Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα

Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.     

Selection and Characterization of Artificial Proteins Targeting the Tubulin α Subunit

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Plasma Levels of Inter-α Inhibitor Proteins in Children with Acute Dengue Virus Infection

Background

Inter-α inhibitor proteins (IaIp) belong to a family of protease inhibitors that are involved in the haemostatic and the vascular system. Dengue viruses (DENV) infections are characterized by coagulopathy and increased vascular permeability. In this study we measured the concentration of IaIp during DENV infections and evaluated its potential as a biomarker.

Methods and Findings

Concentrations of IaIp were measured in patients with acute DENV infections using a quantitative, competitive enzyme linked immunoassay. Concentrations of IaIp measured in pediatric patients suffering from severe DENV infections were significantly lower than in healthy controls.

Conclusions

This is the first report to demonstrate changes in concentration of IaIp during viral infections. The data also highlight the potential of IaIp as a biological marker for severity of DENV infections.     

Targeting of the Gi2α Gene in es cells with Replacement and Insertion Vectors

Abstract

Five replacement vectors (RV) and one insertion vector (IV) were constructed in which ca. 10 kb of genomic G_i2α sequence, flanked on one (IV) or both (RV) sides by a thymidine kinase (TK) marker, were disrupted by a Neo marker inserted into the Ncol site of exon 3. G418^RFIAU^R clones corresponding to ca. 4×10⁸ ES cells electroporated with replacement vectors were analyzed and revealed no targeting event. The insertion vector, however, was integrated by a single reciprocal recombination resulting in a duplication of homology (Hit step; G418^RFIAUS^S, which was lost-together with the plasmid and the TK sequences - by intrachromosomal recombination (Run step; G418^RFIAU^R). Thus, the Hit and Run strategy can be used with a selectable marker disrupting the targeted gene, giving rise to the same targeted product that would have been expected to arise from a double crossover with a replacement vector.     

Yeast Importin-α (Srp1) Performs Distinct Roles in the Import of Nuclear Proteins and in Targeting Proteasomes to the Nucleus

Srp1 (importin-α) can translocate proteins that contain a nuclear localization signal (NLS) into the nucleus. The loss of Srp1 is lethal, although several temperature-sensitive mutants have been described. Among these mutants, srp1-31 displays the characteristic nuclear import defect of importin-α mutants, whereas srp1-49 shows a defect in protein degradation. We characterized these and additional srp1 mutants to determine whether distinct mechanisms were required for intracellular proteolysis and the import of NLS-containing proteins. We determined that srp1 mutants that failed to import NLS-containing proteins (srp1-31 and srp1-55) successfully localized proteasomes to the nucleus. In contrast, srp1 mutants that did not target proteasomes to the nucleus (srp1-49 and srp1-E402Q) were able to import NLS-containing proteins. The proteasome targeting defect of specific srp1 mutants caused stabilization of nuclear substrates and overall accumulation of multiubiquitylated proteins. Co-expression of a member of each class of srp1 mutants corrected both the proteasome localization defect and the import of NLS-containing proteins. These findings indicate that the targeting of proteasomes to the nucleus occurs by a mechanism distinct from the Srp1-mediated import of nuclear proteins.     

Topological Frustration in βα-Repeat Proteins: Sequence Diversity Modulates the Conserved Folding Mechanisms of α/β/α Sandwich Proteins

The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of nonlocal interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of βα-repeat proteins, which are dominated by local interactions, we performed a combined experimental and simulation analysis on two members of the flavodoxin-like, α/β/α sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured submillisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine, and valine (ILV) side chains that form a large sequence-local cluster involving the central β-sheet and helices α2, α3, and α4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side chains in its corresponding hydrophobic cluster. The location of the hydrophobic clusters in the structure may also be related to the differing functional properties of these response regulators. Comparison with the results of previous experimental and simulation analyses on the homologous CheY argues that prematurely folded unproductive intermediates are a common property of the βα-repeat motif.     

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.     

Interactions of Human Mismatch Repair Proteins MutS�� and MutL�� with Proteins of the ATR-Chk1 Pathway

At clinically relevant doses, chemotherapeutic S_N1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSα and MutLα. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutSα and MutLα with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSα with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLα with TopBP1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1, and Chk1 and of MutLα with TopBP1. MutSα-Claspin and MutLα-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα- and MutSα-dependent fashion after N-methyl-N′-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.     

Targeting the ICB2 site of the topoisomerase IIα promoter with a formamido-pyrrole–imidazole–pyrrole H-pin polyamide

The synthesis, DNA binding characteristics and biological activity of an N-formamido pyrrole- and imidazole-containing H-pin polyamide (f-PIP H-pin, 2) designed to selectively target the ICB2 site on the topoIIα promoter, is reported herein. Thermal denaturation, circular dichroism, isothermal titration calorimetry, surface plasmon resonance and DNase I footprinting studies demonstrated that 2 maintained the selectivity of the unlinked parent monomer f-PIP (1) and with a slight enhancement in binding affinity (K_eq = 5 × 10⁵ M^?1) to the cognate site (5′-TACGAT-3′). H-pin 2 also exhibited comparable ability to inhibit NF-Y binding to 1, as demonstrated by gel shift studies. However, in stark contrast to monomer 1, the H-pin did not affect the up-regulation of topoisomerase IIα (topoIIα) in cells (Western blot), suggesting that the H-pin does not enter the nucleus. This study is the first to the authors’ knowledge that reports such a markedly different cellular response between two compounds of almost identical binding characteristics.     

HDAC2 phosphorylation-dependent Klf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs

Abnormal proliferation of vascular smooth muscle cells (VSMCs) occurs in hypertension, atherosclerosis and restenosis after angioplasty, leading to pathophysiological vascular remodeling. As an important growth arrest gene, p21 plays critical roles in vascular remodeling. Regulation of p21 expression by retinoic acid receptor (RAR) and its ligand has important implications for control of pathological vascular remodeling. Nevertheless, the mechanism of RAR-mediated p21 expression in VSMCs remains poorly understood. Here, we show that, under basal conditions, RARα forms a complex with histone deacetylase 2 (HDAC2) and Krüppel-like factor 5 (Klf5) at the p21 promoter to inhibit its expression. Upon RARα agonist stimulation, HDAC2 is phosphorylated by CK2α. Phosphorylation of HDAC2, on the one hand, promotes its dissociation from RARα, thus allowing the liganded-RARα to interact with co-activators; on the other hand, it increases its interaction with Klf5, thus leading to deacetylation of Klf5. Deacetylation of Klf5 facilitates its dissociation from the p21 promoter, relieving its repressive effect on the p21 promoter. Interference with HDAC2 phosphorylation by either CK2α knockdown or the use of phosphorylation-deficient mutant of HDAC2 prevents the dissociation of Klf5 from the p21 promoter and impairs RAR agonist-induced p21 activation. Our results reveal a novel mechanism involving a phosphorylation-deacetylation cascade that functions to remove the basal repression complex from the p21 promoter upon RAR agonist treatment, allowing for optimum agonist-induced p21 expression.     

Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca²⁺ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca²⁺. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca²⁺ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca²⁺ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca²⁺ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K⁺ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.Protein kinase C (PKC),¹ a family of phospholipid-dependent serine/threonine kinases and of which there are at least 12 subspecies, plays an important role in various cellular signal transductions (Nishizuka, 1984, 1988, 1992). Regardless of ubiquitous expression of PKCs in various tissues, the central nervous system abundantly contains several unique PKCs. In particular, the γ-subspecies of PKC (γ-PKC) is present only in the central nervous system and is thought to be involved in many neuronal functions including the formation of neural plasticity and memory (Nishizuka, 1986; Abeliovich et al., 1993a ,b; Tanaka and Nishizuka, 1994).PKC isozymes are divided into three subfamilies based on differences in the regulatory domain: conventional PKC (cPKC), novel PKC (nPKC), and atypical PKC (aPKC). Conventional PKCs have two common regions in the regulatory domain, C1 and C2. The C1 region has two cysteine-rich loops (zinc finger–like motifs) that interact with diacylglycerol (DG) or phorbol esters (Nishizuka, 1988; Ono et al., 1989). The C2 region mediates calcium binding (Ono et al., 1989) and is only present in cPKCs (Ono et al., 1988b ), although a region related to C2 region was recently reported in nPKC, a calcium-independent PKC (Parker and Dekker, 1997). Full activation of cPKCs, including γ-PKC, requires DG and calcium. The C1 region is also present in nPKC, and one of the cysteine-rich loops is found in aPKCs.Conventional PKCs and nPKCs, whose regulatory domains contain C1, are known to be translocated from the cytosol to particulate fraction when activated by DG or phorbol esters (Kraft et al., 1982). Therefore, the translocation of PKCs is a good marker of whether these enzymes are activated. Although this phenomenon is well known, the mechanism and physiological significance of PKC translocation have not yet been clarified. By conventional enzymological or immunohistochemical methods, it is impossible to observe the translocation of PKC in real time, in the same cells, and in living states, except in the investigation using fluorescent probes that directly bind PKC (Chen and Poenie, 1993). In addition, these fluorescent compounds are suggested to inhibit the activity of PKC itself at high concentration.To resolve these problems and to directly observe the translocation of γ-PKC in living cells, we produced a fusion protein of γ-PKC and green fluorescent protein (GFP). The GFP, isolated from jellyfish Aequorea victoria, has fluorescence without additional substrates and cofactors (Cubitt et al., 1995). Recent studies have revealed that GFP is a good candidate as a molecular reporter protein to monitor the alternation of protein localization, gene expression, and protein trafficking in living cells (Cubitt et al., 1995). In this study, we visualized and analyzed the translocation of γ-PKC–GFP fusion protein with confocal laser scanning fluorescence microscopy, using various stimulations, such as phorbol esters, Ca²⁺ ionophore, K⁺ depolarization, and receptor-mediated stimulus.