RESUMEN
Most acute promyelocytic leukemia (APL) cases are associated with recurrent translocations between the gene of retinoic receptor alpha and that of PML (t(15;17)) or PLZF (t(11;17)). PML localizes onto discrete intranuclear domains, the PML-nuclear bodies, and displays anti-oncogenic and pro-apoptotic properties. PLZF encodes a transcription factor belonging to the POZ/domain and Krüppel zinc finger (POK) family which interacts directly with PML. PLZF is related to another POK protein, LAZ3(BCL6), which is structurally altered, and presumably misexpressed, in many non-Hodgkin lymphoma (NHL) cases. PLZF and LAZ3 share many functional properties: both inhibit cell growth, concentrate into punctated nuclear subdomains and are sequence-specific transcriptional repressors recruiting a histone deacetylase-repressing complex. Given these similarities, we tested whether both proteins could be targeted by each other. Here, LAZ3 and PLZF are shown to colocalize onto nuclear dots. Moreover, truncated derivatives of one protein, which display a diffuse nuclear localization, are recruited onto nuclear dots by the full-length other. The colocalization and the reciprocal 'rescue' is the result of a direct interaction between LAZ3 and PLZF, as indicated by yeast two hybrid assays, in vitro immunoprecipitations, and GST pull down experiments. In contrast to LAZ3 homomerization, LAZ3/PLZF heteromerization in yeast does not solely depend on POZ/POZ contacts but rather also relies on interactions between the two zinc finger regions and 'cross' contacts between the zinc finger region and the POZ domain of each partner. Likewise, LAZ3 shows some colocalization with the PLZF partner PML upon stable overexpression of both proteins in CHO cells and interacts with PML in yeast. Finally, endogenous LAZ3 and PLZF are co-induced and partially colocalized in myeloid MDS cells. These data indicate that a physical interaction between LAZ3 and PLZF underlies their simultaneous recruitment onto multiproteic nuclear complexes, presumably involved in transcriptional silencing and whose integrity (for APL) and/or function (for APL and NHL) may be altered in oncogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Linfoma no Hodgkin/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Animales , Células CHO , Células COS , Cricetinae , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Humanos , Factores de Transcripción de Tipo Kruppel , Leucemia Promielocítica Aguda/genética , Linfoma no Hodgkin/genética , Sustancias Macromoleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteína de la Leucemia Promielocítica , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Proteínas Proto-Oncogénicas c-bcl-6 , Eliminación de Secuencia , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos HíbridosRESUMEN
PML is a nuclear matrix protein with growth suppressing properties, whose expression is deregulated during oncogenesis. Moreover, in the t(15;17) translocation of acute promyelocytic leukaemia (APL), PML fusion to the retinoic acid receptor alpha (RAR alpha) is the likely molecular basis of leukaemogenesis. Here we show that interferons (IFNs) alpha, beta, and gamma upregulate PML mRNA expression. Analysis of 5' genomic sequences of the PML gene revealed an IFN-alpha/-beta stimulated response element (ISRE) and an IFN-gamma activation site (GAS) in the untranslated first exon. Binding of IFN signal transducers and activators of transcription (STATs) was demonstrated to be weak for the PML GAS, but strong for the PML ISRE which also seemed to contribute substantially to the IFN-gamma response. Thus, PML is a primary target gene of IFNs and would appear as a suitable candidate for mediating some of their antiproliferative effects. Abnormalities of PML structure, localisation or expression in human malignancy, constitute examples of how an IFN target gene may be altered in oncogenesis.
Asunto(s)
Interferones/farmacología , Proteínas de Neoplasias , Proteínas Nucleares , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Cricetinae , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteína de la Leucemia Promielocítica , Proteínas Supresoras de TumorRESUMEN
The LAZ3/BCL6 gene was identified by its disruption in 3q27 translocations associated with diffuse large cell lymphomas. It is predicted to be a transcription factor as it contains six Krüppel-like Zinc finger motifs and a N-terminal BTB/POZ domain, a protein/protein interaction interface that is widely conserved in Metazoans. Using two antisera raised against non overlapping regions of the predicted ORF, we demonstrate that the LAZ3/BCL6 protein appears as a close ca. 79 kDa doublet in B lymphoid cell lines with either a rearranged or a non rearranged LAZ3/BCL6 locus. By immunofluorescence experiments on transiently transfected COS-1 or NIH3T3 cells, we show that the LAZ3/BCL6 protein displays a punctuated nuclear localisation. This appears to rely on LAZ3/BCL6 proper folding and/or activities as it is impaired in a hormone reversible-fashion through fusion of LAZ3/BCL6 to the ligand-binding domain of the oestrogen receptor. Moreover, deletion of its BTB/POZ domain leads to the disappearance of the nuclear dots although the protein remains nuclear. In addition, by using the yeast two-hybrid system, we show that the LAZ3/BCL6 BTB/POZ domain homomerises in vivo. Thus, the LAZ3/BCL6 BTB/POZ domain has the capability to self-interact and target the protein to discrete nuclear substructures.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Proto-Oncogénicas/fisiología , Factores de Transcripción/fisiología , Dedos de Zinc , Células 3T3 , Animales , Humanos , Ratones , Proteínas Proto-Oncogénicas c-bcl-6RESUMEN
Altered sub-nuclear localisation of the nuclear body-associated PML protein in acute promyelocytic leukaemia, has been proposed to contribute to leukaemogenesis. We have recently shown that PML is a primary target gene of interferons. Here, it is shown that PML has growth suppressive properties and displays an altered expression pattern during human oncogenesis. PML is widely expressed in cell-lines and is cell-cycle regulated. Overexpression of the protein induces a sharp reduction in growth rates in vitro and in vivo. In contrast with cell-lines, in normal tissues (including those that rapidly proliferate) only a few cells have detectable PML levels. However, these can be upregulated by soluble factors (e.g. IFN, estrogens). Human epithelial tumors show a gradual increase of PML levels as the lesion progresses from benign dysplasia to carcinoma. A similar induction is found in the surrounding stroma and vessels, which likely results from paracrine interactions. Strikingly, when malignant cells turn invasive, they loose PML expression, while expression is conserved in the stromal compartment. These observations point to the existence of a consistent deregulation in the expression of the PML growth-suppressor during human oncogenesis.
Asunto(s)
Ciclo Celular , Inhibidores de Crecimiento , Proteínas de Neoplasias , Neoplasias/metabolismo , Proteínas Nucleares , Factores de Transcripción/metabolismo , Animales , Estrógenos/fisiología , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteína de la Leucemia Promielocítica , Piel/metabolismo , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Primary blasts of a t(11;17)(q23;q21) acute promyelocytic leukaemia (APL) patient were analysed with respect to retinoic acid (RA) and arsenic trioxide (As2O3) sensitivity as well as PLZF/RARalpha status. Although RA induced partial monocytic differentiation ex vivo, but not in vivo, As203 failed to induce apoptosis in culture, contrasting with t(15;17) APL and arguing against the clinical use of As203 in t(11;17)(q23;q21) APL. Prior to cell culture, PLZF/RARalpha was found to exactly co-localize with PML onto PML nuclear bodies. However upon cell culture, it quickly shifted towards microspeckles, its localization found in transfection experiments. Arsenic trioxide, known to induce aggregation of PML nuclear bodies, left the microspeckled PLZF/RARalpha localization completely unaffected. RA treatment led to PLZF/RARalpha degradation. However, this complete PLZF/RARalpha degradation was not accompanied by differentiation or apoptosis, which could suggest a contribution of the reciprocal RARalpha/PLZF fusion product in leukaemogenesis or the existence of irreversible changes induced by the chimera.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Proteínas de Unión al ADN/efectos de los fármacos , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusión Oncogénica/efectos de los fármacos , Óxidos/farmacología , Receptores de Ácido Retinoico/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Tretinoina/farmacología , Apoptosis , Trióxido de Arsénico , Western Blotting , Diferenciación Celular/efectos de los fármacos , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 17 , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Técnica del Anticuerpo Fluorescente , Humanos , Factores de Transcripción de Tipo Kruppel , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Proteínas de Fusión Oncogénica/metabolismo , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Factores de Transcripción/metabolismo , Translocación Genética , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1gamma. Similar to TIF1alpha and TIF1beta, the structure of TIF1beta is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain. Although structurally related to TIF1alpha and TIF1beta, TIF1gamma presents several functional differences. In contrast to TIF1alpha, but like TIF1beta, TIF1 does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells. Whereas TIF1alpha and TIF1beta were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1alpha, MODI (HP1beta) and MOD2 (HP1gamma) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1gamma does not interact with either the KRAB domain of KOX1 or the HP1 proteins. Nevertheless, TIF1gamma, like TIF1alpha and TIF1beta, exhibits a strong silencing activity when tethered to a promoter. Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1gamma, this motif likely participates in TIF1 dependent repression.
Asunto(s)
Familia de Multigenes , Secuencia de Aminoácidos , Secuencia de Bases , Homólogo de la Proteína Chromobox 5 , Bandeo Cromosómico , Mapeo Cromosómico , Clonación Molecular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Unión Proteica , ARN Mensajero/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Represoras/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Factores de Transcripción/genética , Transcripción Genética , Transfección , Proteína 28 que Contiene Motivos TripartitoRESUMEN
PML has been identified through its fusion to the RAR alpha gene in acute promyelocytic leukemia (APL). The PML protein is specifically associated to nuclear bodies (NBs) whose alterations in APL were proposed to contribute to leukemogenesis. The role of this nuclear domain (which also harbors the Sp100 autoantigen and the NDP52 protein) is unknown. Here, we show that the PML protein, like Sp100 and NDP52, is induced by interferons (IFNs alpha, beta and gamma) in a large variety of human cells. Interestingly, the NBs that contain the three IFN-induced proteins appear to be associated to speckles labelled by the IFN-mediator Mx1. These observations link NBs to IFN response pathways, which may contribute to the elucidation of the biological role of these structures. In APL cells, IFNs induced both PML and PML/RAR alpha expression, resulting in an increased sequestration of PML and RXRs in the microspeckles induced by the fusion protein. As PML has growth suppressing properties, it may mediate some of the antiproliferative effects of IFN. In APL, inactivation of PML may result in disruption of growth control.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al GTP , Interferones/farmacología , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Neoplasias , Proteínas/metabolismo , Factores de Transcripción/biosíntesis , Western Blotting , Humanos , Microscopía Confocal , Proteínas de Resistencia a Mixovirus , Proteínas Nucleares/biosíntesis , Proteína de la Leucemia Promielocítica , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Metal-binding cysteine and histidine residues are often used to stabilise a protein fold through coordination of zinc ions. These zinc fingers are either involved in nucleic acid binding (TFIIIA, GAL4, nuclear receptors, retroviral gag...) or in yet unidentified biochemical functions (LIM and RING domains). The latter characterized by a unique histidine residue in the zinc binding motif (C2HC5 and C3HC4 for the LIM and RING respectively) may constitute protein/protein interaction interfaces. We have identified a new C4HC3 motif in a variety of proteins including the Drosophila trithorax and its human homologue ALL-1 involved in oncogenic translocations in acute leukaemias. This domain, for which we propose the name TTC (for trithorax consensus) is found in many transcriptional regulators or DNA-binding proteins. Interestingly, TTC was found in several bromodomain containing transcriptional adaptors including the E1A-binding p300 and the CREB-binding CBP proteins. In CBP, this domain does not appear to be involved in DNA, CREB or TFIIB binding. In the chromosomal translocations that involve the 11q23 locus, the C-terminal end of ALL-1 (which contains 4 TTC fingers) is constantly lost. The absence of these motifs in the fusion genes may relate to their leukemogenic potential.
Asunto(s)
Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Drosophila/química , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Translocación GenéticaRESUMEN
The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin conjugating enzyme and is required for DNA repair, DNA-damage-induced mutagenesis and sporulation. Here, we show that RAD6 and the rhp6+ gene from the distantly related yeast Schizosaccharomyces pombe share a high degree of structural and functional homology. The predominantly acidic carboxyl-terminal 21 amino acids present in the RAD6 protein are absent in the rhp6(+)-encoded protein; otherwise, the two proteins are very similar, with 77% identical residues. Like rad6, null mutations of the rhp6+ gene confer a defect in DNA repair, UV mutagenesis and sporulation, and the RAD6 and rhp6+ genes can functionally substitute for one another. These observations suggest that functional interactions between RAD6 (rhp6+) protein and other components of the DNA repair complex have been conserved among eukaryotes.
Asunto(s)
Proteínas Fúngicas/genética , Ligasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Reparación del ADN/genética , ADN de Hongos/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Ácido Nucleico , Esporas Fúngicas/genética , Enzimas Ubiquitina-Conjugadoras , Rayos UltravioletaRESUMEN
This article presents the development of a set of new expression vectors for overproduction of proteins in Escherichia coli. The vectors, pETUBI-ES1, 2 and 3, allow in-frame cloning of any sequence with the ubiquitin gene driven by the strong T7f10 promoter. Combination of the T7 expression system with ubiquitin fusion appears to have a synergistic effect on protein overproduction. Large amounts of stable RNA are produced by T7 RNA polymerase, and fusion of ubiquitin to the N-terminus of target proteins seems to confer more efficient translation, better folding or protection against proteolytic degradation. The ubiquitin part can be utilized for purification of the fusion protein, after which it can be easily removed from the fusion product by ubiquitin-specific proteases. The advantage of combining both systems is demonstrated by the synthesis of large quantities (up to 40-50% of the total protein) of the human ERCC1 protein that hitherto was refractory to overproduction in various other E. coli and yeast expression systems.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , Endonucleasas , Escherichia coli/genética , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Ubiquitinas/metabolismo , Secuencia de Bases , Clonación Molecular , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas/genética , Proteínas/aislamiento & purificación , Mapeo Restrictivo , Fagos T/enzimología , Fagos T/genética , Ubiquitinas/genética , Proteínas ViralesRESUMEN
Human foamy retrovirus (HFV) is found as two proviruses (HFV and delta HFV) which differ by a splice-induced deletion within the bel1 transactivator gene. The defective delta HFV (which lacks a functional Bel1 but harbors an intronless bet gene) is predominantly found in nonlytic infections in vitro as well as in vivo. Here, we show that infection of cell lines stably transduced by delta HFV DNA with the highly lytic HFV leads to chronic infections characterized by an absence of lysis, a balanced ratio of HFV to delta HFV, and a persistent Bet expression accompanied by a shutoff of structural genes. While this system only partially reflects the natural situation, in which target cells are infected by HFV and delta HFV simultaneously, it strongly suggests that delta HFV is a defective interfering retrovirus. Accordingly, previous or concomitant exposure to delta HFV viruses greatly enhances the formation of lysis-resistant clones in culture after HFV infection. The inability of delta HFV proviruses encoding a mutated bet gene to induce chronic infection suggests a role for Bet in this process. Through a specific, splice-induced, genomic deletion, resulting in a switch from Bel1 to Bet expression, the lytic properties of HFV are progressively lost. Such programmed inactivation of a key gene represents a new regulatory mechanism of gene expression in retroviruses.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Virus Defectuosos/genética , Genes Virales , Proteínas de los Retroviridae/biosíntesis , Eliminación de Secuencia , Spumavirus/genética , Transactivadores/biosíntesis , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Amnios , Línea Celular , Mapeo Cromosómico , Células Clonales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Virus Defectuosos/patogenicidad , Humanos , Intrones , Datos de Secuencia Molecular , Infecciones por Retroviridae/fisiopatología , Infecciones por Retroviridae/virología , Proteínas de los Retroviridae/química , Proteínas de los Retroviridae/genética , Homología de Secuencia de Aminoácido , Spumavirus/patogenicidad , Factores de Tiempo , Transactivadores/química , Transactivadores/genética , TransfecciónRESUMEN
The interferon (IFN)-induced promyelocytic leukemia (PML) protein is specifically associated with nuclear bodies (NBs) whose functions are yet unknown. Two of the NB-associated proteins, PML and Sp100, are induced by IFN. Here we show that overexpression of PML and not Sp100 induces resistance to infections by vesicular stomatitis virus (VSV) (a rhabdovirus) and influenza A virus (an orthomyxovirus) but not by encephalomyocarditis virus (a picornavirus). Inhibition of viral multiplication was dependent on both the level of PML expression and the multiplicity of infection and reached 100-fold. PML was shown to interfere with VSV mRNA and protein synthesis. Compared to the IFN mediator MxA protein, PML had less powerful antiviral activity. While nuclear body localization of PML did not seem to be required for the antiviral effect, deletion of the PML coiled-coil domain completely abolished it. Taken together, these results suggest that PML can contribute to the antiviral state induced in IFN-treated cells.
Asunto(s)
Antígenos Nucleares , Virus de la Influenza A , Gripe Humana/metabolismo , Interferones/farmacología , Proteínas de Neoplasias , Infecciones por Rhabdoviridae/metabolismo , Factores de Transcripción/metabolismo , Virus de la Estomatitis Vesicular Indiana , Animales , Autoantígenos/metabolismo , Células CHO , Cricetinae , Susceptibilidad a Enfermedades , Humanos , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme (E2) that is required for DNA repair, damage-induced mutagenesis, and sporulation. We have cloned the two human RAD6 homologs, designated HHR6A and HHR6B. The two 152-amino acid human proteins share 95% sequence identity with each other and approximately 70% and approximately 85% overall identity with the homologs from yeasts (S. cerevisiae and Schizosaccharomyces pombe) and Drosophila melanogaster, respectively. Neither of the human RAD6 homologs possess the acidic C-terminal sequence present in the S. cerevisiae RAD6 protein. Genetic complementation experiments reveal that HHR6A as well as HHR6B can carry out the DNA repair and mutagenesis functions of RAD6 in S. cerevisiae rad6 delta mutants.
Asunto(s)
Reparación del ADN/genética , Genes Fúngicos , Ligasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sondas de ADN , Drosophila/genética , Proteínas Fúngicas/genética , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Poli A/genética , Poli A/aislamiento & purificación , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Homología de Secuencia de Ácido Nucleico , Enzimas Ubiquitina-ConjugadorasRESUMEN
The PML gene is fused to the retinoic acid receptor alpha (RAR alpha) gene in t(15;17) acute promyelocytic leukemia (APL), creating a PML-RAR alpha fusion oncoprotein. The PML gene product has been localized to subnuclear dot-like structures variously termed PODs, ND10s, Kr bodies, or PML nuclear bodies (PML NBs). The present study describes the cloning of a lymphoid-restricted gene, LYSP100, that is homologous to another protein that localizes to PML NBs, SP100. In addition to SP100 homology regions, one LYSP100 cDNA isoform contains a bromodomain and a PHD/TTC domain, which are present in a variety of transcriptional regulatory proteins. By immunofluorescence, LYSP100 was localized to nuclear dots that were surprisingly largely nonoverlapping with PML NBs. However, a minority of LYSP100 nuclear dots exactly colocalized with PML and SP100. We term the LYSP100 structures "LANDs," for LYSP100-associated nuclear domains. Although LYSP100 is expressed only in lymphoid cells, LANDs could be visualized in HeLa cells by transfection of a LYSP100 cDNA. Immunoelectron microscopy revealed LANDs to be globular, electron-dense structures morphologically distinct from the annular structures characteristic of PML NBs. LANDs were most often found in the nucleoplasm, but were also found at the nuclear membrane and in the cytoplasm, suggesting that these structures may traffic between the cytoplasm and the nucleus. By double-immunogold labeling of PML and LYSP100, some LANDs were shown to contain both PML and LYSP100. Thus, PML is localized to a second subnuclear domain that is morphologically and biochemically distinct from PML NBs.
Asunto(s)
Antígenos Nucleares , Núcleo Celular/ultraestructura , Linfocitos/ultraestructura , Proteínas de Neoplasias , Proteínas Nucleares/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Autoantígenos/química , Secuencia de Bases , Compartimento Celular , Clonación Molecular , Cartilla de ADN/química , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa , Humanos , Sustancias Macromoleculares , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Proteínas Supresoras de TumorRESUMEN
In the acute promyelocytic leukemia (APL) cell line NB4, as well as in APL patients' cells, arsenic trioxide (As2O3) leads to incomplete cell maturation, induction of apoptosis, as well as to the degradation of the oncogenic PML/RARalpha fusion protein. We have isolated an arsenic-resistant NB4 subline (NB4-AsR), which fails to undergo apoptosis, but maintains the partial differentiation response to this drug. When grown in the presence of As2O3, NB4-AsR cells degrade PML/RARalpha, slightly differentiate, and become more sensitive to serum deprivation-induced apoptosis. Similarly, in RA-resistant NB4-R1 cells, RA induced a significant PML/RARalpha degradation and yet failed to induce cell maturation. Thus, As2O3- or retinoic acid (RA)-induced PML/RARalpha degradation may be a prerequisite, but is not sufficient for the full differentiative/apoptotic response to these drugs. Strikingly, RA-triggered differentiation and apoptosis were greatly accelerated in As2O3-treated NB4-AsR cells. The synergism between these two agents in this setting could provide an experimental basis for combined or sequential RA/As2O3 therapies.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Óxidos/farmacología , Tretinoina/farmacología , Trióxido de Arsénico , Diferenciación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Humanos , Leucemia Promielocítica Aguda/patología , Microscopía Confocal , Células Tumorales CultivadasRESUMEN
The human excision repair gene ERCC-1 was cloned after DNA mediated gene transfer to the CHO mutant 43-3B, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-1 cDNA and partial characterization of the gene. ERCC-1 has a size of 15 kb and is located on human chromosome 19. The ERCC-1 precursor RNA is subject to alternative splicing of an internal 72 bp coding exon. Only the cDNA of the larger 1.1 kb transcript, encoding a protein of 297 amino acids, was able to confer resistance to ultraviolet light and mitomycin-C on 43-3B cells. Significant amino acid sequence homology was found between the ERCC-1 gene product and the yeast excision repair protein RAD10. The most homologous region displayed structural homology with DNA binding domains of various polypeptides.
Asunto(s)
Aspartato Carbamoiltransferasa , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante) , Deleción Cromosómica , Clonación Molecular , Reparación del ADN , ADN/análisis , Dihidroorotasa , Genes Fúngicos , Genes , Complejos Multienzimáticos , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Mapeo Cromosómico , Cricetinae , Cricetulus , Proteínas de Unión al ADN/análisis , Prueba de Complementación Genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Poli A/metabolismo , Proteínas/análisis , ARN/metabolismo , ARN MensajeroRESUMEN
Nuclear bodies (NBs) are ultrastructurally defined granules predominantly found in dividing cells. Here we show that PML, a protein involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), is specifically bound to a NB. PML and several NB-associated proteins, found as auto-antigens in primary biliary cirrhosis (PBC), are co-localized and co-regulated. The APL-derived PML-RAR alpha fusion protein is shown to be predominantly localized in the cytoplasm, whereas a fraction is nuclear and delocalizes the NB antigens to multiple smaller nuclear clusters devoid of ultrastructural organization. RA administration (which in APL patients induces blast differentiation and consequently complete remissions) causes the re-aggregation of PML and PBC auto-antigens onto the NB, while PML-RAR alpha remains mainly cytoplasmic. Thus, PML-RAR alpha expression leads to a RA-reversible alteration of a nuclear domain. These results shed a new light on the pathogenesis of APL and provide a molecular link between NBs and oncogenesis.
Asunto(s)
Cromosomas Humanos Par 15 , Cromosomas Humanos Par 17 , Gránulos Citoplasmáticos/fisiología , Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias , Proteínas Nucleares , Factores de Transcripción/metabolismo , Translocación Genética , Tretinoina/farmacología , Animales , Células CHO , Cricetinae , Gránulos Citoplasmáticos/efectos de los fármacos , Humanos , Microscopía Inmunoelectrónica , Proteína de la Leucemia Promielocítica , Receptores de Ácido Retinoico/análisis , Receptores de Ácido Retinoico/biosíntesis , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/análisis , Factores de Transcripción/biosíntesis , Transfección , Proteínas Supresoras de TumorRESUMEN
In this report the genomic characterization of the human excision repair gene ERCC-1 is presented. The gene consists of 10 exons spread over approximately 15 kb. By means of a transfection assay the ERCC-1 promoter was confined to a region of +/- 170 bp upstream of the transcriptional start site. Classical promoter elements like CAAT, TATA and GC-boxes are absent from this region. Furthermore, ERCC-1 transcription is not UV-inducible. A possible explanation is provided for the previously reported alternative splicing of exon VIII. Analysis of ERCC-1 cDNA clones revealed the occurrence of differential polyadenylation which gives ERCC-1 transcripts of 3.4 and 3.8 kb in addition to the major 1.1 kb mRNA. Apparent evolutionary conservation of differential polyadenylation of ERCC-1 transcripts suggests a possible role for this mode of RNA processing in the ERCC-1 repair function.
Asunto(s)
Reparación del ADN , Genes , Animales , Secuencia de Bases , Línea Celular , ADN/aislamiento & purificación , Células HeLa/metabolismo , Humanos , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Transfección , Rayos UltravioletaRESUMEN
Previously the human nucleotide excision repair gene ERCC3 was shown to be responsible for a rare combination of the autosomal recessive DNA repair disorders xeroderma pigmentosum (complementation group B) and Cockayne's syndrome (complementation group C). The human and mouse ERCC3 proteins contain several sequence motifs suggesting that it is a nucleic acid or chromatin binding helicase. To study the significance of these domains and the overall evolutionary conservation of the gene, the homolog from Drosophila melanogaster was isolated by low stringency hybridizations using two flanking probes of the human ERCC3 cDNA. The flanking probe strategy selects for long stretches of nucleotide sequence homology, and avoids isolation of small regions with fortuitous homology. In situ hybridization localized the gene onto chromosome III 67E3/4, a region devoid of known D.melanogaster mutagen sensitive mutants. Northern blot analysis showed that the gene is continuously expressed in all stages of fly development. A slight increase (2-3 times) of ERCC3Dm transcript was observed in the later stages. Two almost full length cDNAs were isolated, which have different 5' untranslated regions (UTR). The SD4 cDNA harbours only one long open reading frame (ORF) coding for ERCC3Dm. Another clone (SD2), however, has the potential to encode two proteins: a 170 amino acids polypeptide starting at the optimal first ATG has no detectable homology with any other proteins currently in the data bases, and another ORF beginning at the suboptimal second startcodon which is identical to that of SD4. Comparison of the encoded ERCC3Dm protein with the homologous proteins of mouse and man shows a strong amino acid conservation (71% identity), especially in the postulated DNA binding region and seven 'helicase' domains. The ERCC3Dm sequence is fully consistent with the presumed functions and the high conservation of these regions strengthens their functional significance. Microinjection and DNA transfection of ERCC3Dm into human xeroderma pigmentosum (c.g. B) fibroblasts and group 3 rodent mutants did not yield detectable correction. One of the possibilities to explain these negative findings is that the D.melanogaster protein may be unable to function in a mammalian repair context.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Xerodermia Pigmentosa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Southern Blotting , Clonación Molecular , Secuencia Conservada , ADN , Proteínas de Unión al ADN/metabolismo , Prueba de Complementación Genética , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Glándulas Salivales/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The chromosomal localizations of two closely related human DNA repair genes, HHR6A and HHR6B, were determined by in situ hybridization with biotinylated probes. HHR6A and HHR6B (human homolog of yeast RAD6) encode ubiquitin-conjugating enzymes (E2 enzymes), likely to be involved in postreplication repair and induced mutagenesis. The HHR6B gene was assigned to human chromosome 5q23-q31, whereas the HHR6A gene was localized on the human X chromosome (Xq24-q25). This latter assignment was confirmed with an X-specific human-mouse/hamster somatic cell hybrid panel. Southern blot analysis points to an X and an autosomal localization of HHR6A and HHR6B, respectively, in the mouse. The potential involvement of these genes in human genetic disorders is discussed.