RESUMEN
Tumor-derived mutant forms of p53 compromise its DNA binding, transcriptional, and growth regulatory activity in a manner that is dependent upon the cell-type and the type of mutation. Given the high frequency of p53 mutations in human tumors, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy. In support of this possibility p53 mutants possess a certain degree of conformational flexibility that allows for re-induction of function by a number of structurally different artificial compounds or by short peptides. This raises the question of whether physiological pathways for p53 mutant reactivation also exist and can be exploited therapeutically. The activity of wild-type p53 is modulated by various acetyl-transferases and deacetylases, but whether acetylation influences signaling by p53 mutant is still unknown. Here, we show that the PCAF acetyl-transferase is down-regulated in tumors harboring p53 mutants, where its re-expression leads to p53 acetylation and to cell death. Furthermore, acetylation restores the DNA-binding ability of p53 mutants in vitro and expression of PCAF, or treatment with deacetylase inhibitors, promotes their binding to p53-regulated promoters and transcriptional activity in vivo. These data suggest that PCAF-mediated acetylation rescues activity of at least a set of p53 mutations. Therefore, we propose that dis-regulation of PCAF activity is a pre-requisite for p53 mutant loss of function and for the oncogenic potential acquired by neoplastic cells expressing these proteins. Our findings offer a new rationale for therapeutic targeting of PCAF activity in tumors harboring oncogenic versions of p53.
Asunto(s)
ADN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Cromatina/metabolismo , Neoplasias Colorrectales/metabolismo , Humanos , Ratones , Mutación , Unión Proteica , Proteína p53 Supresora de Tumor/genética , Factores de Transcripción p300-CBP/genéticaRESUMEN
BACKGROUND: The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial. RESULTS: Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin alpha temperature-sensitive yeast mutant, indicating an importin alpha-mediated process. Direct interaction between the full-length IN and importin alpha was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide. CONCLUSION: Our present findings support the view that nuclear import of IN occurs via the importin alpha pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Infecciones por VIH/virología , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/fisiología , Señales de Localización Nuclear/farmacología , Replicación Viral/efectos de los fármacos , Animales , Células COS , Chlorocebus aethiops , Infecciones por VIH/metabolismo , Integrasa de VIH/química , Inhibidores de Integrasa VIH/química , VIH-1/efectos de los fármacos , Células HeLa , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Redes y Vías Metabólicas/efectos de los fármacos , Señales de Localización Nuclear/química , Señales de Localización Nuclear/fisiología , Integración Viral/efectos de los fármacos , alfa Carioferinas/metabolismoRESUMEN
We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).
Asunto(s)
Histonas/química , Membrana Dobles de Lípidos/química , Mycoplasma/metabolismo , Biotina/química , Biotinilación , Western Blotting , Membrana Celular/metabolismo , Colesterol , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Escherichia coli/metabolismo , Humanos , Lípidos/química , Liposomas/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Péptidos/química , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes/química , TemperaturaRESUMEN
Microarray studies revealed that as a first hit the SV40 T/t antigen causes deregulation of 462 genes in mammary gland cells (ME cells) of WAP-SVT/t transgenic animals. The majority of deregulated genes are cell proliferation specific and Rb-E2F dependent, causing ME cell proliferation and gland hyperplasia but not breast cancer formation. In the breast tumor cells a further 207 genes are differentially expressed, most of them belonging to the cell communication category. In tissue culture breast tumor cells frequently switch off WAP-SVT/t transgene expression and regain the morphology and growth characteristics of normal ME cells, although the tumor-revertant cells are aneuploid and only 114 genes regain the expression level of normal ME cells. The profile of retransformants shows that only 38 deregulated genes are tumor-specific, and that none of them is considered to be a typical breast cancer gene.
Asunto(s)
Aneuploidia , Antígenos Transformadores de Poliomavirus/fisiología , Ciclo Celular/fisiología , Perfilación de la Expresión Génica , Glándulas Mamarias Animales/citología , Neoplasias Mamarias Experimentales/etiología , Animales , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada , Transformación Celular Viral , Células Cultivadas , Femenino , Regulación Viral de la Expresión Génica , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/fisiología , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transfección , Transgenes , Células Tumorales CultivadasRESUMEN
Transgenic mice, which selectively express the WAP-HBX transgene in mammary gland epithelial cells (ME-cells), were established in order to elucidate the consequences of HBX gene expression on organ differentiation, cell death program and tumor development. Transgene expression was demonstrable by RT-PCR, Northern and Western blot analysis during pregnancy, lactation and after weaning. HBX synthesis neither affect mammary gland differentiation nor apoptosis in ME-cells. Although breast cancer formation was rare in WAP-HBX animals (<1%), WAP-HBX*p53+/- hybrid animals developed breast tumors at an increased rate (12/85) after a latency period of 8-18 months. We also show here for the first time that HBX can immortalize ME-cells generated from mammary gland tissue segments in a p53-independent fashion. HBX causes cyclin D1 gene overexpression during early pregnancy, and this is maintained in ME-cells isolated either from mammary gland or from breast tumors. Intranuclear cyclin D1 accumulation also occurs in the absence of external growth factors and the BrdU incorporation rate remains high under serum starvation conditions. Finally, both cyclin D1 induction and HBX mitotic activity are dependent on p38 and c-Jun N-terminal kinase, but not on MEK-1 kinase activity.
Asunto(s)
Ciclinas/genética , Neoplasias Mamarias Experimentales/genética , Transactivadores/genética , Animales , Apoptosis , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Ciclina D , Ciclinas/biosíntesis , Regulación Neoplásica de la Expresión Génica , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/fisiología , Neoplasias Mamarias Experimentales/etiología , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Transgénicos , Proteínas de la Leche/genética , Proteínas de la Leche/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 degrees C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.
Asunto(s)
Histonas/metabolismo , Petunia/metabolismo , Protoplastos/metabolismo , Células Cultivadas , Endocitosis , Técnicas de Transferencia de Gen , Vectores Genéticos , Microscopía Fluorescente , Albúmina Sérica Bovina/metabolismoRESUMEN
In spite of recent efforts to elucidate the nuclear import pathway of the human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), its exact route as well as the domains that mediate its import are still unknown. Here, we show that a synthetic peptide bearing the amino acid residues 161-173 of the HIV-1 IN is able to mediate active import of covalently attached bovine serum albumin molecules into nuclei of permeabilized cells and therefore was designated as nuclear localization signal-IN (NLS(IN)). A peptide bearing residues 161-173 in the reversed order showed low karyophilic properties. Active nuclear import was demonstrated by using fluorescence microscopy and a quantitative ELISA-based assay system. Nuclear import was blocked by addition of the NLS(IN) peptide, as well as by a peptide bearing the NLS of the simian virus 40 T-antigen (NLS-SV40). The NLS(IN) peptide partially inhibited nuclear import mediated by the full-length recombinant HIV-1 IN protein, indicating that the sequence of the NLS(IN) is involved in mediating nuclear import of the IN protein. The NLS(IN) as well as the full-length IN protein interacted specifically with importin alpha, binding of which was blocked by the NLS(IN) peptide itself as well as by the NLS-SV40.
Asunto(s)
Transporte Activo de Núcleo Celular , Integrasa de VIH/metabolismo , Carioferinas/metabolismo , Señales de Localización Nuclear , Fragmentos de Péptidos/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Secuencia de Aminoácidos , Integrasa de VIH/química , Células HeLa , Humanos , Microscopía Fluorescente , Fragmentos de Péptidos/síntesis química , Unión Proteica/efectos de los fármacos , Transporte de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
Cancers are clones of autonomous cells defined by individual karyotypes, much like species. Despite such karyotypic evidence for causality, three to six synergistic mutations, termed oncogenes, are generally thought to cause cancer. To test single oncogenes, they are artificially activated with heterologous promoters and spliced into the germ line of mice to initiate cancers with collaborating spontaneous oncogenes. Because such cancers are studied as models for the treatment of natural cancers with related oncogenes, the following must be answered: 1) which oncogenes collaborate with the transgenes in cancers; 2) how do single transgenic oncogenes induce diverse cancers and hyperplasias; 3) what maintains cancers that lose initiating transgenes; 4) why are cancers aneuploid, over- and underexpressing thousands of normal genes? Here we try to answer these questions with the theory that carcinogenesis is a form of speciation. We postulate that transgenic oncogenes initiate carcinogenesis by inducing aneuploidy. Aneuploidy destabilizes the karyotype by unbalancing teams of mitosis genes. This instability thus catalyzes the evolution of new cancer species with individual karyotypes. Depending on their degree of aneuploidy, these cancers then evolve new subspecies. To test this theory, we have analyzed the karyotypes and phenotypes of mammary carcinomas of mice with transgenic SV40 tumor virus- and hepatitis B virus-derived oncogenes. We found that (1) a given transgene induced diverse carcinomas with individual karyotypes and phenotypes; (2) these karyotypes coevolved with newly acquired phenotypes such as drug resistance; (3) 8 of 12 carcinomas were transgene negative. Having found one-to-one correlations between individual karyotypes and phenotypes and consistent coevolutions of karyotypes and phenotypes, we conclude that carcinogenesis is a form of speciation and that individual karyotypes maintain cancers as they maintain species. Because activated oncogenes destabilize karyotypes and are dispensable in cancers, we conclude that they function indirectly, like carcinogens. Such oncogenes would thus not be valid models for the treatment of cancers.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Neoplasias Mamarias Experimentales/genética , Oncogenes , Aneuploidia , Animales , Antígenos Transformadores de Poliomavirus/análisis , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Cariotipificación , Neoplasias Mamarias Experimentales/etiología , Ratones , Fenotipo , Transactivadores/genética , Transgenes , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The aim of our work was to establish a database for breast cancer gene expression data in order to compare human and mouse breast cancer. We identified human and mouse homologues genes and compared the expression profile of 24 human breast tumors with 6 WAP-SVT/t breast tumors (WAP-SVT/t animals, line 8). Our studies confirmed the heterogeneity in gene expression of human as well as mouse breast cancer cells. However, 63 genes were found to be differentially expressed (upregulated: 40; downregulated: 23 genes) in at least 75% of the breast tumors of both species. To differentiate between early and late events in tumor formation, we compared the 63 differentially expressed genes with a mouse data set obtained from hyperplastic mammary glands. This revealed that the majority of the early deregulated genes are cell proliferation specific. These early changes seem to be necessary although not sufficient for breast cancer formation. Late alterations concern mainly genes belonging to the category of cell communication and metabolism. Interestingly, most of the 63 conserved genes are commonly associated with tumorigenesis.
Asunto(s)
Neoplasias de la Mama/genética , Animales , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Neoplasias Mamarias Experimentales , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Homología de Secuencia de Ácido NucleicoRESUMEN
CorrXpression is a stand-alone desktop application for the identification of significant genes within collections of microarrays. The software combines three methods in two steps of analysis: correspondence analysis (CA), ratio analysis and correlation analysis. The graphical interface of CorrXpression visualizes the result of the CA with a biplot and the expression of selected genes in dependency of the experiments as bar diagrams. The CA-plot is an excellent tool for visualization and evaluation of data and results of ratio analysis and correlation analysis. The input data are selected from a database or from appropriate ASCII files.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis por Micromatrices/métodos , Programas Informáticos , Animales , Simulación por Computador , Femenino , Ratones , Interfaz Usuario-ComputadorRESUMEN
The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two approximately 2.6 kb DNAs or a monopartite genome of approximately 3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3-20 of TYLCV CP (CP-NLS) or aa 84-106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA-NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin alpha, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin alpha.
Asunto(s)
Geminiviridae/fisiología , Proteínas Virales/fisiología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Geminiviridae/genética , Geminiviridae/patogenicidad , Células HeLa , Humanos , Técnicas In Vitro , Carioferinas/metabolismo , Datos de Secuencia Molecular , Señales de Localización Nuclear , Plantas/virología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Albúmina Sérica Bovina/metabolismo , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The present work shows that histones are able to directly cross cell plasma membranes and mediate penetration of macromolecules covalently attached to them. Adding a mixture containing the five nucleosomal histones, H1, H2A, H2B, H3 and H4, as well as each of the last four individual histones to intact HeLa and Colo-205 cultured cells resulted in cell penetration and nuclear import of these externally added histones. This was observed by fluorescent and confocal microscopy using fixed and unfixed cells, showing that penetration was not due to the fixation process. Accumulation was also estimated by a quantitative assay that did not require cell fixation and allowed neutralization of surface-bound histones. Translocation into the HeLa and Colo-205 cells occurred at 4 degrees C, in ATP-depleted cells and in cells incubated with sucrose (0.5 M) - conditions that block the endocytic pathway. Furthermore, various endocytosis inhibitors such as colchicine, nocodazole, cytochalasin D, brefeldin A, chloroquine and nystatin did not have any effect on the penetration process. Thus, cellular uptake was mostly due to direct translocation of the histones through the cell plasma membrane and not to endocytosis. The histones were also able to mediate penetration of covalently attached bovine serum albumin (BSA) molecules, indicating their potential as carriers for the delivery of macromolecules into living mammalian cells.
Asunto(s)
Transporte Biológico/fisiología , Membrana Celular/metabolismo , Histonas/metabolismo , Células Cultivadas , Clonación Molecular , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Células HeLa , Humanos , Carioferinas/metabolismo , Linfocitos/metabolismo , Microscopía Fluorescente , Nocodazol/farmacología , Sacarosa/metabolismoRESUMEN
The HIV-1 Rev protein plays a pivotal role in viral replication, and therefore, inhibition of its function should block the progression of the virus-induced immune deficiency syndrome (AIDS). Here, RNA molecules have been shown to inhibit import of the HIV-1 Rev protein into nuclei of permeabilized cells. Nuclear uptake of biotinylated recombinant His-tagged Rev-GFP was assessed in nuclear extracts from digitonin-permeabilized cells by binding to either importin beta-receptors or nickel molecules immobilized on a microtiter plate. Using this method together with fluorescence microscopy, we determined that nuclear import of Rev is inhibited by the addition of a reticulocyte lysate which routinely is used as a source of nuclear import receptors. This inhibition was released by treatment with the RNase enzyme. Also t-RNA molecules and the oligoribonucleotide RRE IIB, namely, the second stem structure of the Rev responsive element (RRE) of the viral RNA, inhibit Rev nuclear import. Similar results were obtained when BSA molecules with covalently attached Rev-arginine rich motif (ARM) peptides were used as a nuclear transport substrate, indicating that the nuclear import inhibition of the Rev protein is due to the presence of the ARM domain. Binding experiments revealed that the RNA molecules inhibit the interaction between the ARM region and importin beta, implying that the RNA prevents the formation of the import complex. The implication of our results for the regulation of the nuclear import of Rev as well as for the use of RNA molecules as antiviral drugs is discussed.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteínas Bacterianas , Núcleo Celular/metabolismo , Productos del Gen rev/fisiología , VIH-1/fisiología , Proteínas/fisiología , ARN/farmacología , Proteínas Represoras/fisiología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Secuencias de Aminoácidos/efectos de los fármacos , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Unión Competitiva , Núcleo Celular/efectos de los fármacos , Núcleo Celular/virología , Citosol/metabolismo , Citosol/fisiología , ADN/farmacología , Productos del Gen rev/antagonistas & inhibidores , Productos del Gen rev/metabolismo , Proteínas Fluorescentes Verdes , VIH-1/efectos de los fármacos , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso , Proteínas Nucleares/farmacología , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Albúmina Sérica Bovina/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Dermaseptins are a family of antimicrobial peptides that lyse target bacterial cells by destabilization of their membranes. Here we present a novel application of a peptide derived from the dermaseptin S4, S4(13). At nontoxic concentrations, fluorescently labeled S4(13) was able to penetrate intact cultured HeLa cells but essentially failed to enter their nuclei despite its low molecular weight. Covalent attachment of nuclear localization signal (NLS) motifs of the SV40-T-antigen and of the HIV-1 Rev protein (ARM) conferred karyophilic properties upon the S4(13). The resulting peptides, which were designated as PV-S4(13) and RR-S4(13) penetrated into intact HeLa cells and were able to accumulate within the cells' nuclei. In studies with digitonin-permeabilized cells, nuclear uptake of the PV-S4(13) and the RR-S4(13) peptides showed the same features that characterize active nuclear import. Nuclear import was observed at 37 degrees C, was ATP-dependent, and was inhibited by the free peptides bearing the SV40 NLS and the Rev and Tat ARMs. Microinjected S4(13) remained in the cytoplasm while microinjected RR-S4(13) was translocated into the cells' nuclei. The new type of cell-permeable "karyophilic" peptides described here may be of potential application as a lead compound for therapeutic purposes, as a tool to study nucleocytoplasmic shuttling in intact cells, and for the delivery of peptides to the nucleus.