Asunto(s)

Perfilación de la Expresión Génica/métodos , Genes ras , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Bases de Datos Factuales , Humanos , Ratones , Fenotipo , Plásmidos , ARN/genética , Receptores de Esteroides/genética , Transcripción Genética , Transfección

Asunto(s)

Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Proteínas/genética , Proteínas Recombinantes/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteína de Unión al GTP rac1/genética

Asunto(s)

Proteínas de Unión al GTP/fisiología , Proteínas de Neoplasias/fisiología , Proteínas/fisiología , Transducción de Señal/fisiología , Células 3T3 , Animales , Membrana Celular/ultraestructura , Mapeo Cromosómico , Cromosomas Humanos Par 21/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/fisiología , Factores de Intercambio de Guanina Nucleótido , Guanosina Trifosfato/fisiología , Humanos , Linfoma de Células T/genética , Linfoma de Células T/patología , Ratones , Modelos Biológicos , Mutagénesis , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/fisiopatología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Proteínas/genética , Homología de Secuencia , Transducción de Señal/genética , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteínas de Unión al GTP rac , Proteína de Unión al GTP rhoA

RESUMEN

We have recently identified the invasion-inducing Tiam1 gene by proviral insertional mutagenesis. The Tiam1 protein shares a Dbl homology (DH) domain with an increasing number of oncoproteins, some of which have been shown to function as GDP dissociation stimulators (GDS) for small GTPases of the Rho family. In vitro and in vivo analyses indicate that Tiam1 activates the Rho like GTPase Rac1. Here we have analysed the consequences of overexpression of several mutant Tiam1 proteins in NIH3T3 fibroblasts. Similar to other proteins containing a DH domain, N-terminal truncation of the Tiam1 protein activates its oncogenic potential, establishing Tiam1 as a proto-oncogene. In addition, we show the sequences N-terminal of the catalytic DH domain are required for morphological transformation accompanied by the formation of membrane ruffling, but not for the induction of an oncogenic phenotype. Overexpression of constitutively active Rac1 (V12Rac1) in NIH3T3 cells produces a similar oncogenic phenotype, suggesting that the oncogenic effects of Tiam1 are a consequence of Rac activation.

Asunto(s)

Transformación Celular Neoplásica/genética , Proteínas de Unión al GTP/genética , Proteínas/genética , Células 3T3 , Animales , ADN Complementario , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido , Ratones , Ratones Desnudos , Proteínas/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Transfección , Proteínas de Unión al GTP rac

RESUMEN

Rho-like GTPases have been implicated in the regulation of the actin cytoskeleton which controls the morphology, adhesion and motility of cells. Like Ras proteins, they become activated when bound GDP is exchanged for GTP, a process catalysed by GDP-dissociation stimulator (GDS) proteins. Several GDS proteins specific for Rho-like GTPases have been identified. Most of these contain a conserved catalytic domain, the DBL-homology (DH) domain, and activate Cdc42 or Rho but not Rac. We have isolated the invasion-inducing Tiam1 gene, which also encodes a protein with a DH domain. Here we show that Tiam1 is a GDS protein for Rho-like GTPases in vitro. In fibroblasts, Tiam1 induces a similar phenotype as constitutively activated (V12)Rac1, including membrane ruffling, and this is inhibited by dominant negative (N17)Rac1. Moreover, T-lymphoma cells expressing V12Rac1 become invasive, indicating that the Tiam1-Rac signalling pathway could be operating in the invasion and metastasis of tumour cells.

Asunto(s)

Proteínas de Unión al GTP/fisiología , Invasividad Neoplásica , Proteínas/fisiología , Células 3T3 , Animales , Línea Celular , Membrana Celular/fisiología , Transformación Celular Neoplásica , Fibroblastos/fisiología , GTP Fosfohidrolasas/fisiología , Glutatión Transferasa/metabolismo , Factores de Intercambio de Guanina Nucleótido , Guanosina 5'-O-(3-Tiotrifosfato)/fisiología , Guanosina Difosfato/fisiología , Ratones , Metástasis de la Neoplasia , Proteínas Recombinantes de Fusión , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Transfección , Células Tumorales Cultivadas , Proteínas de Unión al GTP rac , Proteínas de Unión al GTP rap , Proteína de Unión al GTP rhoA

RESUMEN

By means of proviral tagging in combination with in vitro selection for invasive T-lymphoma variants, we have previously identified the murine invasion- and metastasis-inducing Tiam1 gene. Tiam1 encodes a novel protein which shares a Dbl-homology (DH) domain with GDP dissociation stimulator-(GDS) proteins that activate Rho-like GTPases. We have cloned the human TIAM1 coding sequence and studied its evolutionary conservation and expression pattern. TIAM1 is highly conserved among vertebrates. The close similarity between human TIAM1 and the mouse homologue is indicated by 88% and 95% identity of nucleotides and predicted sequences, respectively. The murine gene is highly expressed in brain and testis and at low or moderate levels in almost all other normal tissues. Interestingly, Tiam1 transcripts were found in virtually all analysed tumor cell lines of human and rodent origin including B- and T-lymphomas, neuroblastomas, melanomas and carcinomas. The evolutionary conservation as well as the broad expression pattern of Tiam1 in most normal tissues, suggests a general function in cellular signaling processes presumably by activation of a Rho-like GTPase that regulates the cytoskeletal organization.

Asunto(s)

Proteínas/genética , Secuencia de Aminoácidos , Evolución Biológica , Clonación Molecular , ADN Complementario/genética , Regulación Neoplásica de la Expresión Génica , Genes , Factores de Intercambio de Guanina Nucleótido , Humanos , Datos de Secuencia Molecular , Invasividad Neoplásica , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Distribución Tisular

RESUMEN

The murine invasion-inducing Tiam1 gene maps to the distal end of chromosome 16, 3.8 cM centromeric of the Ets2 gene. TIAM1, the human homolog of Tiam1, maps to the syntenic region (q22) on human chromosome 21. The gene order in 21q22 is cen-TIAM1-AML1-ERG-ETS2-tel.

Asunto(s)

Cromosomas Humanos Par 21 , Proteínas/genética , Animales , Mapeo Cromosómico , Femenino , Factores de Intercambio de Guanina Nucleótido , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T

RESUMEN

Using proviral tagging in combination with in vitro selection for invasiveness, we have identified a gene, designated Tiam-1, that affects invasion. In the selected invasive T lymphoma variants, proviral insertions were found within coding exons of the Tiam-1 gene, resulting in both truncated 5'-end and 3'-end transcripts that give rise to N- and C-terminal Tiam-1 protein fragments. In one invasive variant, amplification of the Tiam-1 locus was observed with concomitant increase in the amount of normal Tiam-1 protein. Cell clones that were invasive in vitro produced experimental metastases in nude mice, and transfection of truncated Tiam-1 cDNAs into noninvasive cells made these cells invasive. The predicted Tiam-1 protein harbors a Dbl- and Pleckstrin-homologous domain, which it shares with GDP-GTP exchangers for Rho-like proteins that have been implicated in cytoskeletal organization.

Asunto(s)

Linfoma de Células T/genética , Invasividad Neoplásica/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/análisis , ADN de Neoplasias/análisis , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido , Metástasis Linfática , Linfoma de Células T/microbiología , Linfoma de Células T/patología , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/genética , Mutagénesis Insercional , Proteínas Oncogénicas/genética , Proteínas/química , Proteínas Proto-Oncogénicas/genética , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Células Tumorales Cultivadas , Integración Viral/genética , Proteínas de Unión al GTP rap

RESUMEN

By somatic cell fusion studies between noninvasive mouse T-lymphoma cells and invasive human activated normal T-cells we have previously shown that the genetic information responsible for the induction of invasive and metastatic potential in interspecies T-cell hybrids is located on human chromosome 7. Apparently, genes derived from normal activated T-cells are dominantly expressed in the hybrids and control the invasive and, as a consequence, metastatic potential of these T-lymphoma cells. To sublocalize the invasion-inducing locus on chromosome 7 we have generated hybrids that harbor only specific regions of human chromosome 7 with or without a small fragment of human chromosome 21. Analysis of these hybrids revealed that the invasion-inducing locus maps to 7p12----cen. The human DNA complement of the hybrids was confirmed by Southern blot analysis using a large panel of chromosome 7-specific DNA probes. Several of these genes could be further sublocalized. These included: ARAF2 to 7p12----cen, D7S21 to 7pter----p12, ACTB to 7p15----p12, EGFR to 7p12, MDH2 to 7cen----q22, and PDGFA to 7pter----p15.

Asunto(s)

Cromosomas Humanos Par 7 , Invasividad Neoplásica/genética , Southern Blotting , Sondas de ADN , Fluorescencia , Humanos , Células Híbridas , Hibridación de Ácido Nucleico

RESUMEN

Measurements of plasma viscosity (PV) and erythrocyte sedimentation rate (ESR) are supposed to reflect the complex of acute phase reactants in inflammations. Both tests were prospectively studied at the rheumatology out-patient department with regard to their ability to discriminate between inflammatory and non-inflammatory rheumatic diseases in new patients (n = 235). PV and ESR were measured using the Coulter Viscometer II and the Westergren method, respectively. The Receiver Operating Characteristic curve for PV was slightly better than for ESR. However, at the higher cut-off points (PV greater than 1.81 mPa.s, ESR greater than 25 mm/l hr), sensitivities, specificities, predictive values and odds ratios were more favourable for ESR. Furthermore, since the costs of PV measurement are considerably higher, there is no reason for an implementation of PV in the daily routine of the rheumatologist at the outpatient department.

Asunto(s)

Sedimentación Sanguínea , Viscosidad Sanguínea , Enfermedades Reumáticas/diagnóstico , Reumatología/métodos , Adolescente , Adulto , Anciano , Atención Ambulatoria , Diagnóstico Diferencial , Femenino , Humanos , Inflamación/sangre , Inflamación/diagnóstico , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Análisis de Regresión , Enfermedades Reumáticas/sangre

RESUMEN

Point mutations in codon 12 of the K-ras oncogene are frequent in human lung adenocarcinomas. To study the expression of the K-ras gene in these tumors we have developed a mRNA detection technique based on the polymerase chain reaction (PCR). By this technique, K-ras expression can be detected semi-quantitatively in samples of less than 100 ng total RNA. Hybridization of the amplified cDNA sequences with mutation-specific oligonucleotides allows separate quantification of the expression of normal and point-mutated alleles in a single sample. RNA samples from 24 human non-small-cell lung carcinomas (NSCLC), from 2 lung metastases of colonic adenocarcinomas, from 3 human lung adenocarcinoma cell lines, and from normal lung tissue were analyzed. In most tumors, expression of K-ras was detected at levels equal to or several times higher than those found in normal lung tissue. A lung metastasis from a colon adenocarcinoma, known to contain an amplified K-ras gene, highly over-expressed the K-ras gene. In those tumors in which the K-ras oncogene was activated by a point mutation, both alleles of the gene were expressed. Our results show that a high over-expression of K-ras is a rare event in human lung carcinomas, but that a certain degree of over-expression of the mutated allele can be demonstrated in tumors with an activated K-ras gene. With the technique we describe here, adequate estimation of the expression of specific genes in minimal amounts of tumor cells becomes possible.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/genética , Genes ras , Neoplasias Pulmonares/genética , Adenocarcinoma/genética , Alelos , Secuencia de Bases , Carcinoma/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/genética , Línea Celular , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Expresión Génica , Humanos , Immunoblotting , Neoplasias Pulmonares/patología , Datos de Secuencia Molecular , Mutación , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación

RESUMEN

Non-invasive, non-metastatic mouse BW5147 T-lymphoma cells were treated with non-mutagenic concentrations of the hypomethylating agent 5-azacytidine (5-aza-C). Subsequently, invasive variants were selected on monolayers of rat embryo fibroblasts. The estimated frequency of induction of invasive variants was smaller than 1 in 10(6) cells. We obtained several independent clones that were stable in the expression of the invasive phenotype. In contrast to the parental cell line, the highly invasive clones produced widespread metastases upon tail vein injection in all the syngeneic AKR mice tested, whereas clones with an intermediate level of invasiveness formed metastases only in part of the mice tested. DNA analysis using the methylation-sensitive and insensitive restriction enzymes, Hpa-II and Msp-I, respectively, showed that the DNA of the invasive variants remained hypomethylated, up to 6 months after 5-aza-C treatment. 5-aza-C is thus able to induce invasive and metastatic potential in the BW5147 T-lymphoma cells, similar to the activated human c-Ha-ras oncogene or human chromosome 7, as studied previously. The acquisition of invasive and metastatic potential is presumably caused by DNA hypomethylation and thus activation of one or more silent invasion controlling genes.

Asunto(s)

Azacitidina/farmacología , Linfoma de Células T/patología , Invasividad Neoplásica , Metástasis de la Neoplasia , Animales , ADN/metabolismo , Femenino , Metilación , Ratones , Ratones Endogámicos AKR , Oncogenes

Asunto(s)

Linfoma/genética , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Linfocitos T , Animales , Regulación Neoplásica de la Expresión Génica , Ratones , Oncogenes , Transfección/genética

RESUMEN

We have used DNaseI footprinting and gel mobility assays to analyze the upstream region of the human A gamma-globin gene promoter. Four protein factors were found to bind this region. A non-erythroid factor present in the 0.4M KCl fraction of a heparin agarose column binds to the CAC box (-140). A ubiquitous octamer factor present in the 0.2M fraction binds to an ATGCAAT element (-175), but is completed out by the erythroid specific factor NF-E1 (in the 0.4M KCl fraction), which binds a site (-186) immediately flanking the octamer. A novel factor binding to a stretch of 8A around -233, was identified in the 0.2M KCl fraction. This factor is not present in HeLa nuclear extracts. To study the transcriptional importance of these protein binding sites we have used an "A gamma-minilocus", similar to that described for the beta-globin gene (1) in K562 cells. This provides evidence that the NF-E1 and CAC box in the -210 to -122 region of the A gamma-promoter are important for the efficient expression of the gamma-globin gene.

Asunto(s)

Genes , Globinas/genética , Proteínas Nucleares/metabolismo , Transcripción Genética , Secuencia de Bases , Southern Blotting , Línea Celular , Núcleo Celular/metabolismo , Deleción Cromosómica , Desoxirribonucleasa I , Humanos , Datos de Secuencia Molecular , Mapeo Nucleótido , Regiones Promotoras Genéticas , Mapeo Restrictivo

RESUMEN

We have discussed the use of BW5147 T-lymphoma cells as a model system to explore the genetic basis of invasion and metastasis. The degree of invasiveness of these cells in vitro is highly correlated with experimental metastasis formation in vivo upon tail vein injection in syngeneic AKR mice. A powerful in vitro selection system has been developed which allows to select rare invasive cell variants obtained by various experimental manipulations. We found that introduction of the human c-Ha-ras oncogene, the presence of human chromosome 7 from normal activated T-cells, DNA hypomethylation induced by 5-azacytidine treatment, and possibly also retrovirus insertional mutagenesis can convert noninvasive BW5147 T-lymphoma cells into invasive and metastatic cells. Several experimental approaches are discussed to identify the gene(s) involved.

Asunto(s)

Linfoma/genética , Animales , Cromosomas Humanos Par 7 , ADN de Neoplasias/genética , Humanos , Hibridomas/patología , Metilación , Ratones , Mutación , Invasividad Neoplásica/genética , Oncogenes/fisiología , Linfocitos T , Transfección , Células Tumorales Cultivadas

RESUMEN

We have introduced into murine erythroleukaemia (MEL) cells several series of deletion mutants of hybrid genes between the human beta-globin gene and the murine H-2Kb gene. S1 nuclease and transcriptional run-off analysis showed that the human beta-globin gene contains at least three globin specific transcriptional control elements. One is a promoter element and is located 160 bp upstream from the transcription initiation site; it increases the efficiency of the promoter after MEL cells are induced to differentiate. The other two elements are tissue-specific transcriptional enhancers and are located downstream from the transcription initiation site, the first in the structural beta-globin gene and the second in the 3' flanking sequences.

Asunto(s)

Elementos de Facilitación Genéticos , Genes Reguladores , Genes , Globinas/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Deleción Cromosómica , Enzimas de Restricción del ADN , Células HeLa/metabolismo , Humanos , Leucemia Eritroblástica Aguda , Datos de Secuencia Molecular , Transfección

RESUMEN

Metastasis formation is a multistep process that probably requires a complex interplay of a large and heterogeneous group of genes, including genes involved in cellular resistance to immunorejection and genes controlling the invasive potential of cells. Transfection experiments have shown that oncogenes of the ras gene family as well as oncogenes of the kinase group are able to induce invasive and metastatic properties in non-transformed cells as well as in tumorigenic, but non-metastatic, cells. However, these findings are not in agreement with observations on spontaneous human tumours in which no correlation was found between activation or increased expression of ras genes and the invasive and metastatic properties of these cells. Further studies have indicated that in particular cell types nuclear oncogenes like N-myc and adenovirus derived E1A may influence metastasis formation by trans-regulation of other genes, including class I genes of the major histocompatibility complex and genes coding for proteolytic enzymes. The many efforts to identify additional invasion and metastasis associated genes by transfection of high molecular weight metastatic tumour DNA met with little success. Somatic cell fusion studies, however, indicate that such genes exist and that one or more of them are located on human chromosome 7.

Asunto(s)

Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Técnicas Genéticas , Humanos , Oncogenes , Fenotipo , Transfección