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1.
Poult Sci ; 100(1): 26-38, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33357689

ABSTRACT

Duck enteritis virus (DEV) multifunctional tegument protein UL13 is predicted to be a conserved herpesvirus protein kinase; however, little is known about its subcellular localization signal. In this study, through transfection of 2 predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein, 2 bipartite nuclear localization signals (NLS) were identified. We found that ivermectin blocked the NLS-mediated nuclear import of DEV UL13, showing that the nuclear localization signal of DEV UL13 is a classical importin α- and ß-dependent process. We constructed a DEV UL13 mutant strain in which the NLS of DEV UL13 was deleted to explore whether deletion of the NLS affects viral replication. Amino acids 4 to 7 and 90 to 96 were predicted to be NLSs, further proving that nuclear import occurs via a classical importin α- and ß-dependent process. We also found that the NLS of pUL13 had no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results provide significant information regarding the biological function of pUL13 during DEV infection.


Subject(s)
Enteritis , Mardivirus , Nuclear Localization Signals , Protein Kinases , Animals , Antiparasitic Agents/pharmacology , Cells, Cultured , Ducks , Enteritis/physiopathology , Enteritis/veterinary , Enteritis/virology , Intracellular Space/metabolism , Intracellular Space/virology , Ivermectin/pharmacology , Mardivirus/genetics , Mardivirus/metabolism , Mutation , Nuclear Localization Signals/drug effects , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Transport/drug effects , Protein Transport/genetics
2.
Phys Biol ; 16(3): 036003, 2019 03 22.
Article in English | MEDLINE | ID: mdl-30763921

ABSTRACT

Androgen receptor (AR) signaling drives prostate cancer (PC) progression and remains active upon transition to castration resistant prostate cancer (CRPC). Active AR signaling is achieved through the nuclear accumulation of AR following ligand binding and through expression of ligand-independent, constitutively active AR splice variants, such as AR-V7, which is the most commonly expressed variant in metastatic CRPC (mCRPC) patients. Most currently approved PC therapies aim to abrogate AR signaling and activity by inhibiting this ligand-mediated nuclear translocation. In a prospective multi-institutional clinical study, we recently showed that taxane based chemotherapy is also capable of impairing AR nuclear localization (ARNL) in circulating tumor cells (CTCs) from CRPC patients, whereas taxane induced decreases in ARNL were associated with response. Thus, quantitative assessment of ARNL in CTCs can be used to monitor therapeutic response in patients and help guide clinical decisions. Here, we describe the development and implementation of quantitative high throughput (QHT) image analysis algorithms to aid in CTC identification and quantitative assessment of percent ARNL (%ARNL). We applied this algorithm to fifteen CRPC patients at the start of taxane chemotherapy, quantified %ARNL in CTCs, and correlated with expression of AR-V7 mRNA (from CTCs enriched via negative, CD45+ depletion of peripheral blood) and with biochemical (prostate specific antigen; PSA) response to taxane chemotherapy. We found that CTCs from AR-V7 positive patients had higher baseline %ARNL compared to CTCs from AR-V7 negative patients, consistent with the constitutive nuclear localization of AR-V7. In addition, lower %ARNL in CTCs at baseline was associated with biochemical response to taxane chemotherapy. High inter- and intra-patient heterogeneity was also observed. As ARNL is required for active AR signaling, the QHT algorithms described herein can provide prognostic and/or predictive value in future clinical studies.


Subject(s)
Antineoplastic Agents/pharmacology , Bridged-Ring Compounds/pharmacology , Drug Resistance, Neoplasm/drug effects , Neoplastic Cells, Circulating/drug effects , Nuclear Localization Signals/analysis , Prostatic Neoplasms/drug therapy , Receptors, Androgen/metabolism , Taxoids/pharmacology , Algorithms , Cell Nucleus/drug effects , High-Throughput Nucleotide Sequencing , Humans , Male , Neoplastic Cells, Circulating/metabolism , Nuclear Localization Signals/drug effects , Prostatic Neoplasms/secondary , RNA, Messenger/genetics , Tumor Cells, Cultured
3.
Exp Cell Res ; 370(2): 454-460, 2018 09 15.
Article in English | MEDLINE | ID: mdl-29981748

ABSTRACT

YB-1 nuclear translocation/accumulation caused by anticancer agents leads to malignant transformation. Nuclear import of YB-1 requires a nuclear localization signal (YB-NLS). Previously, we identified five nucleocytoplasmic-shuttling proteins as YB-NLS binding proteins, and showed that they co-accumulate in the nucleus with YB-1 in response to treatment with actinomycin D. In addition, another group reported that transportin-1 is the molecule responsible for YB-1 nuclear translocation, binding to a region (PY-NLS) consistent with the YB-NLS. Recently, we found that indirubin 3'-oxime inhibits the nuclear localization of YB-1 in HepG2 cells and increases their sensitivity to actinomycin D. Here, we found that YB-1 nuclear translocation is dependent on the cellular mRNA level and that indirubin 3'-oxime inhibits the interaction between YB-1 and transportin-1. Interestingly, in cells showing inhibition of actinomycin D-induced YB-1 nuclear translocation by the compound, the YB-NLS-binding proteins as well as transportin-1 and its cargos were imported to the nucleus. Furthermore, the compound inhibited nuclear localization of the GFP-conjugated full-length YB-1 but not that of GFP-conjugated YB-NLS. These results indicate that indirubin 3'-oxime is a specific inhibitor of anticancer agent-induced YB-1 nuclear translocation, interacting with YB-1 itself in a region other than the YB-NLS/PY-NLS. This compound would increase the efficacy of cancer therapy.


Subject(s)
Carcinoma, Hepatocellular/drug therapy , Cell Nucleus/drug effects , Indoles/pharmacology , Nuclear Localization Signals/drug effects , Oximes/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/metabolism , Cell Nucleus/metabolism , Hep G2 Cells/drug effects , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , beta Karyopherins/drug effects , beta Karyopherins/metabolism
4.
Nat Commun ; 9(1): 508, 2018 02 06.
Article in English | MEDLINE | ID: mdl-29410405

ABSTRACT

Enhanced glycolysis in cancer cells has been linked to cell protection from DNA damaging signals, although the mechanism is largely unknown. The 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) catalyzes the generation of fructose-2,6-bisphosphate, a potent allosteric stimulator of glycolysis. Intriguingly, among the four members of PFKFB family, PFKFB3 is uniquely localized in the nucleus, although the reason remains unclear. Here we show that chemotherapeutic agent cisplatin promotes glycolysis, which is suppressed by PFKFB3 deletion. Mechanistically, cisplatin induces PFKFB3 acetylation at lysine 472 (K472), which impairs activity of the nuclear localization signal (NLS) and accumulates PFKFB3 in the cytoplasm. Cytoplasmic accumulation of PFKFB3 facilitates its phosphorylation by AMPK, leading to PFKFB3 activation and enhanced glycolysis. Inhibition of PFKFB3 sensitizes tumor to cisplatin treatment in a xenograft model. Our findings reveal a mechanism for cells to stimulate glycolysis to protect from DNA damage and potentially suggest a therapeutic strategy to sensitize tumor cells to genotoxic agents by targeting PFKFB3.


Subject(s)
Acetylation/drug effects , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cisplatin/pharmacology , Glycolysis/drug effects , Phosphofructokinase-2/drug effects , A549 Cells , Adenylate Kinase/drug effects , Adenylate Kinase/metabolism , Cell Line, Tumor , Cytoplasm/drug effects , Cytoplasm/metabolism , HCT116 Cells , HeLa Cells , Humans , Nuclear Localization Signals/drug effects , Nuclear Localization Signals/metabolism , Phosphofructokinase-2/metabolism , Phosphorylation/drug effects
5.
Naunyn Schmiedebergs Arch Pharmacol ; 390(12): 1229-1238, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28875231

ABSTRACT

The development of hybrids from natural products is a promising strategy for drug discovery. In cancer therapy, there is a need to discover novel agents that can induce apoptosis in cancer cells. To contribute to this field of interest, we investigated the effect of a synthetic hybrid from cativic acid and caffeic acid (5) on viability, proliferation, and apoptosis in human neuroblastoma cells (IMR-32). Three hybrids were prepared via Mitsunobu esterification from 17-hydroxycativic acid (1) and natural phenols. Cell viability was analyzed by MTT assay. SYTOX green and LDH leakage were used to determine the cytotoxic effect. Caspase-3 activity, cell cycle phases, and proliferation were analyzed in order to characterize the biological effects of hybrid 5. The mitogen-activated protein kinase (MAPK) status was evaluated for elucidating the potential mechanisms involved in hybrid 5 effect. Hybrid 5 reduced the viability of IMR-32 cells in a time- and concentration-dependent manner (IC50 = 18.0 ± 1.3 µM) as a result of its antiproliferative effect through changes in the cell cycle distribution and induction of apoptosis associated with activation of caspase-3. Exposure to 5 triggered ERK1/2 activation and nuclear translocation. Hybrid 5 also promoted an increase in nuclear localization of the transcription factor c-Jun. Inhibition of ERK1/2 and JNK potentiated 5-induced inhibition of IMR-32 viability. Hybrid 5 displays cell growth inhibition by promoting cell cycle arrest and apoptosis, through ERK1/2 and JNK participation.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Caffeic Acids/pharmacology , Diterpenes/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Apoptosis/drug effects , Caffeic Acids/chemistry , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Diterpenes/chemistry , Grindelia/chemistry , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinases/metabolism , Neuroblastoma/drug therapy , Nuclear Localization Signals/drug effects
6.
Biochem Biophys Res Commun ; 477(4): 1065-1071, 2016 09 02.
Article in English | MEDLINE | ID: mdl-27402273

ABSTRACT

Lung cancer is the most frequently diagnosed malignancy that contributes to high proportion of deaths globally among patients who die due to cancer. Chemotherapy remains the common mode of treatment for lung cancer patients though with limited success. We assessed the biological effects and associated molecular changes of evodiamine, a plant alkaloid, on human lung cancer A549 and H1299 cells along with other epithelial cancer and normal lung SAEC cells. Our data showed that 20-40 µM evodiamine treatment for 24-48 h strongly (up to 73%, P < 0.001) reduced the growth and survival of these cancer cells. However, it also moderately inhibited growth and survival of SAEC cells. A strong inhibition (P < 0.001) was observed on clonogenicity of A549 cells. Further, evodiamine increased (4-fold) mitochondrial membrane depolarization with 6-fold increase in apoptosis and a slight increase in Bax/Bcl-2 ratio. It increased the cytochrome-c release from mitochondria into the cytosol as well as nucleus. Cytosolic cytochrome-c activated cascade of caspase-9 and caspase-3 intrinsic pathway, however, DR5 and caspase-8 extrinsic pathway was also activated which could be due to nuclear cytochrome-c. Pan-caspase inhibitor (z-VAD.fmk) partially reversed evodiamine induced apoptosis. An increase in p53 as well as its serine 15 phosphorylation was also observed. Pifithrin-α, a p53 inhibitor, slightly inhibited growth of A549 cells and under p53 inhibitory condition evodiamine-induced apoptosis could not be reversed. Together these findings suggest that evodiamine is a strong inducer of apoptosis in lung epithelial cancer cells independent of their p53 status and that could involve both intrinsic as well as extrinsic pathway of apoptosis. Thus evodiamine could be a potential anticancer agent against lung cancer.


Subject(s)
Cell Nucleus/metabolism , Cytochromes c/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Quinazolines/administration & dosage , Tumor Suppressor Protein p53/metabolism , A549 Cells , Alkaloids/administration & dosage , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Lung Neoplasms/pathology , Nuclear Localization Signals/drug effects , Tissue Distribution , Treatment Outcome
7.
Mol Pharm ; 13(9): 3141-52, 2016 09 06.
Article in English | MEDLINE | ID: mdl-27458925

ABSTRACT

The major intracellular barriers associated with DNA delivery using nonviral vectors are inefficient endosomal/lysosomal escape and poor nuclear uptake. LAH4-L1, a pH responsive cationic amphipathic peptide, is an efficient DNA delivery vector that promotes the release of nucleic acid into cytoplasm through endosomal escape. Here we further enhance the DNA transfection efficiency of LAH4-L1 by incorporating nuclear localizing signal (NLS) to promote nuclear importation. Four NLSs were investigated: Simian virus 40 (SV40) large T-antigen derived NLS, nucleoplasmin targeting signal, M9 sequence, and the reverse SV40 derived NLS. All peptides tested were able to form positively charged nanosized complexes with DNA. Significant improvement in DNA transfection was observed in slow-dividing epithelial cancer cells (Calu-3), macrophages (RAW264.7), dendritic cells (JAWSII), and thymidine-induced growth-arrested cells, but not in rapidly dividing cells (A549). Among the four NLS-modified peptides, PK1 (modified with SV40 derived NLS) and PK2 (modified with reverse SV40 derived NLS) were the most consistent in improving DNA transfection; up to a 10-fold increase in gene expression was observed for PK1 and PK2 over the unmodified LAH4-L1. Additionally PK1 and PK2 were shown to enhance cellular uptake as well as nuclear entry of DNA. Overall, we show that the incorporation of SV40 derived NLS, in particular, to LAH4-L1 is a promising strategy to improve DNA delivery efficiency in slow-dividing cells and dendritic cells, with development potential for in vivo applications and as a DNA vaccine carrier.


Subject(s)
Active Transport, Cell Nucleus/physiology , Cell Nucleus/metabolism , Peptides/pharmacology , Plasmids/metabolism , A549 Cells , Active Transport, Cell Nucleus/genetics , Animals , Cell Line , Cell Line, Tumor , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Electrophoretic Mobility Shift Assay , Genetic Vectors , Humans , Hydrogen-Ion Concentration , Macrophages/drug effects , Macrophages/metabolism , Mice , Nuclear Localization Signals/drug effects , Peptides/metabolism , Plasmids/genetics , RAW 264.7 Cells , Simian virus 40/genetics , Transfection
8.
Lab Invest ; 95(10): 1157-73, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26192086

ABSTRACT

Epithelial-to-mesenchymal transition (EMT) and apoptosis of peritoneal mesothelial cells are known to be the earliest mechanisms of peritoneal fibrosis in peritoneal dialysis (PD). Endoplasmic reticulum (ER) stress with an unfolded protein response is regarded to have a role in the development of organ fibrosis. To investigate the potential role of ER stress as a target to prevent and/or delay the development of peritoneal fibrosis, we examined the effect of ER stress on EMT or apoptosis of human peritoneal mesothelial cells (HPMCs) and elucidated the mechanisms underlying the protective effect of ER stress preconditioning on TGF-ß1-induced EMT. ER stress inducers, tunicamycin (TM) and thapsigargin (TG), induced EMT with Smad2/3 phosphorylation, an increased nuclear translocation of ß-catenin and Snail expression. Low concentrations of TM and TG did not induce apoptosis within 48 h; however, high concentrations of TM- (>1 ng/ml) and TG- (>1 nM) induced apoptosis at 12 h with a persistent increase in C/EBP homologous protein. TGF-ß1 induced EMT and apoptosis in HPMCs, which was ameliorated by taurine-conjugated ursodeoxycholic acid, an ER stress blocker. Interestingly, pre-treatment with TM or TG for 4 h also protected the cells from TGF-ß1-induced EMT and apoptosis, demonstrating the role of ER stress as an adaptive response to protect HPMCs from EMT and apoptosis. Peritoneal mesothelial cells isolated from PD patients displayed an increase in GRP78/94, which was correlated with the degree of EMT. These findings suggest that the modulation of ER stress in HPMCs could serve as a novel approach to ameliorate peritoneal damage in PD patients.


Subject(s)
Apoptosis , Endoplasmic Reticulum Stress , Epithelial-Mesenchymal Transition , Models, Biological , Peritoneal Dialysis/adverse effects , Peritoneal Fibrosis/pathology , Unfolded Protein Response , Anti-Bacterial Agents/adverse effects , Ascitic Fluid/metabolism , Ascitic Fluid/pathology , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Gene Expression Regulation/drug effects , Humans , Membrane Transport Modulators/adverse effects , Nuclear Localization Signals/drug effects , Nuclear Localization Signals/metabolism , Peritoneal Fibrosis/chemically induced , Peritoneal Fibrosis/etiology , Peritoneal Fibrosis/metabolism , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Snail Family Transcription Factors , Thapsigargin/adverse effects , Transcription Factors/genetics , Transcription Factors/metabolism , Transforming Growth Factor beta1/metabolism , Tunicamycin/adverse effects , beta Catenin/metabolism
9.
Chem Biol ; 22(7): 862-75, 2015 Jul 23.
Article in English | MEDLINE | ID: mdl-26119998

ABSTRACT

Synthetic pyrrole (P)-imidazole (I) containing polyamides can target predetermined DNA sequences and modulate gene expression by interfering with transcription factor binding. We have previously shown that rationally designed polyamides targeting the inverted CCAAT box 2 (ICB2) of the topoisomerase IIα (topo IIα) promoter can inhibit binding of transcription factor NF-Y, re-inducing expression of the enzyme in confluent cells. Here, the A/T recognizing fluorophore, p-anisylbenzimidazolecarboxamido (Hx) was incorporated into the hybrid polyamide HxIP, which fluoresces upon binding to DNA, providing an intrinsic probe to monitor cellular uptake. HxIP targets the 5'-TACGAT-3' sequence of the 5' flank of ICB2 with high affinity and sequence specificity, eliciting an ICB2-selective inhibition/displacement of NF-Y. HxIP is readily taken up by NIH3T3 and A549 cells, and detected in the nucleus within minutes. Exposure to the polyamide at confluence resulted in a dose-dependent upregulation of topo IIα expression and enhanced formation of etoposide-induced DNA strand breaks.


Subject(s)
DNA Probes/pharmacology , Fluorescent Dyes/pharmacology , Nylons/pharmacology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Benzimidazoles/pharmacology , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cells, Cultured , DNA Probes/genetics , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Gene Expression/drug effects , Humans , Imidazoles/pharmacology , Mice , NIH 3T3 Cells , Nuclear Localization Signals/drug effects , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Promoter Regions, Genetic , Protein Binding , Pyrroles/pharmacology
10.
Am J Physiol Renal Physiol ; 305(3): F362-9, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23720348

ABSTRACT

Activation of the transcription factor NFAT5 by high NaCl involves changes in phosphorylation. By siRNA screening, we previously found that protein targeting to glycogen (PTG), a regulatory subunit of protein phosphatase1 (PP1), contributes to regulation of high NaCl-induced NFAT5 transcriptional activity. The present study addresses the mechanism involved. We find that high NaCl-induced inhibition of PTG elevates NFAT5 activity by increasing NFAT5 transactivating activity, protein abundance, and nuclear localization. PTG acts via a catalytic subunit PP1γ. PTG associates physically with PP1γ, and NaCl reduces both this association and remaining PTG-associated PP1γ activity. High NaCl-induced phosphorylation of p38, ERK, and SHP-1 contributes to activation of NFAT5. Knockdown of PTG does not affect phosphorylation of p38 or ERK. However, PTG and PP1γ bind to SHP-1, and knockdown of either PTG or PP1γ increases high NaCl-induced phosphorylation of SHP-1-S591, which inhibits SHP-1. Mutation of SHP-1-S591 to alanine, which cannot be phosphorylated, increases inhibition of NFAT5 by SHP-1. Thus high NaCl reduces the stimulatory effect of PTG and PP1γ on SHP-1, which in turn reduces the inhibitory effect of SHP-1 on NFAT5. Our findings add to the known functions of PTG, which was previously recognized only for its glycogenic activity.


Subject(s)
Protein Phosphatase 1/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/antagonists & inhibitors , Sodium Chloride/pharmacology , Transcription Factors/metabolism , Blotting, Western , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , MAP Kinase Signaling System/physiology , Nuclear Localization Signals/drug effects , Plasmids , Polymerase Chain Reaction , Protein Phosphatase 1/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 6/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering , Transfection , p38 Mitogen-Activated Protein Kinases/metabolism
11.
Biochim Biophys Acta ; 1833(3): 583-92, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23123190

ABSTRACT

The apoptosis signal-regulating kinase 1 (ASK1) is activated in response to a wide variety of extracellular stressors. Consequently, dysregulation of ASK1 is associated with multiple pathologies. Here, we show that ASK1 translocates from the cytoplasm to the nucleus in HEK293 cells and human cardiomyocytes in response to hydrogen peroxide (H(2)O(2)) or angiotensin respectively. Immunoprecipitation and mass spectrometry experiments reveal that ASK1 physically interacts with the karyopherin α2/ß1 heterodimer in response to stress and genetic knockdown experiments confirm that this association mediates H(2)O(2)-induced ASK1 nuclear translocation. In addition, we have identified a nuclear localization signal (NLS)-like motif within the primary amino acid sequence of ASK1 composed of two clusters of basic amino acids separated by an intervening 16 amino acid spacer, KR[ACANDLLVDEFLKVSS]KKKK. Mutation of the downstream lysine cluster markedly reduces the H(2)O(2)-induced ASK1-karyopherin α2/ß1 interaction and inhibits ASK1 nuclear translocation. Furthermore, we demonstrate that nuclear ASK1 is active and participates in H(2)O(2)-induced ASK1-mediated cell death. Collectively, our findings have identified a functional interaction between ASK1 and the karyopherin α2/ß1 heterodimer and have also revealed a novel mechanism by which nuclear trafficking regulates the apoptotic function of ASK1 in response to stress.


Subject(s)
Apoptosis/drug effects , Hydrogen Peroxide/pharmacology , MAP Kinase Kinase Kinase 5/metabolism , Myocytes, Cardiac/drug effects , Oxidative Stress/drug effects , alpha Karyopherins/metabolism , beta Karyopherins/metabolism , Amino Acid Sequence , Angiotensins/pharmacology , Blotting, Western , Cell Nucleus/metabolism , Cell Proliferation , Cells, Cultured , Cytoplasm/metabolism , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , MAP Kinase Kinase Kinase 5/genetics , Molecular Sequence Data , Mutation/genetics , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Nuclear Localization Signals/drug effects , Oxidants/pharmacology , Protein Binding , Protein Multimerization , Sequence Homology, Amino Acid , Signal Transduction/drug effects , alpha Karyopherins/genetics , beta Karyopherins/genetics
12.
Toxicol Appl Pharmacol ; 248(2): 111-21, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20678512

ABSTRACT

Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.


Subject(s)
Breast/drug effects , Epigenesis, Genetic/drug effects , Epithelial Cells/drug effects , Estrogens, Non-Steroidal/toxicity , Phenols/toxicity , Adolescent , Adult , Benzhydryl Compounds , Breast/metabolism , Cell Line, Tumor , Epithelial Cells/metabolism , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Gene Expression/drug effects , Humans , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nuclear Localization Signals/drug effects , Young Adult
13.
Stem Cells ; 28(3): 390-8, 2010 Mar 31.
Article in English | MEDLINE | ID: mdl-19998373

ABSTRACT

There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self-renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin alpha V beta 3-dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of beta-catenin nuclear localization. Blocking integrin alpha V beta 3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Neoplasms/metabolism , Neoplastic Stem Cells/metabolism , Vitronectin/metabolism , Animals , Biomarkers/analysis , Biomarkers/metabolism , Blood Proteins/metabolism , Blood Proteins/pharmacology , Breast Neoplasms/chemically induced , Breast Neoplasms/metabolism , Breast Neoplasms/physiopathology , Carcinoma/chemically induced , Carcinoma/metabolism , Carcinoma/physiopathology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic/drug effects , Chromatography, Liquid , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , Integrin alphaVbeta3/drug effects , Integrin alphaVbeta3/metabolism , Male , Mass Spectrometry , Mice , Neoplasms/chemically induced , Neoplasms/physiopathology , Neoplastic Stem Cells/drug effects , Nuclear Localization Signals/drug effects , Nuclear Localization Signals/metabolism , Prostatic Neoplasms/chemically induced , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/physiopathology , Vitronectin/pharmacology , beta Catenin/drug effects , beta Catenin/metabolism
14.
J Virol ; 79(20): 13028-36, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16189005

ABSTRACT

Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.


Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , Oxadiazoles/pharmacology , Anti-HIV Agents/chemistry , Cells, Cultured , Computer-Aided Design , Dose-Response Relationship, Drug , Gene Products, gag/chemistry , HIV Antigens/chemistry , HIV-1/chemistry , Humans , Models, Molecular , Monocytes , Nuclear Localization Signals/drug effects , Oxadiazoles/chemistry , Viral Proteins/chemistry , Virus Replication/drug effects , gag Gene Products, Human Immunodeficiency Virus
15.
J Cell Biochem ; 94(5): 944-53, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15578567

ABSTRACT

Protein tyrosine phosphatase SHP-1 plays a critical role in the regulation of a variety of intracellular signaling pathways. SHP-1 is predominantly expressed in the cells of hematopoietic origin, and is recognized as a negative regulator of lymphocyte development and activation. SHP-1 consists of two Src homology 2 (SH2) domains and one protein tyrosine phosphatase (PTP) domain followed by a highly basic C-terminal tail containing tyrosyl phosphorylation sites. It is unclear how the C-terminal tail regulates SHP-1 function. We report the examination of the subcellular localization of a variety of truncated or mutated SHP-1 proteins fused with enhanced green fluorescent protein (EGFP) protein at either the N-terminal or the C-terminal end in different cell lines. Our data demonstrate that a nuclear localization signal (NLS) is located in the C-terminal tail of SHP-1 and the signal is primarily defined by three amino-acid residues (KRK) at the C-terminus. This signal is generally blocked in the native protein and can be exposed by fusing EGFP at the appropriate position or by domain truncation. We have also revealed that this NLS of SHP-1 is triggered by epidermal growth factor (EGF) stimulation and mediates translocation of SHP-1 from the cytosol to the nucleus in COS7 cell lines. These results not only demonstrate the importance of the C-terminal tail of SHP-1 in the regulation of nuclear localization, but also provide insights into its role in SHP-1-involved signal transduction pathways.


Subject(s)
Epidermal Growth Factor/pharmacology , Nuclear Localization Signals/drug effects , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Animals , Cell Line , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins , Microscopy, Electron , Molecular Sequence Data , Protein Transport , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/chemistry , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism
16.
J Control Release ; 98(3): 379-93, 2004 Aug 27.
Article in English | MEDLINE | ID: mdl-15312994

ABSTRACT

A regulatable fusion protein was constructed for controlling the localization of plasmid products. A ligand-inducible nuclear localization signal, nuclear export signal (NES) and a truncated form of the ligand binding domain of the progesterone receptor were attached to the desired protein. Enhanced green fluorescent protein (EGFP) was used as a model protein and its trafficking between the nucleus and cytoplasm was studied using fluorescence microscopy in response to the ligand, mifepristone. It was found that the protein trafficking into the nucleus was dose dependent with ligand concentration. Increasing the ligand dose from 1 to 100 nM enhanced import and reduced the rate of export of the fusion protein from the nucleus to the cytoplasm. This study demonstrates the feasibility of using an export signal and a ligand-inducible nuclear import signal as a bi-directional on/off switch with potential use for controlled targeting of therapeutic proteins to subcellular compartments.


Subject(s)
Drug Delivery Systems/methods , Recombinant Proteins/metabolism , Subcellular Fractions/metabolism , Animals , Cell Line, Tumor , Cloning, Molecular , Cytoplasm/metabolism , Dose-Response Relationship, Drug , Green Fluorescent Proteins/metabolism , Hormone Antagonists/pharmacology , Ligands , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Mifepristone/pharmacology , Nuclear Localization Signals/drug effects , Plasmids/genetics , Protein Binding , Receptors, Progesterone/metabolism
17.
Bioconjug Chem ; 15(3): 482-90, 2004.
Article in English | MEDLINE | ID: mdl-15149175

ABSTRACT

Gold nanoparticles modified with nuclear localization peptides were synthesized and evaluated for their subcellular distribution in HeLa human cervical epithelium cells, 3T3/NIH murine fibroblastoma cells, and HepG2 human hepatocarcinoma cells. Video-enhanced color differential interference contrast microscopy and transmission electron microscopy indicated that transport of nanoparticles into the cytoplasm and nucleus depends on peptide sequence and cell line. Recently, the ability of certain peptides, called protein transduction domains (PTDs), to transclocate cell and nuclear membranes in a receptor- and temperature-independent manner has been questioned (see for example, Lundberg, M.; Wikstrom, S.; Johansson, M. (2003) Mol. Ther. 8, 143-150). We have evaluated the cellular trajectory of gold nanoparticles carrying the PTD from HIV Tat protein. Our observations were that (1) the conjugates did not enter the nucleus of 3T3/NIH or HepG2 cells, and (2) cellular uptake of Tat PTD peptide-gold nanoparticle conjugates was temperature dependent, suggesting an endosomal pathway of uptake. Gold nanoparticles modified with the adenovirus nuclear localization signal and the integrin binding domain also entered cells via an energy-dependent mechanism, but in contrast to the Tat PTD, these signals triggered nuclear uptake of nanoparticles in HeLa and HepG2 cell lines.


Subject(s)
Cell Nucleus/metabolism , Gold/pharmacokinetics , Nuclear Localization Signals/metabolism , Peptides/pharmacokinetics , 3T3 Cells , Animals , Biological Transport, Active/physiology , Cell Line, Tumor , Cell Nucleus/drug effects , Gene Products, tat/chemistry , Gold/chemistry , HeLa Cells , Humans , Macromolecular Substances , Mice , Nuclear Localization Signals/drug effects , Peptides/chemistry , Protein Structure, Tertiary/physiology , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/pharmacokinetics , Temperature , Transduction, Genetic
18.
Blood Cells Mol Dis ; 28(1): 63-74, 2002.
Article in English | MEDLINE | ID: mdl-11987243

ABSTRACT

SHP-1 protein tyrosine phosphatase is a critical regulator of signaling in hematopoietic cells as illustrated by the lethal hematopoietic disorders in SHP-1-deficient mice. We and others have shown in previous studies that SHP-1 regulates membrane receptor signaling: it binds via its N-terminal region SH2 domains to tyrosine phosphorylated membrane receptors to dephosphorylate key substrates in the receptor complexes. Here we demonstrate that the SHP-1 C-terminal region contains a bipartite NLS that mediates SHP-1 nuclear localization in response to cytokines. This NLS was located within amino acids 576-595 of the PTPase and, when fused by itself to EGFP, targeted the fluorescent protein into the nuclei of transiently transfected NIH3T3 fibroblasts and Bac1.2f5 macrophage cells. When positioned within SHP-1, the activity of the NSL was under tight regulation as indicated by the predominant cytoplasmic distribution of the EGFP/SHP-1 fusion protein in NIH3T3 transfectants and the exclusive cytoplasmic localization of the endogenous SHP-1 in hematopoietic cell line PBLC-1. Activation of the NLS in SHP-1 by IL-4 was demonstrated by increased nuclear localization of the EGFP/SHP-1 fusion protein in NIH3T3 transfectants and of the endogenous SHP-1 protein in PBCL-1 cells at 4, 6 and 8 h post-IL-4 stimulation. SHP-1 nuclear localization in PBCL-1 cells was also induced by IL-7 in a similar manner, suggesting it as a common event in cytokine signaling. In comparison to that of the wild-type phosphatase, an SHP-1 mutant lacking the NLS showed only approximately half of the activity in inhibiting proliferation of NIH3T3 transfectants. These results provide evidence of cytokine-regulated SHP-1 nuclear localization mediated by a bipartite NLS and suggest that SHP-1 regulates nuclear signaling in cell growth control.


Subject(s)
Active Transport, Cell Nucleus , Cytokines/pharmacology , Nuclear Localization Signals/physiology , Protein Tyrosine Phosphatases/metabolism , 3T3 Cells , Active Transport, Cell Nucleus/drug effects , Animals , Base Sequence , Cell Division/drug effects , Hematopoietic Stem Cells/metabolism , Interleukin-4/pharmacology , Interleukin-7/pharmacology , Intracellular Signaling Peptides and Proteins , Mice , Microscopy, Fluorescence , Nuclear Localization Signals/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection
19.
J Cell Sci ; 114(Pt 24): 4621-7, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11792826

ABSTRACT

Protein-NLS-coated gold particles up to approximately 250 A in diameter are transported through the nuclear pores in normal, proliferating BALB/c 3T3 cells. This size can increase or decrease, depending on cellular activity. It has been suggested that increases in functional pore size are related to a reduction in the amount of available p53. To further test this hypothesis, we investigated the effects of cycloheximide and pifithrin-alpha, which inhibits p53-dependent transcriptional activation, on nuclear transport. After 3 hours in cycloheximide, there was a significant increase in the size of the gold particles that entered the nucleoplasm. When the incubation period was extended to 6 hours or longer, transport capacity returned to the control level. By using proteasome inhibitors, it was shown that the cycloheximide-dependent increase in functional pore size was due to the inhibition of protein synthesis, consistent with the fact that p53 is a short-lived protein, and requires the activity of at least two different factors. Although cycloheximide increases the functional diameter of the channel available for signal-mediated transport by approximately 60 A, it had no significant effect on either the import rate of small NLS-containing substrates (FITC-BSA-NLS), or passive diffusion of fluorescent-labeled proteins across the envelope. This suggests that changes in transport capacity were not caused by an increase in overall pore diameter but instead are due to a transient increase in pore size that accompanies signal-mediated transport. Pifithrin-alpha also caused an increase in functional pore diameter without altering the import rate of FITC-BSA-NLS, providing further support for the view that p53 can initiate changes in nuclear transport capacity.


Subject(s)
Fibroblasts/physiology , Fibroblasts/ultrastructure , Nuclear Pore/physiology , 3T3 Cells , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Animals , Benzothiazoles , Cycloheximide/pharmacology , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Diffusion/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Gold Colloid/metabolism , Leupeptins/pharmacology , Mice , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Nuclear Localization Signals/drug effects , Nuclear Localization Signals/physiology , Nuclear Pore/drug effects , Nuclear Pore/metabolism , Proteasome Endopeptidase Complex , Thiazoles/pharmacology , Toluene/analogs & derivatives , Toluene/pharmacology , Tumor Suppressor Protein p53/physiology
20.
Mol Cell ; 6(3): 539-50, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11030334

ABSTRACT

NFAT transcription factors are highly phosphorylated proteins that are regulated by the calcium-dependent phosphatase calcineurin. We show by mass spectrometry that NFAT1 is phosphorylated on fourteen conserved phosphoserine residues in its regulatory domain, thirteen of which are dephosphorylated upon stimulation. Dephosphorylation of all thirteen residues is required to mask a nuclear export signal (NES), cause full exposure of a nuclear localization signal (NLS), and promote transcriptional activity. An inducible phosphorylation site in the transactivation domain contributes to transcriptional activity. Our data suggest that dephosphorylation promotes NFAT1 activation by increasing the probability of an active conformation, in a manner analogous to that by which depolarization increases the open probability of voltage-gated ion channels. This conformational switch paradigm may explain modification-induced functional changes in other heavily phosphorylated proteins.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Nuclear Proteins , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcriptional Activation/physiology , Animals , Carcinogens/pharmacology , Conserved Sequence , DNA-Binding Proteins/genetics , Humans , Ionomycin/pharmacology , Ionophores/pharmacology , Jurkat Cells , Kidney/cytology , Mice , Molecular Sequence Data , Mutagenesis/physiology , NFATC Transcription Factors , Nuclear Localization Signals/drug effects , Nuclear Localization Signals/physiology , Phosphorylation , Phosphoserine/metabolism , Protein Conformation , Sequence Homology, Amino Acid , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factors/genetics , Transcriptional Activation/drug effects
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