HeLa Cell Nuclei Research Articles

Importin β1 Protein-mediated Nuclear Localization of Death Receptor 5 (DR5) Limits DR5/Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Cell Death of Human Tumor Cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5)-mediated cell death plays an important role in the elimination of tumor cells and transformed cells. Recently, recombinant TRAIL and agonistic anti-DR5 monoclonal antibodies have been developed and applied to cancer therapy. However, depending on the type of cancer, the sensitivity to TRAIL has been reportedly different, and some tumor cells are resistant to TRAIL-mediated apoptosis. Using confocal microscopy, we found that large amounts of DR5 were localized in the nucleus in HeLa and HepG2 cells. Moreover, these tumor cells were resistant to TRAIL, whereas DU145 cells, which do not have nuclear DR5, were highly sensitive to TRAIL. By means of immunoprecipitation and Western blot analysis, we found that DR5 and importin β1 were physically associated, suggesting that the nuclear DR5 was transported through the nuclear import pathway mediated by importin β1. Two functional nuclear localization signals were identified in DR5, the mutation of which abrogated the nuclear localization of DR5 in HeLa cells. Moreover, the nuclear transport of DR5 was also prevented by the knockdown of importin β1 using siRNA, resulting in the up-regulation of DR5 expression on the cell surface and an increased sensitivity of HeLa and HepG2 cells to TRAIL. Taken together, our findings suggest that the importin β1-mediated nuclear localization of DR5 limits the DR5/TRAIL-induced cell death of human tumor cells and thus can be a novel target to improve cancer therapy with recombinant TRAIL and anti-DR5 antibodies.

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

Lipoic Acid Modified Low Molecular Weight Polyethylenimine Mediates Nontoxic and Highly Potent in Vitro Gene Transfection

The clinical success of gene therapy intimately relies on the development of safe and efficient gene carrier systems. We found here that 1.8 kDa polyethylenimine (PEI) following hydrophobic modification with lipoic acid (LA) mediated nontoxic and highly potent in vitro gene transfection in both HeLa and 293T cells. 1.8 kDa PEI-LA conjugates were prepared with controlled degree of substitution (DS) by coupling LA to PEI using carbodiimide chemistry. Gel electrophoresis measurements showed that the DNA binding ability of 1.8 kDa PEI was impaired by lipoylation, in which an N/P ratio of 2/1 and 4-6/1 was required for 1.8 kDa PEI and 1.8 kDa PEI-LA conjugates, respectively, to completely inhibit DNA migration. Interestingly, dynamic light scattering measurements (DLS) revealed that PEI-LA conjugates condensed DNA into much smaller sizes (183-84 nm) than unmodified 1.8 kDa PEI (444-139 nm) at N/P ratios ranging from 20/1 to 60/1. These polyplexes revealed similar surface charges of ca. +22 to +30 mV. 1.8 kDa PEI-LA(2) polyplexes formed at an N/P ratio of 10/1 were stable against exchange with 12-fold excess of negatively charged dextran sodium sulfate (DSS) relative to DNA phosphate groups while 1.8 kDa PEI controls dissociated at 6-fold excess of DSS, indicating that lipoylation of 1.8 kDa PEI resulted in stronger binding with DNA. Importantly, DNA was released from 1.8 kDa PEI-LA(2) polyplexes upon addition of 10 mM dithiothreitol (DTT). Reduction-triggered unpacking of 1.8 kDa PEI-LA(2) polyplexes was also confirmed by DLS. MTT assays demonstrated that all PEI-LA conjugates and polyplexes were essentially nontoxic to HeLa and 293T cells up to a tested concentration of 50 μg/mL and an N/P ratio of 80/1, respectively. The in vitro gene transfection studies in HeLa and 293T cells showed that lipoylation of 1.8 kDa PEI markedly boosted its transfection activity. For example, 1.8 kDa PEI-LA(2) polyplexes displayed 400-fold and 500-fold higher levels of gene expression than unmodified 1.8 kDa PEI controls, which were ca. 2-fold and 3-fold higher than 25 kDa PEI controls, in serum-free and 10% serum media, respectively. The transfection efficiency decreased with increasing DS, following an order of 1.8 kDa PEI-LA(2) > 1.8 kDa PEI-LA(4) > 1.8 kDa PEI-LA(6) ≫ 1.8 kDa PEI. Confocal laser scanning microscopy (CLSM) studies corroborated that 1.8 kDa PEI-LA(2) delivered and released DNA into the nuclei of HeLa cells more efficiently than 25 kDa PEI. These nontoxic 1.8 kDa PEI-LA conjugates form a superb basis for the development of targeting, biocompatible and highly efficient carriers of gene delivery.

Non-viral gene transfection in vitro using endosomal pH-sensitive reversibly hydrophobilized polyethylenimine

Reversibly hydrophobilized 10 kDa polyethylenimine (PEI) based on rapidly acid-degradable acetal-containing hydrophobe was designed for nontoxic and highly efficient non-viral gene transfer. Water soluble PEI derivatives with average 5, 9 and 14 units of pH-sensitive 2,4,6-trimethoxybenzylidene-tris(hydroxymethyl)ethane (TMB-THME) hydrophobe per molecule, denoted as PEI- g-(TMB-THME) n, were readily obtained by treating 10 kDa PEI with varying amounts of TMB-THME-nitrophenyl chloroformate. Gel retardation assays showed that all PEI- g-(TMB-THME) n derivatives could effectively condense DNA at an N/P ratio of 5/1. Notably, polyplexes of PEI- g-(TMB-THME) n derivatives had smaller sizes (about 100∼170 nm) and higher surface charges (+25 ∼ +43 mV) than the parent 10 kDa PEI at the same N/P ratios ranging from 10/1 to 40/1. MTT assays revealed that these PEI- g-(TMB-THME) n derivatives were practically non-toxic at polymer concentrations used in transfection experiments. The acetal degradation of PEI- g-(TMB-THME) 9 was shown to be highly pH dependent in which half lives of 1.3, 2.8 and 11 h were determined for pH 4.0, 5.0 and 6.0, respectively, while negligible hydrolysis (<12%) was observed after 24 h at pH 7.4. Gel electrophoresis, dynamic light scattering (DLS) and zeta potential analyses indicated that polyplexes formed at an N/P ratio of 10/1 were dissociated following 5 h incubation at pH 5.0, highlighting the importance of hydrophobic TMB-THME moieties in DNA condensation and supporting that acetal hydrolysis in endosomes would facilitate DNA release. Notably, in vitro transfection experiments performed at N/P ratios of 10/1 and 20/1 in HeLa, 293T, HepG2 and KB cells using plasmid pGL3 expressing luciferase as the reporter gene showed that reversibly hydrophobilized PEIs had superior transfection activity to 25 kDa PEI control. For example, polyplexes of PEI- g-(TMB-THME) 14 showed about 235-fold and 175-fold higher transfection efficiency as compared to 10 kDa PEI in HeLa cells in serum-free and 10% serum media, respectively, which were approximately 7-fold and 16-fold higher than 25 kDa PEI formulation at its optimal N/P ratio under otherwise the same conditions. Confocal laser scanning microscope (CLSM) studies confirmed that PEI- g-(TMB-THME) 14 efficiently delivered Cy5-labeled DNA to the nuclei of HeLa cells. These endosomal pH-sensitive reversibly hydrophobilized PEIs have great potentials for safe and efficient non-viral gene transfection.

Discrete foci containing RNAse A are found in nucleoli of HeLa cells after aging in culture

We have studied by means of ultrastructural immunocytochemistry the localization of RNase A in nuclei of HeLa cells in control conditions and following cell ageing in culture. We have found that roundish, electron dense foci, which contain a significant amount of RNase A, can be detected within nucleoli of aged cells. These bodies also contain RNA and lack ribosomal S3 proteins, and may represent either simple storage sites or areas where RNA degradation takes place.

A cyano analogue of boswellic acid induces crosstalk between p53/PUMA/Bax and telomerase that stages the human papillomavirus type 18 positive HeLa cells to apoptotic death

The p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV) in over 90% of cervical cancers. HPV E6 protein promotes the degradation of p53 thereby inhibiting its stabilization and activation. This study demonstrates that treatment with a novel cyano derivative of 11-keto-β-boswellic acid, i.e. butyl 2-cyano-3, 11-dioxours-1,12-dien-24-oate (BCDD) reduced the viral E6 mRNA expression and lead to the accumulation of transcriptionally active p53 in the nucleus of HPV18 HeLa cells following DNA damage. Western blot analysis showed that BCDD robustly up regulated time-dependent expression of p53/PUMA/p21 whereas it deprived cells essentially of p-AKT and NF-κB cell survival signalling cascade. BCDD appeared to gear up PUMA activation through p53 pathway and that both p53 and p21 translocated heavily into the nucleus. Simultaneously, it inhibited anti-apoptotic Bcl-2, augumented Drp-1 expression, disrupted mitochondrial functions causing the activation of proapoptotic proteins and caspases activation. Additionally, BCDD inhibited telomerase expression that's likely to result in a marked reduction of the tumorigenic potential of high-grade cervical cancers. Consequently BCDD caused apoptotic death in cervical cancer cells as evidenced by DNA fragmentation and PARP-cleavage. Further, BCDD did not affect the extrinsic signalling transduction pathway as depicted by its null effect on caspase-8. The in vivo anticancer activity of BCDD was investigated in Ehrlich Ascites carcinoma model where it exhibited tumor regression by 48% at 30mg/kg, i.p., in mice. These findings indicated that BCDD is a potential candidate that may be found useful in the management of cervical cancer.

A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody ( H3Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat 48–60 peptide (GRKKRRQRRRPPQ). H3Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.

Nutrient stress does not cause retrograde transport of cytoplasmic tRNA to the nucleus in evolutionarily diverse organisms

Intracellular trafficking of tRNA was long thought to be a one-way trip from the site of biogenesis in the nucleus to the translation machinery in the cytoplasm. This view has recently been challenged, however, by the discovery that tRNA can move retrograde from the cytoplasm back to the nucleus in Saccharomyces cerevisiae and rat hepatoma H4IIE cells during nutrient stress and in S. cerevisiae after intron-containing pre-tRNAs are spliced in the cytoplasm. Contrary to studies reported, we present data suggesting that nutrient stress does not cause retrograde transport of cytoplasmic tRNAs to the nucleus in rat hepatoma H4IIE cells, human HeLa and HEK293 cells, and the yeasts Kluyveromyces lactis and S. cerevisiae. However, the efficiency of nuclear re-export of retrograded spliced tRNA was severely affected in S. cerevisiae and two other Saccharomyces species deprived of nutrient. Collectively, the data suggest that nutrient stress does not cause nuclear import of cytoplasmic tRNA; instead, nutrient stress specifically regulates nuclear re-export of retrograded spliced tRNAs but not nuclear export of tRNAs made from intronless pre-tRNAs in Saccharomyces species. Furthermore, we provide evidence suggesting that Mtr10p and the Gsp1pGTP/Gsp1pGDP cycle are not involved in nuclear tRNA import in S. cerevisiae during nutrient stress.

Cytotoxic Effects of Native and Recombinant Frutalin, a Plant Galactose‐Binding Lectin, on HeLa Cervical Cancer Cells

Frutalin is the α-D-galactose-binding lectin isolated from breadfruit seeds. Frutalin was obtained from two different sources: native frutalin was purified from its natural origin, and recombinant frutalin was produced and purified from Pichia pastoris. This work aimed to study and compare the effect of native and recombinant frutalin on HeLa cervical cancer cells proliferation and apoptosis. Furthermore, the interaction between frutalin and the HeLa cells was investigated by confocal microscopy. Despite having different carbohydrate-binding affinities, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cells growth (IC50~100 μg/mL) and equally induced cell apoptosis. The interaction studies showed that both lectins were rapidly internalised and targeted to HeLa cell's nucleus. Altogether, these results indicate that frutalin action is not dependent on its sugar-binding properties. This study provides important information about the bioactivity of frutalin and contributes to the understanding of the plant lectins cytotoxic activity.

Fibrous-structured magnetic and mesoporous Fe3O4/silica microspheres: synthesis and intracellular doxorubicin delivery

A novel, fibrous-structured bifunctional (magnetic and mesoporous) Fe3O4/silica microsphere was successfully synthesized through a simple and economical self-assembled process in which hydrophobic 9 nm-Fe3O4nanocrystals were directly used without modifications. The obtained material is performed as a drug delivery carrier to investigate the in vitro and intracellular delivery properties of doxorubicin hydrochloride (DOX). X-Ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), N2adsorption/desorption, Fourier transform infrared spectroscopy (FT-IR), and superconducting quantum interference device (SQUID) were employed to characterize the composite. The results reveal that the novel composite exhibits typical mesoporous structure, narrow size distribution, good monodispersity, and superparamagnetic features. Notably, confocal laser scanning microscopy (CLSM) images indicate that the DOX-loaded sample could deliver DOX into the nuclei of HeLa cells to kill cells. Also, MTT assay confirms that the DOX-loaded sample leads to pronounced and efficient cytotoxic effects to L929 fibroblast cells, even similar to that of free DOX at high concentrations, whereas the pure material is non-toxic. Therefore, the novel material is expected to have potential application for targeted cancer therapy.

Activation of stress-regulated transcription factors by triethylene glycol dimethacrylate monomer

Triethylene glycol dimethacrylate (TEGDMA) is a resin monomer available for short exposure scenarios of oral tissues due to incomplete polymerization processes of dental composite materials. The generation of reactive oxygen species (ROS) in the presence of resin monomers is discussed as a common mechanism underlying cellular reactions as diverse as disturbed responses of the innate immune system, inhibition of dentin mineralization processes, genotoxicity and a delayed cell cycle. Yet, the signaling pathway through a network of proteins that finally initiates the execution of monomer-induced specific cell responses is unknown so far. The aim of the present study was to extend the knowledge of molecular mechanisms of monomer-induced cell death as a basis for reasonable therapy strategies. Thus, the monomer-induced expression and phosphorylation of stress-related transcription factors was analyzed in various cell lines. The time-related induction of apoptosis was investigated as well. The expression of p53 increased in HeLa cell cultures treated with camptothecin (positive control) for 24h, and the formation of p53Ser15 and p53Ser46 was detected in cell nuclei by Western blotting. TEGDMA (3 m m) appeared to stimulate p53 expression only slightly, but increased p21 expression was found in cell nuclei and cytoplasm. Both camptothecin and TEGDMA increased p53 expression to some extent in the nuclear fraction in human transformed pulp-derived cells (tHPC), and similar effects were detected in RAW264.7 macrophages. No clear induction of c-Jun and phospho-c-Jun by TEGDMA was detected in HeLa cell nuclei, and the expression of ATF-2 and phospho-ATF-2 was inhibited in the presence of the monomer. ATF-3 expression was found only in the nuclear fraction of camptothecin-treated HeLa cultures. TEGDMA seemed to inhibit the formation of phospho-c-Jun and phospho-ATF-2 in tHPC, and the monomer acted negatively on the expression of c-Jun, ATF-2 and ATF-3 in RAW264.7 macrophages. These changes in the expression and activation of stress-related transcription factors were time-related to the induction of apoptosis by TEGDMA in all cell lines. The present study provides experimental evidence that TEGDMA interferes with the regulation of cellular pathways through transcription factors activated as a consequence of DNA damage like p53 or initiated downstream of MAPK (mitogen-activated protein kinases) like c-Jun, ATF-2 and ATF-3. The direct causal correlation between DNA damage, activation or inhibition of MAPKs and transcription factors, and apoptosis is under current investigation. However, the induction of apoptosis in different cell lines in the presence of monomers like TEGDMA may be subject to a higher level of complexity than currently suggested by simple linear models.

Contactless determination of nuclear compressibility using 3D image‐ and model‐based analysis of drug‐induced cellular deformation

Mechanical properties of the chromatin-bearing nucleus in normal and pathological cells are of general interest for epigenetics and medicine. Conventional techniques for quantitative measurements of material properties of cellular matter are based on application of controlled forces onto the cellular or nuclear boundary and do not allow probing intracellular structures that are not directly accessible for physical contact inside the living cell. In this work, we present a novel approach for contactless determination of the nuclear compressibility (i.e. the Poisson's ratio ν) in living cells by means of image- and model-based analysis of drug-induced cell deformation. The Poisson's ratio of the HeLa cell nucleus is determined from time-series of 3D images as a parameter of constitutive model that minimizes the dissimilarity between the numerically predicted and experimentally observed images.

Nucleolar Follistatin Promotes Cancer Cell Survival under Glucose-deprived Conditions through Inhibiting Cellular rRNA Synthesis

Solid tumor development is frequently accompanied by energy-deficient conditions such as glucose deprivation and hypoxia. Follistatin (FST), a secretory protein originally identified from ovarian follicular fluid, has been suggested to be involved in tumor development. However, whether it plays a role in cancer cell survival under energy-deprived conditions remains elusive. In this study, we demonstrated that glucose deprivation markedly enhanced the expression and nucleolar localization of FST in HeLa cells. The nucleolar localization of FST relied on its nuclear localization signal (NLS) comprising the residues 64-87. Localization of FST to the nucleolus attenuated rRNA synthesis, a key process for cellular energy homeostasis and cell survival. Overexpression of FST delayed glucose deprivation-induced apoptosis, whereas down-regulation of FST exerted the opposite effect. These functions depended on the presence of an intact NLS because the NLS-deleted mutant of FST lost the rRNA inhibition effect and the cell protective effect. Altogether, we identified a novel nucleolar function of FST, which is of importance in the modulation of cancer cell survival in response to glucose deprivation.

A role for TREX components in the release of spliced mRNA from nuclear speckle domains

The TREX complex, which functions in mRNA export, is recruited to mRNA during splicing. Both the splicing machinery and the TREX complex are concentrated in 20-50 discrete foci known as nuclear speckle domains. In this study, we use a model system where DNA constructs are microinjected into HeLa cell nuclei, to follow the fates of the transcripts. We show that transcripts lacking functional splice sites, which are inefficiently exported, do not associate with nuclear speckle domains but are instead distributed throughout the nucleoplasm. In contrast, pre-mRNAs containing functional splice sites accumulate in nuclear speckles, and our data suggest that splicing occurs in these domains. When the TREX components UAP56 or Aly are knocked down, spliced mRNA, as well as total polyA+ RNA, accumulates in nuclear speckle domains. Together, our data raise the possibility that pre-mRNA undergoes splicing in nuclear speckle domains, before their release by TREX components for efficient export to the cytoplasm.

State of oncomarker protein B23/nucleophosmin in HeLa cells

Western blot after SDS-PAGE for protein separation showed two immunoreactive bands corresponding to monomers (38-40 kDa) and oligomers (210-230 kDa) of nucleophosmin in HeLa cell lysates. Decreasing the buffer ionic strength during the incubation of cells and nuclei destabilized these oligomers. We also showed the existence of two B23/nucleophosmin pools in nuclei of HeLa cells with different sensitivity to hypotonic buffer treatment: one extractable from the nucleus and the other non-extractable and tightly bound to the nucleus. A detailed structural analysis of the extractable B23 pool was carried out: two closely related nucleophosmin isoforms (B23.1 and B23.2) were identified as a result of analysis of C-terminal amino acid sequences using carboxypeptidase hydrolysis; the N-termini of both isoforms are blocked by an acetyl group. As a result of sequencing of the deacetylated proteins, it has been established that the N-terminal amino acid sequence of nucleophosmin in these preparations is truncated by nine amino acid residues and the acetylated residue is Ser. The truncated monomer of nucleophosmin (represented only by the extractable part of the protein) on addition of magnesium ions to low ionic strength buffer or increase in buffer ionic strength was shown to form oligomers with molecular weights (210-230 kDa) similar to those revealed in the total cell lysate. It should be noted that the set of oligomers in this case differs from the one in total cell lysate. Our strategy of characterization of B23 forms for HeLa cells can be applied for other tumor cells.

PEGylated PEI-based biodegradable polymers as non-viral gene vectors

Novel functional biodegradable gene vectors, poly(L-succinimide)-g-polyethylenimines-g-poly(ethylene glycol) (PSI-g-PEI-g-PEGs) were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG, M(w)=750 Da) to PEI segments (M(w)=800 Da) of PSI-g-PEI. The physicochemical properties of PSI-g-PEI-g-PEGs, including buffering capability, pDNA binding ability, cytotoxicity, zeta potential and the particle size of polymer/pDNA complexes, were explored. The influence of PEGylation was discussed based on a comparative study of PSI-g-PEI-g-PEGs, PSI-g-PEI and PEI25k (M(w)=25 kDa). SEM images revealed that PSI-g-PEI-g-PEG/pDNA particles have a regular shape with the diameter ranging from 70 to 170 nm. PEGylation could suppress the aggregation occurrence between complexes, resulting in a reduction of the polymer/pDNA complex size. PSI-g-PEI-g-PEGs exhibited remarkably lower cytotoxicity compared to PSI-g-PEI and PEI25k. In 293T and HeLa cells, the obtained PSI-g-PEI-g-PEGs showed very high transfection efficiency compared to PEI25k. Fluorescent confocal microscopy demonstrated that PSI-g-PEI-g-PEGs could effectively transport pGL-3 plasmids into the nuclei of HeLa cells. Taking into account the continued high transfection efficacy and decreased toxicity after PEG modification, PSI-g-PEI-g-PEGs show great potential as the non-viral vectors for gene transfection.

Role of nuclear tubule on the apoptosis of HeLa cells induced by femtosecond laser

It was found that nuclear tubules (NTs) would develop inside the nuclei of HeLa cells when they were irradiated by a femtosecond laser at the wavelength of 1554 nm for 40 s or longer. These NTs provided a pathway for the excess calcium generated by the laser to diffuse from the cytoplasm to the nucleus. Concurrently, the NTs served to spread mitochondria deep inside the nucleus so that they could initiate DNA fragmentations in regions covered by the NTs in the apoptotic cell. The role of NT as the precursor and passage to apoptosis is firmly established.

Identification and Characterization of Mitochondrial Targeting Sequence of Human Apurinic/Apyrimidinic Endonuclease 1

Dually targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS), which makes them difficult to predict by current bioinformatics approaches. Human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the cellular response to oxidative stress. It is a dually targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. However, the mitochondrial translocation mechanism of APE1 is not well characterized because it harbors an unconventional MTS that is difficult to predict by bioinformatics analysis. Two experimental approaches were combined in this study to identify the MTS of APE1. First, the interactions between the peptides from APE1 and the three purified translocase receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. Consequently, the intracellular distribution of green fluorescent protein-tagged, truncated, or mutated APE1 proteins was traced by tag detection. The results demonstrated that the only MTS of APE1 is harbored within residues 289-318 in the C terminus, which is normally masked by the intact N-terminal structure. As a dually targeted mitochondrial protein, APE1 possesses a special distribution pattern of different subcellular targeting signals, the identification of which sheds light on future prediction of MTSs.

Abstract 3157: Subcellular localization and mRNA targets of a novel human RNA-binding protein, KIAA0020

Abstract Human KIAA0020 belongs to the PUF family of RNA-binding proteins. Most of family members contain eight RNA-binding Pumilio repeats and are known to regulate mRNA stability and translation. In fact, hunchback, Cyclin B, and HO endonuclease mRNAs are known targets for certain PUF family members in human and other species. Currently little is known about human KIAA0020, which contains six instead of eight Pumilio repeats and shares low homology with its family members. In this study, we characterized this protein and found that wild-type KIAA0020 was localized to multiple foci within the nucleus of HeLa and HEK293T cells. Deletion of C- terminus and, to a less extent, N-terminus of KIAA0020, permitted partial cytosolic localization. Furthermore, we have found a number of mRNA targets that were regulated by KIAA0020 in cells containing ectopic overexpression of KIAA0020 variants or shRNA-mediated knockdown. Based on microarray data analysis from Oncomine, KIAA0020 appears to be deregulated in a variety of human cancer types. Understanding the subcellular localization and mRNA targets of KIAA0020 will improve our knowledge of the function of this protein and its relevance to cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3157.

Isolation and characterization of a novel zinc finger gene, ZNFD, activating AP1(PMA) transcriptional activities

ZFPs (Zinc Finger Proteins) play important roles in various cellular functions, including transcriptional activation, transcriptional repression, cell proliferation, and development. C(2)H(2) (Cys-Cys-His-His motif) ZFPs are the most abundant proteins among the founding members of the ZFP super family in eukaryotes. In this study, we isolate a novel C(2)H(2) ZNF (Zinc Finger) gene ZNFD. It contains an ORF (Open Reading Frame) with a length of 990 bp, encoding 329 amino acids. The predicted protein contains a C(2)H(2) zinc finger. RT-PCR analysis in 18 human adult tissues indicated that it was expressed in five human adult tissues. Green fluorescence protein localization analysis showed that human ZNFD was located in the nucleus of Hela cells. Overexpression of ZNFD in the COS7 cells activates the transcriptional activities of AP1(PMA) (Activator of protein 1, that responds specifically to phorbol ester). Together the data indicate that ZNFD is probably a new type of C(2)H(2) ZFP and the ZNFD protein may act as a transcriptional activator in PKC (protein kinase C) signal pathway to mediate cellular functions.