RESUMEN

Cell cycle withdrawal involves several transcription factors such as E2Fs members that play a key role in cell growth control. Here we describe a novel putative bZIP transcription factor isolated from the retina and involved in neuronal proliferation arrest at the terminal differentiation: PATF (Proliferation Arrest Transcription Factor). We show that PATF associates with E2F4 protein and interacts with the E2F consensus site. PATF expression increases with establishment of quiescent state. Furthermore, the nuclear PATF localization like E2F4, depends on cell growth arrest. The decrease of PATF amount, using a retroviral antisense strategy, results in pursued neuroretina cell mitosis. Our results indicate that PATF could be a new molecular signal implicated in the final neuronal cell cycle withdrawal.

Asunto(s)

Proteínas Aviares , Neuronas/citología , Retina/embriología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , División Celular , Núcleo Celular/metabolismo , Embrión de Pollo , Clonación Molecular , Secuencia de Consenso , ADN sin Sentido/farmacología , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F4 , Proteínas del Ojo/genética , Proteínas del Ojo/fisiología , Humanos , Datos de Secuencia Molecular , Neuronas/metabolismo , Células PC12 , ARN Mensajero/biosíntesis , Conejos , Ratas , Retina/citología , Factores de Transcripción/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas

RESUMEN

Neuroblastoma is a very common solid tumor which arises in childhood and shows an extreme heterogeneity at the clinical, histological and genetic levels. Besides age and stage, N-myc amplification and 1p deletion are prognostic factors of the disease: in Europe, these genetic markers are used to conduct therapy. In France, N-myc amplification is a factor of bad prognosis which leads, in all forms of the disease including localised forms and metastatic forms of children aged of less than 1 year, to a myeloablative treatment with autologous hematopoietic stem cells transplantation. By contrast, N-myc amplification has no impact on the survival of children aged of more than 1 year with a poor prognosis (30% overall survival, 5 years) but this genetic abnormality is taken into account to treat primary tumor of these patients. In an attempt to find out prognostic factors of these aggressive forms of the disease, various pathways (apoptosis, differentiation angiogenesis, detoxication, immune response) have been recently surveyed, but studies have been carried out on a limited number of genes. Moreover, experimental models of human metastatic neuroblastoma have been obtained in which variations of genes transcript levels involved in these pathways, are observed. The current break-through of cDNA microarrays allows to develop a dynamic transcriptomic scanning of these models as well as of tumors and bone marrows from patients upon conventional chemotherapy. This technology will enable: i) to define molecular entities of the metastatic disease; ii) to apply adapted treatment; iii) to develop new therapeutic strategies.

Asunto(s)

Genes myc/genética , Neuroblastoma/genética , Factores de Edad , Animales , Niño , Preescolar , Amplificación de Genes , Humanos , Lactante , Ratones , Modelos Animales , Proteínas de Neoplasias/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/secundario , Pronóstico

RESUMEN

The metabolic conversion of n-3 fatty acids was studied in the human Y79 retinoblastoma cell line. Cultured cells were exposed to increasing concentrations of either 18:3n-3, 22:5n-3, or 22:6n-3, and their phospholipid fatty acid composition was analyzed after 72 hr. Cells internalized the supplemental fatty acids and proceeded to their metabolic conversion. Supplemental 22:6n-3 was directly esterified into cell phospholipids, at levels typical for normal neural retinas (41% by weight of phosphatidylethanolamine fatty acids, and 24% of phosphatidylcholine fatty acids). In contrast, 18:3n-3 was mainly converted to 20:5n-3 and 22:5n-3, both of which appeared in cell phospholipids after exposure to low external concentrations of 18:3n-3 (10 microg/ml). Y79 cells can proceed to the metabolic conversion of 18:3n-3 through elongation and Delta6- and Delta5-desaturation. When cells were exposed to high external concentrations of 18:3n-3 (30 microg/ml), the supplemental fatty acid was directly incorporated, and its relative content increased in both phospholipid classes to the detriment of all other n-3 fatty acids. Cells cultured in the presence of 22:5n-3 did not incorporate 22:6n-3 into their phospholipids but did incorporate 20:5n-3 and 22:5n-3. The data suggest that Y79 cells can proceed to the microsomal steps of n-3 metabolism, involving elongation, desaturation, and chain shortening of 22C fatty acids. Although Y79 cells avidly used supplemental 22:6n-3 for phospholipid incorporation at levels typical for normal photoreceptor cells, they failed to match such levels through metabolic conversion of n-3 parent fatty acids. The terminal step of the very long-chain polyunsaturated fatty acid synthesis, consisting in Delta6-desaturation followed by peroxisomal chain shortening of 24C-fatty acids, could be rate-limiting in Y79 cells.

Asunto(s)

Ácidos Grasos Omega-3/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfolípidos/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Retinoblastoma , Factores de Tiempo , Células Tumorales Cultivadas , Ácido alfa-Linolénico/metabolismo

Asunto(s)

Proteínas de Unión al ADN/fisiología , Genes de Retinoblastoma/fisiología , Neuroblastoma/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Represoras/fisiología , Proteína de Retinoblastoma/fisiología , Factores de Transcripción , Animales , Humanos , Lactante , Proteína 2 Inhibidora de la Diferenciación , Ratones , Transcripción Genética

RESUMEN

Fibroblast growth factor 1 (FGF1) is a multipotent factor in the development and differentiation of the central nervous system. Recent studies in PC12 cells attribute these effects to high endogenous FGF1 expression. To examine the differentiation mechanisms induced by FGF1, we performed studies in SH-SY5Y human neuroblastoma cells. We monitored the impact of FGF1 overexpression in SH-SY5Y either after addition of exogenous FGF1 and heparin or after stable transfection with the FGF1 eukaryotic expression vector. Under both conditions, the FGF1 endogenous rise caused SH-SY5Y cell differentiation with morphological changes (appearance of neuritic extensions), increased GAP-43 gene expression, decreased of N-myc gene expression, and prolonged long-term survival in serum-free media. These modifications were correlated with Bcl-2 upregulation. These results suggest that there is a link between the endogenous FGF1 signaling pathway and Bcl-2 in neuronal survival modulation.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Bases , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Cartilla de ADN , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteína GAP-43/genética , Genes myc , Humanos , Neuritas/efectos de los fármacos , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , Células Tumorales Cultivadas

RESUMEN

Alpha1-Microglobulin and bikunin are two plasma glycoproteins encoded by a gene for alpha1-microglobulin/bikunin precursor (AMBP). The strict liver-specific transcription of the AMBP gene is controlled by an elaborate and remote enhancer made of six clustered boxes numbered 1 to 6 (core enhancer) that are binding sites for the hepatocyte-enriched nuclear factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3 and HNF-4 respectively. Three further boxes, 7 to 9, have now been found in the enhancer area in a position 5' of box 2, 5' of box 1 and 3' of box 6, respectively. Electrophoretic mobility-shift assays with nuclear extracts from the HepG2 hepatoma cell line demonstrated that boxes 7 and 8 are both functional HNF-4-binding sites of high and low affinity respectively, whereas no binding capacity of box 9 was detected by this method. Transfection of HepG2 and Chinese hamster ovary cells with chloramphenicol acetyltransferase constructs harbouring the core or extended AMBP enhancer with wild-type or mutated boxes and co-transfection with expression plasmids for a wild-type or defective HNF-4 identified box 7 as an essential element for the basal activity of this enhancer. The response of boxes 7 and 8 varies with the level of HNF-4 in cells. Box 9 exhibits a repressor activity that can be detected when box 8 is ablated. In vivo this corresponds to conditions of low box 8 occupancy when the intracellular level of HNF-4 is limited. These results reinforce the view that the AMBP enhancer is a quite elaborate and unusual example of a modular enhancer whose activity is fine-tuned by the level of cognate nuclear factors in the cell.

Asunto(s)

alfa-Globulinas/genética , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Glicoproteínas/genética , Hígado/metabolismo , Glicoproteínas de Membrana , Fosfoproteínas/metabolismo , Precursores de Proteínas/genética , Factores de Transcripción/metabolismo , Inhibidor de la Tripsina de Soja de Kunitz , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión/genética , Células CHO , Línea Celular , Cricetinae , ADN/genética , ADN/metabolismo , Factor Nuclear 4 del Hepatocito , Humanos , Cinética , Datos de Secuencia Molecular , Activación Transcripcional , Transfección

RESUMEN

Inter-alpha-inhibitor (IalphaI) and related molecules, collectively referred to as the IalphaI family, are a group of plasma protease inhibitors. They display attractive features such as precursor polypeptides that give rise to mature chains with quite distinct fates and functions, and inter-chain glycosaminoglycan bonds within the various molecules. The discovery of an ever growing number of such molecules has raised pertinent questions about their pathophysiological functions. The knowledge of this family has long been structure-oriented, whereas the structure/function and structure/regulation relationships of the family members and their genes have been largely ignored. These relationships are now being elucidated in events such as gene transcription, precursor processing, changes in plasma protein levels in health and disease and binding capacities that involve hyaluronan as well as other plasma proteins as ligands. This review presents some recent progress made in these fields that paves the way for an understanding of the functions of IalphaI family members in vivo. Finally, given the wealth of heterogeneous, complicated and sometimes contradictory nomenclatures and acronyms currently in use for this family, a new, uniform, nomenclature is proposed for IalphaI family genes, precursor polypeptides and assembled proteins.

Asunto(s)

alfa-Globulinas/fisiología , Inhibidores de Tripsina/farmacología , alfa-Globulinas/genética , alfa-Globulinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Quimotripsina/antagonistas & inhibidores , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Relación Estructura-Actividad , Inhibidores de Tripsina/genética , Inhibidores de Tripsina/metabolismo

RESUMEN

The inter-alpha-inhibitor (I alpha I) family is comprised of the plasma protease inhibitors I alpha I, inter-alpha-like inhibitor (I alpha LI), pre-alpha-inhibitor (P alpha I) and bikunin. I alpha I, I alpha LI and P alpha I are distinct assemblies of bikunin with one of three heavy (H) chains designated H1, H2 and H3. These H chains and bikunin are respectively encoded by a set of three H genes and an alpha 1-microglobulin/bikunin precursor (AMBP) gene. All four gene products undergo maturation steps from precursor polypeptides. The full-length cDNAs for the H1-, H2- and H3-chain precursors were cloned from a mouse liver cDNA library and sequenced. Extensive searches of amino acid sequence similarities to other proteins in databanks revealed (i) a highly significant similarity of the C-terminal sequence in the three H-chain precursors to the multicopper-binding domain in the group of multicopper oxidase proteins and (ii) the presence of von Willebrand type-A domains in the mature H chains. Amino acid sequence comparisons between the three mouse H1-, H2- and H3-chain precursors and their human counterparts allowed us to appraise the timing and order of occurrence of the three H-chain genes from a shared ancestor during mammalian evolution. Owing to a multiple alignment of the six mouse and human nucleotide sequences for these H-chain precursors, a reverse transcriptase PCR assay with degenerate oligonucleotides was designed, allowing us to (i) present evidence that no mRNAs for further H genes exist in mouse liver and (ii) demonstrate a previously undescribed transcription of the H2- and H3-chain mRNAs in mouse brain, which contrasts with the expression of all four, H1, H2, H3 and AMBP, mRNAs in liver.

Asunto(s)

alfa-Globulinas/genética , Encéfalo/metabolismo , Hígado/metabolismo , Oxidorreductasas/química , Precursores de Proteínas/genética , Transcripción Genética , alfa-Globulinas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Química Encefálica , ADN Complementario/química , Humanos , Hígado/química , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/química , ARN Mensajero/análisis , ADN Polimerasa Dirigida por ARN , Homología de Secuencia , Factor de von Willebrand/química

RESUMEN

Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4.

Asunto(s)

alfa-Globulinas/genética , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos/genética , Glicoproteínas/genética , Glicoproteínas de Membrana , Proteínas Nucleares , Fosfoproteínas , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Inhibidor de la Tripsina de Soja de Kunitz , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Células CHO , Cricetinae , ADN/metabolismo , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Factor Nuclear 4 del Hepatocito , Humanos , Hígado/citología , Hígado/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Precursores de Proteínas/genética , Análisis de Secuencia de ADN , Transfección , Células Tumorales Cultivadas

RESUMEN

The inter-alpha-inhibitor family is composed of the plasma-protease inhibitors inter-alpha-inhibitor, pre-alpha-inhibitor and bikunin. Inter-alpha-inhibitor and pre-alpha-inhibitor are distinct assemblies of bikunin with distinct sets from three heavy (H) chains designated H1, H2 and H3. These H chains are encoded by a set of three evolutionarily related H genes, and bikunin by an alpha-1-microglobulin/bikunin precursor gene (AMBP). This precursor is cleaved to yield bikunin, a member of the Kunitz-type protease-inhibitor superfamily, and alpha-1-microglobulin, which belongs to the lipocalin superfamily. Northern-blot experiments with RNAs obtained from various tissues in fetal and in adult mice indicated that the transcription of the four AMBP and H genes is liver-restricted, although there is expression of H3 in brain. An analysis of the H1, H2, H3 and AMBP transcripts, as well as of transcripts for other control genes, in liver during development showed a progressive increase in the amounts of the H1, H2, H3 and AMBP RNAs, which all peak transiently at day 5 after birth. This was shown by a nuclear run-on experiment to originate from a change in transcription rate. The transient and postnatal increase in transcription could be explained neither by the liver-restricted expression nor by a common origin of these four genes, nor by a perinatal requirement for many lipocalins or protease inhibitors. This suggests that all four genes are perinatally triggered at the level of similar elements in their transcriptional regulatory regions, a conclusion strengthened by the weak expression of the four genes that is seen in a mutant mouse strain (albino) that is deficient in some liver-specific transcription factors.

Asunto(s)

Envejecimiento/fisiología , alfa-Globulinas/genética , Regulación de la Expresión Génica , Hígado/metabolismo , Glicoproteínas de Membrana , Ratones Endogámicos , Familia de Multigenes , Inhibidor de la Tripsina de Soja de Kunitz , Inhibidores de Tripsina/genética , Albinismo/genética , alfa-Globulinas/biosíntesis , Animales , Animales Recién Nacidos , Femenino , Feto , Eliminación de Gen , Edad Gestacional , Glicoproteínas/genética , Humanos , Hígado/embriología , Hígado/crecimiento & desarrollo , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos BALB C/genética , Ratones Endogámicos C3H/genética , Ratones Endogámicos C57BL/genética , Ratones Endogámicos/genética , Precursores de Proteínas/genética , Ratas , Transcripción Genética

Asunto(s)

Cloranfenicol O-Acetiltransferasa/genética , beta-Galactosidasa/genética , Automatización , Células Cultivadas , Técnicas Genéticas , Sensibilidad y Especificidad , Transfección

RESUMEN

alpha 1-Microglobulin (A1M) and bikunin are plasma proteins which are present both as free molecules and as complexes with either IgA heavy chains for A1M or the H1, H2, and H3 heavy chains of the inter-alpha-inhibitor family for bikunin. Mature A1M and bikunin originate from the cleavage of an A1M/bikunin precursor (ABP) synthesized from a single gene with liver-specific expression. Five kilobases of the 5'-flanking region of the human ABP gene were sequenced. Deletion mutants of this region subcloned upstream of a CAT reporter gene were transfected into HepG2 hepatoma cells. A segment covering the -2.7- to -2.8-kb area is required for full activity of the ABP gene. This segment contains a cluster of six elements (boxes 1-6, 5' to 3') which are potential binding sites for the liver-enriched trans-acting factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3, and HNF-4, respectively. This cluster enhances the activity of heterologous minimal promoters in a position- and distance-independent fashion in HepG2 cells. This enhancer activity is restricted to liver cells as the cluster is unable to activate promoters in Chinese hamster ovary (CHO) or HeLa cells. By band-shift experiments we have shown that the liver-enriched transcription factors HNF-1, or HNF-3, do bind to boxes 1 and 4, or 3, respectively. The combination of a weak promoter and a strong distant and liver-specific enhancer distinguishes the ABP gene from most other plasma protein genes expressed in hepatocytes.

Asunto(s)

alfa-Globulinas/genética , Elementos de Facilitación Genéticos , Glicoproteínas/genética , Hígado/fisiología , Glicoproteínas de Membrana , Inhibidores de Proteasas , Transcripción Genética , Inhibidor de la Tripsina de Soja de Kunitz , Secuencia de Bases , Carcinoma Hepatocelular , Núcleo Celular/fisiología , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , Expresión Génica , Humanos , Neoplasias Hepáticas , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Regiones Promotoras Genéticas , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Eliminación de Secuencia , Transfección , Células Tumorales Cultivadas

RESUMEN

The inter-alpha-inhibitor (I alpha I) and pre-alpha-inhibitor (P alpha I) family is composed of three plasma protease inhibitors, I alpha I, P alpha I, and bikunin, whose chains are encoded by a set of three evolutionarily related heavy (H) chain genes designated H1, H2, and H3 and a fourth gene, the so-called alpha 1-microglobulin/bikunin precursor (AMBP) gene. The latter codes for a precursor that splits into: (i) alpha 1-microglobulin, which belongs to the lipocalin superfamily; and (ii) bikunin, which is made up of two tandemly arranged protease inhibitor domains and belongs to the superfamily of Kunitz-type protease inhibitors. The bikunin chain is found in I alpha I and P alpha I molecules and it is also present as a free molecule in plasma. In human, the AMBP and H2 genes have been mapped to 9q32-q34 and 10p14-p15, respectively, while the H1 and H3 genes are tandemly located at 3p21.1-p21.2. In situ hybridization mappings indicate that the mouse AMBP gene (Intin-4) is located at 4C1----C4, and the H1 (Intin-1) and H3 (Intin-3) genes are colocated at 14A2----C1. In interspecific backcrosses (C57BL/6Pas x Mus spretus) a TaqI restriction variant in (and/or near) the H2 (Intin-2) gene identified a linkage of this gene with other polymorphic loci, which assigns Intin-2 to the centromeric area of chromosome 2. All such assignments are in conserved chromosomal regions between human and mouse. Therefore the genetic events that gave rise to the four I alpha I family genes took place prior to the divergence between human and mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

alfa-Globulinas/genética , Ratones/genética , Precursores de Proteínas/genética , Inhibidores de Tripsina/genética , Animales , Southern Blotting , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 9 , Sondas de ADN , Electroforesis en Gel de Agar , Haplotipos , Humanos , Ratones Endogámicos C57BL , Muridae , Hibridación de Ácido Nucleico