Synergistic Targeting of the Regulatory and Catalytic Subunits of PI3Kδ in Mature B-cell Malignancies.

Aberrant activation of the B-cell receptor (BCR) is implicated in the pathogenesis of mature B-cell tumors, a concept validated in part by the clinical success of inhibitors of the BCR-related kinases BTK (Bruton's tyrosine kinase) and PI3Kδ. These inhibitors have limitations, including the paucity of complete responses, acquired resistance, and toxicity. Here, we examined the mechanism by which the cyclic-AMP/PDE4 signaling axis suppresses PI3K, toward identifying a novel mechanism-based combinatorial strategy to attack BCR-dependency in mature B-cell malignancies.

MicroRNA 155 control of p53 activity is context dependent and mediated by Aicda and Socs1.

In biological processes, the balance between positive and negative inputs is critical for an effective physiological response and to prevent disease. A case in point is the germinal center (GC) reaction, wherein high mutational and proliferation rates are accompanied by an obligatory suppression of the DNA repair machinery. Understandably, when the GC reaction goes awry, loss of immune cells or lymphoid cancer ensues.

Metabolites from Aspergillus versicolor, an endolichenic fungus from the lichen Lobaria retigera.

Three new anthraquinone derivatives (1-3) and one new artifact (4) were isolated, along with six known anthraquinone derivatives (5-10) and three xanthones (11-13), from a culture of an endolichenic fungus, Aspergillus versicolor, that was isolated from the lichen Lobaria retigera. The structures of these substances were determined on the basis of 1D and 2D (COSY, HMQC, and HMBC) NMR and MS analyses. The substances 1-4 were also tested for their cytotoxic activity.

MicroRNA-155 controls RB phosphorylation in normal and malignant B lymphocytes via the noncanonical TGF-β1/SMAD5 signaling module.

MicroRNA-155 (miR-155) plays pleiotropic roles in the biology of normal and malignant B lymphocytes, including the modulation of the transforming growth factor β (TGF-β) pathway via the targeting of SMAD5. However, the extent of the miR-155-mediated disruption of the TGF-β1/SMAD5 axis remains to be elucidated. To address this issue, we used the miR-155 knockout (KO) mouse and diffuse large B-cell lymphoma (DLBCL) cell lines ectopically expressing miR-155.

A capture-sequencing strategy identifies IRF8, EBF1, and APRIL as novel IGH fusion partners in B-cell lymphoma.

The characterization of immunoglobulin heavy chain (IGH) translocations provides information on the diagnosis and guides therapeutic decisions in mature B-cell malignancies while enhancing our understanding of normal and malignant B-cell biology. However, existing methodologies for the detection of IGH translocations are labor intensive, often require viable cells, and are biased toward known IGH fusions. To overcome these limitations, we developed a capture sequencing strategy for the identification of IGH rearrangements at nucleotide level resolution and tested its capabilities as a diagnostic and discovery tool in 78 primary diffuse large B-cell lymphomas (DLBCLs).

MicroRNAs miR-125a and miR-125b constitutively activate the NF-κB pathway by targeting the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20).

Constitutive activation of the NF-κB pathway is associated with diffuse large B-cell lymphoma (DLBCL) pathogenesis, but whether microRNA dysfunction can contribute to these events remains unclear. Starting from an integrative screening strategy, we uncovered that the negative NF-κB regulator TNFAIP3 is a direct target of miR-125a and miR-125b, which are commonly gained and/or overexpressed in DLBCL. Ectopic expression of these microRNAs in multiple cell models enhanced K63-linked ubiquitination of proximal signaling complexes and elevated NF-κB activity, leading to aberrant expression of its transcriptional targets and the development of a proproliferative and antiapoptotic phenotype in malignant B cells.

Laminin-α1 LG4-5 domain binding to dystroglycan mediates muscle cell survival, growth, and the AP-1 and NF-κB transcription factors but also has adverse effects.

In our previous studies, we showed laminin binds α-dystroglycan in the dystrophin glycoprotein complex and initiates cell signaling pathways. Here, differentiated C2C12 myocytes serve as a model of skeletal muscle. C2C12 cells have a biphasic response to the laminin-α(1) laminin globular (LG) 4-5 domains (1E3) dependent on the concentration used; at low concentrations of 1E3 (<1 μg/ml), myoblast proliferation is increased while higher concentrations (>1 μg/ml) cause apoptosis in myoblasts and differentiated myotubes.

CCR5 promoter haplotype transcription complex characterization.

CC chemokine receptor 5 (CCR5) is a major coreceptor for cell entry of human immunodeficiency virus (HIV); its expression is highly associated with virus replication and susceptibility. Single nucleotide polymorphisms (SNPs) in the CCR5 promoter play a critical role in CCR5 transcriptional regulation. HHA and HHE represent two contrasting haplotypes of CCR5 with only two base pair differences in the promoter.

Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis.

Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps.

Repeated probing of Southwestern blots using alkaline phosphatase stripping.

Southwestern blotting is when a DNA sequence is used to probe DNA-binding proteins on an electrophoretic gel blot. It would be highly desirable to be able to probe a blot repeatedly with different DNA sequences. Alkaline phosphatase can remove 5'-phosphoryl groups from DNA and radiolabeled 5'-(32)P-DNA probes are commonly used in Southwestern blotting.

Methods for proteomic analysis of transcription factors.

Investigation of the transcription factor (TF) proteome presents challenges including the large number of low abundance and post-translationally modified proteins involved. Specialized purification and analysis methods have been developed over the last decades which facilitate the study of the TF proteome and these are reviewed here. Generally applicable proteomics methods that have been successfully applied are also discussed.

Two-dimensional southwestern blotting and characterization of transcription factors on-blot.

Two-dimensional Southwestern blotting (2D-SW) described here combines several steps. Proteins are separated by two-dimensional gel electrophoresis and transferred to nitrocellulose (NC) or polyvinylidene fluoride (PVDF) membrane. The blotted proteins are then partially renatured and probed with a specific radiolabeled oligonucleotide for Southwestern blotting (SW) analysis.

Purification and identification of positive regulators binding to a novel element in the c-Jun promoter.

A putative response element, GAGCCTC, was observed years ago in footprinting analysis of the c-jun promoter, and here we investigate its function in regulating c-jun expression and identify a protein complex that binds there. Electrophoretic mobility shift assays demonstrate a sequence-specific binding complex with this element in HEK293 cells. Additionally, unlabeled consensus AP-1 element DNA, but not a similar NF-jun element DNA, competes with complex formation.

Trapping of transcription factors with symmetrical DNA using thiol-disulfide exchange chemistry.

Green fluorescent protein (GFP) fused to the C-terminal 100 amino acids of CAAT enhancer binding protein (C/EBP) also containing an N-terminal (His)(6) tag (GFP-C/EBP) was used as a transcription factor model to test whether thiol-disulfide exchange reactions could be used to successfully purify transcription factors. A symmetrical dithiol oligonucleotide with dual CAAT elements was constructed with 5' and 3' thiols. Upon reduction, circular dichroism confirms it spontaneously anneals with its internally complementary sequence to form the hairpin structure: 5'-HS-GCAGATTGCGCAATCTGC 3'-HS-CGTCTAACGCGTTAGACG The specific GFP-C/EBP protein-DNA complex, formed in solution at nM concentrations, could then be recovered (trapped) via thiol-disulfide exchange with a disulfide thiopropyl-Sepharose and eluted with dithiothreitol.

Laminin-induced activation of Rac1 and JNKp46 is initiated by Src family kinases and mimics the effects of skeletal muscle contraction.

Binding of laminin to dystroglycan in the dystrophin glycoprotein complex causes signaling through dystroglycan-syntrophin-grb2-SOS1-Rac1-PAK1-JNK. Laminin binding also causes syntrophin tyrosine phosphorylation to initiate signaling. The kinase responsible was investigated here.

Cyanogen bromide-activated coupling: DNA catalytic chromatography purification of EcoRI endonuclease.

A method to purify enzymes utilizing their specific biological affinity and catalytic specificity is described. For this chromatographic technique, an enzyme binds immobilized substrate coupled to a column in the absence of a cofactor required for catalysis but permissive for substrate binding. After washing, the missing cofactor is added to the column mobile phase, and the enzyme converts substrate into product and elutes from the column.

Promoter trapping of c-jun promoter-binding transcription factors.

A new method called promoter trapping was developed to purify promoter-protein complex using the c-jun promoter (-200+81) as a model, which was shown to have significant promoter activity. Polymerase chain reaction (PCR), lambda exonuclease digestion combined with (AC)(5)-Sepharose DNA affinity chromatography were used to produce c-jun promoter with a (GT)(5) tail at each 3' end. The intact promoter and different length pieces with one or two (GT)(5) tails had almost the same capacity to bind with (AC)(5)-Sepharose.

Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node.

A new C-type lectin-like gene encodes 293 amino acids and maps to chromosome 19p13.3 adjacent to the previously described C-type lectin genes, CD23, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and DC-SIGN-related protein (DC-SIGNR). The four genes form a tight cluster in an insert size of 105 kb and have analogous genomic structures.

[Study on differentially expressed molecules influencing the metastatic potential between highly and poorly metastatic human lung giant cell carcinoma].

Objective: To study the metastasis-associated molecules differentially expressed in highly and poorly metastatic sublines and the mechanism of metastasis in lung giant cell carcinoma.

Methods: Highly and poorly metastatic sublines (PLA801D and PLA801C)were used as metastasis model. Cell motility and invasion assay in vitro were first compared between the two sublines.

[Function of IL-18 in promoting metastasis of lung cancer].

Objective: To study the function of IL-18 in promoting metastasis of lung cancer.

Methods: The differential expression of IL-18 protein or mRNA level between highly and poorly metastatic sublines of human lung giant cell carcinoma metastatic model was detected by Western blot, semi-quantitative RT-PCR and northern blot analysis. The poorly metastatic PLA801C subline or highly metastatic PLA801D subline was transfected with constructed IL-18 sense or IL-18 antisense expressed plasmid by lipofectamine stable transfection technique.

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis.

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro.