Chloramphenicol Acetyltransferase Activity Research Articles

Osteoblast-specific gene expression after transplantation of marrow cells: Implications for skeletal gene therapy

Somatic gene therapies require targeted transfer of the therapeutic gene(s) into stem cells that proliferate and then differentiate and express the gene in a tissue-restricted manner. We have developed an approach for gene therapy using marrow cells that takes advantage of the osteoblast specificity of the osteocalcin promoter to confine expression of chimeric genes to bone. Adherent marrow cells, carrying a reporter gene [chloramphenicol acetyltransferase (CAT)] under the control of a 1.7-kilobase rat osteocalcin gene promoter, were expanded ex vivo. After transplantation by intravenous infusion, engrafted donor cells in recipient mice were detected by the presence of the transgene in a broad spectrum of tissues. However, expression of the transgene was restricted to osteoblasts and osteocytes, as established by biochemical analysis of CAT activity and immunohistochemical analysis of CAT expression at the single cell level. Our data indicate that donor cells achieved long-term engraftment in various tissues of the recipients and that the CAT gene under control of the osteocalcin promoter is expressed specifically in bone. Thus, transplantation of multipotential marrow cells containing the osteocalcin promoter-controlled transgene provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for treatment of bone disorders or tumor metastasis to the skeleton.

Role of intron 1 in smooth muscle α-actin transcriptional regulation in activated mesangial cells in vivo

The activation of glomerular mesangial cells is one of the early, important features of progressive glomerular disease. Smooth muscle alpha-actin (SMalphaA) is an excellent marker of activated mesangial cells. However, the mechanisms of SMalphaA regulation are only available from in vitro investigation. We examined in vivo promoter analysis of the SMalphaA gene-utilizing transgenic mice harboring different promoter regions of the SMalphaA gene fused to chloramphenicol acetyl transferase (CAT). CAT activities were tested in primary cultured mesangial cells and in glomerular legions of Habu venom glomerulonephritis. The DNA sequence -891 to +3828, which contains exon 1, intron 1, and the first 14 bp of exon 2 in addition to the 5'-flanking sequence of the SMalphaA gene, induced high levels of transcription in activated mesangial cells in in vivo habu venom glomerulonephritis and in cultured mesangial cells derived from transgenic mice. The DNA region -891 to -124 was a positive element in mesangial cells derived from transgenic mice. Deletions (3316 or 137 bp) in intron 1 reduced transcription to undetectable levels. The 137 bp sequence is highly conserved among several species, containing one CArG box element, which is one of the key motifs for transcriptional activation of contractile-related proteins. In vitro transfection analysis failed to demonstrate these positive effects of intron 1 and region -891 to -124. Conclusions. In vivo promoter analysis of the SMalphaA gene provided new information about the transcriptional regulation of SMalphaA in activated mesangial cells. The DNA region -891 to -124 has a positive effect on SMalphaA transcription in cultured mesangial cells. The intron 1 region (+1088 to +1224) plays a pivotal role in SMalphaA transcription in activated mesangial cells in vivo. Further analysis of this conserved region in intron 1, including the CArG motif, will be of great value in understanding the molecular mechanisms of mesangial activation.

CCAAT Enhancer-binding Protein β and GATA-4 Binding Regions within the Promoter of the Steroidogenic Acute Regulatory Protein (StAR) Gene Are Required for Transcription in Rat Ovarian Cells

Steroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the -1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshly prepared cells. FSH inducibility determined over a 6-h incubation was 10-40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR promoter constructs: a non-consensus binding sequence (-81/-72) for CCAAT enhancer-binding protein beta (C/EBPbeta) and a consensus motif for GATA-4 binding (-61/-66). Western analyses showed that GATA-4 is constitutively expressed in the granulosa cells, while all isoforms of C/EBPbeta were markedly inducible by FSH. Site-directed mutations of both binding sequences practically ablated both basal and hormone-driven chloramphenicol acetyltransferase activities to less than 5% of the parental -96/+6 construct. Unlike earlier notions, elimination of potential binding sites for steroidogenic factor-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBPbeta and GATA-4 represent a novel combination of transcription factors capable of conferring an acute response to hormones upon their concomitant binding to the StAR promoter.

Selective activation of the probasin androgen-responsive region by steroid hormones.

Glucocorticoid and androgen receptors have been shown to function through the same palindromic glucocorticoid response element (GRE) and yet have differential effects on gene transcription. In this study, we examined the functional and structural relationship of the androgen and glucocorticoid receptors with the androgen responsive region (ARR) of the probasin (PB) gene containing two androgen receptor binding sites, ARBS-1 and ARBS-2. Transfection studies indicated that one copy of each cis-acting DNA element was essential for maximal androgen-induced chloramphenicol acetyltransferase (CAT) activity and that androgen selectivity was maintained when multiple copies of the minimal wild type (wt) androgen responsive region containing both ARBS-1 and ARBS-2 (-244 to -96) were subcloned in front of the thymidine kinase promoter. Furthermore, replacing the androgen response region with 1, 2 or 3 copies of either ARBS-1 or ARBS-2 restored less than 4% of the biological activity seen with the wt PB ARR. Multiple copies of either ARBS-1 or ARBS-2 did not result in glucocorticoid-induced CAT gene activity. By comparison, 1 or 2 copies of the tyrosine aminotransferase (TAT) GRE, as well as the mouse mammary tumour virus GRE, were strong inducers of CAT activity in response to both androgen and glucocorticoid treatment. In addition, band shift assays demonstrated that although the synthetic glucocorticoid receptor, GR-DNA binding domain (GR-DBD), and the synthetic androgen receptor, AR2, could interact with the TAT GRE (dissociation constants Kd of 63.9 and 14.1 respectively), only AR2 but not GR-DBD binding could be detected on ARBS-1 and ARBS-2. Our findings provide further evidence that androgen-induced regulation of gene transcription can occur through androgen-specific DNA binding sites that are distinct from the common GRE.

Isotopic Assays for Reporter Gene Activity

This unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA to chloramphenicol. Transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material. The second reporter system uses a kit to perform a simple two-site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with 125I-labeled antibody specific for hGH, and immune complexes are collected by an avidin-coated bead. The quantity of hormone is determined based on comparison with a standard curve.

Structural and functional heterogeneity of nuclear bodies.

The nuclear body is a cellular structure that appears to be involved in the pathogenesis of acute promyelocytic leukemia and viral infection. In addition, the nuclear body is a target of autoantibodies in patients with the autoimmune disease primary biliary cirrhosis. Although the precise function of the nuclear body in normal cellular biology is unknown, this structure may have a role in the regulation of gene transcription. In a previous investigation, we identified a leukocyte-specific, gamma interferon (IFN-gamma)-inducible autoantigen designated Sp140. The objectives of the present study were to investigate the cellular location of Sp140 with respect to the nuclear-body components PML and Sp100 and to examine the potential role of Sp140 in the regulation of gene transcription. We used adenovirus-mediated gene transfer to express Sp140 in human cells and observed that the protein colocalized with PML and Sp100 in resting cells and associated with structures containing PML during mitosis. In cells infected with the adenovirus expressing Sp140 and incubated with IFN-gamma, the number of PML-Sp100 nuclear bodies per cell increased but immunoreactive Sp140 was not evenly distributed among the nuclear bodies. Sp140 associated with a subset of IFN-gamma-induced PML-Sp100 nuclear bodies. To examine the potential effect of Sp140 on gene transcription, a plasmid encoding Sp140 fused to the DNA-binding domain of GAL4 was cotransfected into COS cells with a chloramphenicol acetyltransferase (CAT) reporter gene containing five GAL4-binding sites and a simian virus 40 enhancer region. The GAL4-Sp140 fusion protein increased the expression of the reporter gene. In contrast, Sp100 fused to the GAL4 DNA-binding domain inhibited CAT activity in transfected mammalian cells. The results of this study demonstrate that Sp140 associates with a subset of PML-Sp100 nuclear bodies in IFN-gamma-treated cells and that Sp140 may activate gene transcription. Taken together, these observations suggest that the nuclear bodies within a cell may be heterogeneous with respect to both composition and function.

Hepatocyte growth factor activates the AP-1 complex: a comparison between normal and transformed rat hepatocytes

Background/Aims: Stimulation of activator protein-1 (AP-1), a Fos/Jun complex, is a key event in the cell response to growth factors. We have investigated whether hepatocyte growth factor (HGF) induces differential AP-1 responses in normal and transformed rat hepatocytes, the 7777 cells. Methods: Primocultures of isolated hepatocytes or 7777 cells were stimulated with HGF. Gene expression was evaluated by ribonuclease protection assay and Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay. Identification of the proteins bound to the probes was made by supershift assays with specific antibodies. Cells were electroporated with plasmids containing an AP-1-dependent chloramphenicol acetyl transferase (CAT) gene, and CAT activity was measured 24 h after treatment with medium alone or HGF. Results: In both cell types, HGF triggered the same program of jun family mRNA activation, but distinct Fos/Jun proteins accumulated in the nucleus. HGF increased DNA-binding activity to the phorbol 12-O-tetradecanoate-13-acetate responsive element (TRE) in both cell types, but distinct TRE-binding proteins were recruited in the AP-1 dimers. HGF also increased consistently binding to a cAMP responsive element (CRE) in hepatocytes only. Finally, HGF triggered TRE- and CRE-dependent gene activations in hepatocytes but TRE-dependent gene activation alone in 7777 cells. Conclusions: HGF-induced AP-1 activation leads to the formation of distinct dimers with different functional capacities in normal and transformed hepatocytes. These data suggest the importance of qualitative abnormalities of the AP-1 complex for the establishment or maintainance of a transformed phenotype.

Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus.

Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.

Quantitative Aspects of the Use of Bacterial Chloramphenicol Acetyltransferase as a Reporter System in the YeastSaccharomyces cerevisiae

The quantitative aspects of the use of the reporter system chloramphenicol acetyltransferase (CAT) has been evaluated in the yeastSaccharomyces cerevisiae.It was found that the CAT activity measured with a radiosotopic fluor diffusion assay was strongly dependent on the amount of yeast extract applied, both when CAT was expressed endogenously and when a purifiedEscherichia colienzyme was investigated. Desalting the yeast extract by gel filtration partly eliminated the problem, indicating that some low-molecular-weight compound was involved in the phenomenon. However, the extract still exhibited stability problems on ice. An immunological CAT assay was tested and found to yield satisfactory quantitative result.

Identification of an oxygen-responsive element in the 5′-flanking sequence of the rat cytosolic phosphoenolpyruvate carboxykinase-1 gene, modulating its glucagon-dependent activation

The glucagon-stimulated transcription of the cytosolic phosphoenolpyruvate carboxykinase-1 (PCK1) gene is mediated by cAMP and positively modulated by oxygen in primary hepatocytes. Rat hepatocytes were transfected with constructs containing the first 2500, 493 or 281 bp of the PCK1 5ʹ-flanking region in front of the chloramphenicol acetyltransferase (CAT) reporter gene. With all three constructs glucagon induced CAT activity with decreasing efficiency maximally under arterial pO2 and to about 65% under venous pO2. Rat hepatocytes were then transfected with constructs containing the first 493 bp of the PCK1 5ʹ-flanking region in front of the luciferase (LUC) reporter gene, which were block-mutated at the CRE1 (cAMP-response element-1; -93/-86), putative CRE2 (-146/-139), promoter element (P) 1 (-118/-104), P2 (-193/-181) or P4 (-291/-273) sites. Glucagon induced LUC activity strongly when the P1 and P2 sites were mutated and weakly when the P4 site was mutated; induction of the P1, P2 and P4 mutants was positively modulated by the pO2. Glucagon also induced LUC activity strongly when the putative CRE2 site was altered; however, induction of the CRE2 mutant was not modulated by the pO2. Glucagon did not induce LUC activity when the CRE1 site was modified. These experiments suggested that the CRE1 but not the putative CRE2 was an essential site necessary for the cAMP-mediated PCK1 gene activation by glucagon and that the putative CRE2 site was involved in the oxygen-dependent modulation of PCK1 gene activation. To confirm these conclusions rat hepatocytes were transfected with simian virus 40 (SV40)-promoter-driven LUC-gene constructs containing three CRE1 sequences (-95/-84), three CRE2 sequences (-148/-137) or three CRE1 sequences plus two CRE2 sequences of the PCK1 gene in front of the SV40 promoter. Glucagon induced LUC activity markedly when the CRE1, but not when the CRE2, sites were in front of the SV40-LUC gene; however, induction of the (CRE1)3SV40-LUC constructs was not modulated by the pO2. Glucagon also induced LUC activity very strongly when the CRE1 and CRE2 sites were combined; induction of the (CRE1)3(CRE2)2SV40-LUC constructs was positively modulated by the pO2. These findings corroborated that sequences of the putative CRE2 site were responsible for the modulation by oxygen of the CRE1-dependent induction by glucagon of PCK1 gene transcription.

Identification of an oxygen-responsive element in the 5′-flanking sequence of the rat cytosolic phosphoenolpyruvate carboxykinase-1 gene, modulating its glucagon-dependent activation

The glucagon-stimulated transcription of the cytosolic phosphoenolpyruvate carboxykinase-1 (PCK1) gene is mediated by cAMP and positively modulated by oxygen in primary hepatocytes. Rat hepatocytes were transfected with constructs containing the first 2500, 493 or 281 bp of the PCK1 5'-flanking region in front of the chloramphenicol acetyltransferase (CAT) reporter gene. With all three constructs glucagon induced CAT activity with decreasing efficiency maximally under arterial pO2 and to about 65% under venous pO2. Rat hepatocytes were then transfected with constructs containing the first 493 bp of the PCK1 5'-flanking region in front of the luciferase (LUC) reporter gene, which were block-mutated at the CRE1 (cAMP-response element-1; -93/-86), putative CRE2 (-146/-139), promoter element (P) 1 (-118/-104), P2 (-193/-181) or P4 (-291/-273) sites. Glucagon induced LUC activity strongly when the P1 and P2 sites were mutated and weakly when the P4 site was mutated; induction of the P1, P2 and P4 mutants was positively modulated by the pO2. Glucagon also induced LUC activity strongly when the putative CRE2 site was altered; however, induction of the CRE2 mutant was not modulated by the pO2. Glucagon did not induce LUC activity when the CRE1 site was modified. These experiments suggested that the CRE1 but not the putative CRE2 was an essential site necessary for the cAMP-mediated PCK1 gene activation by glucagon and that the putative CRE2 site was involved in the oxygen-dependent modulation of PCK1 gene activation. To confirm these conclusions rat hepatocytes were transfected with simian virus 40 (SV40)-promoter-driven LUC-gene constructs containing three CRE1 sequences (-95/-84), three CRE2 sequences (-148/-137) or three CRE1 sequences plus two CRE2 sequences of the PCK1 gene in front of the SV40 promoter. Glucagon induced LUC activity markedly when the CRE1, but not when the CRE2, sites were in front of the SV40-LUC gene; however, induction of the (CRE1)3SV40-LUC constructs was not modulated by the pO2. Glucagon also induced LUC activity very strongly when the CRE1 and CRE2 sites were combined; induction of the (CRE1)3(CRE2)2SV40-LUC constructs was positively modulated by the pO2. These findings corroborated that sequences of the putative CRE2 site were responsible for the modulation by oxygen of the CRE1-dependent induction by glucagon of PCK1 gene transcription.

Nitric Oxide Regulation of Gene Transcription via Soluble Guanylate Cyclase and Type I cGMP-dependent Protein Kinase

Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2'-(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism.

Increase in incidence of resistance to ampicillin, chloramphenicol and trimethoprim in clinical isolates of Salmonella serotype Typhimurium with investigation of molecular epidemiology and mechanisms of resistance.

Antimicrobial resistance patterns of Salmonella serotype Typhimurium isolates obtained during the period 1987-1994 were examined and the molecular epidemiology and the mechanisms of resistance to ampicillin, chloramphenicol and trimethoprim were investigated in 24 strains isolated during 1994. Resistance to ampicillin increased from 18% to 78%, to chloramphenicol from 15% to 78%, to tetracycline from 53% to 89% and to co-trimoxazole from 3% to 37%, whereas resistance to norfloxacin remained at 0%. Of Salmonella serotype Typhimurium strains isolated during 1994, all ampicillin-resistant strains had an MIC > 256 mg/L, except one strain in which the MIC was 64 mg/L. Twelve strains (52%) had a TEM-type beta-lactamase, nine (39%) a CARB-type beta-lactamase and two strains (8%) had an OXA-type beta-lactamase. Chloramphenicol acetyl-transferase activity was detected in only nine (47%) of 19 chloramphenicol resistant strains, whereas all eight trimethoprim-resistant strains produced a dihydrofolate reductase type Ia enzyme. Three different epidemiological groups were defined by either low-frequency restriction analysis of chromosomal DNA and pulsed-field gel electrophoresis or repetitive extragenic palindromic-PCR. The latter technique provided an alternative, rapid and powerful genotyping method for S. Typhimurium. Although quinolones provide a good therapeutic alternative, the multiresistance of S. Typhimurium is of public health concern and it is important to continue surveillance of resistance levels and their mechanisms.

Calcium-dependent Regulation of Cytochromec Gene Expression in Skeletal Muscle Cells: IDENTIFICATION OF A PROTEIN KINASE C-DEPENDENT PATHWAY

Mitochondrial biogenesis can occur rapidly in mammalian skeletal muscle subjected to a variety of physiological conditions. However, the intracellular signal(s) involved in regulating this process remain unknown. Using nuclearly encoded cytochrome c, we show that its expression in muscle cells is increased by changes in cytosolic Ca2+ using the ionophore A23187. Treatment of myotubes with A23187 increased cytochrome c mRNA expression up to 1.7-fold. Transfection experiments using promoter-chloramphenicol acetyltransferase constructs revealed that this increase could be transcriptionally mediated since A23187 increased chloramphenicol acetyltransferase activity by 2.5-fold. This increase was not changed by KN62, an inhibitor of Ca2+/calmodulin-dependent kinases II and IV, and it was not modified by overexpression of protein kinase A and cAMP response element-binding protein, demonstrating that the A23187 effect was not mediated through Ca2+/calmodulin-dependent kinase- or protein kinase A-dependent pathways. However, treatment of myotubes with staurosporine or 12-O-tetradecanoylphorbol-13-acetate reduced the effect of A23187 on cytochrome c transactivation by 40-50%. Coexpression of the Ca2+-sensitive protein kinase C isoforms alpha and betaII, but not the Ca2+-insensitive delta isoform, exaggerated the A23187-mediated response. The short-term effect of A23187 was mediated in part by mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) since its activation peaked 2 h after A23187 treatment, and cytochrome c transactivation was reduced by PD98089, a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor. These results demonstrate the existence of a Ca2+-sensitive, protein kinase C-dependent pathway involved in cytochrome c expression and implicate Ca2+ as a signal in the up-regulation of nuclear genes encoding mitochondrial proteins.

Strength and regulation of the different promoters for chromosomal beta-lactamases of Klebsiella oxytoca.

The two groups of chromosomal beta-lactamases from Klebsiella oxytoca (OXY-1 and OXY-2) can be overproduced 73- to 223-fold, due to point mutations in the consensus sequences of their promoters. The different versions of promoters from blaOXY-1 and blaOXY-2 were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene of pKK232-8, and their relative strengths were determined in Escherichia coli and in K. oxytoca. The three different mutations in the OXY beta-lactamase promoters resulted in a 4- to 31-fold increase in CAT activity compared to that of the wild-type promoter. The G-->T transversion in the first base of the -10 consensus sequence caused a greater increase in the promoter strength of the wild-type promoter than the two other principal mutations (a G-to-A transition of the fifth base of the -10 consensus sequence and a T-to-A transversion of the fourth base of the -35 sequence). The strength of the promoter carrying a double mutation (transition in the Pribnow box and the transversion in the -35 hexamer) was increased 15- to 61-fold in comparison to that of the wild-type promoter. A change from 17 to 16 bp between the -35 and -10 consensus sequences resulted in a ninefold decrease of the promoter strength. The expression of the blaOXY promoter in E. coli differs from that in K. oxytoca, particularly for promoters carrying strong mutations. Furthermore, the blaOXY promoter appears not to be controlled by DNA supercoiling or an upstream curved DNA, but it is dependent on the gene copy number.

C-myc Intron Element-binding Proteins Are Required for 1,25-Dihydroxyvitamin D3 Regulation of c-myc during HL-60 Cell Differentiation and the Involvement of HOXB4

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) suppresses c-myc expression during differentiation of HL-60 cells along the monocytic pathway by blocking transcriptional elongation at the first exon/intron border of the c-myc gene. In the present study, the physiological relevance of three putative regulatory protein binding sites found within a 280-base pair region in intron 1 of the c-myc gene was explored. HL-60 promyelocytic leukemia cells were transiently transfected with three different c-myc promoter constructs cloned upstream of a chloramphenicol acetyltransferase (CAT) reporter gene. With the wild-type c-myc promoter construct (pMPCAT), which contains MIE1, MIE2, and MIE3 binding sites, 1,25-(OH)2D3 was able to decrease CAT activity by 45.4 +/- 7.9% (mean +/- S.E., n = 8). The ability of 1, 25-(OH)2D3 to inhibit CAT activity was significantly decreased to 18. 5 +/- 4.3% (59.3% reversal, p < 0.02) when examined with a MIE1 deletion construct (pMPCAT-MIE1). Moreover, 1,25-(OH)2D3 was completely ineffective at suppressing CAT activity in cells transfected with pMPCAT-287, a construct without MIE1, MIE2, and MIE3 binding sites (-6.5 +/- 10.9%, p < 0.002). MIE1- and MIE2-binding proteins induced by 1,25-(OH)2D3 had similar gel shift mobilities, while MIE3-binding proteins migrated differently. Furthermore, chelerythrine chloride, a selective protein kinase C (PKC) inhibitor, and a PKCbeta antisense oligonucleotide completely blocked the binding of nuclear proteins induced by 1,25-(OH)2D3 to MIE1, MIE2, and MIE3. A 1,25-(OH)2D3-inducible MIE1-binding protein was identified to be HOXB4. HOXB4 levels were significantly increased in response to 1,25-(OH)2D3. Taken together, these results indicate that HOXB4 is one of the nuclear phosphoproteins involved in c-myc transcription elongation block during HL-60 cell differentiation by 1,25-(OH)2D3.

Transcriptional regulation of fatty acid synthase gene by insulin/glucose, polyunsaturated fatty acid and leptin in hepatocytes and adipocytes in normal and genetically obese rats.

Transcriptional regulation of the fatty acid synthase (FAS) gene by insulin/glucose, polyunsaturated fatty acids and leptin was investigated in hepatocytes and adipocytes of Wistar fatty rats and their lean littermates. The sequence spanning nucleotides -57 to -35 of FAS gene, which is responsive to insulin/glucose stimulation [Fukuda, H., Iritani, N. & Noguchi, T. (1997) FEBS Lett. 406, 243-248], was linked to a reporter gene containing a heterologous promoter and transfected into rat hepatocytes or adipocytes. The activity of the reporter, chloramphenicol acetyltransferase, in the presence of glucose alone was similar in the primary cultured cells from the lean and obese rats. In the presence of insulin/glucose, however, chloramphenicol acetyltransferase activity was markedly increased in hepatocytes of lean rats, but was not significantly increased in those of obese rats. The stimulation by insulin/glucose was reduced in arachidonic acid-treated cells of lean rats. Similarly, the stimulation by insulin/glucose was reduced in leptin-treated cells and in cells from lean rats containing an expression vector encoding leptin. However, neither polyunsaturated fatty acids nor leptin-treated cells from obese rats responded to insulin-stimulation. The same effects were observed at endogenous FAS mRNA and enzyme levels. Similar results were seen in adipocytes, although the stimulation and suppression were much smaller than in hepatocytes. The insulin-binding capacities of the receptors of liver and adipose tissue were reduced in the presence of leptin or polyunsaturated fatty acids. Leptin and polyunsaturated fatty acids appeared to suppress the insulin stimulation of FAS transcription by reducing the insulin-binding capacities of receptors. Leptin converged on the insulin/glucose response element of FAS gene and suppressed the transcription.

Activation of HIV-1 enhancer sequence by vaccinia virus.

To investigate whether vaccinia virus (VV) can augment gene expression of human immunodeficiency virus type 1 (HIV-1), co-transfection experiments were carried out in which recombinant plasmids containing various portions of the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into cultured cells. A high level of enhancement in CAT activity directed by the HIV-1 LTRs containing the enhancer sequence was observed in cells infected with VV, as in the cells infected with type 1 herpes simplex virus (HSV-1). The sequence responsible for this augmentation of CAT activity was different from that recognized by HIV-1 Tat. These data clearly demonstrated that VV transactivates HIV-1 LTR through a mechanism distinct from that of activation by HIV-1 Tat.

Hepatitis B virus X protein transactivates the inducible nitric oxide synthase promoter.

The capability of hepatitis B virus (HBV) to increase the transcription of the human hepatic inducible nitric oxide synthase (iNOS) by transactivating its promoter has been studied. We have observed by reverse-transcription polymerase chain reaction (RT-PCR) that although the mRNA for the iNOS was almost undetectable in the human hepatoblastoma cell line, HepG2, it was constitutively expressed in the 2.2.15 cell line (a derivative of the HepG2 that produces complete HBV particles). Transfection of HepG2 and 2.2.15 cells with the p1iNOS-CAT plasmid (containing a 1.1-kb fragment of the iNOS promoter) resulted in an increase in chloramphenicol acetyl transferase (CAT) activity in 2.2.15 cells. Similar results were observed when HepG2 and Chang liver cell lines were cotransfected with the p1iNOS-CAT plasmid and the complete HBV genome. It was shown that pX was responsible for the transactivation by cotransfection of HepG2 cells with the p1iNOS-CAT and plasmids expressing the HBV-encoded pX protein, core antigen, and e antigen. Cotransfection of HepG2 cells with the pX expression plasmids and a series of deletion mutants of the 1.1-kb iNOS promoter fragments established that transactivation by pX depends on the presence of at least one nuclear factor-kappaB (NF-kappaB) binding site. This was further confirmed by cotransfecting cells with a plasmid expressing the NF-kappaB inhibitor, IkappaB.

The glucocorticoid properties of the synthetic steroid pregna-1,4-diene-11 β-ol-3,20-dione (ΔHOP) are not entirely correlated with the steroid binding to the glucocorticoid receptor

The natural steroid 11 β-hydroxyprogesterone is not only a modulator of 11 β-hydroxy-steroid dehydrogenase activity, but also an efficient inducer of tyrosine aminotransferase activity in hepatocytes. In contrast with the low affinity for the mineralocorticoid receptor, 11 β-hydroxyprogesterone binds well to both the glucocorticoid receptor and the carrier protein transcortin. It is accepted that the introduction of a 1:ene double bond into 3-keto 4:ene steroids increases the glucocorticoid potency, so that 3-keto-1,4:diene steroids show improved chemical stability and are more potent glucocorticoids than their respective 4:ene analogs. The steroid pregna-1,4-diene-11 β-ol-3,20-dione (ΔHOP) had previously been described as an anti-inflamatory compound and an inhibitor of macromolecular biosynthesis in thymocytes and lymphocytes. In such studies, ΔHOP also exhibited some particular glucocorticoid properties which made it attractive as a tool for the study of the mechanism of action of glucocorticoids. In the present paper we show that ΔHOP possesses some classical biological actions of glucocorticoids such as deposition of glycogen in rat liver, induction of TAT activity in hepatocytes, and inhibition of the uptake of leucine and thymidine by thymocytes. It also exhibits minimal sodium-retaining properties. Consistent with these biological effects, ΔHOP shows a 70 times lower relative binding affinity for the mineralocortioid receptor than aldosterone, but a reasonable affinity for the glucocorticoid receptor, and is as efficient as dexamethasone in dissociating the 90 kDa heat shock protein from the glucocorticoid receptor heterocomplex. However, the inhibition of the uptake of amino acids and nucleotides observed in the presence of ΔHOP is not efficiently blocked when thymocytes are coincubated in the presence of steroids with known antiglucocorticoid activity. ΔHOP is similarly inefficient in inducing chloramphenicol–acetyl transferase activity in cells transfected with a plasmid that possesses two canonical glucocorticoid-responsive elements. Unlike most glucocorticoids, ΔHOP does not induce the fragmentation of DNA in a regular pattern characteristic of apoptosis and it does not reduce thymus weight. This unusual dissociation of glucocorticoid parameters makes ΔHOP a useful tool to discriminate between mechanisms of action by which steroids can exert their biological effects.