Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli.

BACKGROUND: The occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. METHODS: To understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV-vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. RESULTS: We observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37 µM. CONCLUSIONS: It was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. GENERAL SIGNIFICANCE: Understanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.

Selection of scFv phages specific for chloramphenicol acetyl transferase (CAT), as alternatives for antibodies in CAT detection assays.

Reporter gene assays are commonly used in applied toxicology to measure the transcription of genes involved in toxic responses. In these reporter gene assays, transgenic cells are used, which contain a promoter-operator region of a gene of interest fused to a reporter gene. The transcription of the gene of interest can be measured by the detection of the reporter protein. Chloramphenicol acetyl transferase (CAT) is frequently used as a reporter protein in mammalian reporter gene assays. Although CAT can be measured by different detection systems, like enzymatic and immune assays, most of these tests are expensive, time-consuming and labor-intensive. The excellent characteristics of phages, like their high affinity and specificity, their fast, cheap and animal-friendly manufacturing process with low batch-to-batch variations and their stability, make them appropriate as alternatives for antibodies in detection assays. Therefore, in this study single-chain variable fragment (scFv) phages were selected with affinity for CAT. Several scFv phages were selected that showed affinity towards CAT in a screening ELISA. Surface plasmon resonance analyses showed that the tested scFv phages have an affinity for CAT with a dissociation constant (K(d)) around 1 µM. The selected scFv phages in this study could be used as capture elements in a highly sensitive sandwich ELISA to detect CAT concentration as low as 0.1 ng ml⁻¹ or 4 pM. This low detection limit demonstrates the potential of the scFv phages as an alternative for capturing antibodies in a highly sensitive detection test to measure CAT concentrations in reporter gene assays.

Src family kinase members have a common response to histone deacetylase inhibitors in human colon cancer cells.

Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation and/or apoptosis in numerous cancer cell types and have shown promise in clinical trials. These agents are particularly novel, given their ability to selectively influence gene expression. Previously, we demonstrated that the HDIs butyrate and trichostatin A (TSA) directly repress c-Src proto-oncogene expression in many cancer cell lines. Activation and/or overexpression of c-Src have been frequently observed in numerous malignancies, especially of the colon. Therefore, our observation was particularly interesting since butyrate is a naturally abundant component of the large intestine and has been suggested to be a cancer-preventive agent. However, c-Src is not the only Src family kinase (SFK) member to be implicated in the development of human cancers, including those of the colon. Therefore, the relative expression levels of known SFKs were examined in a panel of human colon cancer cell lines. We found a surprisingly diverse expression pattern but noted that most cell lines expressed relatively high levels of at least 2 SFKs. When the effects of butyrate and TSA were examined in representative cell lines, the expression of all SFKs was repressed in a dose- and time-dependent manner. Further, detailed examination of Lck, Yes and Lyn demonstrated that this repression had a direct effect on transcription and was independent of new protein synthesis. These results mirror our earlier data obtained with c-Src and suggest that SFKs are a major target of HDIs and likely account in part for the anticancer effects of these promising new drugs.

Chloromycetin resistance of clinically isolated E coli is conversed by using EGS technique to repress the chloromycetin acetyl transferase.

AIM: To explore the possibility of repression of chloromycetin (Cm) acyl transferase by using external guided sequence (EGS) in order to converse the clinical E coli isolates from Cm- resistant to Cm- sensitive. METHODS: EGS directed against chloromycetin acetyl transferase gene (cat) was cloned to vector pEGFP-C1 which contains the kanamycin (Km) resistance gene. The recombinant plasmid pEGFP-C1+EGScat1+cat2 was constructed and the blank vector without EGS fragment was used as control plasmids. By using the CaCl(2) transformation method, the recombinant plasmids were introduced into the clinically isolated Cm resistant but Km sensitive E coli strains. Transformants were screened on LB agar plates containing Km. Extraction of plasmids and PCR were applied to identify the positive clones. The growth curve of EGS transformed bacteria cultured in broth with Cm resistance was determined by using spectrophotometer at A(600). Drug sensitivity was tested in solid culture containing Cm by using KB method. RESULTS: Transformation studies were carried out on 16 clinically isolated Cm-resistant (250 microg/mL of Cm) E coli strains by using pEGFP-C1-EGScat1cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after the transformation using EGS. Of the 16 tested strains, 4 strains were transformed successfully. Transformants with EGS plasmid showed growth inhibition when grown in liquid broth culture containing 200 microg/mL of Cm. In drug sensitivity test, these strains were sensitive to Cm on LB-agar plates containing 200 microg/mL of Cm. Extraction of plasmids and PCR amplification showed the existence of EGS plasmids in these four transformed strains. These results indicated that the Cat of the four clinical isolates had been suppressed and the four strains were converted to Cm sensitive ones. CONCLUSION: The EGS directed against Cat is able to inhibit the expression of Cat, and hence convert Cm-resistant bacteria to Cm-sensitive ones. Thus, the EGS has the capability of converting the phenotype of clinical drug-resistant isolates strains to drug-sensitive ones.

Effect of antidepressant drugs on the human corticotropin-releasing-hormone gene promoter activity in neuro-2A cells.

In order to test if antidepressant drugs can directly influence corticotropin-releasing hormone (CRH) gene expression, their effect on CRH gene promoter activity was evaluated in neuro-2A cells stably transfected with a human CRH - chloramphenicol acetyl-transferase plasmid. Forskolin (an activator of adenylate cyclase), but not phorbol 12-myristate 13-acetate (an activator of protein kinase C), ca. 3-fold increased reporter gene activity, which confirms the critical role of the cAMP-responsive element in regulation of the CRH gene. Imipramine and fluoxetine present in the medium for 5 days, in a concentration-dependent manner (3-30 microM) inhibited the basal activity of CRH gene promoter, while tianeptine was inactive. The obtained results indicate that inhibition of the human CRH gene promoter activity by imipramine and fluoxetine, but not tianeptine, may play a role in a mechanism by which the former drugs attenuate HPA axis activity.

Transcriptional regulation of CD28 expression by CD28GR, a novel promoter element located in exon 1 of the CD28 gene.

CD28 provides an essential costimulatory signal required for Ag-mediated T cell activation via the TCR. Although accumulating evidence exists for the signaling properties of CD28, less is known regarding the regulation of CD28 expression. In this study, we have identified a novel promoter element of CD28, CD28GR (GGGGAGGAGGGG), which is located between +181 and +192 in exon 1 of the CD28 gene. Mutations within the 12-bp CD28GR sequence abolished its transcriptional activity. CD28GR contains a Sp1/EGR-1 binding site, which was found to act as the predominant functional element for regulating CD28 gene expression in Jurkat cells. Exon 1/CD28GR-driven transcription in Jurkat cells was augmented by cotransfection with Sp1 or EGR-1 expression plasmid. Similar augmentation was also shown with pharmacologic activation. This study is the first to identify a regulatory element that is critical for conferring constitutive and activation-induced transcriptional activation of the CD28 gene. Furthermore, our results proposed potential involvement of Sp1 in regulating CD28 expression. The linkage between Sp1 and the expression of CD28 has important implications in how viral infections, such as HIV, can induce immunosuppression.

Anti-gene effect in live cells of AG motif triplex-forming oligonucleotides containing an increasing number of phosphorothioate linkages.

The murine Ki-ras promoter contains a unique polypurine--polypyrimidine [poly(R.Y)] sequence between -290 and -320 from the 3' boundary of exon phi. Previously we demonstrated triplex formation and transcription inhibition promoted by GT and AG oligonucleotides directed against this site [Alunni-Fabbroni et al. (1996) Biochemistry 35, 16361--16369]. In this work, we have investigated triplex formation and anti-gene activity of five 20-mer AG motif triplex-forming oligonucleotides specific for the Ki-ras poly(R.Y) target, derived from 5'-AGGGAGGGAGGAAGGGAGGG (20AG) by replacing an increasing number of phosphodiester linkages with phosphorothioate linkages (S(i)-20AG; i = 2, 3, 4, 5, 19). Electrophoretic mobility-shift experiments (EMSA) showed that four thioate oligonucleotides, S(i)-20AG (i = 2, 3, 4, 5), recognized the Ki-ras target and exhibited dissociation constants similar to that of 20AG: K(d) = 12 +/- 2 nM, while the all-thioate S(19)-20AG exhibited a K(d) of 128 +/- 15 nM. Moreover, the binding between the Ki-ras promoter and oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) was characterized by DMS/piperidine and DNase I footprinting experiments. We observed that the introduction in the phosphodiester oligonucleotide 20AG of sulfur atoms reduced its aggregation significantly and increased its nuclease resistance. Transient transfection experiments using preformed triplexes with a recombinant plasmid containing the reporter chloramphenicol acetyltransferase (CAT) gene under the control of Ki-ras promoter showed that oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) promote a strong inhibition of up to 75% of the CAT expression when compared with control Ki-ras unspecific oligonucleotides. Taken together, these data provide a guideline for designing triplex-forming effector molecules capable of controlling Ki-ras expression in vivo.

Transactivation of progestin- and estrogen-responsive promoters by 19-nor progestins in African Green Monkey Kidney CV1 cells.

New and more potent progestins and antiprogestins suitable for reproductive therapy and contraception are currently the target of intensive research. The design of such drugs has been hampered by the complex technology required for screening these compounds at the molecular level. To solve this problem, we developed an in vitro cell system that allows detection of the progestagenic effects of a given compound using a PRE2-TATA-CAT reporter vector transiently introduced in a cell line stably transfected with the rabbit progesterone receptor (PR). The African Green Monkey Kidney CV1 (AGMK-CV1) cell line was chosen because these cells do not express endogenous steroid receptors; the selected clone stably expressing the rabbit PR has been maintained in our laboratory for more than 2 yr without detectable losses in PR content and progestagenic response. The presence and function of the PR were assessed by immunohistochemical and saturation analyses as well as by monitoring transactivation of the PRE2-TATA-CAT reporter gene. In this cell line, the PR is expressed at a concentration of 0.170 fmol/mg of protein, and the receptor is localized within the cell nucleus in either the presence or absence of the potent synthetic progestin R5020. This PR-expressing cell system allowed study of the in vitro progestational activity of several 19-nor progestins. The antiprogestin RU486 inhibited CAT activity induced by R5020; norethisterone (NET), levonorgestrel (LNG), and gestodene (GSD) induced PRE2-TATA-CAT activity at concentrations similar to those of R5020, whereas NET A-ring-reduced metabolites induced CAT activity at an extent lower than (5alpha-NET) or similar (3beta,5alpha-NET) to that of the precursor compound. The PRE2-TATA-CAT induction by 17beta-estradiol was also analyzed and no crossreactivity was detected. However, when the ERE-VitA2-TK-CAT (estrogen-responsive element-vitellogenin A2-thymidine kinase promoter-CAT) reporter vector and the estradiol receptor alpha or beta were cotransfected, CAT activity was induced in the presence of 17beta-estradiol, and NET tetrahydro-reduced derivatives. The results indicate that this AGMK-CV1-PR cell assay system appears to be suitable for measuring the effects of different synthetic progestins at the transcriptional level. In this assay system, NET, LNG, and GSD exhibit potent progestational effects at the transcriptional level. In the particular case of NET, the assay system allowed us to determine that the single or multiple hormonal transcriptional effects of this compound are partially mediated by its A-ring-reduced derivatives.

Inhibition of Tat-mediated transactivation of HIV-1 LTR transcription by polyamide nucleic acid targeted to TAR hairpin element.

Tat, an essential human immunodeficiency virus type 1 protein interacts with the transactivation response element (TAR) and stimulates transcription from the viral long-terminal repeat (LTR). Blockage of Tat-TAR interaction halts viral transcription and hence replication. We have found that polyamide nucleic acid (PNA), targeted to the TAR sequences of viral RNA genome is able to prevent Tat-TAR interaction by efficient sequestration of the TAR. Anti-TAR PNA competes for TAR and prevents Tat-mediated stimulation of HIV-1 LTR transcription in vitro but has no influence on the basal level of transcription in the absence of Tat. Using a reporter gene construct pHIV LTR-CAT and pCMV-Tat in cell culture, we have further shown that anti-TAR PNA is able to block Tat-mediated transactivation of HIV-1 LTR transcription in vivo as judged by the extent of LTR driven CAT gene expression in the absence and presence of anti-TAR PNA. Supplementation of 100 nM of anti-TAR PNA into the culture medium further enhances the suppression of transactivation. Nonspecific scrambled PNA had no influence on Tat-TAR interaction and LTR-driven CAT gene expression in cell culture. These results suggest that PNA targeted to the TAR sequence of the viral genome may be a potential inhibitor of HIV-1 gene expression.

SP-A 3'-UTR is involved in the glucocorticoid inhibition of human SP-A gene expression.

The synthetic glucocorticoid dexamethasone has a major inhibitory effect on human surfactant protein A1 (SP-A1) and SP-A2 gene expression that occurs at both the transcriptional and posttranscriptional levels. Toward the identification of cis-acting elements that may be involved in the dexamethasone regulation of SP-A mRNA stability, chimeric chloramphenicol acetyltransferase (CAT) constructs that contained various portions of SP-A1 or SP-A2 cDNA in place of the native CAT 3'-untranslated region (UTR) were transiently transfected into the lung adenocarcinoma cell line NCI-H441. CAT activity was reduced in NCI-H441 cells by exposure to 100 nM dexamethasone only for the chimeric CAT constructs that contained the SP-A 3'-UTR. Moreover, the inhibitory response seen with dexamethasone was greater for the 3'-UTR derived from the SP-A1 allele 6A3 than with the 3'-UTR derived from either the SP-A1 allele 6A2 or SP-A2 allele 1A0, indicating differential regulation between SP-A genes and/or alleles.

The role of p38 mitogen-activated protein kinase in IL-1 beta transcription.

Several reports have shown that bicyclic imidazoles, specific inhibitors of the p38 mitogen-activated protein kinase (MAPK), block cytokine synthesis at the translational level. In this study, we examined the role of p38 MAPK in the regulation of the IL-1beta cytokine gene in monocytic cell lines using the bicyclic imidazole SB203580. Addition of SB203580 30 min before stimulation of monocytes with LPS inhibited IL-1beta protein and steady state message in a dose-dependent manner in both RAW264.7 and J774 cell lines. The loss of IL-1beta message was due mainly to inhibition of transcription, since nuclear run-off analysis showed an approximately 80% decrease in specific IL-1 RNA synthesis. In contrast, SB203580 had no effect on the synthesis of TNF-alpha message. LPS-stimulated p38 MAPK activity in the RAW264.7 cells was blocked by SB203580, as measured by the inhibition of MAPKAP2 kinase activity, a downstream target of the p38 MAPK. CCAATT/enhancer binding protein (C/EBP)/NFIL-6-driven chloramphenicol acetyltransferase (CAT) reporter activity was sensitive to SB203580, indicating that C/EBP/NFIL-6 transcription factor(s) are also targets of p38 MAPK. In contrast, transfected CAT constructs containing NF-kappaB elements were only partially inhibited (approximately 35%) at the highest concentration of SB203580 after LPS stimulation. As measured by EMSA, LPS-stimulated NF-kappaB activation was not affected by SB203580. Overall, the results demonstrate, for the first time, a role for p38 MAPK in IL-1beta transcription by acting through C/EBP/NFIL-6 transcription factors.

Esterases in serum-containing growth media counteract chloramphenicol acetyltransferase activity in vitro.

The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that of B. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility of E. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. coli bearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo.

Immunosuppressive leflunomide metabolite (A77 1726) blocks TNF-dependent nuclear factor-kappa B activation and gene expression.

Leflunomide is a novel immunosuppressive and antiinflammatory agent currently being tested for treatment of autoimmune diseases and transplant rejection. NF-kappa B is a transcription factor activated in response to a wide variety of inflammatory stimuli, including TNF, but whether leflunomide blocks NF-kappa B activation is not known. In the present report we demonstrate that treatment of a human T cell line (Jurkat) with leflunomide blocks TNF-mediated NF-kappa B activation in a dose- and time-dependent manner, with maximum inhibition at 5-10 microM. Inhibition was not restricted to TNF-induced activation, because leflunomide also inhibited NF-kappa B activation induced by other inflammatory agents, including phorbol ester, LPS, H2O2, okadaic acid, and ceramide. Leflunomide blocked the degradation of I kappa B alpha and subsequent nuclear translocation of the p65 subunit, steps essential for NF-kappa B activation. This correlated with inhibition of dual specificity-mitogen-activated protein kinase kinase as well as an Src protein tyrosine kinase, p56lck, by leflunomide. Reducing agents did not reverse the effect of leflunomide. Leflunomide also suppressed the TNF-activated NF-kappa B-dependent reporter gene expression. Our results thus indicate that leflunomide is a potent inhibitor of NF-kappa B activation induced by a wide variety of inflammatory stimuli, and this provides the molecular basis for its anti-inflammatory and immunosuppressive effects.

The redox-sensitive human antioxidant responsive element induces gene expression under low oxygen conditions.

Transient transfection studies of human HepG2 and mouse Hepa hepatocarcinoma cells with a reporter gene construct regulated by a human antioxidant responsive element (ARE) from the NQO1 gene demonstrated that the element is responsive to low oxygen conditions. The antioxidant N-acetyl L-cysteine (NAC) strongly inhibited basal aerobic reporter gene activity in HepG2 cells without obviously affecting the hypoxic induction, as is consistent with ARE sensitivity to oxidative stress in aerobic cultures. Electrophoretic mobility shift (EMS) assays of nuclear extracts of HepG2 and Hepa cells lysed under aerobic or hypoxic conditions or after exposure to the phenolic compound 3-(2)-tert-butyl-4-hydroxyanisole (BHA), showed specific and constitutive protein binding to the ARE under all of these conditions. Taken together, these findings show that the ARE can mediate gene expression in response to low oxygen conditions. Co-ordinately regulated expression of ARE-dependent genes, such as phase II detoxification enzymes, may be an important phenotype of solid tumors containing significant regions of pathophysiological hypoxia.

Interleukin-1 represses COLIA1 promoter activity in calvarial bones of transgenic ColCAT mice in vitro and in vivo.

Interleukin-1 (IL-1) inhibits collagen synthesis in osteoblastic cell lines and primary osteoblast-like cells. However, promoter elements regulating type I collagen A1 (COLIA1) expression in vivo and in organ culture may differ from those regulating expression in cell culture. We have examined the effects of IL-1 on reporter gene activity in neonatal transgenic mouse calvariae bearing COLIA1 promoter-chloramphenicol acetyltransferase (ColCAT) fusion genes. The parent construct, ColCAT 3.6, contains 3.5 kb of 5' flanking sequence and 115 bp of 5' untranslated region fused to the CAT reporter. In 48-h calvarial organ cultures, IL-1 repressed ColCAT 3.6 promoter activity and collagen synthesis in a dose-related manner, with a maximal inhibition of 40-65%. This repression was retained in 5' deletion constructs truncated to-1719 bp. The inhibition of transgene mRNA was blocked by cycloheximide, indicating a requirement for new protein synthesis. Pretreatment with indomethacin diminished the inhibitory effect of IL-1 on CAT activity and collagen synthesis, suggesting partial mediation by prostaglandins. Local in vivo injection of IL-1 (500 ng) decreased calvarial transgene mRNA after 8 h, an effect that was partially blocked by indomethacin. ColCAT transgenic mice represent a useful model for in vitro and in vivo assessment of COLIA promoter regulation by cytokines and other factors.

Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene.

Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completeness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild-type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5 alpha-dihydrotestosterone (5 alpha-DHT) (10(-8) M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 +/- 73 (mean +/- SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 +/- 395 functional hARs/nucleus in hFOB/AR-2 cells, and 3,987 +/- 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5 alpha-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5 alpha-DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.

Growth hormone modulates insulin regulation of hepatic insulin-like growth factor binding protein-1 transcription.

Hepatic transcription of insulin-like growth factor-binding protein-1 (IGFBP-1) is enhanced in hypophysectomized (hypox) rats and can be rapidly down-regulated by GH administration. Here we examined the effect of insulin on IGFBP-1 messenger RNA abundance in hypox rats and the effects of insulin and GH on IGFBP-1/chloramphenicol acetyltransferase (CAT) reporter plasmids transiently transfected into isolated hepatocytes from pituitary-intact and hypox rats. Unlike GH, administration of insulin to hypox rats in doses of 10 or 50 micrograms/100 g BW had no effect on hepatic IGFBP-1 messenger RNA abundance. Insulin at 10(-7) M resulted in a 42.1 +/- 9.8% suppression of CAT activity in hepatocytes from pituitary-intact animals transfected with a CAT reporter plasmid containing 1671 bp of the 5'-flanking region of the rat IGFBP-1 gene. In the same assay, GH at a concentration of 2.3 x 10(-8) M significantly reduced CAT activity. In contrast, insulin had no effect on CAT activity in hepatocytes from hypox rats, whereas GH resulted in comparable suppression of CAT activity in hepatocytes from hypox rats and pituitary-intact rats, 13.6 +/- 2.3% vs. 18.2 +/- 3.2%. Deletional analysis and mobility shift assays were used to identify the GH-responsive regions in the IGFBP-1 gene. GH suppression of CAT activity was lost when the IGFBP-1 5'-flanking region was deleted down to -277 bp, whereas insulin suppression was retained for all but the smallest fragment of the IGFBP-1 gene. Mobility shift assays were used to compare nuclear extracts from sham-operated, hypox, and GH-treated hypox rats. When hepatic nuclear extracts from hypox rats were incubated with the -277 to -82 and the -556 to -368 bp fragments, retarded bands were apparent that were not present in the extracts from sham-operated rats. GH treatment of hypox rats 15 or 30 min before death completely normalized the retardation pattern seen with the -277 to -82 bp fragment, but did not affect the pattern seen with the -556 to -368 bp fragment. A 20-bp fragment corresponding to the previously identified insulin response element, -108 to -89 bp, was also analyzed. An additional retarded band, not seen with nuclear extracts from sham-operated rats, was apparent when nuclear extracts of hypox rats or GH-treated hypox rats were used. These data provide the first in vitro evidence that GH directly regulates transcription of IGFBP-1 expression. In addition, our findings suggest that GH modulates insulin regulation of IGFBP-1 transcription, possibly by altering the milieu of trans-acting factors that interact with both the insulin response element and distinct upstream sites.

Specific inhibition of c-fos proto-oncogene expression by triple-helix-forming oligonucleotides.

The promoter region of the c-fos oncogene 5' flanking sequence contains enhancer elements crucial for binding nuclear factors that regulate transcription following cell proliferation and differentiation. Single-stranded deoxyoligonucleotides were chosen for modulation of c-fos protooncogene expression because of their high-affinity binding to specific nucleotide sequences. We designed two oligonucleotides that form a triple-helix complex on the retinoblastoma gene product-responsible element of the c-fos oncogene. Modification of the DNA triplex with dimethyl sulfate and affinity cleaving assays demonstrate that the predicted oligonucleotides form a DNA triplex structure with the c-fos promoter in a sequence-specific manner. Tumorigenic and non-tumorigenic fibroblasts were transiently transfected with fos-CAT plasmid modified with alkylating triplex-forming oligonucleotide reagents. A dramatic depression of CAT activity was found when the cross-linked triple helix complex at the retinoblastoma gene product-related site of the c-fos promoter was used. These experiments suggest that transcription of individual genes can be selectively modulated in cell culture by sequence specific triplex formation in regulatory enhancer sequences.

Kinetic mechanism of chloramphenicol acetyltransferase: the role of ternary complex interconversion in rate determination.

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol (Cm) by a ternary complex mechanism and with a random order of addition of substrates. A closer examination of the mechanism of the reaction catalyzed by the type III CAT variant (CATIII) has included the measurement of the individual rate constants by stopped-flow fluorimetry at 5 degrees C. Under all conditions employed, product release from the binary complexes in both forward and reverse reactions was found to be too slow to account for the observed overall rate of turnover for the reaction. Additional, faster routes for product release are achieved via the formation of the nonproductive ternary complexes (CAT:3-acetyl-Cm:acetyl-CoA and CAT:CoA:Cm). The release of 3-acetyl-Cm from the binary complex is 5-fold slower than kcat (135 s-1 at 5 degrees C), whereas the dissociation rate constants of 3-acetyl-Cm from the ternary complexes with CoA and acetyl-CoA are 120 and 200 s-1, respectively. Arrhenius plots of dissociation rate constants indicate a slow release of products over a broad temperature range. Computer simulations based on the rate constants of CATIII applied to a ternary complex mechanism, assuming random order of substrate addition and product release, yielded nonlinear initial rates of product formation unless both nonproductive ternary complexes were included in the model. Simulated steady-state kinetic analyses based on the latter assumption yielded kinetic parameters that compared favorably with those determined experimentally.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced activity by alpha-naphthoflavone and 6-methyl-1,3,8-trichlorodibenzofuran using an aryl hydrocarbon (Ah)-responsive construct.

Rat hepatoma H4IIE and mouse hepatoma Hepa 1c1c7 cells were transiently transfected with a plasmid construct that contained the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the mouse mammary tumor virus promoter and one copy of the dioxin responsive element. Treatment of transfected H4IIE and Hepa 1c1c7 cells with 10(-13) to 10(-6) M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a concentration-dependent increase in transient CAT activity. Maximum CAT activity was induced in both cell lines by exposure to 10(-9) M TCDD. The induction of CAT activity correlated well with the TCDD-induced, P4501A1-dependent ethoxyresorufin O-deethylase activity. Cotreatment of transfected cells with 10(-9) M TCDD and 10(-8) to 10(-6) M alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) resulted in a concentration-dependent reduction of TCDD-induced CAT activity. Treatment of cells with 10(-6) M alpha NF or MCDF alone resulted in only minimal induction of CAT activity. Both antagonists inhibited the induction of genes under the control of the CYP1A1 and mouse mammary tumor virus promoters, which indicates that the alpha NF- and MCDF-mediated antagonism of TCDD-induced, aryl hydrocarbon receptor-dependent gene transcription does not depend on promoter context.