RESUMEN
Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the digestive tract, are believed to arise from the interstitial cells of Cajal. GISTs are characterized by mutations in the proto-oncogene KIT that lead to constitutive activation of its tyrosine kinase activity. The tyrosine kinase inhibitor STI571, active against the BCR-ABL fusion protein in chronic myeloid leukemia, was recently shown to be highly effective in GISTs. We used 13,826-element cDNA microarrays to define the expression patterns of 13 KIT mutation-positive GISTs and compared them with the expression profiles of a group of spindle cell tumors from locations outside the gastrointestinal tract. Our results showed a remarkably distinct and uniform expression profile for all of the GISTs. In particular, hierarchical clustering of a subset of 113 cDNAs placed all of the GIST samples into one branch, with a Pearson correlation >0.91. This homogeneity suggests that the molecular pathogenesis of a GIST results from expansion of a clone that has acquired an activating mutation in KIT without the extreme genetic instability found in the common epithelial cancers. The results provide insight into the histogenesis of GIST and the clinical behavior of this therapeutically responsive tumor.
Asunto(s)
Neoplasias Gastrointestinales/genética , Mutación , Proteínas Proto-Oncogénicas c-kit/genética , Sarcoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Femenino , Neoplasias Gastrointestinales/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Sarcoma/metabolismo , Células del Estroma/patologíaRESUMEN
Insulin-like growth factor-binding protein 6 (IGFBP6), an extracellular protein with preferential affinity for insulin-like growth factor (IGF) II, belongs to a family of binding proteins with at least six members. We have characterized the genomic structure and the chromosomal location of the human IGFBP6, which is present in the human genome as a single-copy gene spanning 4.7 kb. It consists of four exons, encoding the translated regions, with sizes of 334, 146, 120, and 123 bp, while the intervening introns are 2661, 182, and 844 bp. Three mRNA cap sites were localized 101, 100, and 96 bp upstream of the ATG translation start codon as determined by S1 nuclease analysis. The proximal 5'-flanking region does not have any TATA or CAAT consensus sequences. The IGFBP6 was localized to Chr 12 by analysis of somatic cell hybrids and regionalized to 12q13 by fluorescence DNA in situ hybridization.
Asunto(s)
Cromosomas Humanos Par 12 , Genoma Humano , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Secuencia de Aminoácidos , Mapeo Cromosómico , Exones/genética , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , ARN Mensajero/genética , Análisis de SecuenciaRESUMEN
The insulin response element (IRE) of the human insulin-like growth factor-binding protein-1 (IGFBP-1) promoter contains a palindrome of the T(A/G)TTT sequence crucial to hormonal regulation of many genes. In initial studies of how this IRE participates in hormonal regulation, the electromobility shift assay was used under a variety of conditions to identify IRE-binding proteins. An exhaustive search identified five proteins that specifically bind this IRE; purified proteins were used to show that all five are related to either the high mobility group I/Y (HMGI/Y) or hepatic nuclear factor 3 (HNF3) protein families. Further studies used purified HNF3 and HMGI proteins to show: 1) eah protects the IGFBP-1 IRE from deoxyribonuclease I (DNaseI) digestion; and 2) HNF3 but not HMGI/Y binds to the related phosphoenolpyruvate carboxykinase and Apo CIII IREs. A series of IRE mutants with variable responsiveness to insulin were used to show that the presence of a TGTTT sequence in the mutants did parallel, but HMGI/Y and HNF3 binding to the mutants did not parallel, the ability of the mutants to confer the inhibitory effect of insulin. In contrast, HNF3 binding to these IRE mutants roughly correlates with response of the mutants to glucocorticoids. The way by which HNF3 and/or other as yet unidentified IRE-binding proteins confer insulin inhibition to IGFBP-1 transcription and the role of HMGI/Y in IRE function have yet to be established.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción , Secuencia de Bases , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patología , ADN/análisis , ADN/química , ADN/genética , Proteínas de Unión al ADN/análisis , Proteína HMGA1a , Factor Nuclear 3-alfa del Hepatocito , Proteínas del Grupo de Alta Movilidad/análisis , Humanos , Insulina/farmacología , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patología , Mutación , Proteínas Nucleares/análisis , Oligonucleótidos/análisis , Oligonucleótidos/química , Oligonucleótidos/genética , Unión Proteica , Transfección , Células Tumorales CultivadasRESUMEN
Recent genome searches suggest a putative linkage of many loci to susceptibility to type I diabetes. The chromosome 2q31-35 region is reported to be linked to susceptibility to type I diabetes and is thought to contain several diabetes susceptibility loci. These candidate genes include the HOXD gene cluster, BETA2, CTLA4, CD28, IGFBP2, and IGFBP5. Association studies in populations and families are required to confirm and/or identify the actual susceptibility loci. We hereby report several previously unknown DNA polymorphisms for HOXD8, BETA2, and IGFBP5, which we have used along with previously known polymorphisms of HOXD8 and CTLA4 to test whether these candidate loci are the susceptibility genes on chromosome 2q31-35. Using a case-control design with a subsequent family-association approach to confirm associations, we find no evidence that these candidate genes are associated with susceptibility to type I diabetes.
Asunto(s)
Cromosomas Humanos Par 2/genética , Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 1/genética , Proteínas de Homeodominio/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Transactivadores/genética , Factores de Transcripción/genética , Alelos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Antígenos CD28/genética , Estudios de Casos y Controles , Mapeo Cromosómico , Cartilla de ADN/química , Diabetes Mellitus Tipo 1/inmunología , Familia , Frecuencia de los Genes , Ligamiento Genético , Antígenos HLA-DR/inmunología , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Repeticiones de Microsatélite/genética , Familia de Multigenes , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
A complementary DNA (cDNA) clone encoding chicken insulin-like growth factor binding protein-5 (cIGFBP-5) was isolated. The nucleotide sequence of the 1236-bp clone encodes a mature polypeptide of 251 amino acids with a calculated molecular weight of approximately 28.2 kDa. The deduced amino acid sequence is 83% identical to human IGFBP-5. Labeled cIGFBP-5 cDNA detected a single mRNA transcript of approximately 6 kb by Northern blot analysis of various tissues obtained from embryonic and 6 weeks post-hatch chickens; interestingly, adult heart showed an approximately 10-fold increase in cIGFBP-5 mRNA relative to embryonic heart. The pattern of IGFBP-5 mRNA expression in chicken tissues was similar to that found in mammals during fetal and extrauterine life. In addition, IGFBP-5 mRNA was abundant in primary cultures of chicken myoblasts throughout in vitro differentiation and fusion. The conservation of IGFBP-5 primary structure and expression pattern across vertebrate species suggests conservation of important functions during evolution, particularly in muscle differentiation.
Asunto(s)
Pollos/fisiología , Evolución Molecular , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Diferenciación Celular/genética , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Secuencia Conservada , ADN Complementario , Regulación del Desarrollo de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/citología , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , ARN Mensajero/biosíntesis , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Vertebrados/genéticaRESUMEN
An insulin response element (IRE) has been identified approximately 100 base pairs (bp) 5' to the transcription start site of the human insulin-like growth factor binding protein-1 (hIGFBP-1) gene. This cis element appears crucial to the multihormonal regulation of hIGFBP-1 expression in liver, since (i) an intact IRE is required for maximal stimulation of hIGFBP-1 promoter activity by dexamethasone, and (ii) the IRE confers insulin inhibition of both basal and dexamethasone-stimulated hIGFBP-1 promoter activity. Further progress in understanding how the IRE confers insulin and glucocorticoid effects requires identification of transcription factors confering effects of these hormones. D-site binding protein (DBP), and members of the hepatic nuclear factor 3 (HNF 3) and high mobility group I/Y (HMG I/Y) protein families, each known to bind DNA elements similar in sequence to the IRE, were tested for IRE binding. DBP, HMGI and HNF 3 beta each protected the hIGFBP-1 IRE from DNAseI digestion. Additional studies are required to establish whether binding of any of these proteins to the IRE is important to the regulation of hIGFBP-1 expression by insulin and/or glucocorticoids.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Proteína HMGA1a , Factor Nuclear 3-beta del Hepatocito , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Leucina Zippers , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The insulin-like growth factor binding proteins (IGFBPs) have conserved characteristics of their genomic organization, including similar locations of exon borders relative to nucleotides encoding conserved cysteine residues. Furthermore, the human IGFBP genes, as well as the human homeobox (HOX) genes, are localized to chromosomes 2, 7, 12, and 17. Although little is known about the evolution of the IGFBP genes, the association of human IGFBP and homeobox (HOX) genes at four chromosomal loci may indicate that their ancestral genes were linked prior to the first duplication of chromosomal DNA containing the ancestral HOX cluster. The hypothesis that IGFBPs are ancient proteins is supported by the reported detection of IGFBP activity in serum from the Agnathan species, Geotria australis, a primitive vertebrate. Further studies of IGFBPs in different species are needed to understand the evolution of this protein/gene family. Chicken provides a good intermediate model, since birds diverged from mammals approximately 300 million years ago. A complementary DNA (cDNA) clone encoding chicken insulin-like growth factor binding protein-5 (cIGFBP-5) was isolated. The deduced amino acid sequence is 83% identical to human IGFBP-5 and encodes a mature polypeptide of 251 amino acids. The conservation of IGFBP-5 primary structure across vertebrate species suggests maintenance of important functions during evolution.
Asunto(s)
Evolución Molecular , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/química , Secuencia de Aminoácidos , Animales , Pollos , Mapeo Cromosómico , Secuencia Conservada , Genes Homeobox , Humanos , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Ratas , Somatomedinas/química , Somatomedinas/genética , XenopusRESUMEN
Glucocorticoids stimulate, while insulin inhibits, the hepatic transcription of insulin-like growth factor-binding protein-1 (IGFBP-1). In the present studies, human HEP G2 hepatoma cells were transiently transfected with human (h)IGFBP-1 promoter constructs. Activity of a construct containing the first 1205 base pairs (bp) of the hIGFBP-1 promoter was stimulated 6-9.5-fold by dexamethasone, and this increase was inhibited approximately 76% by insulin. Deletion and site-directed mutations of the hIGFBP-1 promoter (a) identified two glucocorticoid response elements, located within the first 200 bp of the promoter, which are essential for dexamethasone-stimulated promoter activity and which specifically bind human glucocorticoid receptor; (b) showed that a recently characterized insulin-responsive element, located approximately 110 bp 5' to the transcription start site (Suwanichkul, A., Morris, S.L., and Powell, D. R. (1993) J. Biol. Chem. 268, 17063-17068), confers the entire inhibitory effect of insulin not only on basal but also on glucocorticoid-stimulated promoter activity; and (c) showed that this insulin-responsive element is essential for maximal glucocorticoid-stimulated activity. These studies suggest that the interaction of proteins that bind to a cluster of cis elements located in the first 200 bp of the hIGFBP-1 promoter are of major importance in modulating the opposing effects of glucocorticoids and insulin on hepatic hIGFBP-1 expression.
Asunto(s)
Dexametasona/metabolismo , Regulación de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/genética , Insulina/fisiología , Regiones Promotoras Genéticas , Secuencia de Bases , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Receptores de Glucocorticoides/metabolismo , Células Tumorales CultivadasRESUMEN
To better understand the regulation of insulin-like growth factor binding protein-5 (IGFBP-5) expression, we cloned the IGFBP-5 gene from human genomic libraries and identified a region in the 5' flanking sequence which functions as a promoter. The human IGFBP-5 gene is divided into four exons which, primarily due to a first intron of approximately 25 kilobases, span approximately 33 kilobases of DNA. Southern analysis identified a single copy of the IGFBP-5 gene in the haploid human genome, and several independent mapping strategies found this gene tightly linked with, and in opposite transcriptional orientation to, the IGFBP-2 gene at chromosomal region 2q33-34. Primer extension studies identified the IGFBP-5 mRNA cap site 772 base pairs (bp) 5' to the first nucleotide of the translation start codon. Analysis of the 5'-flanking sequence identified a potential TATA element beginning 33 bp 5' to the mRNA cap site. When a DNA fragment containing this cap site and 461 bp of upstream sequence was placed 5' to the chloramphenicol acetyltransferase reporter gene and transfected into MDA-MB-468 human breast cancer cells, it directed chloramphenicol acetyltransferase expression in an orientation-specific manner, suggesting that this region contains elements essential for IGFBP-5 promoter activity.
Asunto(s)
Proteínas Portadoras/genética , Cromosomas Humanos , Regiones Promotoras Genéticas , Somatomedinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/metabolismo , Células Cultivadas , Mapeo Cromosómico , ADN Complementario , Humanos , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina , Datos de Secuencia Molecular , ARN Mensajero/metabolismoRESUMEN
The insulin-like growth factor binding proteins (IGFBPs) constitute a structurally related protein family with six known members. We have mapped and regionally localized the genes coding for human IGFBP1, 2, 3, and 4, by in situ hybridization and somatic cell hybrid analysis. The IGFBP2 gene maps to chromosomal region 2q33-q34 whereas the genes for IGFBP1 and 3 are localized in a tail-to-tail fashion on chromosome 7 region p14-p12. The IGFBP4 gene is located in the chromosomal region 17q12-21.1. Structural characterization of the genes coding for IGFBP-1, 2, 3, and 5 showed that the translated parts are divided into four exons. The exons are similar both in size and sequence in all studied genes.
Asunto(s)
Proteínas Portadoras/genética , Mapeo Cromosómico , Somatomedinas , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/análisis , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 7 , ADN/análisis , ADN/aislamiento & purificación , Humanos , Células Híbridas , Hibridación in Situ , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Insulin-like growth-factor-binding proteins (IGFBPs) constitute a family of structurally related proteins that specifically bind insulin-like growth factors and modulate their functions. In this study, the chromosomal localization was determined for the gene encoding IGFBP4, i.e. inhibitory-IGFBP. A polymerase chain reaction (PCR) fragment corresponding to the previously published cDNA sequence was used to isolate overlapping cosmid clones. By fluorescent in situ hybridization to metaphase chromosomes, the IGFBP4 gene was then localized to chromosomal region 17q21-21.1. This result was in agreement with PCR analysis of a panel of somatic cell hybrids.