytes was shown to be controlled at the level of transcription. However, adipocytes stably transfected with constructs that include -3.3 kb upstream of the C/EBP gene do not express the reporter genes in a differentiation-specific manner. We detected several DNA-binding proteins that interact with the upstream sites of the C/EBP gene. Those include two labile and two heat-stable site-specific DNA-binding proteins that are present in nuclear extracts from several tissues and cultured cell lines.

Adipose Tissue↗
PubMed1992

Transcriptional control in hepatocytes of normal and c14CoS albino deletion mice.

The transcription rates of the albumin and alpha-fetoprotein (alpha FP) genes were reduced to marginally detectable levels in livers of newborn or fetal c14CoS albino deletion mutant mice, which lack the hepatocyte specific developmental regulation (hsdr-1) locus on chromosome 7 and die shortly after birth. However, steady-state levels of these two mRNAs in livers of mutant mice were similar to those in normal mice, where these genes are actively transcribed. In c14CoS mice, transcription rates of transcription factor genes HNF-1, C/EBP and HNF-4 were reduced, albeit to different extents. These effects are specific because transcription of the HNF-3, DBP, LAP and Jun-B genes remained normal in mutant mice. Steady-state levels of all of these mRNAs reflected the transcriptional activities. Levels of HNF-1 and HNF-4 mRNAs showed much greater depression than that of C/EBP in mutant liver. The availability of this group of transcription factors may be reduced in c14CoS hepatocytes and therefore caused depressed transcription rates of their target genes such as those encoding albumin and alpha FP. However, the normal steady-state levels of albumin and alpha FP mRNAs in mutant mice remains unexplained. Fetal c14CoS hepatocytes in primary culture did acquire competence for glucocorticoid inducible transcription of the albumin, alpha FP, HNF-4 and metallothionein genes but not of the tyrosine aminotransferase (TAT) gene. These results indicate that the hsdr-1 locus is dispensable for the glucocorticoid induced transcription of these genes but not of TAT. The effects caused by the c14CoS deletion are pleiotropic in controlling the expression of numerous genes at distinct levels in the liver.

Albinism↗
PubMed1991

The different tissue transcription patterns of genes for HNF-1, C/EBP, HNF-3, and HNF-4, protein factors that govern liver-specific transcription.

The transcription factors that act in hepatocyte-specific gene expression include proteins that are present mainly in liver cells (HNF-1/LFB1, C/EBP, HNF-3, HNF-4) (HNF, hepatocyte nuclear factor; C/EBP, rat enhancer binding protein) and proteins that are widely distributed (AP-1, NF-1, NF-Y/ACF). We show here that the genes encoding each of these liver-enriched factors exhibit different patterns of transcriptional control in different tissues. In addition, there were several instances in which transcription was detected (e.g., for HNF-1) when no mRNA or specific DNA binding protein was found, suggesting the importance of posttranscriptional control in some instances for these factors. These experiments identify C/EBP, HNF-3, and HNF-4, and perhaps also HNF-1, as targets for the study of cascades of transcriptionally controlled transcription factors in differentiated cells.

Base Sequence↗
PubMed1990

Fetal and adult localization of C/EBP: evidence for combinatorial action of transcription factors in cell-specific gene expression.

C/EBP is a positive-acting transcription factor important for hepatocyte-specific expression present not only in hepatocytes but also a limited number of other cell types in adult mice. By Northern blot analysis and in situ hybridization experiments with mouse embryos and adult tissues, we first detected C/EBP mRNA in hepatocytes at the 13th gestational day, when no other cell types give detectable signals and thus by this test C/EBP is, at least in the embryo, a 'liver-specific' factor. Only trace amounts of C/EBP were seen in the yolk sac and no mRNA was detectable in choroid plexus in either embryos or adult animals. Both these cell types produce some proteins (e.g. albumin, transthyretin, alpha-1 antitrypsin and others) that are also made in the liver where C/EBP is important for their production; thus either fewer factors or different factors govern yolk sac and choroid plexus production of these proteins. C/EBP mRNA was not detected in fetal brain but was present in several regions of the adult mouse brain again emphasizing that this factor does not appear to have a very early embryologic role. In the adult brain, it was most concentrated in CA1 to CA4 regions of the hippocampus, in cerebellar Purkinje cells, and in layer II and III of the cortex.

Animals↗
PubMed1989

Cell-specific transcriptional control of the mouse DNA-binding protein mC/EBP.

The mRNA encoding the mouse homolog of C/EBP, a rat DNA-binding protein that participates in activating a number of genes in hepatocytes, is present in liver cells at a far higher concentration than in most other cells, including spleen, kidney, muscle, and the majority of the brain. However, fat cells and intestinal cells contain 25-50% as much mRNA as liver cells. "Run-on" experiments show that the basis for the restricted cellular distribution of the mouse C/EBP mRNA is transcriptional regulation of the gene. We also show that disruption of cell-cell contacts incident to liver cell dispersion results in a prompt and extensive reduction in mouse C/EBP transcription as we had earlier shown to be the case for a group of 10 genes transcribed in a hepatocyte-specific fashion. In contrast, breaking cell contacts and plating the hepatocytes in culture leads to a prolonged increase in transcription of the Jun-B gene that encodes a widely distributed transcription factor. These results illustrate that the regulation of expression of a mammalian regulatory protein with limited tissue distribution is controlled at the level of transcription and depends on cell contacts.

Animals↗
PubMed1989

A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation.

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.

8-Bromo Cyclic Adenosine Monophosphate↗