Mobility Shift Experiments Research Articles

Diamide stress induces a metallothionein BmtA through a repressor BxmR and is modulated by Zn-inducible BmtA in the cyanobacterium Oscillatoria brevis

A cysteine-rich metal binding protein MT (metallothionein) (named BmtA) is induced upon exposure to multiple heavy metal ions in the freshwater cyanobacterium Oscillatoria brevis. The SmtB/ArsR family repressor BxmR from O. brevis represses the expression of an operon encoding bmtA and bxmR. In the present study, the expression of bmtA was induced in vivo by diamide, a specific thiol oxidant, in O. brevis cells. In vitro electrophoretic gel mobility shift experiments revealed that the incubation with diamide induces disassembly of the BxmR- bxmR/bmtA operator (O)/promoter (P) complex [multiple resolvable complexes of BxmR with oligonucleotide (named P5) containing a single 12-2-12 inverted repeat derived from the O/P region of bxmR/bmtA]. Thus, the exposure to diamide induces MT mRNA in O. brevis, and this induction is associated with diamide-mediated inhibition of BxmR-P5 complex. BxmR is more sensitive to diamide than to H 2O 2. Furthermore, pretreatment of O. brevis with Zn decreased intracellular peroxidation products caused by diamide. Thus, these results imply that MT induced by Zn-pretreatment functions to protect O. brevis cells against diamide stress.

KLF6 is one transcription factor involved in regulating acid ceramidase gene expression

Acid ceramidase (AC; E.C.3.5.1.23) activity is required to hydrolyze ceramide into sphingosine. An inherited deficiency of this enzymatic activity leads to the lipid storage disorder, Farber Lipogranulomatosis. Aberrant AC activity also has been demonstrated in several human cancers. We have characterized a 1931-bp putative promoter region of the murine AC gene by Luciferase reporter assays, electrophoretic mobility shift assays, and mutational analysis. A 143-bp sequence essential for AC promoter activity was found, and mobility shift and super-shift experiments using nuclear extracts of NIH3T3 cells demonstrated that a 34-bp, GC-rich sub-region could bind the transcription factors KLF6, Sp1, and AP2. Transient over-expression of KLF6 in NIH3T3 cells significantly increased the activity of a co-transfected Luciferase reporter construct containing the wild-type AC promoter, and a positive correlation was observed between AC and KFL6 RNA and protein expression in two different human cancer cell lines in which KLF6 expression was either “knocked-down” by RNAi or increased by retroviral-mediated gene transfer. Northern blot analysis also revealed a correlation of KLF6 and AC gene expression in various human tissues. These results provide the first characterization of the AC promoter from any species and demonstrate that KLF6 is one transcription factor involved in the regulation of AC gene expression.

Redox Regulation of Retinoic Acid Receptor Function in Myeloid Leukemia Cell Differentiation and Apoptosis.

Ape1/ref-1 is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFkB, PAX, HIF-1a, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that Ape1/ref-1 expression is related to apoptosis. To further define this relationship, we used retroviral gene transduction to over-express HA-tagged Ape1/ref-1 in HL-60 myeloid leukemia cells. We observed that the RA-induced growth inhibition of HL-60 cells over-expressing Ape1/ref-1 was significantly enhanced compared to wild type HL-60 cells. To determine if the growth inhibition was related to enhanced programmed cell death and differentiation, we treated Ape1/ref-1 transduced and vector-only (LXSN) transduced HL-60 cells with RA and evaluated the expression of Ape1/ref-1 and the development of apoptosis and markers of differentiation.Results: 1) RA induced expression of the retroviral Ape1/ref-1 construct as determined by Western blot resulting in a higher (ie retroviral + endogenous Ape1/ref-1) overall expression of Ape1/ref-1 compared to control cells; 2) analysis of RA-treated cells for apoptosis by propidium iodide, TUNEL, and Annexin V staining as well as morphology, unexpectedly demonstrated enhanced programmed cell death in cells expressing the transduced Ape1/ref-1; 3) Ape-1 over-expression enhanced the retinoid differentiation response by morphology and expression of CD11b. Additional mobility shift experiments demonstrated the redox dependence of retinoic acid receptor binding to retinoid response elements mediated by Ape-1/ref-1. In conclusion, our data supports the contention that Ape1/ref-1 expression may be important for mediating RA-induced myeloid differentiation and programmed cell death.

Inhibition of in vitro RNA binding and replicase activity by phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus

Tombusviruses, which are small plus-strand RNA viruses of plants, require the viral-coded p33 replication co-factor for template selection and recruitment into replication in infected cells. As presented in the accompanying paper [Shapka, N., Stork, J., Nagy, P.D., 2005. Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication. J. Virol. 343, 65–78.], p33 can be phosphorylated in vitro at serine and threonine residues adjacent to its arginine–proline-rich RNA binding site. To test the effect of phosphorylation on p33 function, in this paper, we used phosphorylation-mimicking aspartic acid mutants of Cucumber necrosis virus (CNV) p33 and in-vitro-phosphorylated p33 in gel mobility shift experiments. We found that phosphorylation inhibited the ability of p33 to bind to the viral RNA. In contrast, the nonphosphorylation-mimicking alanine mutants of p33 bound to viral RNA as efficiently as the nonphosphorylated wild type p33 did. In vitro assays with purified CNV replicase preparations revealed that phosphorylation-mimicking mutants of p33 did not support the assembly of functional CNV replicase complexes in yeast, a model host. Based on these results, we propose that the primary function of reversible phosphorylation of p33 is to regulate the RNA binding capacity of p33, which could affect the assembly of new viral replicase complexes, recruitment of the viral RNA template into replication and/or release of viral RNA from replication. Thus, phosphorylation of p33 might help in switching the role of the viral RNA from replication to other processes, such as viral RNA encapsidation and cell-to-cell movement in infected hosts.

Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.

Highly Stable DNA Triplexes Formed with Cationic Phosphoramidate Pyrimidine α‐Oligonucleotides

The ability of cationic phosphoramidate pyrimidine alpha-oligonucleotides (ONs) to form triplexes with DNA duplexes was investigated by UV melting experiments, circular dichroism spectroscopy and gel mobility shift experiments. Replacement of the phosphodiester linkages in alpha-ONs with positively charged phosphoramidate linkages results in more efficient triplex formation, the triplex stability increasing with the number of positive charges. At a neutral pH and in the absence of magnesium ions, it was found that a fully cationic phosphoramidate alpha-TFO (triplex-forming oligonucleotide) forms a highly stable triplex that melts at a higher temperature than the duplex target. No hysteresis between the annealing and melting curves was noticed; this indicates fast association. Moreover, the recognition of a DNA duplex with a cationic alpha-TFO through Hoogsteen base pairing is highly sequence-specific. To the best of our knowledge, this is the first report of stable triplexes in the pyrimidine motif formed by cationic alpha-oligonucleotides and duplex targets.

Microarray Analysis and Motif Detection Reveal New Targets of theSalmonella entericaSerovar Typhimurium HilA Regulatory Protein, IncludinghilAItself

DNA regulatory motifs reflect the direct transcriptional interactions between regulators and their target genes and contain important information regarding transcriptional networks. In silico motif detection strategies search for DNA patterns that are present more frequently in a set of related sequences than in a set of unrelated sequences. Related sequences could be genes that are coexpressed and are therefore expected to share similar conserved regulatory motifs. We identified coexpressed genes by carrying out microarray-based transcript profiling of Salmonella enterica serovar Typhimurium in response to the spent culture supernatant of the probiotic strain Lactobacillus rhamnosus GG. Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. They are known to antagonize intestinal pathogens in vivo, including salmonellae. S. enterica serovar Typhimurium causes human gastroenteritis. Infection is initiated by entry of salmonellae into intestinal epithelial cells. The expression of invasion genes is tightly regulated by environmental conditions, as well as by many bacterial factors including the key regulator HilA. One mechanism by which probiotics may antagonize intestinal pathogens is by influencing invasion gene expression. Our microarray experiment yielded a cluster of coexpressed Salmonella genes that are predicted to be down-regulated by spent culture supernatant. This cluster was enriched for genes known to be HilA dependent. In silico motif detection revealed a motif that overlaps the previously described HilA box in the promoter region of three of these genes, spi4_H, sicA, and hilA. Site-directed mutagenesis, beta-galactosidase reporter assays, and gel mobility shift experiments indicated that sicA expression requires HilA and that hilA is negatively autoregulated.

Regulation of CAPRICE Transcription by MYB Proteins for Root Epidermis Differentiation in Arabidopsis

Epidermal cell differentiation in Arabidopsis root is studied as a model system for understanding cell fate specification. Two types of MYB-related transcription factors are involved in this cell differentiation. One of these, CAPRICE (CPC), encoding an R3-type MYB protein, is a positive regulator of hair cell differentiation and is preferentially transcribed in hairless cells. We analyzed the regulatory mechanism of CPC transcription. Deletion analyses of the CPC promoter revealed that hairless cell-specific transcription of the CPC gene required a 69 bp sequence, and a tandem repeat of this region was sufficient for its expression in epidermis. This region includes two MYB-binding sites, and the epidermis-specific transcription of CPC was abolished when base substitutions were introduced in these sites. We showed by gel mobility shift experiments and by yeast one-hybrid assay that WEREWOLF (WER), which is an R2R3-type MYB protein, directly binds to this region. We showed that WER also binds to the GL2 promoter region, indicating that WER directly regulates CPC and GL2 transcription by binding to their promoter regions.

Molecular Mechanism of Inhibition of Survivin Transcription by the GC-rich Sequence-selective DNA Binding Antitumor Agent, Hedamycin: EVIDENCE OF SURVIVIN DOWN-REGULATION ASSOCIATED WITH DRUG SENSITIVITY

Expression of the antiapoptotic protein survivin is associated with cancer cell viability and drug resistance. Thus, control of its expression in cancer cells has significant consequences for cancer therapeutics. Here we have shown that hedamycin, a GC-rich DNA binding drug, down-regulated survivin expression. Using a series of survivin promoter-luciferase constructs, we have identified an 86-bp GC-rich DNA element (-124 to -39) that mediates the ability of hedamycin to down-regulate survivin expression. Furthermore, both in vivo foot-printing and in vitro gel mobility shift experiments revealed that hedamycin bound to a 21-bp GC-rich DNA element (-115 to -95) in the survivin promoter. This drug-DNA interaction abrogated the binding of Sp-1 or Sp1-like proteins to the 21-bp cis-acting DNA element, and mutagenesis of this region consistently diminished survivin promoter activity. Finally, down-regulation of survivin transcription by hedamycin modulated the viability of cancer cells. These data suggest that abrogation of Sp-1 or Sp1-like protein binding to the 21-bp DNA element in the survivin promoter contributes at least in part to the inhibitory effect of hedamycin on survivin gene transcription. Drug-induced modulation of survivin gene expression may provide novel approaches for cancer therapeutics.

Activation of the vrg6 Promoter of Bordetella pertussis by RisA

The BvgAS two-component system positively regulates the expression of the virulence genes of Bordetella pertussis and negatively regulates a second set of genes whose function is unknown. The BvgAS-mediated regulation of the bvg-repressed genes is accomplished through the activation of expression of the negative regulator, BvgR. A second two-component regulatory system, RisAS, is required for expression of the bvg-repressed surface antigens VraA and VraB. We examined the roles of BvgR and RisA in the regulation of four bvg-repressed genes in B. pertussis. Our analyses demonstrated that all four genes are repressed by the product of the bvgR locus and are activated by the product of the risA locus. Deletion analysis of the vrg6 promoter identified the upstream and downstream boundaries of the promoter and, in contrast to previously published results, demonstrated that sequences downstream of the start of transcription are not required for the regulation of expression of vrg6. Gel mobility-shift experiments demonstrated sequence-specific binding of RisA to the vrg6 and vrg18 promoters, and led to the identification of two putative RisA binding sites. Finally, transcriptional analysis and Western blot analysis demonstrated that BvgR regulates neither the expression nor the stability of RisA.

Role of USF1 and USF2 as potential repressor proteins for human intestinal monocarboxylate transporter 1 promoter

Butyrate, a short-chain fatty acid, is the major energy fuel for the colonocytes. We have previously reported that monocarboxylate transporter isoform 1 (MCT1) mediates uptake of butyrate by human colonic Caco-2 cells. To better understand the mechanisms of MCT1 expression and regulation in the human intestine, we examined the activity and regulation of MCT1 promoter in Caco-2 cells. The transcription initiation site in the MCT1 promoter was identified as a guanine nucleotide 281 bp upstream from the translation initiation site and is surrounded by a guanine-cytosine-rich area. The promoter was found to be highly active when transfected into Caco-2 cells, and its activity decreased with deletions at its 5'-end. Gel mobility shift experiments showed binding of the transcription factors upstream stimulatory factor (USF)1 and 2 to the site -114 to -119 of the MCT1 promoter. With the use of site-directed mutagenesis and promoter activity in Caco-2 cells, the USF proteins appeared to have a repressor role on the MCT1 promoter, which was further confirmed by cotransfecting expression vectors encoding USF1 and 2 in Caco-2 cells and determining endogenous MCT1 expression in USF2 overexpressed cells. The two potential SP1 binding sites found in the same region of the promoter were found not to be involved in its regulation.

Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli

katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli. katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA-ankA transcript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli-like sigma70 -35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H2O2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA, confirming the essential role of the gene in protecting X. campestris from H2O2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H2O2 and the phenotype could be complemented by plasmid-borne expression of ankA. Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris.

Recognition and binding of mismatch repair proteins at an oncogenic hot spot

BackgroundThe current investigation was undertaken to determine key steps differentiating G:T and G:A repair at the H-ras oncogenic hot spot within the nuclear environment because of the large difference in repair efficiency of these two mismatches.ResultsElectrophoretic mobility shift (gel shift) experiments demonstrate that DNA containing mismatched bases are recognized and bound equally efficiently by hMutSα in both MMR proficient and MMR deficient (hMLH1-/-) nuclear extracts. Competition experiments demonstrate that while hMutSα predictably binds the G:T mismatch to a much greater extent than G:A, hMutSα demonstrates a surprisingly equal ratio of competitive inhibition for both G:T and G:A mismatch binding reactions at the H-ras hot spot of mutation. Further, mismatch repair assays reveal almost 2-fold higher efficiency of overall G:A repair (5'-nick directed correct MMR to G:C and incorrect repair to T:A), as compared to G:T overall repair. Conversely, correct MMR of G:T → G:C is significantly higher (96%) than that of G:A → G:C (60%).ConclusionCombined, these results suggest that initiation of correct MMR requires the contribution of two separate steps; initial recognition by hMutSα followed by subsequent binding. The 'avidity' of the binding step determines the extent of MMR pathway activation, or the activation of a different cellular pathway. Thus, initial recognition by hMutSα in combination with subsequent decreased binding to the G:A mismatch (as compared to G:T) may contribute to the observed increased frequency of incorrect repair of G:A, resulting in the predominant GGC → GTC (Gly → Val) ras-activating mutation found in a high percentage of human tumors.

The Effects of Lead and Cadmium on Erythroid Differentiation and Gata-1 Regulated Gene Expression.

Lead (Pb) and Cadmium (Cd) are among the most toxic of industrial pollutants. Anemia is one of the pathologic effects caused by exposure to these heavy metals. While previous investigations have identified the inhibition of heme synthetic enzymes by Pb and the inhibition of erythropoietin production by Cd as likely causes for these toxin-induced anemias, we were intrigued by recent reports that heavy metals, including Pb and Cd are able to replace coordinating zinc (Zn) atoms in Zn finger transcription factors (e.g., P53, TFIIIA and Sp1), disrupting their function. These reports lead us to consider the hypothesis that another cause of the anemias associated with Pb and Cd poisoning might be the displacement of Zn atoms from the two Zn fingers of the erythroid-specific transcription factor GATA-1. GATA-1 is required for normal erythropoiesis and binding sites for GATA-1 are found in the regulatory elements of erythroid-specific genes. While most of the previous studies had been performed in cell-free systems, addressing our hypothesis in a cell-based system, could add weight to pathophysiologic relevance of Zn replacement by heavy metals. To test our hypothesis, we used the model of murine erythroleukemia (MEL) cell differentiation in response to dimethylsulfoxide (DMSO). We first treated MEL cells with increasing concentrations of Pb acetate (0, 0.1, 1, 4 10 and 100 uM) or Cd acetate at the same concentrations and then measured cell viability by trypan blue staining. Decreased cell viability was seen with Pb concentrations above 4 uM by 4 days in culture. Decreased viability was not seen at any Cd concentration through 7 days. We next examined the effect of the same concentrations of Pb and Cd on differentiation of MEL cells as measured by benzidine staining. Control cells treated only with DMSO showed 0% positive staining on day 0 and 90% staining by day 7 of the experiment. Pb treatment at all concentrations had no effect on differentiation. Cd, at concentrations of 10 and 100uM, appeared to slow the kinetics of differentiation but the proportion of benzidine positive cells by day 7 was only slightly reduced. This lack of effect on our model of differentiation lead us ask whether Pb or Cd could be affecting the expression of individual genes which require GATA-1 for proper expression. We used quantitative real-time PCR to measure the levels of mRNA for β-globin, α-globin, erythropoietin receptor and GATA-1 genes daily over the 7 day course of the differentiation experiment. The β-actin gene served as a control for these experiments. As expected, levels of β- and α-globin mRNAs increased 5–10 fold during the experiment. Levels of the other genes, including β-actin, decreased over the time course. No dose-dependent changes in the levels of gene expression were observed with either Pb or Cd treatments. Given these findings, we wanted to determine whether Pb and Cd were capable of inhibiting the ability of GATA-1 to bind DNA. Following pre-incubation with the Pb or Cd, gel mobility shift experiments were conducted using MEL nuclear extract and an oligonucleotide known to bind GATA-1 from the β-globin LCR. No changes in binding were seen at the concentrations used in our experiments. Only at concentrations of 1mM or higher of either metal was DNA binding inhibited. These findings suggest that our hypothesis was incorrect and that at concentrations associated with Pb and Cd toxicity in humans there apear to be no significant effects on erythroid differentiation or GATA-1 function.

Short polypyrimidine/purine sequences modulate tissue-specific control of a cell cycle variant alfalfa histone H3 gene promoter

Plant and animal cells produce a form of histone H3 protein that is deposited in the chromatin in a cell cycle-independent manner. Genes for such replacement histone H3 are highly and constitutively expressed despite the presence of replication-associated histone promoter elements. Previously, we have shown that polypyrimidine/polypurine (PPY/PPU) sequences in replacement histone H3 promoters and introns may be required for this constitutive expression. Here, we report that gel mobility shift experiments detect proteins in plant nuclear extracts that can specifically interact with double and single stranded PPY/PPU elements located in the 5′ untranslated region (UTR) of alfalfa replacement histone H3.2 genes. GUS reporter gene analyses of transgenic tobacco seedlings demonstrate that insertion of parts of these sequence elements into the 5′UTR of a replication variant alfalfa histone H3.1 gene results in H3.1 promoter activities that are no longer confined to tissues where replication of DNA occurs. Similar spatial distribution of activity was observed when the replacement variant histone H3.2 promoter was used as a control. We speculate that the PPY/PPU-rich elements of replacement H3 genes may alter chromatin structure and may have been a critical factor in the emergence of replacement H3 histone genes.

1,25-Dihydroxyvitamin D3 Down-regulation of PHEX Gene Expression Is Mediated by Apparent Repression of a 110 kDa Transfactor That Binds to a Polyadenine Element in the Promoter

The PHEX gene encodes an endopeptidase expressed in osteoblasts that inactivates an uncharacterized peptide hormone, phosphatonin, which suppresses bone mineralization as well as renal phosphate reabsorption and vitamin D bioactivation. We demonstrate that 1alpha-25-dihydroxyvitamin D (1,25(OH)2D3), the, active renal vitamin D metabolite, decreases PHEX mRNA in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. Promoter/reporter construct analysis of the murine PHEX gene in transfected UMR-106 cells localized the repressive effect of 1,25(OH)2D3 to the -133 to -74 bp region, and gel mobility shift experiments revealed that 1,25(OH)2D3 treatment of the cells diminished the binding of a nuclear protein(s) to a stretch of 17 adenines from bp -116 to -100 in the proximal PHEX promoter. Either overexpression of a dominant-negative vitamin D receptor (VDR) or deletion of this sequence of 17 A-T base pairs abolished the repressive effect of 1,25(OH)2D3 by attenuating basal promoter activity, indicating that this region mediates the 1,25(OH)2D3 response and is involved in basal transcription. South-western blot analysis and DNA affinity purification show that an unidentified 110 kDa nuclear protein binds to the poly(A) element. Because 1,25(OH)2D3-liganded VDR neither binds to the polyadenine region of the PHEX promoter nor directly influences the association of the 110 kDa transfactor, we conclude that 1,25(OH)2D3 indirectly decreases PHEX expression via VDR-mediated repression (or modification) of this novel transactivator. Thus, we have identified a cis-element required for PHEX gene transcription that participates in negative feedback control of PHEX expression and thereby modulates the actions of phosphatonin.

Fibronectin stimulates human lung carcinoma cell proliferation by suppressing p21 gene expression via signals involving Erk and Rho kinase

The cyclin-dependent kinase inhibitor p21 WAF-1/CIP1/MDA-6 (p21) plays a key role in cell cycle inhibition and apoptosis, and is negatively regulated during cell proliferation. Extracellular matrices can affect cellular proliferation, but their effects on p21 have not been entirely elucidated. Herein, we explore the effects of the matrix glycoprotein fibronectin on p21 expression in human lung carcinoma cells. Our studies show that fibronectin stimulates cell proliferation, and that this effect is associated with suppression of p21 and stimulation of cyclin D1 mRNA and protein levels in human lung non-small lung cell carcinoma cells (H1838). In contrast, the matrix protein collagen type 1 had no effect. The suppression of p21 by fibronectin was blocked by inhibitors of the extracellular signal-regulated kinase pathway (PD98095), and the Rho-kinase pathway (Y-27632). Fibronectin stimulated the phosphorylation of Erk and increased Rho protein expression. To determine the molecular mechanism(s) responsible for the inhibitory effects of fibronectin on p21 expression, transient transfection assays were performed with cells expressing a wild-type human p21 promoter construct. In these cells, fibronectin reduced p21 gene promoter activity. Finally, electrophoresis mobility shift experiments revealed that fibronectin decreased nuclear Sp1 binding activity in the promoter region of the p21 gene promoter, and a Sp1 competing oligonucleotide inhibited the fibronectin response. Taken together, our results suggest that fibronectin stimulates lung cancer carcinoma cell growth by reducing the cyclin-dependent kinase inhibitor p21 and by inducing cyclin D1 gene expression. The reduction of p21 by fibronectin appears to be mediated through Erk and Rho-kinase signaling and DNA–protein interactions at the Sp1 site in the p21 gene promoter. These observations unveil a novel mechanism for p21 gene regulation by fibronectin in lung carcinoma cell growth that represents a potential target for therapy.

Transcriptional regulation of Vibrio cholerae hemagglutinin/protease by the cyclic AMP receptor protein and RpoS.

Vibrio cholerae secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HA/protease), which is encoded by hapA and displays a broad range of potentially pathogenic activities. Production of HA/protease requires transcriptional activation by the quorum-sensing regulator HapR. In this study we demonstrate that transcription of hapA is growth phase dependent and specifically activated in the deceleration and stationary growth phases. Addition of glucose in these phases repressed hapA transcription by inducing V. cholerae to resume exponential growth, which in turn diminished the expression of a rpoS-lacZ transcriptional fusion. Contrary to a previous observation, we demonstrate that transcription of hapA requires the rpoS-encoded sigma(s) factor. The cyclic AMP (cAMP) receptor protein (CRP) strongly enhanced hapA transcription in the deceleration phase. Analysis of rpoS and hapR mRNA in isogenic CRP+ and CRP- strains suggested that CRP enhances the transcription of rpoS and hapR. Analysis of strains containing hapR-lacZ and hapA-lacZ fusions confirmed that hapA is transcribed in response to concurrent quorum-sensing and nutrient limitation stimuli. Mutations inactivating the stringent response regulator RelA and the HapR-controlled AphA regulator did not affect HA/protease expression. Electrophoretic mobility shift experiments showed that pure cAMP-CRP and HapR alone do not bind the hapA promoter. This result suggests that HapR activation of hapA differs from its interaction with the aphA promoter and could involve additional factors.

Hyper-responsiveness to Stimulation of Human Immunodeficiency Virus-infected CD4+ T Cells Requires Nef and Tat Virus Gene Products and Results from Higher NFAT, NF-κB, and AP-1 Induction

A chronic state of immune hyperactivation is a feature of human immunodeficiency virus type-1 (HIV-1) infection. Studies on the molecular mechanisms by which HIV-1 can modulate the activation state of T cells indicate that both Nef and Tat can alter T cell activation. However, the vast majority of data has been obtained from experiments performed with vectors encoding a single virus protein. We demonstrate that infection of human CD4(+) T lymphocytes with fully infectious HIV-1 leads to a hyper-responsiveness of the interleukin-2 promoter. Hypersensitivity in HIV-1-infected T cells was observed upon stimulation with various agents that are engaging different signal transduction pathways. Experiments performed with recombinant heat stable antigen-encoding HIV-1 indicated that the virus-infected cells are the cells with an enhanced response. Both Nef and Tat are involved in this virus-mediated enhancing effect on interleukin-2 promoter activity. Interestingly, whereas Nef seems to be acting mainly through hyperactivation of nuclear factor of activated T cells (NFAT), Tat acts in an NFAT-independent manner. Mobility shift experiments demonstrated that the HIV-1-associated priming of human T cells for stimulation results in a greater induction of transcription factors recognized as essential players in T cell activation, i.e. NFAT, NF-kappaB, and AP-1. A hyper-responsive state was also established upon HIV-1 infection of a more natural cellular reservoir, i.e. primary CD4(+) T lymphocytes. Considering that the HIV-1 life cycle is tightly regulated by the T cell signaling machinery, the priming for activation of a major viral reservoir represents a means by which this retrovirus can create an ideal cellular microenvironment for its propagation and maintenance.

Exploring Hoogsteen and reversed-Hoogsteen duplex and triplex formation with tricyclo-DNA purine sequences.

The duplex- and triplex-formation properties of the tricyclo-DNA purine decamer 5'p-gagaaggaaa-3' as a single strand or as part of a hairpin duplex with corresponding parallel and antiparallel pyrimidine DNA and RNA complements, as well as with antiparallel purine DNA and RNA complements, were investigated by UV melting curve analysis, circular dichroism spectroscopy, and gel mobility shift experiments. It was found that tricyclo-DNA forms very stable duplexes with the pyrimidine RNA and DNA complements not only in the Watson-Crick pairing mode, but also in the Hoogsteen one. Below pH 6.0, the tc-DNA/DNA and tc-DNA/RNA Hoogsteen duplexes were found to be more stable than the corresponding Watson-Crick DNA duplexes. Triplexes of the hairpin structure with parallel pyrimidine complements revealed even stronger Hoogsteen pairing relative to the duplexes, presumably due to structural preorganization phenomena. Triplex formation with antiparallel pyrimidine and purine third strands (reversed-Hoogsteen motif) could not be observed and seem to be unstable.