Nuclear Protein Fractions Research Articles

Estradiol-mediated improvements in adipose tissue insulin sensitivity are related to the balance of adipose tissue estrogen receptor α and β in postmenopausal women.

We recently demonstrated that short-term estradiol (E2) treatment improved insulin-mediated suppression of lipolysis in postmenopausal women, but to a greater extent in those who were late compared to early postmenopausal. In this follow-up study we tested whether subcutaneous adipose tissue (SAT) expression of estrogen receptors (ER) α and β differs between early and late postmenopausal women. We further tested whether the balance of ERα to ERβ in SAT determined the effect of E2 on SAT insulin sensitivity. The present study included 35 women who were ≤6 years past menopause (EPM; n = 16) or ≥10 years past menopause (LPM; n = 19). Fasted SAT samples were taken following 1-week transdermal E2 treatment or placebo (PL) in a random cross-over design. Samples were analyzed for nuclear/cytosolic protein content and mRNA expression using Western blot and qPCR, respectively. While ESR1 increased slightly (~1.4-fold) following E2 treatment in both groups, ERα and ERβ protein expression did not differ between groups at baseline or in response to E2. However, the balance of ERα/ERβ protein in the SAT nuclear fraction increased 10% in EPM compared to a 25% decrease in LPM women (group x treatment interaction, p<0.05). A greater proportion of ERα/ERβ protein in the nuclear fraction of SAT at baseline (placebo day) was associated with greater reduction in SAT insulin resistance (i.e., better suppression of lipolysis, EC50) in response to E2 (r = -0.431, p<0.05). In conclusion, there do not appear to be differences in the proportion of adipose tissue ERα/ERβ protein in late, compared to early, postmenopausal women. However, the balance of ERα/ERβ may be important for E2-mediated improvement in adipose tissue insulin sensitivity.Trial Registration: Clinical Trials#: NCT01605071

Inhibition of radiation-induced lung inflammation and NF-kB activation by soy isoflavones.

Abstract Background Lung cancer patients treated with radiotherapy (RT) develop late respiratory distress as a result of inflammatory responses and lung fibrosis. We previously demonstrated that soy isoflavones (SIF) inhibited RT-induced inflammation, pneumonitis and fibrosis. NF-kB is a major transcription factor responsible for driving inflammatory responses. We have now investigated the effects of SIF on histopathology and status of NF-kB in lungs and BEAS-2B normal lung epithelial line treated with RT. Methods C57BL/6 and BALB/c mice were treated with 10Gy thoracic RT and oral SIF given daily for up to 18 weeks. Lung tissues were processed for histology and western blot protein analysis. In-vitro BEAS-2B cells were treated with SIF and 3Gy RT. NF-kB-p65 was assessed by western blot of cytoplasmic and nuclear protein fractions. Results H&amp;E and Masson’s trichrome staining for fibrosis showed SIF reduced RT-induced inflammation and collagen fiber accumulation in lung parenchyma. SIF inhibited the infiltration and activation of macrophages and neutrophils in irradiated lungs. Analysis of NF-kB-p65 subunit, a marker of NF-kB activation, showed that RT induced expression of p65 was reduced by SIF in the lungs at 1–12 weeks after RT. In-vitro studies showed that while RT caused an increase in nuclear p65 at 3 and 24 hours post-RT, treatment with SIF and RT showed reduced p65 translocation to the nucleus. Conclusions SIF treatment mitigated radiation-induced histopathological alterations and p65 activation in-vivo and in-vitro. These findings suggest a trend for SIF to regulated NF-kB activation and the inflammatory response after RT. Future studies will decipher the signaling pathways involved in SIF-mediated radioprotection.

Identification of β-catenin-interacting proteins in nuclear fractions of native rat collecting duct cells.

The gene encoding the aquaporin-2 water channel is regulated transcriptionally in response to vasopressin. In the renal collecting duct, vasopressin stimulates the nuclear translocation and phosphorylation (at Ser552) of β-catenin, a multifunctional protein that acts as a transcriptional coregulator in the nucleus. The purpose of this study was to identify β-catenin-interacting proteins that might be involved in transcriptional regulation in rat inner medullary collecting duct (IMCD) cells, using experimental and computational approaches. We used a standard chromatin immunoprecipitation procedure coupled to mass spectrometry (ChIP-MS) in a nuclear fraction isolated from rat IMCD suspensions. Over four biological replicates, we reproducibly identified 43 β-catenin-binding proteins, including several known β-catenin-binding partners as well as novel interacting proteins. Multiple proteins involved in transcriptional regulation were identified (Taf1, Jup, Tdrd3, Cdh1, Cenpj, and several histones). Many of the identified β-catenin-binding partners were found in prior studies to translocate to the nucleus in response to vasopressin. There was only one DNA-binding transcription factor (TF), specifically Taf1, part of the RNA-polymerase II preinitiation complex. To identify sequence-specific TFs that might interact with β-catenin, Bayes' theorem was used to integrate data from several information sources. The analysis identified several TFs with potential binding sites in the Aqp2 gene promoter that could interact with β-catenin in the regulation of Aqp2 gene transcription, specifically Jun, Junb, Jund, Atf1, Atf2, Mef2d, Usf1, Max, Pou2f1, and Rxra. The findings provide information necessary for modeling the transcriptional response to vasopressin.

Angiomotin to regulate prostate cancer cell proliferation by signaling through Hippo-YAP pathway.

e559 Background: Angiomotin (AMOT) is a family of proteins found to be a component of the apical junctional complex of vertebrate epithelial cells and is recently found to play important roles in neurofibromatosis type 2 (NF-2). Whether AMOT plays a role in prostate cancer (PCa) is unknown. Methods: Purified GST-AMOTp80 was used as immunogen for antibody generation. Real-time PCR, western blot and immunohistochemistry were used to identify the expression of AMOT. To study the function of AMOT, retroviral vector were constructed, also shRNA was used to knockdown AMOT in cells. Cell migration and invasion assays were performed by using transwell chambers. Nuclear and cytoplasmic protein fractions were prepared by using NE-PER reagents (Pierce). The SPSS 19.0 software was used for statistical analysis. Chi-square test and t test were used for the comparisons between groups. Results: AMOT is expressed as two isoforms, AMOTp80 and AMOTp130, which has a 409 aa N-terminal domain that is absent in AMOTp80. Both AMOTp80 and AMOTp130 are expressed in LNCaP and C4-2B4, but at a low to undetectable level in PC3 cells. Further study showed that AMOTp130 and AMOTp80 have distinct functions in PCa cells. We found that AMOTp80 functioned as a tumor promoter by enhancing PCa cell proliferation while AMOTp130 did not. Mechanistic studies showed that AMOTp80 signaled through the Hippo pathway by promoting the nuclear translocation of YAP, resulting in an increased expression of YAP target protein BMP4. Moreover, inhibition of BMP receptor activity by LDN-193189 abrogates AMOTp80-mediated cell proliferation. Conclusions: Together, this study reveals a novel mechanism whereby the AMOTp80-Merlin-MST1-LATS-YAP-BMP4 pathway leads to AMOTp80-induced tumor cell proliferation.

Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry

Reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with mass spectrometry (MS) is typically employed for mapping modifications in proteins and peptides. Here we applied a low-flow capillary electrophoresis (CE) −electrospray ionization interface coupled to Orbitrap mass spectrometers to analyze challenging modifications such as asparagine deamidation, aspartate isomerization, arginine citrullination, and phosphopeptide isomers. We achieved excellent resolution of asparagine (Asn), aspartic acid (Asp) and isoaspartic acid (iso-Asp) containing peptides using a synthetic peptide mixture. The migration order in CE enabled a clear assignment of in vitro deamidation/isomerization sites in a protein standard mixture of intermediate complexity (48 proteins) as well as the determination of the in vivo deamidation rate of histone H1.0 directly in a crude nuclear protein fraction. Besides these well-known modifications citrullination, a post-translational modification which changes the positively charged guanidinium group of arginine to the uncharged ureido group of citrulline, was investigated. Applying CE-MS for fast and sensitive analyses of various post-translational modifications of intact and enzymatically digested histone H4, we were able to detect a variety of citrullinated proteoforms. MS/MS analysis with electron transfer dissociation (ETD) fragmentation identified the presence of deiminated Arg at position 3 and 17 of histone H4. Moreover, based on CE-MS, isobaric mono-phosphorylated peptides obtained in the course of a kinase activity study were separated and individual positional isomers quantified.

An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation.

Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis.

In Chronic Lymphocytic Leukemia ΔCT7544-7545 Mutant NOTCH1 Maintains Transcription Factor Activity with Longer Lasting Effects Due to Slower Degradation

The NOTCH1 gene locusencodes a cell surface receptor which is recurrently mutated in chronic lymphocytic leukemia (CLL), with a rising incidence in higher risk cases. Upon ligand-binding two cleavage events occur. As a consequence the receptor’s intracellular domain (NICD) is released and shifts to the nucleus, where it functions as a transcription factor. Since in CLL NOTCH1 mutations mainly affect the protein’s PEST-domain, which is involved in NICD degradation, they were assumed to be activating mutations. However, to date this has only been proven for mutations that lead to the loss of the complete PEST-domain or major parts of it, whereas the hotspot mutation in CLL (ΔCT7544-7545; E2515fs) leads to the loss of only 39 amino acids.To prove the activating nature of the CLL hotspot mutation, we developed a cell culture based model using lentiviral transduction of B-cell lymphoma cell lines. A full-length NOTCH1 construct was cloned into a pCCL transfer vector and the CLL hotspot mutation was introduced. The cell lines SU-DHL4, Raji and Daudi were transduced with wild-type NOTCH1 (NOTCH1WT), ΔCT-mutant NOTCH1 (NOTCH1ΔCT) and empty vector.For validation experiments, primary CLL samples were screened for PEST-domain NOTCH1 mutations by targeted next-generation sequencing of exon 34.Activation of NOTCH1 was induced via treatment of cells with the Ca2+ chelator EGTA (1.0 mM, 1 hour). Liberated NICD was semi-quantitatively assessed by Western Blot and image densitometry from whole cell lysates or nuclear protein fractions. In addition, the NICD was visualized by intracellular immunofluorescent staining along with nuclear staining for DAPI and histone H3K27me / histone H3K27ac. Expression of the NOTCH1 target gene HES1 was quantified using TaqMan® RT-PCR analysis.Treatment with EGTA robustly activated NOTCH1 signalling; with the highest levels of NICD seen in the nucleus 2 to 4 hours after activation.Immunofluorescent staining of the NICD in NOTCH1 transduced cells 3 hours after activation revealed a speckled pattern in the nucleus without differences between NICDWT and NICDΔCT. Speckles co-localized with areas of relaxed chromatin, with NICD staining coincident with histone H3K27ac staining and separate from histone H3K27me staining. Co-localization of NICD speckles with areas of relaxed chromatin was confirmed in primary CLL samples where it was again observed independent of NOTCH1 mutation status.In Western Blot analyses, NICDWT and NICDΔCT were distinguishable due to their different molecular weight. Without prior activation, NOTCH1WT transduced cell lines displayed low levels of NICD. In contrast, NOTCH1ΔCT transduced cell lines presented with considerably higher levels of NICDΔCT despite comparable cell surface levels of transduced NOTCH1. After activation of NOTCH1WT and NOTCH1ΔCT with EGTA, the levels of NICDWT and NICDΔCT rose to a similar maximum, but a subsequent time-course showed a slower degradation of the NICDΔCT compared to the NICDWT. The slower degradation of mutant NICD was confirmed in primary CLL samples including samples with truncating mutations of the PEST-domain other than the hotspot mutation E2515fs (Q2440*, S2486*, Q2307*).HES1 is an established NOTCH1 target gene activated by nuclear NICD. Baseline expression levels of HES1 did not differ between NOTCH1WT and NOTCH1ΔCT transduced SU-DHL4 cells. After NOTCH1 activation, HES1 expression was up-regulated ~2.9 fold compared to baseline. Peak expression levels were seen around 3 hours after activation. HES1 expression levels fell more slowly in NOTCH1ΔCT transduced SU-DHL4 cells suggesting longer lasting effects on target genes by NICDΔCT than by NICDWT.In summary, our data demonstrates for the first time that the ΔCT7544-7545 mutant of NOTCH1 maintains transcriptional activity for longer due to slower degradation of its NICD. Our understanding about NICD target genes remains incomplete, but their identification will be crucial to understand the role of NOTCH1 mutations in CLL pathogenesis. DisclosuresTausch:Gilead: Other: Travel support, Speakers Bureau; Celgene: Other: Travel support; Amgen: Other: Travel support. Stilgenbauer:Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding. Cragg:Roche: Consultancy, Research Funding; GSK: Research Funding; Bioinvent International: Consultancy, Research Funding; Baxalta: Consultancy; Gilead Sciences: Research Funding.

Protein kinase A activation by retinoic acid in the nuclei of HL60 cells

BackgroundActivation of protein kinase A (PKA) occurs during retinoic acid (RA)-induced granulocytic differentiation of human promyelocytic leukemia HL60 cells. It is known that the RIIα regulatory subunit of PKA, is modified by RA (retinoylated) in the early stages of differentiation. We have investigated the effects of RA on PKA during cell differentiation in order to understand the potential significance of this process in the retinoylation of RIIα subunits. MethodsImmunoblotting, immunoprecipitation, confocal microscopy, PCR, and PKA activity assays were employed for characterizing the effects of RA on PKA. ResultsWe found that RA induces intracellular mobility of RIIα and the activation of PKA in HL60 cells. Increases in RIIα levels were observed in RA-treated HL60 cells. RA treatment altered intracellular localization of the PKA subunits, RIIα and Cα, and increased their protein levels in the nuclei as detected by both immunoblotting and immunostaining analyses. Coincident with the increase in nuclear Cα, RA-treated HL60 cells showed increases in both the protein phosphorylation activity of PKA and the levels of phosphorylated proteins in nuclear fractions as compared to control cells. In addition, RIIα protein was stabilized in RA-treated HL60 cells as compared to control cells. ConclusionsThese results suggest that RA stabilizes RIIα protein and activates PKA in the nucleus, with a resultant increase in the phosphorylation of nuclear proteins. General significanceOur evidence suggests that retinoylation of PKA might contribute to its stabilization and activation and that this could potentially participate in RA's ability to induce granulocytic differentiation of HL60 cells.

Filamin A is reduced and contributes to the CASR sensitivity in human parathyroid tumors.

Parathyroid tumors display reduced sensitivity to extracellular calcium ([Ca2+]o). [Ca2+]o activates calcium-sensing receptor (CASR), which interacts with the scaffold protein filamin A (FLNA). The study aimed to investigate: (1) the FLNA expression in human parathyroid tumors, (2) its effects on the CASR mRNA and protein expression, and (3) on ERK signaling activation, (4) the effect of the carboxy-terminal CASR variants and (5) of the treatment with the CASR agonist R568 on FLNA-mediated ERK phosphorylation in HEK293 cells. Full-length FLNA immunostaining was variably reduced in parathyroid tumors. Immunofluorescence showed that FLNA localized in membrane and cytoplasm and co-localized with CASR in parathyroid adenomas (PAds)-derived cells. Cleaved C-terminus FLNA fragment could also be detected in PAds nuclear protein fractions. In HEK293 cells transfected with 990R-CASR or 990G-CASR variants, silencing of endogenous FLNA reduced CASR mRNA levels and total and membrane-associated CASR proteins. In agreement, FLNA mRNA levels positively correlated with CASR expression in a series of 74 PAds; however, any significant correlation with primary hyperparathyroidism severity could be detected and FLNA transcript levels did not differ between PAds harboring 990R or 990G CASR variants. R568 treatment was efficient in restoring 990R-CASR and 990G-CASR sensitivity to [Ca2+]o in the absence of FLNA. In conclusion, FLNA is downregulated in parathyroid tumors and parallels the CASR expression levels. Loss of FLNA reduces CASR mRNA and protein expression levels and the CASR-induced ERK phosphorylation. FLNA is involved in receptor expression, membrane localization and ERK signaling activation of both 990R and 990G CASR variants.

Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling.

The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca2+ dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca2+ ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca2+ concentration. Neither increasing nor decreasing intracellular Ca2+ concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2−/− heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca2+. Thus, KChIP2 does not function as a conventional transcription factor in the heart.

Contribution of Human Fibroblasts and Endothelial Cells to the Hallmarks of Inflammation as Determined by Proteome Profiling

In order to systematically analyze proteins fulfilling effector functionalities during inflammation, here we present a comprehensive proteome study of inflammatory activated primary human endothelial cells and fibroblasts. Cells were stimulated with interleukin 1-β and fractionated in order to obtain secreted, cytoplasmic and nuclear protein fractions. Proteins were submitted to a data-dependent bottom up analytical platform using a QExactive orbitrap and the MaxQuant software for protein identification and label-free quantification. Results were further combined with similarly generated data previously obtained from the analysis of inflammatory activated peripheral blood mononuclear cells. Applying a false discovery rate of less than 0.01 at both, peptide and protein level, a total of 8370 protein groups assembled from 117,599 peptides was identified; mass spectrometry data have been made fully accessible via ProteomeXchange with identifier PXD003406 to PXD003417.Comparative proteome analysis allowed us to determine common and cell type-specific inflammation signatures comprising novel candidate marker molecules and related expression patterns of transcription factors. Cardinal features of inflammation such as interleukin 1-β processing and the interferon response differed substantially between the investigated cells. Furthermore, cells also exerted similar inflammation-related tasks; however, by making use of different sets of proteins. Hallmarks of inflammation thus emerged, including angiogenesis, extracellular matrix reorganization, adaptive and innate immune responses, oxidative stress response, cell proliferation and differentiation, cell adhesion and migration in addition to monosaccharide metabolic processes, representing both, common and cell type-specific responsibilities of cells during inflammation.

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone displays suppression of inflammatory responses via inhibition of Src, Syk, and NF-κB.

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone (MPP) is an aldol condensation product resulting from pyrrole-2-carbaldehyde and m- and p- substituted acetophenones. However, its biological activity has not yet been evaluated. Since it has been reported that some propenone-type compounds display anti-inflammatory activity, we investigated whether MPP could negatively modulate inflammatory responses. To do this, we employed lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and examined the inhibitory levels of nitric oxide (NO) production and transcriptional activation, as well as the target proteins involved in the inflammatory signaling cascade. Interestingly, MPP was found to reduce the production of NO in LPS-treated RAW264.7 cells, without causing cytotoxicity. Moreover, this compound suppressed the mRNA levels of inflammatory genes, such as inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-α. Using luciferase reporter gene assays performed in HEK293 cells and immunoblotting analysis with nuclear protein fractions, we determined that MPP reduced the transcriptional activation of nuclear factor (NF)-κB. Furthermore, the activation of a series of upstream signals for NF-κB activation, composed of Src, Syk, Akt, and IκBα, were also blocked by this compound. It was confirmed that MPP was able to suppress autophosphorylation of overexpressed Src and Syk in HEK293 cells. Therefore, these results suggest that MPP can function as an anti-inflammatory drug with NF-κB inhibitory properties via the suppression of Src and Syk.

The kin17 Protein in Murine Melanoma Cells.

kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma.

Abstract 5347: SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells

Abstract Although differentiation-inducing agents have significantly improved the management of acute promyelocytic leukemia, no significant progress has been made in the treatment of other acute myeloid leukemias (AML). Numerous proteins involved in tumor development have so-called allosteric disulfide bonds amenable to modifications affecting protein structure and function. We have developed SK053, a small molecule and mechanism-selective inhibitor of enzymes involved in allosteric disulfide bonds formation such as thioredoxin, thioredoxin reductase and protein disulfide isomerase (PDI). The aim of our studies was to evaluate anti-leukemic activity of SK053 in human AML cells. To validate if SK053 targets PDI, a binding assay and an insulin turbidimetric activity assay were used. Cytostatic/cytotoxic effects in HL60, NB4, KG-1 and MOLM14 cells as well as in primary AML cells were assessed with trypan blue exclusion. Differentiation of AML cells was studied with May-Grünwald-Giemsa staining, nitro blue tetrazolium reduction assay and flow cytometry analysis of CD11b, CD14 and CD15 levels and by RNA sequencing, qRT-PCR and western blotting (WB). We observed covalent binding of SK053 to PDI and inhibition of its enzymatic activity with IC50 of 10 μM. Since PDI blocks translation of CCAAT enhancer binding protein alpha (CEBPA), a transcription factor crucial for neutrophils maturation, we evaluated the potential of SK053 to induce differentiation and cytostatic/cytotoxic effects in human AML cells. SK053 exerts significant cytostatic/cytotoxic activity in human AML cells (HL60, NB4, KG-1 and MOLM14), and induces differentiation of AML blasts into more mature myeloid cells. Incubation of AML cells with SK053 induced expression of CEBPA and hexokinase 3 mRNA in quantitative RT-PCR and increased amount of CEBPA protein in nuclear fraction measured in WB. Finally, SK053 induces differentiation of primary leukemic cells freshly isolated from AML patients. RNA-seq analysis revealed that incubation of HL60 cells with SK053 down-regulates mRNA for MYC and ID1 oncogenes as well as for histone proteins. Expression of other genes of mature myeloid lineage such as adhesion molecules (collagen type XV, fibronectin I, MAC-1), hydrolytic enzymes (carboxypeptidase, proteinase 3, CA12 anhydrase, ADAM19 metalloprotease), proteoglycan 2 (core of eosinophilic granules) and PGLYRP3 (peptidoglycan recognition protein 3) was significantly up-regulated. The GeneOntology analysis done with the RNAseq results revealed enrichment of gene transcripts regulating myeloid cells differentiation. SK053 exerts potent anti-leukemic activity and induces differentiation of numerous types of human AML cells. Targeting allosteric disulfide bonds with small molecule inhibitors presents a promising therapeutic strategy in AML. Citation Format: Dominika Nowis, Justyna Chlebowska, Pawel Gaj, Michal Lazniewski, Malgorzata Firczuk, Karolina Furs, Radoslaw Sadowski, Pawel Leszczynski, Piotr Stawinski, Szymon Klossowski, Ryszard Ostaszewski, Krzysztof Giannopoulos, Rafal Ploski, Dariusz Plewczynski, Jakub Golab. SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2015-5347

HER4 tumor expression in breast cancer patients randomized to treatment with or without tamoxifen.

The human epidermal growth factor receptor (HER) 4 is a relative of HER2 and has been associated to endocrine breast cancer and prediction of tamoxifen response. In addition to PI3K/Akt and MAPK pathway activation, ligand binding to HER4 triggers proteolytic cleavage and release of an intracellular receptor domain (4ICD) with signaling properties. The aim of the present study was to analyze HER4 protein expression and intracellular localization in breast cancer tissue from patients randomized to treatment with or without adjuvant tamoxifen. To investigate HER4 expression and localization in response to estradiol (E2) and 4-hydroxytamoxifen (4-OHT) exposure, we also performed in vitro studies. Cytoplasmic, nuclear and membrane expression of HER4 protein was evaluated by immunohistochemical staining in tumor tissue from 912 breast cancer patients. Three different breast epithelia cancer cell lines were exposed to E2 and 4-OHT and mRNA expression was analyzed using qPCR. Further, nuclear and cytoplasmic proteins were separated and analyzed with western blotting. We found an association between nuclear HER4 protein expression and ER-positivity (P=0.004). Furthermore, significant association was found between cytoplasmic HER4 and ER-negativity (P<0.0005), PgR-negativity (P<0.0005), tumor size >20 mm (P=0.001) and HER2-negativity (P=0.008). However, no overall significance of HER4 on recurrence-free survival was found. After E2 exposure, HER4 mRNA and protein expression had decreased in two cell lines in vitro yet no changes in nuclear or cytoplasmic protein fractions were seen. In conclusion, nuclear HER4 seem to be co-located with ER, however, we did not find support for overall HER4 expression in independently predicting response of tamoxifen treatment. The possible influence of separate isoforms was not tested and future studies may further evaluate HER4 significance.

Abstract A04: Changes in matrix metalloproteinase expression in SN-38 resistant colorectal cancer cells

Abstract The matrix metalloproteinases (MMPs) are a complex family of zinc-dependent proteolytic enzymes, which collectively are capable of degrading all components of the extracellular matrix (ECM). The ECM provides structure and support for normal tissues, and acts both as a barrier to and support for cancer cell invasion and metastasis. MMPs are therefore believed to play an important role in regulating the rate of cancer progression. However, the repeated failure of MMP inhibitors in clinical trials, as well as recent discoveries of novel MMP activities and localizations, has led to a re-evaluation of the roles of MMPs to the cancer process. In colorectal cancer (CRC) the outlook following disease relapse after surgery and initial chemotherapy is poor, due both to drug resistance and further dissemination of disease. We investigated the involvement of MMPs and related molecules in this context. We have generated a series of derivatives of the human CRC cell line HT29 that are resistant to SN-38, the active metabolite of the chemotherapeutic agent irinotecan. The principal representative cell line, HT29-S, has a slower proliferation rate than parental HT29 cells, and yet forms denser outgrowths in monolayer culture. The abundance and localization of several MMPs and tissue inhibitors of metalloproteinases (TIMPs) have been assessed in both HT29 and HT29-S cells by western blotting of cytosolic and nuclear protein fractions,and by immunofluorescence. HT29-S cells showed a lower expression of MMP1, MMP7, and MMP9, but also TIMP1 and TIMP2, compared to their parental counterparts, with some differences in cellular distribution. In particular, MMP1 gave a strong nuclear signal by both methods in HT29 cells, which was strongly reduced in HT29-S. A reduction in MMP expression will allow for an increased accumulation of ECM components. This may act through integrin receptors to activate survival pathways and contribute to the chemoresistance observed in HT29-S cells, and may also facilitate the spread of cancer cells by refining the ECM framework in advanced CRC. Citation Format: Spencer I T Berg, Murray J. Cutler, Jr., Jonathan Blay. Changes in matrix metalloproteinase expression in SN-38 resistant colorectal cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A04.

Arabidopsis NAP-related proteins (NRPs) are soluble nuclear proteins immobilized by heat

Nucleosome assembly protein-related proteins (NRPs) are multifunctional proteins having histone chaperone and phosphatase inhibitor properties. Although it is believed that these proteins are nuclear and bind the chromatin, they can be detected in the cytoplasmic but not in the nuclear protein fraction by immunoblotting analysis. It is shown here that under normal conditions, NRPs are nuclear but soluble and leak out of the nuclei during their purification. However, under elevated temperatures (above 42 °C), NRPs display significantly reduced mobility and are retained in the nuclei during purification probably due to binding other immobile macromolecules in the nucleus. Our observations highlight the necessity to use different techniques in parallel to unambiguously determine the intracellular localization of proteins. As heat adapted (38 °C, 2 h followed by 2 h recovery) and heat shocked (45 °C, 1 h), Arabidopsis seedlings were found to have phenotypes similar to those observed in the NRP loss-of-function mutants nrp1-1 nrp2-1 (short, branching roots, increased bleomycin sensitivity), it was also investigated whether the immobilization of NRPs by heat results in disturbed NRP functions. The results indicated, however, that heat affected the investigated traits independent on the presence of NRPs.

Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C

Biochemical fractionation of membrane receptors in the nucleus.

Fractionation of cytoplasmic and nuclear proteins is a well-recognized biochemical technique to detect the intracellular distribution and expression level of proteins of interest. In the last decade, accumulating evidence shows that various types of cell surface receptors, such as receptor tyrosine kinases (RTKs), peptide hormone receptors, and cytokine receptors, are detected in the nuclei. Therefore, subcellular fractionation, including nonnuclear/nuclear extraction and the subsequent subnuclear fractionation without detectable cross-contamination during the process, is critical for studying membrane receptors that transit from the cell surface to the nucleus. Here, we utilize the epidermal growth factor receptor (EGFR) tyrosine kinase as an example of a comprehensive biochemical protocol for isolating membrane receptors in the nuclei of cancer cells.

Human histone acetyltransferase 1 (Hat1) acetylates lysine 5 of histone H2A in vivo

The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74% decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50%, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions.