Functional characterization of transcription regulators that interact with the electrophile response element.

The electrophile response element (EpRE), also referred to as the antioxidant responsive element (ARE), is found in the 5'-regulatory region of a number of genes encoding phase II, drug-metabolizing enzymes. Gene knockout studies have demonstrated the primary regulatory role that an Nrf2:Maf dimer plays by binding to nucleotides within the EpRE consensus sequence. Current models of transcription regulation have also shown the involvement of higher-order transcriptional coactivators, proteins that nucleate around DNA sequence-specific transcription factors, enhancing transcription of the target gene by interacting with components of the basal transcriptional apparatus and by enabling chromatin remodeling. Here, we hypothesized that multiple transcriptional regulators, including: (i) a primary Nrf2-Maf heterodimer, (ii) a proposed secondary, EpRE-specific, p160 family coactivator, ARE-binding protein-1, and (iii) a tertiary coactivator, CBP/p300, nucleate to form a complex at the EpRE that regulates transcription of the dependent gene. To test this hypothesis, we constructed a HepG2 cell line which contains a stably integrated green fluorescent protein (GFP) gene; its inducible expression is regulated by a synthetic TK promoter containing a linked EpRE. To identify the involvement of specific, primary and higher-order transcriptional regulators in the EpRE-mediated regulation of the GFP reporter gene, we microinjected antibodies directed against specific transcription factors into the HepG2/GFP cells and determined their effect upon tBHQ-induced expression of the GFP gene. The results demonstrate that microinjected antibodies directed against Nrf2, MafK, CBP and p300 could each, individually, significantly inhibit tBHQ-induced GFP expression. This directly demonstrates the role that the tertiary regulators, CBP or p300, play in mediating EpRE activation of phase II genes, and also implicates the involvement of secondary, p160 family coactivators. Moreover, we found that the same anti-MafK antibody that blocked induction of the EpRE-regulated GFP gene completely ablated the gel-shift complex that we hypothesize contains an Nrf2:Maf dimer, ARE-binding protein-1, and CBP or p300.

Polymorphic electrophile response elements in the mouse glutathione S-transferase GSTa1 gene that confer increased induction.

Induced transcription of a battery of stress response genes in mammals, including several phase I and phase II drug-metabolizing enzymes, is regulated by the electrophile responsive element (EpRE). Because previous directed mutagenesis of nucleotide motifs within the large, composite EpRE were shown to affect transcription factor binding and associated induced expression of dependent genes, we hypothesized that naturally-occurring variation or polymorphism in the EpRE sequence, if found, could affect the induced expression of important protective genes like glutathione S-transferases, and that this could be an important determinant of cancer risk in humans and other mammals. To determine whether this occurred in nature, 32 strains and species of inbred mice were screened to examine the EpRE sequence present in the mGSTa1 promoter. Two species, Mus caroli and Mus spretus, showed TGAC-->TGGC mutations in the tandem TGAC motif. Inducibility (15-fold) of the variant Mus spretus EpRE sequence in a reporter gene construct in HepG2 cells was significantly increased versus the wild-type EpRE sequence (8-fold). A comparison of mGSTa1-induced expression in the livers of Mus spretus, Mus caroli, and BALB/cJ mice showed the highest level of mGSTa1 mRNA in livers from the Mus spretus and Mus carolimice. This naturally-occurring polymorphism within the EpRE domain is the first mutation with an associated phenotype to be reported within a promoter regulatory element of a drug metabolizing gene.

Development of a green fluorescent protein microplate assay for the screening of chemopreventive agents.

Here we develop a rapid, cell-based, functional assay to screen and identify naturally occurring or synthetic chemicals with chemopreventive activity. We constructed a reporter gene that consists of the gene-encoding green fluorescent protein (GFP) under the transcriptional control of the thymidine kinase (TK) promoter adjacent to which concatamerized EpRE regulatory elements were inserted. Human hepatoma HepG2 cells were transfected with the EpRE/TK-GFP reporter plasmid, and clones with low GFP background expression and high tBHQ-induced GFP expression were isolated. These GFP reporter cells were seeded into a 96-well microtiter plate, incubated for 24 h, and then treated with test compounds for an additional 24 h. The GFP level and DNA content (as an internal cell survival control) of cells in the 96-well plate were measured subsequently using a fluorescence plate reader. Known inducers of phase II enzymes, such as tert-butylhydroquinone, beta-naphthoflavone, and sulforaphane, significantly increased the GFP level in the HepG2 reporter cells. In an initial screening of a chemical library, we identified a synthetic compound whose inducing ability significantly exceeds (1.6-fold) that of the best currently known phase II enzyme inducers. The experimental results indicate that this cell system makes possible a new high throughput screening approach to identify novel chemopreventive molecules.

p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

Phase II drug-metabolizing enzymes, such as glutathione S-transferase and quinone reductase, play an important role in the detoxification of chemical carcinogens. The induction of these detoxifying enzymes by a variety of agents occurs at the transcriptional level and is regulated by a cis-acting element, called the antioxidant response element (ARE) or electrophile-response element. In this study, we identified a signaling kinase pathway that negatively regulates ARE-mediated gene expression. Treatment of human hepatoma HepG2 and murine hepatoma Hepa1c1c7 cells with tert-butylhydroquinone (tBHQ) stimulated the activity of p38, a member of mitogen-activated protein kinase family. Inhibition of p38 activation by its inhibitor, SB203580, enhanced the induction of quinone reductase activity and the activation of ARE reporter gene by tBHQ. In contrast, SB202474, a negative analog of SB203580, had little effect. Consistent with this result, interfering with the p38 kinase pathway by overexpression of a dominant-negative mutant of p38 or MKK3, an immediate upstream regulator of p38, potentiated the activation of the ARE reporter gene by tBHQ, whereas the wild types of p38 and MKK3 diminished such activation. In addition, inhibition of p38 activity augmented the induction of ARE reporter gene activity by tert-butylhydroxyanisole, sulforaphane, and beta-naphthoflavone. Thus, p38 kinase pathway functions as a negative regulator in the ARE-mediated induction of phase II detoxifying enzymes.

Binding of ETS family members is important for the function of the c-sis/platelet-derived growth factor-B TATA neighboring sequence in 12-O-tetradecanoylphorbol-13-acetate-treated K562 cells.

The c-sis/platelet-derived growth factor (PDGF)-B TATA neighboring sequence (TNS) is a promoter element that is required for the full induction of this gene in K562 erythroleukemia cells undergoing 12-O-tetradecanoylphorbol-13-acetate-mediated megakaryoblastic differentiation. Nuclear factors from K562 cells can bind to the c-sis/PDGF-B TNS, generating four complexes in electrophoretic mobility shift assays. One of these complexes was previously shown to contain Sp family members. In this work, we provide evidence implicating two of the remaining complexes as belonging to the ETS family of transcription factors. This includes the identification of a novel constitutive TNS-binding complex containing the ETS family member ELK-1. The binding of both ETS-like complexes was disrupted by mutations in a central CCGGAA core within the TNS and, for one of the complexes, could be promoted by bringing the sequences flanking the core closer to a consensus ETS binding site. The molecular weights of these TNS-binding factors were estimated by UV cross-linking analysis and found to be consistent with those of several ETS family transcription factors, including ELK-1. A consensus ELK-1 binding site could compete for the binding of both putative ETS-like factors, and the novel complex could be disrupted by the addition of an antibody raised against ELK-1. Transient transfection analysis using mutant TNS promoter-reporter constructs demonstrated a strong correlation between the binding of the ETS-like factors and the transcriptional activity of the TNS. In contrast, mutations that prevented the binding of Sp family transcription factors had no effect on promoter activity. Thus, ETS family members, such as ELK-1, are not only capable of binding to the TNS but seem to be necessary for the function of this element in differentiating K562 cells.

Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells.

Methylglyoxal is an alpha-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 microM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 microM, 0.02% of cellular amino acid residues could be modified. As few as one or two "hits" with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.

CBP/cycA, a CCAAT-binding protein necessary for adhesion-dependent cyclin A transcription, consists of NF-Y and a novel Mr 115,000 subunit.

Cells of most tissues, with the exception of hematopoietic cells, require adhesion to an appropriate surface to grow. Cyclin A is needed for cell cycle progression at the G1-S transition, and appearance of cyclin A mRNA and protein in late G1 has been shown to be dependent on adhesion-initiated signals in normal rat kidney fibroblasts. Previously, we have reported that the adhesion-dependent activation of cyclin A transcription in late G1 is mediated by CBP/cycA (CCAAT-binding protein for cyclin A gene), a novel CCAAT-binding protein. Specific binding of CBP/cycA, a Mr 30,000/40,000/115,000 heterotrimeric protein complex, to the CCAAT element of the cyclin A promoter was detectable in growing but not in G0-arrested or nonadherent normal rat kidney cells. Here, we demonstrate that the Mr 30,000/40,000 subunits of CBP/cycA are identical with NF-YA and NF-YB, the two subunits of NF-Y. In addition, we show that, aside from CBP/cycA, NF-Y itself also binds to the CCAAT element of the cyclin A promoter. But, whereas the binding of CBP/cycA is adhesion and cell cycle dependent and correlates with the expression of cyclin A in late G1 phase, NF-Y itself seems to bind in a cell cycle-independent manner.

Structures of class pi glutathione S-transferase from human placenta in complex with substrate, transition-state analogue and inhibitor.

BACKGROUND: Glutathione S-transferases (GSTs) are detoxification enzymes, found in all aerobic organisms, which catalyse the conjugation of glutathione with a wide range of hydrophobic electrophilic substrates, thereby protecting the cell from serious damage caused by electrophilic compounds. GSTs are classified into five distinct classes (alpha, mu, pi, sigma and theta) by their substrate specificity and primary structure. Human GSTs are of interest because tumour cells show increased levels of expression of single classes of GSTs, which leads to drug resistance. Structural differences between classes of GST can therefore be utilised to develop new anti-cancer drugs. Many mutational and structural studies have been carried out on the mu and alpha classes of GST to elucidate the reaction mechanism, whereas knowledge about the pi class is still limited. RESULTS: We have solved the structures of the pi class GST hP1-1 in complex with its substrate, glutathione, a transition-state complex, the Meisenheimer complex, and an inhibitor, S-(rho-bromobenzyl)-glutathione, and refined them to resolutions of 1.8 A, 2.0 A and 1.9 A, respectively. All ligand molecules are well-defined in the electron density. In all three structures, an additionally bound N-morpholino-ethansulfonic acid molecule from the buffer solution was found. CONCLUSIONS: In the structure of the GST-glutathione complex, two conserved water molecules are observed, one of which hydrogen bonds directly to the sulphur atom of glutathione and the other forms hydrogen bonds with residues around the glutathione-binding site. These water molecules are absent from the structure of the Meisenheimer complex bound to GST, implicating that deprotonation of the cysteine occurs during formation of the ternary complex which involves expulsion of the inner bound water molecule. The comparison of our structures with known mu class GST structures show differences in the location of the electrophile-binding site (H-site), explaining the different substrate specificities of the two classes. Fluorescence measurements are in agreement with the position of the N-morpholino-ethansulfonic acid, close to Trp28, identifying a possible ligandin-substrate binding site.

The tax protein of human T-cell leukemia virus type 1 mediates the transactivation of the c-sis/platelet-derived growth factor-B promoter through interactions with the zinc finger transcription factors Sp1 and NGFI-A/Egr-1.

Transcriptional up-regulation of the c-sis/platelet-derived growth factor-B (PDGF-B) proto-oncogene by the Tax protein of human T-cell leukemia virus type 1 has been implicated as one possible mechanism of cellular transformation by human T-cell leukemia virus type 1. In previous work, we identified an essential site in the c-sis/PDGF-B promoter, Tax-responsive element 1 (TRE1), necessary for transactivation by Tax. We also identified Sp1, Sp3, and NGFI-A/Egr-1 as the primary nuclear transcription factors binding to TRE1 which mediate Tax responsiveness. In the present work, we have investigated the mechanism(s) whereby Tax transactivates the c-sis/PDGF-B proto-oncogene. In vitro transcription assays showed that Tax was able to significantly increase the transcriptional activity of a template containing the -257 to +74 region of the c-sis/PDGF-B promoter. Electrophoretic mobility shift assay analysis showed that Tax increased the DNA binding activity of both Sp1 and NGFI-A/Egr-1 using a TRE1 probe. Analysis of Tax mutants showed that two mutants, IEXC29S and IEXL320G, were unable to significantly transactivate the c-sis/PDGF-B promoter. Finally, co-immunoprecipitation analysis revealed that Tax is able to stably bind to both Sp1 and NGFI-A/Egr-1. Interestingly, co-immunoprecipitation analysis also revealed that Tax mutant IEXC29S is unable to interact with NGFI-A/Egr-1, whereas Tax mutant IEXL320G is able to interact with NGFI-A/Egr-1.

c-sis/platelet-derived growth factor-B promoter requirements for induction during the 12-O-tetradecanoylphorbol-13-acetate-mediated megakaryoblastic differentiation of K562 human erythroleukemia cells.

Platelet-derived growth factor (PDGF), a powerful mitogen and chemoattractant, is composed of two subunits, A and B, which are synthesized by normal megakaryocytes. We have studied the transcriptional regulation of the c-sis/PDGF-B gene in human K562 erythroleukemia cells that have been induced to undergo megakaryoblastic differentiation by treatment with 12-O-tetradecanoylphorbol-13-acetate. Upon differentiation of these cells, c-sis/PDGF-B transcription is increased 50-100-fold. We show here that a minimal c-sis/PDGF-B promoter region, spanning nucleotides -64 to +6, retains full inducibility. Linker scanning mutagenesis within this minimal region identified four segments that were important for expression in differentiating K562 cells: a previously defined sis proximal element (SPE; -64 to -45), the TATA box, the 10 bp immediately downstream of the TATA box [TATA neighboring sequence (TNS); -24 to -15], and the mRNA start site region. Combined mutation of the SPE and TNS resulted in a greater impairment of induction than did mutation of either sequence alone. In contrast, combined mutation of the SPE and the start site or of the TNS and the start site did not lower induction beyond that displayed by the least inducible single mutants. The combination of the SPE and the TNS was sufficient to confer wild-type levels of inducibility to a heterologous promoter. Both the SPE and the TNS were sensitive to alterations in the helical spacing between these elements and the TATA box. Using the electrophoretic mobility shift assay, we demonstrated binding of Sp family members and of two additional unidentified nuclear factors to the TNS in both 12-O-tetradecanoylphorbol-13-acetate-treated and untreated cells. The TNS, therefore, appears to represent a target for a constitutively bound factor(s) that is required for cooperation with a differentiation-specific factor bound at the SPE to drive efficient c-sis/PDGF-B transcription in TPA-treated K562 cells.

Comprehensive analysis of proteins which interact with the antioxidant responsive element: correlation of ARE-BP-1 with the chemoprotective induction response.

Transcriptional activation of the mouse glutathione S-transferase Ya gene by chemoprotective molecules is mediated through the interaction of trans-acting factors with an antioxidant responsive element (ARE) in the promoter region of this gene. In a step toward identifying those factors which bind productively to the GST Ya ARE, all of the discernible, specific ARE-binding proteins (ARE-BP) in nuclear extracts from HepG2 cells were systematically characterized. By gel-mobility-shift analysis, seven specific ARE-BPs, termed ARE-BP-1 through 7 in order of increasing mobility, were observed that did not vary in concentration or migration between induced and uninduced cell extracts. The molecular weights of the individual ARE-BP subunits were determined by a two-dimensional electrophoresis protocol. Ferguson gel analysis of native protein size indicated that several of the ARE-BP-DNA complexes are composed of multiple protein subunits. Wild-type AREs and GST Ya ARE fragments and mutant sequences were evaluated for their ability to mediate induction in a reporter gene system in HepG2 cells. This same panel of sites was tested in an in vitro binding assay for the ability to compete for the ARE-BPs. A binding profile for each ARE-BP was compiled. Correlation between the ARE-BP binding profiles and induction results indicated that: (i) the ARE-BP-1 and ARE-BP-2 complexes formed only with AREs that supported induction, and (ii) the ARE-BP-4 complex formed with all inducible AREs, but it also bound to ARE mutants that failed to support induction. Based on the studies, an early composite regulatory element model for ARE-mediated expression is presented. ARE-BP-1 is proposed to be the mediator of the ARE's unique induction response to chemoprotective agents.

High-level production and purification of biologically active proteins from bacterial and mammalian cells using the tandem pGFLEX expression system.

Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST pi protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial production of biologically active recombinant proteins in E. coli and mammalian cells.

Functional antioxidant responsive elements.

Exposure of human and rodent cells to a wide variety of chemoprotective compounds confers resistance against a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes like glutathione S-transferases (GST). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system. Past studies identified a necessary ARE "core" sequence of RTGACnnnGC, but this sequence alone is insufficient to mediate induction. In this study, the additional sequences necessary to define a sufficient, functional ARE are identified through systematic mutational analysis of the murine GST Ya ARE. Introduction of the newly identified necessary nucleotides into the regions flanking a nonresponsive, ARE-like, GST-Mu promoter sequence produced an inducible element. A screen of the GenBank database with the newly identified ARE consensus identified 16 genes which contained the functional ARE consensus sequence in their promoters. Included within this group was an ARE sequence from the murine ferritin-L promoter that mediated induction when tested. In an electrophoretic mobility-shift assay, the ferritin-L ARE was bound by ARE-binding protein 1, a protein previously identified as the likely mediator of the chemoprotective response. A three-level ARE classification system is presented to account for the distinct induction strengths observed in our mutagenesis studies. A model of the ARE as a composite regulatory site, where multiple transcription factors interact, is presented to account for the complex characteristics of ARE-mediated chemoprotective gene expression.

Adhesion-dependent control of cyclin E/cdk2 activity and cell cycle progression in normal cells but not in Ha-ras transformed NRK cells.

Loss of adhesion of NRK fibroblasts to an appropriate surface leads to cell cycle arrest in late G1 and failure to produce cyclin A. Previously, we showed that adhesion-dependent expression of cyclin A is transcriptionally regulated. In an effort to identify elements of the adhesion-mediated signal transduction cascade upstream of cyclin A activation, we investigated the expression of cyclin E and its associated kinase activity in adherent and suspended NRK cells. Expression of cyclin E was found to be unaffected by suspension. However, cyclin E complexes immunoprecipitated from extracts prepared from NRK cells 12 h after release from G0 arrest were found to be catalytically inactive in suspended but not in adherent cells. This suspension-induced inhibition of cyclin E-associated kinase activity was not observed in NRK cells transformed by a c-Ha-ras oncogene containing a G12V mutation. When G0-synchronized NRK cells were transfected with a cyclin A promoter:luciferase reporter construct along with expression vectors for either wild-type cdk2 or a dominant-negative cdk2 mutant, transcriptional activation of cyclin A was found to be dependent on catalytically active cdk2. Inhibition of cyclin E/cdk2 complexes has frequently been attributed to association of the cdk inhibitors p21(Cip1) and p27(Kip1). However, no differences between adherent and suspended cells could be observed for either expression or cdk2 association of p21(Cip1) or p27(Kip1), nor were any proteins specifically associated with cdk2 or cyclin E in immunoprecipitates from metabolically labeled cell extracts. These results define a pathway through which an adhesion-generated signal controls cyclin A expression by modulating cyclin E/cdk2 activity.

Determination of a binding site for a non-substrate ligand in mammalian cytosolic glutathione S-transferases by means of fluorescence-resonance energy transfer.

To determine the location of the non-substrate-ligand-binding region in mammalian glutathione S-transferases, fluorescence-resonance energy transfer was used to calculate distances between tryptophan residues and protein-bound 8-anilinonaphthalene 1-sulphonate (an anionic ligand) in the human class-alpha glutathione S-transferase, and in a human Trp28-->Phe mutant class-pi glutathione S-transferase. Distance values of 2.21 nm and 1.82 nm were calculated for the class-alpha and class-pi enzymes, respectively. Since glutathione S-transferases bind one non-substrate ligand/protein dimer, the ligand-binding region, according to the calculated distances, is found to be located in the dimer interface near the twofold axis. This region is the same as that in which the parasitic helminth Schistosoma japonicum glutathione S-transferase binds praziquantel, a non-substrate drug used to treat schistosomiasis [McTigue, M. A., Williams, D. R. & Tainer, J. A. (1995) J. Mol. Biol. 246, 21-27]. Since the overall folding topology is conserved and certain features at the dimer interface are similar throughout the superfamily, it is reasonable to expect that all cytosolic glutathione S-transferases bind non-substrate ligands in the amphipathic groove at the dimer interface.

Functional analysis of the SIS proximal element and its activating factors: regulated transcription of the c-SIS/PDGF-B gene in human erythroleukemia cells.

The SIS proximal element (SPE) is essential for the basal transcription of the c-sis/PDGF-B gene as well as the lineage-specific, activated transcription of this gene seen in megakaryocytes. In gel mobility shift analyses, the SPE element forms three gel-shift complexes; the t(op) and b(ottom) complexes were detected in nuclear extracts from both untreated and phorbol 12-myristate 13-acetate ('tetradecanoylphorbol acetate', TPA) treated K562 cells, whereas the m(iddle) complex was detected only in nuclear extracts from TPA-treated K562 cells. Site-directed mutagenesis of the SPE revealed a CCACCC motif that was essential for promoter activity as well as the formation of all three SPE gel-shift complexes. Nested-deletion analyses showed that the SPE was required for TPA-inducibility of c-sis/PDGF-B transcription. Antibody supershift analyses demonstrated that the t gel-shift complex contained both Sp1 and Sp3, and that the b complex contained only Sp3. In vitro transcription assays demonstrated that both Sp1 and Sp3 could support c-sis/PDGF-B transcription independent of each other in untreated K562 cells. However, overexpression of Sp1/Sp3 failed to significantly increase the c-sis/PDGF-B transcription in K562 cells.

Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture.

Methylglyoxal is present at low levels in most cells as a by-product of glycolysis and a product of lipid and amino acid catabolism. The most widely accepted method for measurement of methylglyoxal involves the derivatization of methylglyoxal with 1,2-diaminobenzene derivatives, such as o-phenylenediamine, followed by quantification of the resulting quinoxaline with high-performance liquid chromatography (HPLC). Here we describe the modification of this procedure for the measurement of free intra- and extracellular methylglyoxal in animal cells grown in culture. Cell harvest and sample volume measurement techniques were developed. Solid-phase extraction prior to methylglyoxal derivatization reduced interferences unique to cell culture, such as the phenol red indicator dye used in most cell culture media, and extended the useful life of the HPLC column. In addition, this extraction step significantly lessened the interference represented by oxidative degradation of nucleic acids to methylglyoxal by perchloric acid under assay conditions. The concentration of free intracellular methylglyoxal in Chinese hamster ovary (CHO) cells grown in culture ranged from 0.7 +/- 0.3 microM (mean +/- 2 standard deviations; n = 4) to 1.2 +/- 0.3 microM (mean +/- 2 standard deviations; n = 7). The concentration of free extracellular methylglyoxal in the growth medium was 0.07 +/- 0.02 microM (mean +/- 2 standard deviations; n = 4), severalfold less than that found inside the cell. A possible explanation for the difference between measured free intracellular and extracellular methylglyoxal levels is that the assay for free intracellular methylglyoxal also measures some reversibly bound methylglyoxal.

c-sis/PDGF-B promoter transactivation by the Yax protein of human T-cell leukemia virus type 1.

The human c-sis proto-oncogene promoter is transactivated by the human T-cell leukemia virus type 1 Tax protein in human Jurkat T-cells. Transactivation was >7-fold in Jurkat cells stably expressing the Tax protein (Jurkat-Tax) than in Jurkat E6.1 cells and was further enhanced in Jurkat-Tax cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and the calcium ionophore, ionomycin. Deletion analysis showed that a 167-base pair promoter fragment retained full Tax responsiveness. Insertion of this minimal Tax-responsive region into a heterologous, minimal promoter resulted in approximately a 7-fold increase of transcriptional activation in the presence of Tax. Linker-scanning insertion analysis of this region identified Tax-responsive elements at nucleotides -64 to -45 (TRE1) and -34 to -15 (TATA box region). TRE1 contains a consensus binding site for the Sp family of transcription factors. The TATA box region corresponds to the TATA box and its 3'-neighboring sequence. Gel-shift and antibody supershift analysis of TRE1-binding proteins in unstimulated Jurkat E6.1 and Jurkat-Tax nuclear extracts identified Sp1 and Sp3 as the main TRE1 binding factors. Nuclear extracts from stimulated Jurkat E6.1 and Jurkat-Tax cells identified an additional TRE1 binding factor, Egr-1. These studies define a novel mechanism whereby Tax transactivates the c-sis promoter.

Detection of methylglyoxal as a degradation product of DNA and nucleic acid components treated with strong acid.

The 1,2-diaminobenzene derivation assay for methylglyoxal in biological systems involves the use of perchloric acid, both as a deproteinizing agent and to prevent the spontaneous formation of methylglyoxal from glycolytic pathway intermediates. However, while using a modification of the standard literature assay to measure methylglyoxal in Chinese hamster ovary cells, we found that oxidation of nucleic acids and related compounds by perchloric or trichloroacetic acid results in the formation of methylglyoxal. Compounds containing 2-deoxyribose gave higher levels of methylglyoxal than those containing ribose; purine nucleotides and deoxynucleotides gave more methylglyoxal than did the pyrimidines. Nucleic acids were the most susceptible to degradation, with 12-fold more methylglyoxal being formed from DNA than RNA. Oxidation of nucleic acids increased with higher temperatures and with decreasing nucleic acid fragment size. Another product of nucleic acid oxidation was 2,3-butanedione, the 1,2-diaminobenzene derivative of which is sometimes used as an internal standard during methylglyoxal measurement. Unless accounted for during the assay procedure, the generation of methylglyoxal and 2,3-butanedione due to the oxidation of nucleic acids may lead to substantial errors in the determination of methylglyoxal concentrations in biological systems.

Transcriptional regulation of the SIS/PDGF-B gene in human osteosarcoma cells by the Sp family of transcription factors.

Expression of PDGF-B, the gene encoding the platelet-derived growth factor B chain, has been implicated as a participant in an autocrine growth loop in the human osteosarcoma cell line U2-OS. In previous work, we identified a primary site in the PDGF-B promoter, the SIS proximal element (SPE), which is critical for transcription of the PDGF-B gene in U2-OS cells. We also identified Sp1 as one of the SPE-binding proteins in U2-OS nuclear extracts. In the present work, we have identified another SPE-binding protein to be Sp3. Gel mobility shift assays showed that both Sp1 and Sp3 require the CACCC motif within the SPE for binding. In vitro transcription assays showed that Sp1 or/and Sp3 is necessary for transcription of the PDGF-B gene. Cotransfection experiments functionally demonstrated that Sp1 and Sp3 can independently or additively activate the PDGF-B promoter through the SPE as well as a synthetic promoter. However, the CACCC motif within the SPE is not the only site within the minimal PDGF-B promoter through which Sp1/Sp3 acts; additional nested deletion analyses showed that multiple cis-acting elements within the minimal promoter are required for full level transcription of the PDGF-B gene in U2-OS cells.