Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes.

Protein kinases (PKs) play an important role in the development and progression of cancer by regulating cell growth, survival, invasion, metastasis, and angiogenesis. Dasatinib (BMS-354825), a dual Src/Abl inhibitor, is a promising therapeutic agent with oral bioavailability. It has been used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). Most kinase inhibitors, including Dasatinib, inhibit multiple cellular targets and do not possess exquisite cellular specificity. Recent efforts in kinase research thus focus on the development of large-scale, proteome-wide chemical profiling methods capable of rapid identification of potential cellular (on- and off-) targets of kinase inhibitors. Most existing approaches, however, are still problematic and in many cases not compatible with live-cell studies. In this work, we have successfully developed a cell-permeable kinase probe (DA-2) capable of proteome-wide profiling of potential cellular targets of Dasatinib. In this way, highly regulated, compartmentalized kinase-drug interactions were maintained. By comparing results obtained from different proteomic setups (live cells, cell lysates, and immobilized affinity matrix), we found DA-2 was able to identify significantly more putative kinase targets. In addition to Abl and Src family tyrosine kinases, a number of previously unknown Dasatinib targets have been identified, including several serine/threonine kinases (PCTK3, STK25, eIF-2A, PIM-3, PKA C-α, and PKN2). They were further validated by pull-down/immunoblotting experiments as well as kinase inhibition assays. Further studies are needed to better understand the exact relevance of Dasatinib and its pharmacological effects in relation to these newly identified cellular targets. The approach developed herein should be amenable to the study of many of the existing reversible drugs/drug candidates.

High matrix metalloproteinase-to-E-cadherin ratio measured by bicolor fluorescent in situ hybridization is associated with lymphangiogenesis and lymph node metastasis in prostate cancer.

OBJECTIVE: The colorimetric in situ hybridization (CISH)-based matrix metalloproteinase (MMP)-to-E-cadherin (ECD) ratio (MER) has been revealed as an excellent marker for the disease stage in prostate cancer. The one aim of this study was investigating a new method for estimation of MER by bicolor fluorescent ISH (bicolor FISH) with a computerized fluorescence detector-based system. Another aim was examination of relation of MER by bicolor FISH with expression of vascular endothelial growth factor-C (VEGF-C). METHODS: The bicolor FISH technique used cyanin 5 (cy5)-labeled MMP-2 and -9 probes, and a cyanin 3 (cy3)-labeled ECD probe on needle biopsy specimens from 67 prostate cancer cases. The ISH was followed by computerized detection of the signal intensities and cy5-to-cy3 ratios using a fluorescence detector. VEGF-C expression was examined using cy5-labeled VEGF-C by computerized detection. RESULTS: The bicolor FISH-based MER was well correlated with CISH-based MER (P < 0.0001). The bicolor FISH-based MER correlated with Gleason score and pathologic stage of the cases. VEGF-C mRNA expression was associated with the pathologic stage and maximum lymph vessel density (LVD). The LVD was associated with VEGF-C expression at the tumor area where the maximum MER was detected (P < 0.0001). CONCLUSION: The MER was correlated with the VEGF-C expression and LVD, indicating lymph node metastasis of prostate cancer. Therefore, this computer-assisted MER is a useful marker for preoperative prediction of disease stage, especially lymph node metastasis, of prostate cancer.

Thermally cross-linked superparamagnetic iron oxide nanoparticles: synthesis and application as a dual imaging probe for cancer in vivo.

We report the fabrication and characterization of thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) and their application to the dual imaging of cancer in vivo. Unlike dextran-coated cross-linked iron oxide nanoparticles, which are prepared by a chemical cross-linking method, TCL-SPION are prepared by a simple, thermal cross-linking method using a Si-OH-containing copolymer. The copolymer, poly(3-(trimethoxysilyl)propyl methacrylate-r-PEG methyl ether methacrylate-r-N-acryloxysuccinimide), was synthesized by radical polymerization and used as a coating material for as-synthesized magnetite (Fe3O4) SPION. The polymer-coated SPION was further heated at 80 degrees C to induce cross-linking between the -Si(OH)3 groups in the polymer chains, which finally generated TCL-SPION bearing a carboxyl group as a surface functional group. The particle size, surface charge, presence of polymer-coating layers, and the extent of thermal cross-linking were characterized and confirmed by various measurements, including dynamic light scattering, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. The carboxyl TCL-SPION was converted to amine-modified TCL-SPION and then finally to Cy5.5 dye-conjugated TCL-SPION for use in dual (magnetic resonance/optical) in vivo cancer imaging. When the Cy5.5 TCL-SPION was administered to Lewis lung carcinoma tumor allograft mice by intravenous injection, the tumor was unambiguously detected in T2-weighted magnetic resonance images as a 68% signal drop as well as in optical fluorescence images within 4 h, indicating a high level of accumulation of the nanomagnets within the tumor site. In addition, ex vivo fluorescence images of the harvested tumor and other major organs further confirmed the highest accumulation of the Cy5.5 TCL-SPION within the tumor. It is noteworthy that, despite the fact that TCL-SPION does not bear any targeting ligands on its surface, it was highly effective for tumor detection in vivo by dual imaging.

Photochemically-activated probes of protein-protein interactions.

The activity of light-activatable ("caged") compounds can be temporally and spatially controlled, thereby providing a means to interrogate intracellular biochemical pathways as a function of time and space. Nearly all caged peptides contain photocleavable groups positioned on the side chains of key residues. We describe an alternative active site targeted strategy that disrupts the interaction between the protein target (SH2 domain, kinase, and proteinase) and a critical amide NH moiety of the peptide probe.

99mTc-Sestamibi, a sensitive probe for in vivo imaging of P-glycoprotein inhibition by modulators and mdr1 antisense oligodeoxynucleotides.

PURPOSE: We tested the suitability of (99m)Tc-sestamibi to image the inhibition of P-glycoprotein (Pgp)-mediated multidrug resistance in tumor cells and xenografts after antisense treatment and/or inhibition with a novel Pgp modulator WK-X-34. PROCEDURE: Pgp inhibition was measured by daunorubicin transport assays and fluorescence microscopy in resistant A2780/Adr cells treated with WK-X-34 and antisense. A2780/Adr xenograft mice were dosed with mdr1 antisense oligodeoxynucleotides intratumorally for three days; next, mice were treated with WK-X-34, followed by (99m)Tc-sestamibi injection. Mice were imaged, sacrificed, and tissues collected. Images and isolated tissues were analyzed for (99)Tc distribution. Pgp expression was analyzed by immunofluorescence and reverse transcription-polymerase chain reaction. RESULTS: Both WK-X-34 and mdr1 antisense treatments significantly inhibited Pgp activity in vitro and in xenografts. Biodistribution results correlated with results from the (99m)Tc-sestamibi images. Mdr1 mRNA and Pgp were significantly down-regulated by antisense treatments. CONCLUSIONS: (99m)Tc-sestamibi is a sensitive probe to monitor Pgp inhibition by different mechanisms in vivo in tumor xenografts.

Molecular cloning of an oxytocin-like receptor expressed in the chicken shell gland.

The avian homologs of arginine vasopressin (AVP) and oxytocin (OT) are arginine vasotocin (AVT) and mesotocin (MT), respectively. In birds, AVT shares many of the functions of AVP including regulation of fluid balance, blood pressure regulation and the stress response. AVT also plays an oxytocin-like reproductive role in birds by stimulating uterine (shell gland) contraction during oviposition. The role of MT in avian reproduction is not clear. Here, we report the cloning of a third neuropeptide receptor in the chicken (Gallus gallus). Parsimony analysis reveals that the new receptor has highest homology to mammalian OT receptors and the MT receptors of non-mammalian vertebrates. Moreover, the receptor bears far less homology to the two avian VT receptors that have been cloned. Reverse transcription-polymerase chain reaction and in in situ hybridization analyses reveal the receptor is expressed in both the endometrium and myometrium of the shell gland. The expression pattern and high homology to OT receptors suggest that the receptor may stimulate myometrial contraction and therefore play a critical role in oviposition.

The interferon-inducible p202a protein modulates NF-kappaB activity by inhibiting the binding to DNA of p50/p65 heterodimers and p65 homodimers while enhancing the binding of p50 homodimers.

p202a is a member of the interferon-inducible murine p200 family of proteins. These proteins share 1 or 2 partially conserved 200 amino acid segments of the a or the b type. The known biological activities of p202a include among others the regulation of muscle differentiation, cell proliferation, and apoptosis. These biological activities of p202a can be correlated with the inhibition of the activity of several transcription factors. Thus, the binding of p202a results in the inhibition of the sequence-specific binding to DNA of the c-Fos, c-Jun, E2F1, E2F4, MyoD, myogenin, and c-Myc transcription factors. This study concerns the mechanisms by which p202a inhibits the activity of NF-kappaB, a transcription factor involved among others in host defense, inflammation, immunity, and the apoptotic response. NF-kappaB consists of p50 and p65 subunits. We demonstrate that p202a can inhibit in vitro and in vivo the binding to DNA of p65 homodimers and p50/65 heterodimers, whereas it increases the binding of p50 homodimers. Thus p202a can impair NF-kappaB activity both by inhibiting the binding to DNA of the transcriptionally active p65 homodimers and p50/p65 heterodimers and by boosting the binding of the repressive p50 homodimers. p202a can bind p50 and p65 in vitro and in vivo, and p202a can be part of the p50 homodimer complex bound to DNA. p50 binds in p202a to the a type segment, whereas p65 binds to the b type segment. Transfected ectopic p202a increases the apoptotic effect of tumor necrosis factor (at least in part) by inhibiting NF-kappaB and its antiapoptotic activity.

Molecular identification of the skin transformation center of anuran larval skin using genes of Rana adult keratin (RAK) and SPARC as probes.

Anuran larval skin undergoes a process of metamorphosis into pre-adult and adult skin. Basal skein, larval basal and adult basal cells are basement membrane-attaching cells in the larval, pre-adult and adult epidermis, respectively, and are identified as cells expressing genes of RLK (Rana larval keratin), both RLK and RAK (Rana adult keratin), and RAK. Larval to pre-adult skin conversion takes place in the histological entity called the skin transformation center (STC). The present study performed a cDNA subtractive gene screening on cDNA of the larval and the pre-adult skin, and cloned the secreted protein acidic and rich in cysteine (SPARC) gene as an upregulated gene in the larva to pre-adult skin conversion. RAK gene-positive basal skein cells and fibroblasts in and around the STC were weakly and strongly sparc-positive, respectively. Using sparc and rak, we redefined the STC and visualized it on a histological section as an approximately 150 microm-long region that contained about 20 rak-negative and weakly sparc-positive basal cells. Intense sparc expression was observed in basal skein cells, but not in larval basal cells, suggesting that SPARC acts as a suppressor of rak during epidermal differentiation. This suggestion was tested by investigating the effect of SPARC on cultured larval basal cells. We observed that SPARC suppressed the expression of rak, but not rlk.

Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate.

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/.../MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/.../MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase-interacting and spindle-stabilizing protein).

Differential effects of haloperidol and clozapine on [(3)H]cAMP binding, protein kinase A (PKA) activity, and mRNA and protein expression of selective regulatory and catalytic subunit isoforms of PKA in rat brain.

The present study was undertaken to examine whether the mechanism of action of typical and atypical antipsychotics is related in their ability to regulate key phosphorylating enzyme of adenylyl cyclase-cAMP pathway, i.e., protein kinase A (PKA). For this purpose, regulatory (R) and catalytic (Cat) activities of PKA and expression of various isoforms of regulatory and catalytic subunits were examined in rat brain after single or chronic (21-day) treatment with haloperidol (HAL, 1 mg/kg) or clozapine (CLOZ, 20 mg/kg). It was observed that chronic but not acute treatment of CLOZ significantly decreased [(3)H]cAMP binding to the regulatory subunit of PKA as well as catalytic activity of PKA in particulate and cytosol fractions of the rat cortex, hippocampus, and striatum. In these fractions, CLOZ significantly decreased protein levels of selective RII alpha-, RII beta-, and Cat beta-subunit isoforms of PKA. These decreases were accompanied by decreases in their respective mRNA expression. In contrast, chronic but not acute treatment of HAL significantly increased [(3)H]cAMP binding and the catalytic activity of PKA in particulate and cytosol fractions of only the striatum brain area. In addition, chronic treatment of HAL significantly increased mRNA and protein levels of RII alpha- and RII beta-subunit isoforms in the striatum. None of the antipsychotics caused any change in the expression of the Cat alpha-, RI alpha-, or RI beta-subunit isoform. These results, thus, suggest that HAL and CLOZ differentially regulate PKA catalytic and regulatory activities and the expression of selective catalytic and regulatory subunit isoforms of PKA, which may be associated with their mechanisms of action.

Effects of spectator ligands on the specific recognition of intrastrand platinum-DNA cross-links by high mobility group box and TATA-binding proteins.

The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by [Pt(1R,2R-diaminocyclohexane)](2+), cis-[Pt(NH(3))(cyclohexylamine)](2+), [Pt(ethylenediamine)](2+), cis-[Pt(NH(3))(cyclobutylamine)](2+), and cis-[Pt(NH(3))(2-picoline)](2+) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum. The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG) site. Double-stranded oligonucleotides containing the AG*G*C sequence, where the asterisks denote the sites of platination, with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTG*G*A is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl(2)] and cis-[Pt(NH(3))(cyclohexylamine)Cl(2)] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing of cis-[Pt(NH(3))- (cyclohexylamine)](2+), but not [Pt((1R,2R)-diaminocyclohexane)](2+), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G*A probes but not with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAG*G*C oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular proteins.

An age-related decline in interleukin-10 may contribute to the increased expression of interleukin-6 in brain of aged mice.

OBJECTIVES: The DNA-binding activity of nuclear factor kappaB (NFkappaB) is elevated in brain of aged mice, resulting in elevated levels of the inflammatory cytokine interleukin (IL)-6. The purpose of this study was to determine if in the brain of aged mice a decrease in the anti-inflammatory cytokine IL-10 contributes to the increase in IL-6. METHODS: In initial studies coronal brain sections and glial cells from adult (6-months-old) and aged (24-months-old) mice were incubated in the presence or absence of lipopolysaccharide (LPS) and the concentrations of IL-6 and IL-10 in supernatants were determined. In subsequent studies, the effect of recombinant murine IL-10 on constitutive and inducible NFkappaB activity, IL-6 mRNA expression and IL-6 protein secretion by glia cultured from brains of adult and aged mice was determined. RESULTS: Coronal brain sections and glia from aged mice secreted more IL-6 and less IL-10 than brain sections and glia from adults. This effect of age was evident in the absence and presence of LPS and suggested that a decrease in IL-10 production permitted increased IL-6 production. Consistent with this idea, treatment of glia from aged mice with recombinant IL-10 decreased both constitutive and inducible binding of NFkappaB to the IL-6 gene promoter. The decrease in NFkappaB activity was associated with a reduction of IL-6 mRNA and protein. Exogenous IL-10, however, had no effect on NFkappaB activity, which was undetectable in unstimulated glia from adult mice, and did not decrease steady-state levels of IL-6 mRNA or IL-6 protein secretion. CONCLUSIONS: Collectively, these studies suggest that IL-10 constrained IL-6 gene expression in the adult brain, but in the aged brain it decreased and thus enabled a cascade of intracellular events that increased expression of the IL-6 gene.

Down-regulation of rat hepatic microsomal cytochromes P-450 in microvesicular steatosis induced by orotic acid.

Microvesicular steatosis is an important component of the overall pathogenesis of drug-mediated liver injury. Although mitochondrial damage has a role in the development of microvesicular steatosis, the consequences of fatty change for hepatic gene function are unclear. The present study was undertaken to evaluate hepatic cytochrome P-450 (CYP) function in a rat model of microvesicular steatosis produced by the intake of diets containing 1% orotic acid (OA) that were administered for 5, 10, or 21 days. Hepatic triglyceride levels were increased to 3-fold of control after 5 days and were elevated further at 10 and 21 days. Cholesterol and phospholipid contents were increased after 10 and 21 days but not by 5 days of feeding. Microsomal androst-4-ene-3,17-dione hydroxylation activities mediated by CYP2C11 (16alpha-hydroxylation) and CYP3A2 (6beta-hydroxylation) were decreased in liver from OA-fed rats for only 5 days, whereas CYP2A1/2-mediated steroid 7alpha-hydroxylation was decreased after 10 days; these observations were complemented by immunoblot analysis that demonstrated the impaired expression of the corresponding CYP proteins. CYP2C11 mRNA, the major CYP in male rat liver, was down-regulated in steatotic liver to 52 +/- 4% of control. Thus, microvesicular steatosis induced by short-term intake of OA-containing diets is histologically similar to that produced by hepatotoxic drugs and produces the rapid down-regulation of constitutive CYPs in rat liver. Analogous processes of lipid deposition in human liver after drug- or disease-related injury could precipitate adverse effects during subsequent drug therapy.

Pituitary adenylate cyclase-activating polypeptide (PACAP) protects dorsal root ganglion neurons from death and induces calcitonin gene-related peptide (CGRP) immunoreactivity in vitro.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a recently discovered neuropeptide which is present both in the central and peripheral nervous system of adult rats. Here we show that PACAP is also expressed by dorsal root ganglion sensory neurons of embryonic and newborn rats. To characterize the effects of PACAP on dorsal root ganglion (DRG) neurons, dissociated cultures were established and incubated in the absence or presence of this neuropeptide. The results show that PACAP increases the survival of cultured DRG neurons, and the effect was comparable to that of nerve growth factor (NGF). In DRG explants, PACAP induces the immunoreactivity for the neuropeptide calcitonin gene-related peptide (CGRP). PACAP also promoted the outgrowth of neurites in the DRG cultures. The present results show that PACAP acts as a trophic factor for DRG neurons and that it is able to modulate the expression of another neuropeptide in the ganglia. The presence of PACAP in normal DRG and after nerve lesions suggests that PACAP acts in a autocrine/paracrine manner possibly in conjunction with other neurotrophic factors such as nerve growth factor.

A Bcl-2 antisense oligonucleotide increases alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) toxicity in cortical cultures.

Both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated neurotoxicity and the induction of death-regulatory genes have been implicated in the pathophysiology of delayed ischemic neuronal injury. To assess the role of the antiapoptotic gene Bcl-2 in the modulation of AMPA toxicity, we exposed neuron-enriched cultures from rat cerebral cortex to AMPA, in the absence or presence of an antisense oligodeoxynucleotide (ODN) directed against Bcl-2. AMPA produced concentration-dependent toxicity detected by a decrease in fluorescence of the redox indicator Alamar blue and by an increase in lactic acid dehydrogenase release. This effect was accompanied by the induction of Bcl-2 protein expression, with maximal induction at 100 microM AMPA. A phosphorothioate antisense ODN against Bcl-2 reduced the AMPA-stimulated induction of Bcl-2 protein levels, detected by western blotting, by about 70%. In the presence of the antisense ODN, but not sense or scrambled ODNs, the toxicity of 100 microM AMPA was increased by about 60%. These findings suggest that induction of Bcl-2 expression by AMPA may have a protective role to limit AMPA receptor-mediated neuronal damage and that modifying Bcl-2 expression could have therapeutic potential in ischemia.

Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis.

A human bronchial xenograft model was used to characterize the molecular basis for the previously described defect in bacterial killing that is present in the cystic fibrosis (CF) lung. Airway surface fluid from CF grafts contained abnormally high NaCl and failed to kill bacteria, defects that were corrected with adenoviral vectors. A full-length clone for the only known human beta-defensin (i.e., hBD-1) was isolated. This gene is expressed throughout the respiratory epithelia of non-CF and CF lungs, and its protein product shows salt-dependent antimicrobial activity to P. aeruginosa. Antisense oligonucleotides to hBD-1 ablated the antimicrobial activity in airway surface fluid from non-CF grafts. These data suggest that hBD-1 plays an important role in innate immunity that is compromised in CF by its salt-dependent inactivation.

The anti-craving drug acamprosate reduces c-fos expression in rats undergoing ethanol withdrawal.

Acamprosate (Ca salt of N-acetylhomotaurine) is a novel anti-craving substance which a double-blind placebo-controlled study has proven to be therapeutically useful in the prevention of relapses in weaned alcoholics. In the present study the expression of the immediate-early gene c-fos in rat hippocampal and cerebellar neurons was used to monitor the modulatory effect of acamprosate on neuronal excitability during ethanol withdrawal. Several hybridization techniques were employed to investigate the effect of acamprosate on c-fos expression. Acamprosate (200 mg/kg; intraperitoneally) reduced the elevated c-fos mRNA levels in the hippocampus and the cerebellum following 24 h of ethanol withdrawal, or the application of the convulsant pentylenetetrazole. The effect of ethanol withdrawal on c-fos expression was more pronounced in the cerebellum than in the hippocampus. In the hippocampus (CA1) and the cerebellum acamprosate alone induced a significant increase in c-fos expression in drug-naive animals. Only in the hippocampus did co-administration of pentylenetetrazole during ethanol withdrawal induce a further increase in c-fos expression. The present findings support the notion that acamprosate elicits its preventive effect on relapse by reducing the hyperexcitability of central neurons during withdrawal, following long-term ethanol consumption.

Activation of the interleukin-5 promoter by cAMP in murine EL-4 cells requires the GATA-3 and CLE0 elements.

Interleukin-5 (IL-5) plays a central role in the growth and differentiation of eosinophils and contributes to several disease states including asthma. Accumulating evidence suggests a role for cAMP as an immunomodulator; agents that increase intracellular cAMP levels have been shown to inhibit production of cytokines predominantly produced by T helper (Th) 1 cells such as IL-2 and interferon gamma (IFN-gamma). In contrast, the production of IL-5, predominantly produced by Th2 cells, is actually enhanced by these agents. In this report, we have performed transient transfection experiments with IL-5 promoter-reporter gene constructs, DNase I footprinting assays, and electrophoretic mobility shift assays to investigate the key regulatory regions necessary for activation of the IL-5 promoter by dibutyryl cAMP and phorbol esters in the mouse thymoma line EL-4. Taken together, our data demonstrate the critical importance of two sequences within the IL-5 5'-flanking region for activation by these agents in EL-4 cells: one, a highly conserved 15-base pair element present in genes expressed by Th2 cells, called the conserved lymphokine element 0 (CLE0; located between -53 and -39 in the IL-5 promoter), and the other, two overlapping binding sites for the transcription factor GATA-3 (but not GATA-4) between -70 and -59. Taken together, our data suggest that activation via the unique sequence combination GATA/CLE0 results in selective expression of the IL-5 gene in response to elevated levels of intracellular cAMP.

Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins.

Attack by reactive oxygen intermediates, common to many kinds of cell/tissue injury, has been implicated in the development of diabetic and other vascular diseases. Such oxygen-free radicals can be generated by advanced glycation end products (AGEs), which are nonenzymatically glycated and oxidized proteins. Since cellular interactions of AGEs are mediated by specific cellular binding proteins, receptor for AGE (RAGE) and the lactoferrin-like polypeptide (LF-L), we tested the hypothesis that AGE ligands tethered to the complex of RAGE and LF-L could induce oxidant stress. AGE albumin or AGEs immunoisolated from diabetic plasma resulted in induction of endothelial cell (EC) oxidant stress, including the generation of thiobarbituric acid reactive substances (TBARS) and resulted in the activation of NF-kappa B, each of which was blocked by antibodies to AGE receptor polypeptides and by antioxidants. Infusion of AGE albumin into normal animals led to the appearance of malondialdehyde determinants in the vessel wall and increased TBARS in the tissues, activation of NF-kappa B, and induction of heme oxygenase mRNA. AGE-induced oxidant stress was inhibited by pretreatment of animals with either antibodies to the AGE receptor/binding proteins or antioxidants. These data indicate that interaction of AGEs with cellular targets, such as ECs, leads to oxidant stress resulting in changes in gene expression and other cellular properties, potentially contributing to the development of vascular lesions. Further studies will be required to dissect whether oxidant stress occurs on the cell surface or at an intracellular locus.

EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA.

We have previously reported the purification and characterization of the transcription factor EBP-80 (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, 21915-21922). EBP-80 mediates the DNA methylation effect on transcription from an endogenous proviral long terminal repeat. Here we show that EBP-80 is very similar if not identical to the Ku autoantigen, a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (Mimori, T., Akizuki, M., Yamagata, H., Inada, S., Yoshida, S., and Homma, M. (1981) J. Clin. Invest. 68, 611-620). A number of laboratories have shown that the Ku protein complex binds to free double-stranded DNA ends. In this study, we have examined the binding properties of EBP-80. EBP-80 binds single-stranded DNA with low affinity. Binding to random sequence double-stranded DNA depends on the length of the duplex and is optimal with oligomers of 30 and 32 base pairs; the protein complexes formed with these oligomers have Kd values of 15-20 pM. It binds with comparable high affinities to blunt-ended duplex DNA, to duplex DNA ending in hairpin loops, and to constructs in which an internal segment of duplex DNA is flanked by single-strand extensions. EBP-80 also interacts effectively with circular duplex molecules containing a 30-nucleotide single-stranded region (gap) or a double-stranded segment of nonhomology (bubble), but only weakly with the corresponding closed circular construct made up entirely of duplex DNA. EBP-80 prefers A/T to G/C ends. The binding properties of EBP-80 are consistent with the hypothesis that is recognizes single- to double-strand transitions in DNA. A model is presented for the interaction of EBP-80 with its target sequence in the proviral long terminal repeat.