Cell Nuclear Extracts Research Articles

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads.

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

Sulfonated calix[4]arene functionalized SiO2@TiO2 for recognition of lysine methylation

Lysine methylations are common protein post-translational modifications (PTMs), that play significant roles in regulating gene activities. Studies of their functions and connections with diseases have important values. However, due to the small variations from their native structures and very low component proportions, it is very difficult to extract methylated peptides from biological mixtures. In this research, a new material that utilizes sulfonated calix[4]arene (SC4A) as the recognition unit and silica coated with TiO2 as carrier, denoted as SiO2@TiO2@SC4A, was synthesized. The equilibrium binding experiments demonstrated that SiO2@TiO2@SC4A can identify lysine and arginine methylation and peptides with these methylated residues. The maximum isotherm binding capacities are 70.0, 55.9, 31.4 and 24.8 μmol g−1 for Lys(Me3), Lys(Me)2, Lys(Me) and Lys, respectively. It demonstrated that the higher the degree of methylation, the stronger the interactions.In addition, the analyses of high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) demonstrated that peptides with methylated lysine or arginine can be selectively extracted from spiked histone trypsin digestion. The recoveries for the spiked GGAK(Me)R, GGAKR(Me)2 and GGAK(Me)3R are 83%, 78%, and 84% respectively. The experiments from the nuclear extracts of HeLa cells also illustrated that SiO2@TiO2@SC4A holds a potential in the enrichment and identification of lysine methylations.

Fluorescent Detection of O-GlcNAc via Tandem Glycan Labeling

O-GlcNAcylation is a reversible serine/threonine glycosylation on cytosolic and nuclear proteins that are involved in various regulatory pathways. However, the detection and quantification of O-GlcNAcylation substrates have been challenging. Here, we report a highly efficient method for the identification of O-GlcNAc modification via tandem glycan labeling, in which O-GlcNAc is first galactosylated and then sialylated with a fluorophore-conjugated sialic acid residue, therefore enabling highly sensitive fluorescent detection. The method is validated on various proteins that are known to be modified by O-GlcNAcylation including CK2, NOD2, SREBP1c, AKT1, PKM, and PFKFB3, and on the nuclear extract of HEK293 cells. Using this method, we then report the evidence that hypoxia-inducible factor HIF1α is a potential target for O-GlcNAcylation, suggesting a possibly direct connection between the metabolic O-GlcNAc pathway and the hypoxia pathway.

Abstract 4098: Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance

Abstract Background: Although enzalutamide (ENZ) is an effective treatment for prostate cancer (PC), development of ENZ resistance is inevitable. One mechanism for resistance is upregulation of ARV7, a splice variant of the full-length androgen receptor (FLAR) that lacks the androgen binding and hinge region domains. Nuclear translocation of FLAR requires the hinge region for coupling to tubulin or importin. ARV7 gains nuclear entry independent of the canonical pathway. FLAR has been reported to bind to Hypoxia Inducible Factor-1α (HIF1α) under normoxic conditions in the presence of dihydrotestosterone (DHT). We hypothesized that ARV7 may also heterodimerize with HIF1α in the cytoplasm, enabling nuclear localization and transcriptional activity of both transcription factors. We reported that topotecan (TPT) blocks HIF1α mRNA translation by targeting topoisomerase I (TOPO1) resident on HIF1α mRNA. We determined if ARV7 was bound to HIF1α in ENZ resistant cells and if inhibition of HIF1α translation by topotecan (TPT) could block ARV7 nuclear localization, thereby resensitizing ARV7 expressing cells to ENZ. We also evaluated if TOPO1 resided on FLAR/ARV7 mRNA, providing an additional target for TPT. Methods: 22Rv1 ENZ resistant PC cells that express FLAR and ARV7 protein were treated with the hypoxia mimetic cobalt chloride to stabilize HIF1α. Cytoplasmic and nuclear extracts from 22Rv1 cells were used for co-immunoprecipitation (Co-IP) using anit-HIF1α antibody. The effect of DHT, ENZ, and TPT on HIF1α and AR/ARV7 expression, and interactions between Topo-1 and HIF1α and AR mRNA were determined by western blot and RNA-IP. The effects of ENZ and TPT on 22Rv1 cell viability were evaluated using the XTT viability assay. Results: Topo-I RNA IP demonstrated its linkage to HIF-1α, FLAR and ARV7 mRNA. Co-IP of 22Rv1 whole cell lysates and nuclear extracts with HIF1α-specific antibodies yielded both full length AR and ARV7 protein bands on western blots under normoxic conditions in the presence of DHT and under hypoxic conditions. TPT and ENZ treatment of 22Rv1 cells under hypoxic conditions resulted in significantly reduced HIF1α, FLAR and ARV7 nuclear localization. TPT had an IC50 of 20 nM in normoxia and 91 nM in hypoxia. ENZ had an IC50 of 24.4 uM in normoxia and 388.7 uM in hypoxia. Dose-response curves for TPT / ENZ combinations at concentrations achieved clinically demonstrated synergistic activity, with combination index values of 0.54 under normoxic and 0.19 under hypoxic conditions. Conclusions: Our data indicate that Topo I resides on HIF-1α, FLAR and ARV7 mRNA, providing a potential target for TPT-mediated inhibition of their translation. Clinically achievable TPT concentrations caused HIF1α knockdown in hypoxic 22Rv1 cells in association with diminished nuclear localization of ARV7. 22Rv1 cells showed a synergistic dose-response to the combination of TPT and enzalutamide. These findings support exploration of TPT in combination with ENZ for treatment of PC. Citation Format: John P. Fruehauf, Diana C. Gomez, Leslie Spitalny, Jai-Hyun Kim. Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4098.

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking.

Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein-protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.

Arsenite suppresses the transcriptional activity of EVI1 through the binding to CCHC-type Zn finger domain

Transcription factor EVI1 is essential for normal hematopoiesis in embryos but is aberrantly elevated in bone marrow cells of myelodysplastic syndrome (MDS) patients. EVI1 and its downstream GATA-2 appear to be a possible therapeutic target of MDS. Here we found that treatment of EVI1-expressing K562 cells with arsenite (As(III)) reduced the mRNA and protein levels of EVI1 and GATA-2. A gel shift assay using the nuclear extract of K562 cells showed that As(III) suppressed the DNA-binding activity of EVI1. The DNA-binding activity of the recombinant EVI1 protein was also suppressed by As(III) but was recovered by excess amounts of dithiothreitol, suggesting the involvement of cysteine residues of EVI1. Since the 7th Zn finger domain of EVI1, having a motif of CCHC, is known to be involved in DNA-binding, the synthetic peptide of 7th Zn finger domain was reacted with As(III) and subjected to MALDI-TOF-MS analysis. The results showed that As(III) binds to this peptide via three cysteine residues. As(III)-induced reduction of the DNA-binding activity of the recombinant EVI1 was abolished by the mutations of each of three cysteine residues to alanine in the 7th Zn finger domain. These results demonstrate that As(III) causes the down-regulation of EVI1 and GATA-2 by inhibiting the transcriptional activity of EVI1 through the binding to the cysteine residues of CCHC-type Zn finger domain.

Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the "Framed Mirror" Method.

We propose an improved earlier described “mirror” method for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to the misincorporation of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). This method is based on the synthesis of a complementary chain (“mirror”) by nuclear extracts of different mice organs on a template containing 8-oxoG and dideoxycytidine residue (ddC) at the 3′‑end. The “mirror” was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the “framed mirror” products. The misincorporation of dAMP in “framed mirror” products forms an EcoRI restriction site. The restriction analysis of double-stranded “framed mirror” products allows a quantification of the mutation frequency in nuclear extracts. The data obtained show that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos.

Potential Inhibitor Against Phase Separation, 1,6-hexanediol Specifically Binds to Beta Actin in Nuclear Extract of Human Cell Line

RNA-binding protein (RBP) TLS/FUS plays a major role in induction of phase separation/phase transition and aggregation in relation to familial amyotrophic lateral sclerosis (ALS). Recently, organelles without lipid-bilayer membrane including stress granule, Cajal body, and nucleolus are found to be formed by the phase separation. The phase separation is an event that solutions with two solvents separate into two distinctive phases. The phase separation is prone to have solid phase and forms harmful precipitation or aggregation against living cells, indicating that the phase separation has both benefit and risk on cellular programs. Thus, it is essential for utilization of the phase separation in divergent cellular programs to control or inhibit the undesirable precipitation in living cells. Here, we analyze an inhibitory mechanism of the phase separation and precipitation. Inhibition of the phase separation is one of a critical regulatory step to prevent dysregulation of the phase separation and resulting deleterious precipitations. An inhibitory agent against the phase separation, amphiphilic alcohol, 1,6-hexanediol (1,6-HD) has been reported to examine function of phase separations. Thus, affinity chromatography of biotinylated 1,6-HD is employed to identify an initial event induced by 1,6-HD. Upon successful synthesis of biotinylated compounds of 1,6-HD (bio-1,6-HD), the affinity chromatography with bio-1,6-HD has been established at this study. The bio-1,6-HD captured protein bands on SDS-PAGE gel from HeLa cell nuclear extracts. The bands were analyzed with mass spectrometric analyses, showing that the proteins should be cytoskeleton related proteins. Further analysis using specific antibodies revealed one of the bands as human beta actin. It has been shown that beta actin is involved in divergent cellular activities including the phase separation and also neuronal functions like long-term potentiation. Therefore, beta actin might initiate the 1,6-HD-induced inhibition of the precipitation, although more experiments should be required to test whether it actually works in living cells.

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Detailed Functional Characterization of Splenic Marginal Zone Lymphoma: Uncovering Links between the Epigenetic and the Signaling Machinery

Splenic marginal-zone lymphoma (SMZL) ontogeny and evolution is a complex process, involving, amongst others, (super)antigenic stimulation, molecular deregulation of genes involved in the physiological differentiation of splenic marginal zone B cells (e.g. NOTCH2 and KLF2), and epigenetic alterations leading to silencing of different tumor suppressor genes and overexpression of oncogenes, including the PRC2-complex (EZH2, EED, and SUZ12). However, despite important recent progress, advances in the fundamental understanding of SMZL have not yet been translated in improved patient management, highlighting the need for further molecular investigations. In this context, here we aimed at detailed characterization of: (i) epigenetic modifiers, particularly the histone methyltransferase EZH2, a marker of clinical aggressiveness and druggable target in lymphoma; (ii) immune signaling capacity of SMZL B cells; and, (iii) antigen reactivity profile of the SMZL clonotypic B Cell Receptor immunoglobulin (BcR IG). Quantification of EZH2 mRNA levels using quantitative Real-time PCR (n=26 SMZL patients) and protein levels using western blotting (WB) (n=14) revealed that EZH2 was expressed in all cases yet in a heterogeneous manner (2^-ΔCt range: 0.079-1.38; ΕΖΗ2+ cells: 3.11%-44.8%). Interestingly, higher proliferation rates (Ki67+ cells) were observed in cases with high EZH2 protein levels (n=4) compared to cases with low EZH2 (n=6) (FD=2.6; p<0.05). Stimulation of SMZL CD19+ B cells (n=8) through i) the BcR with anti-IgM ii) Toll-Like receptor 9 (TLR9) with CpG and iii) BcR/TLR for 60 minutes affected pBTK, pERK and pNF-kB levels as revealed by WB and flow cytometry (FCM). Although, great heterogeneity was observed in the responses between different cases, categorization of SMZL cases based on EZH2 levels showed that, at basal level, EZH2high cases expressed higher pBTK, pERK and pNF-κB compared to EZH2low cases (FC=2.4, FC=6,1, FC=12.7, respectively; p<0.05 for all comparisons). BcR and TLR9 activation and, especially, co-stimulation with anti-IgM/CpG induced the expression of pBTK, pERK and pNF-κB in EZH2low cases, while the opposite was observed in EZH2high cases. Moreover, stimulation through both the BcR and BcR/TLR9 for 24 hours could also induce EZH2 expression compared to the unstimulated control cells (FC=1.6; p<0.05 for both comparisons). To gain insight into the type of antigenic stimuli that could activate BcR signaling through the BcR we investigated the antigen binding profile of 10 SMZL clonotypic BcR IGs expressed as recombinant human IgM monoclonal antibodies (mAbs). Cases were representative of the most common IGHV genes in SMZL (IGHV1-2*04, IGHV4-34 and IGHV3-23). Reactivity (at 15 μg/ml) against various autoantigens was evaluated by ELISA. Five of 9 (56%), 4/7 (57%), 5/10 (50%), 2/9(22%), 2/9 (22%), 3/9 (33%), 3/8 (38%), 4/8 (50%) and 4 of 9 (44%) SMZL IgM mAbs displayed reactivity against native DNA, nuclear Hep-2 cell extract, actin, myosin, thyroglobulin, β-amyloid, carbonic anhydrase, F(ab')2 and the hapten trinitrophenyl respectively, with optical density (OD) values greater than the cut-off point. Moreover, 5/10 (50%) SMZL IgM mAbs recognized more than 2 antigens and were characterized as polyreactive. Each of these SMZL mAbs exhibited a distinct antigen polyreactivity profile, while heterogeneity was observed even amongst cases expressing the same gene or cases with similar IGHV gene mutational load. Recognition of epitopes on the surface of viable MEC1 B and HEK293 cells was assessed by FCΜ. Two of 3 and 5/11 mAbs bound to viable MEC1 B cells and HEK293 cells, respectively: binding to HEK293 cells was more pronounced for minimally mutated/unmutated versus mutated mAbs (p<0.05). Importantly, stimulation of SMZL CD19+ B cells with the SMZL BcR cognate antigens actin and myosin resulted in regulation of pERK/pPLCγ2 in 3/6 and 1/2 cases, respectively, as revealed by WB. Overall, SMZL cells can be activated by microenvironmental signals albeit in a different manner depending on EZH2 levels, highlighting links between the epigenetic and the signaling machinery with potential therapeutic implications. Moreover, the recognition of a wide range of autoantigens by the SMZL mAbs indicates that SMZL B cell clones may arise from polyreactive B cells, likely resident in the normal splenic marginal zone. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

Changes in N-Glycosylation Induced By Somatic Hypermutation Modulate the Antigen Reactivity of the Immunoglobulin Receptors in CLL Stereotyped Subset #201

The IGHV4-34 gene is intrinsically autoreactive due to carrying a germline(GL)-encoded (super)antigenic motif binding various self (and exogenous) antigens, while it is one of the few IGHV genes that contain a GL-encoded N-glycosylation (N-glyc) site. IGHV4-34 is overrepresented in chronic lymphocytic leukemia (CLL), particularly in cases expressing B cell receptor immunoglobulin (BcR IG) with a significant load of somatic hypermutation (SHM; 'mutated' CLL, M-CLL). Moreover, a large fraction of IGHV4-34 M-CLL cases are clustered in different stereotyped subsets, of which the best studied is subset #4, the largest within M-CLL, defined by the expression of IgG-switched IGHV4-34/IGKV2-30 BcR IG with a distinctive SHM imprint. Considerably smaller than subset #4 is subset #201, defined by the expression of IGHV4-34/IGLV1-44 BcR IG of the IgMD isotype. Subset #201 is noteworthy owing to recurrent replacement SHMs that frequently lead to the creation of novel N- glyc motifs within the VH domain. This may be functionally relevant, considering that N-linked glycosylation is a widespread post-translational modification that is largely SHM-induced during antigen-specific immune responses and can modulate antibody (Ab) affinity towards antigen. That said, nothing is yet known about the antigen reactivity of subset #201 BcR IG and whether/how it could be affected through SHM-induced changes of N-linked glycosylation. In order to obtain insight into this issue, 4 subset #201 clonotypic IGs were expressed as recombinant monoclonal Abs (mAbs) of the mu isotype in HEK293 human cells, in either the authentic SHM state ('wildtype', WT-mAbs) or after reverting specific SHMs that altered N-glyc sites (R-mAbs) by site-directed mutagenesis. Since not all N-glyc motifs are eventually glycosylated, we used the NetNglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) for the prediction of N-glycan occupancy. Binding to MEC1 B CLL, Jurkat T and HEK293 cells was assessed by flow cytometry. Reactivity against nuclear Hep-2 cell extract, nDNA, actin, myosin, thyroglobulin (TG), β-amyloid, carbonic anhydrase, F(ab')2 and the non-self hapten trinitrophenyl was tested by ELISA. Non-subset #201 M-CLL mAbs (n=14, including 3 subset #4 mAbs), were used as controls. None of the subset #201 WT-mAbs displayed reactivity in any of the ELISAs. However, unlike most CLL mAbs, all subset #201 WT-mAbs bound to live MEC1 cells, while also exhibiting reactivity to HEK293 cells that was significantly higher when compared to non-subset #201 M-CLL (p=0.0095) or subset #4 (p=0.05); additionally, 1/4 subset #201 mAb displayed weak binding to Jurkat T cells. Three of 4 subset #201 mAbs bore a novel N-glyc site introduced by SHM in codons VL CDR1 36-38 of the clonotypic lambda light chains. Reversion to the GL in one such mAb resulted in enhanced binding to all 3 cell lines [fold change (FC) of binding of the R- vs WT-mAb to MEC1, Jurkat and HEK293: 1.3, 7.9 and 3.3, respectively) and in strong anti-TG activity. The GL-encoded N-glyc site in VH CDR2 57-59, that has been reported to be mostly unoccupied, was targeted by SHM in 2/4 subset #201 mAbs: reversion to GL decreased binding to both MEC1 and HEK293 cells (FC: -8 and -1.4 respectively). Finally, in 2/4 cases, SHM at codons VH FR3 67-68 inserted an N-glyc site that, however, is not predicted to acquire N-glycans. Reversion to GL enhanced the binding of one of these mAbs to MEC1 and HEK293 cells (FC: 2.1 and 5.6, respectively). The same mAb bore an additional predicted N-glyc site introduced by SHM at VH FR3 90-92; reversion of this change to GL augmented binding to both MEC1 and HEK293 cells (FC: 4.1 and 9.7, respectively). Double reversion of both aforementioned SHMs conferred further increased binding than any of the single reversions, implying a synergistic effect. Acquisition of novel N-glyc sites is not an intrinsic characteristic of either M-CLL in general or IGHV4-34 M-CLL in particular and its high incidence in subset #201 implies a selective process likely due to distinct (auto)antigenic pressure. Indeed, subset #201 mAbs exhibit an antigen reactivity profile that differs from that of typical polyreactive mAbs, including natural autoantibodies and other CLL mAbs, binding selectively to viable lymphoblastoid cell line cells and human HEK293 epithelial cells. These results further emphasize the importance of SHM in shaping the distinct (auto)antigenic recognition profile of CLL mAbs. Disclosures Chatzidimitriou: Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Constitutively Activated STAT3 Induces the Expression of Gli1 in Chronic Lymphocytic Leukemia Cells

Constitutively Activated STAT3 Induces the Expression of Gli1 in Chronic Lymphocytic Leukemia Cells

Constitutively Activated STAT3 Induces the Production of PTX3 That Contributes to the Induction of Bone Marrow Reticulin Fibrosis in Patient with CLL

Constitutively Activated STAT3 Induces the Production of PTX3 That Contributes to the Induction of Bone Marrow Reticulin Fibrosis in Patient with CLL

PS971 AT1413 ANTIBODY DERIVED FROM A CURED AML PATIENT RECOGNIZES A UNIQUE CD43 EPITOPE SHARED BY AML, MDS AND MELANOMA AND HAS IN VIVO ACTIVITY AS UNMODIFIED ANTIBODY AND AS BISPECIFIC T CELL ENGAGER

Background: Nuclear mislocalization of proteins can interfere with normal cellular function and cooperatively drive tumor development. To understand how this process mediates AML development, we analyzed the nuclear proteome of AML blasts in comparison with normal human CD34+ cells to identify misregulated nuclear proteins. In a preliminary study, we identified S100A4 to be over-expressed in 73% (11/15) AML patients and mislocalized to the nucleus in AML blasts. S100A4 belongs to the S100 multigene family of calcium-binding proteins of the EF-hand type and has been implicated in tumor progression and metastasis in many solid tumours but little is known of its role in hematological malignancy. Aims: To validate S100A4 overexpression and mislocalization in primary AML blasts. To determine the functional role of S100A4 on hematopoietic cell development, growth and survival in normal human CD34+ cells and AML cell lines. Methods: S100A4 expression was determined by western blot. Lentiviral vectors were used to ectopically express S100A4 and shRNA was used to knock down S100A4 expression. Effects on cell growth, survival and differentiation were determined by flow cytometry. To determine S100A4 interacting proteins, S100A4 was co-immunoprecipitated with its binding partners using Ca+2 enriched conditions from nuclear extracts of ME-1 cells followed by LC-MS/MS analysis. Results: Using western blotting, S100A4 protein expression was observed in the nucleus of AML blasts (73%; 24/33) whilst normal CD34+ or CD14+ differentiated monocytic controls have shown only cytosolic expression of S100A4. Upregulated S100A4 is also supported by transcriptome analysis indicating that overexpression arises at least partly at a transcriptional level. An independent dataset (TCGA) supports the overexpression of S100A4 mRNA in AML and suggests that overexpression may confer a poor prognosis (p = 0.0118). To determine whether ectopic expression of nuclear S100A4 can affect the growth and survival of CD34+ cells, we attempted to overexpress nuclear-targeted S100A4 in normal human hematopoietic cells. Overexpression of nuclear S100A4 could not be demonstrated in transduced CD34+ or in normal differentiated cells (probably due to rapid degradation of ectopically expressed S100A4 in these cells). To examine the importance of S100A4 expression in normal and leukaemic cells we next knocked-down S100A4 expression. In CD34+ cells we observed no significant effect on the growth or lineage development of these cells suggesting S100A4 is not required for normal hematopoiesis. We next examined the consequences of knocking down S100A4 expression in AML cell lines (NOMO-1, TF-1, THP-1, and OCI-AML2). S100A4 knockdown severely impaired the growth TF-1 and OCI-AML2 (p < 0.05). Significant apoptosis was observed in OCI-AML-2, TF-1, and NOMO-1 (p < 0.05). Using co-immunoprecipitation coupled with LC/MS to identify binding partners of S100A4, we identified heterogeneous nuclear Ribonucleoprotein M (hnRNPM) as novel binding partner of S100A4 AML. Interestingly, hnRNPM has been implicated in metastasis in breast cancer through mediating alternative splicing. Summary/Conclusion: We found that S100A4 is mislocalized to the nucleus in AML blasts compared to normal hematopoeitic cells and is essential for AML cells growth and survival. HnRNPM emerged as novel binding partner to S100A4 and we hypothesise that S100A4 mediates its effect through forming a splicing complex with known leukemic splicing factors such as SF3B1 and SRSF2 (also been pulled down with S100A4 co-IP).

Release of transglutaminase 2 from respiratory syncytial virus-infected airway epithelial cells and its role in disease pathogenesis

Abstract Respiratory syncytial virus (RSV) is the common cause of severe lower respiratory tract infections in young children, the elderly and the immunocompromised, for which no effective treatment or vaccine is currently available. RSV infections have also been linked to both the development and severity of asthma. Oxidative stress plays a major role in the pathogenesis of many lung inflammatory diseases. Our studies have shown that oxidative stress responses in the airways play a major role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium levels, which in turn activate calcium dependent enzymes, including transglutaminase 2 (TG2). TG2 is a transamidating acyltransferase that catalyzes Ca2+-dependent protein modifications and has been implicated in the pathogenesis of many diseases. This study’s objective was to investigate the release of TG2 in airway epithelial cells in response to RSV infection and its potential mechanism. Studies were conducted in airway epithelial cells, a human lung carcinoma cell line and normal small alveolar epithelial cells. The changes in the TG2 levels were measured by Western blot and RT-PCR after cells were pretreated with antioxidants and infected with RSV. TG2 levels were significantly increased in the secretome with a concomitant decrease in the cell lysates, cytoplasmic and nuclear extracts in response to RSV infection. Antioxidants treatment significantly inhibited RSV-induced TG2 release. TG2 release was induced only by RSV but not RSV-induced cytokines or chemokines. These results suggest that modulation of oxidative stress represents a potential novel pharmacological approach to ameliorate virus-induced acute airway disease through TG2 release blockade.

MEF2B is a member of the BCL6 gene transcriptional complex and induces its expression in diffuse large B-cell lymphoma of the germinal center B-cell-like type

Myocyte enhancer-binding factor 2B (MEF2B) has been implicated as a transcriptional regulator for BCL6. However, details about the interaction between MEF2B and BCL6 during expression, as well as the relationship of MEF2B to the expression of other germinal center (GC) markers, have not yet been fully explained. Using germinal center B-cell-like diffuse large B-cell lymphoma (GC-DLBCL) and activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines, we analyzed the expression of MEF2B and its associations with BCL6, CD10, and ERK. Furthermore, small interfering RNA (siRNA) was used to study the possible effects of MEF2B knockdown on these proteins and cell growth. Analysis of the BCL6 transcriptional complex was performed using electrophoretic mobility shift assay. The correlation between MEF2B expression and the genetic type of DLBCL was assessed using immunohistochemistry on 111 patient samples, and via in silico analysis of publicly available microarray (Gene Expression Omnibus (GEO)) datasets. Our results indicate that the expression of MEF2B protein is important for the growth of GC-DLBCL cells, as evidenced by MEF2B knockdown inhibition of cell growth and the subsequent suppression of BCL6, CD10, and ERK phosphorylation. Analysis of BCL6 transcription factors in nuclear extracts of MEF2-expressing DLBCL cells showed involvement of MEF2B with AP-2α and BCL6 proteins in the formation of the BCL6 gene transcriptional complex. Indeed, differential expression of MEF2B in the GC-DLBCL is statistically significant compared to the ABC-DLBCL in the GEO datasets, as well as in tissue microarray, as indicated via immunohistochemistry (Visco–Young algorithm). Our findings indicate that MEF2B is an essential component of the BCL6 gene transcriptional complex for the regulation of DLBCL growth via the promotion of BCL6 expression. Beyond its regulatory role in DLBCL growth, MEF2B expression correlated positively with BCL6 and CD10 expression, and was preferentially expressed in the GBC-DLBCL group.

Extracellular signal-regulated kinase-1 phosphorylates early growth response-1 at serine 26

Egr-1, an immediate-early gene product and master regulator was originally described as a phosphoprotein following its discovery in the 1980s. However specific residue(s) phosphorylated in Egr-1 remain elusive. Here we phosphorylated recombinant Egr-1 in vitro with ERK1 prior to mass spectrometry, which identified phosphorylation of Ser12 and Ser26 with the latter ∼12 times more abundant than Ser12. Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser26>Ala26 mutant Egr-1) revealed that Ser26 accounts for the majority of phosphorylation of Egr-1 by ERK1. N-FGSFPH(pS)PTMDNYC-C was used as an antigen to generate mouse monoclonal antibodies (pS26 MAb). pS26 MAb recognised ERK1-phosphorylated Egr-1 but not Egr-1 bearing a point mutation at Ser26. pS26 MAb recognised inducible ∼75 kDa and 100 kDa species in nuclear extracts of cells exposed to FGF-2. Peptide blocking revealed both inducible species were phosphosite-specific. Immunoprecipitation of nuclear extracts of cells exposed to FGF-2 with pS26 MAb followed by SDS-PAGE and mass spectrometry identified Egr-1 sequences corresponding to the ∼75 kDa species but not ∼100 kDa species. This study identifies a specific amino acid phosphorylated in endogenous Egr-1.

In Situ Oxidative Stress and Atrial Cell Deaths in Patients with Valve Disease.

Left ventricular hypertrophy and myocardial remodeling occur with aortic valve disease and may lead to heart failure. Although increased oxidative stress and inflammatory factors have been implicated in heart failure, their role in the progression of valve disease remains unclear. We investigated the role of oxidative stress and inflammatory factors in valve disease whether this relates to cell death. Blood samples were taken from 24 patients with valve disease before surgery and the results were compared with those from blood samples from 30 control healthy subjects. Myocardial biopsies from patients with valve disease were also collected before cannulation of the right atrial appendage. NF-κB activities in atrial and mononuclear cells nuclear extracts were determined by electrophoretic mobility shift assay. Nuclear factor kappaB activities were significantly greater in mononuclear cells from AVD patients compared with healthy controls and the antigens were detectable in atrial tissues valve disease patients. Plasma C-reactive protein, B-natriuretic peptides, plasma tumor necrosis factor alpha and soluble tumor necrosis factor receptor 1 and 3-nitrotyrosine levels were significantly higher in valve disease patients. Inducible nitric oxide and 3-nitrotyrosine antigens and cells expressing CD45 antigens were detected within atrial tissues obtained from valve disease patients suggesting oxidative stress originated from in situ leukocytes. The findings suggest that oxidative stress originating from in situ leukocytes within the atrial myocardium may be the potential trigger for excessive transcriptional activities and apoptotic cell death within the atrial myocardium of valve disease patients. This represents a potential therapeutic target.

Peptidylarginine deiminase 4 overexpression resensitizes MCF-7/ADR breast cancer cells to adriamycin via GSK3β/p53 activation.

BackgroundAdriamycin (ADR) is widely used in the clinical chemotherapy against breast cancer. But its efficacy is strongly limited due to the acquisition of multidrug resistance (MDR). Therefore, acquisition of the resistance to ADR is still a major cause of chemotherapy failure in breast cancer patients. Peptidylarginine deiminase IV (PAD4) is reported to target non-histone proteins for citrullination, regulate their substrate activities, and thereby play critical roles in maintaining cell phenotype in breast cancer cells. However, whether PAD4 is involved in the development of MDR in breast cancer is poorly understood.Materials and methodsWe examined the expression of PAD family members, including PAD4 in ADR-resistant MCF-7 cells compared with the parental control cells by real-time PCR and Western blotting analyses. Rescue of PAD4 expression in MCF-7/ADR cells was performed to assess whether PAD4 could restore the sensitivity of MCF-7/ADR cells to ADR treatment with cell counting kit-8, flow cytometry, TUNEL, nuclear and cytoplasmic extract preparations, and immunofluorescence staining analyses.ResultsBoth PAD2 and PAD4 were significantly decreased in ADR-resistant cells. However, only PAD4 overexpression can increase the sensitivity of MCF-7/ADR cells to ADR treatment and decrease MDR1 gene expression. Overexpression of PAD4 in MCF-7/ADR cells inhibited cell proliferation by inducing cell apoptosis. Under ADR treatment, overexpression of PAD4 promoted nuclear accumulation of glycogen synthase kinase-3β and p53, which further activated proapoptotic gene expression and downregulated MDR1 expression. Moreover, PAD4 activity was required for activating proapoptotic gene transcripts.ConclusionWe demonstrate the previously unappreciated role of PAD4 in reversing ADR resistance in MCF-7/ADR cells and help establish PAD4 as a candidate biomarker of prognosis and chemotherapy target for MDR in breast cancers.

TNFα Induces Multidrug Resistance-Associated Protein 4 Expression through p38-E2F1-Nrf2 Signaling in Obstructive Cholestasis.

PurposeTo explore the molecular mechanism of the upregulation of multidrug resistance-associated protein 4 (MRP4) in cholestasis.Materials and MethodsThe mRNA and protein levels of MRP4 in liver samples from cholestatic patients were determined by quantitative real-time PCR and Western blot. In human hepatoma HepG2 cells, electrophoretic mobility shift assay (EMSA) was used to determine the affinity of nuclear factor-E2-related factor (Nrf2) binding to MRP4 promoter. Dual-luciferase reporter assay was used to detect the binding of tumor necrosis factor α (TNFα) to the promotor of E2F1. The bile duct ligation mouse models were established using male C57BL/6 mice.ResultsThe mRNA and protein levels of MRP4 were significantly increased in cholestatic patients. TNFα treatment induced the expression of MRP4 and Nrf2 and enhanced cell nuclear extract binding activity to MRP4 promoter, as demonstrated by EMSA. Nrf2 knockdown reduced MRP4 mRNA levels in both HepG2 and Hep-3B cells. In addition, TNFα increased Rb phosphorylation and expression of MRP4 and Nrf2 and activated E2F1 and phosphorylated p38 in HepG2 and Hep-3B cells. These effects were markedly inhibited by pretreatment with E2F1 siRNA. Dual-luciferase reporter assay validated that TNFα induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects.ConclusionOur findings indicated that TNFα induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.