Asunto(s)
Antígenos CD4/genética , Modelos Inmunológicos , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos CD4/metabolismo , Linaje de la Célula , Silenciador del Gen , Ratones , Secuencias Reguladoras de Ácidos Nucleicos , Procesos Estocásticos , Linfocitos T Colaboradores-Inductores/inmunología , Timo/inmunologíaRESUMEN
A particular feature of gammadelta T cell biology is that cells expressing T cell receptor (TCR) using specific Vgamma/Vdelta segments are localized in distinct epithelial sites, e.g., in mouse epidermis nearly all gammadelta T cells express Vgamma3/Vdelta1. These cells, referred to as dendritic epidermal T cells (DETC) originate from fetal Vgamma3+ thymocytes. The role of gammadelta TCR specificity in DETC's migration/localization to the skin has remained controversial. To address this issue we have generated transgenic (Tg) mice expressing a TCR delta chain (Vdelta6.3-Ddelta1-Ddelta2-Jdelta1-Cdelta), which can pair with Vgamma3 in fetal thymocytes but is not normally expressed by DETC. In wild-type (wt) Vdelta6.3Tg mice DETC were present and virtually all of them express Vdelta6.3. However, DETC were absent in TCR-delta(-/-) Vdelta6.3Tg mice, despite the fact that Vdelta6.3Tg gammadelta T cells were present in normal numbers in other lymphoid and nonlymphoid tissues. In wt Vdelta6.3Tg mice, a high proportion of in-frame Vdelta1 transcripts were found in DETC, suggesting that the expression of an endogenous TCR-delta (most probably Vdelta1) was required for the development of Vdelta6.3+ epidermal gammadelta T cells. Collectively our data demonstrate that TCR specificity is essential for the development of gammadelta T cells in the epidermis. Moreover, they show that the TCR-delta locus is not allelically excluded.
Asunto(s)
Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Piel/inmunología , Linfocitos T/fisiología , Animales , Movimiento Celular , Células Dendríticas/fisiología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Células Madre/fisiología , Timo/citologíaRESUMEN
To study the adaptation of natural killer (NK) cells to their major histocompatibility complex (MHC) class I environment we have established a novel mouse model with mosaic expression of H-2D(d) using a Cre/loxP system. In these mice, we noticed that NK cells expressing the inhibitory receptor for D(d), Ly49A, were specifically underrepresented among cells with low D(d) levels. That was due to the acquisition of D(d) molecules by the Ly49A+ NK cells that have lost their D(d) transgene. The uptake of H-2D molecules via the Ly49A receptor was restricted to strong ligands of Ly49A. Surprisingly, when Ly49A+ NK cells were D(d+), uptake of the alternative ligand D(k) was not detectable. Similarly, one anti-Ly49A mAb (A1) bound inefficiently when Ly49A was expressed on D(d+) NK cells. Concomitantly, functional assays demonstrated a reduced capacity of Ly49A to inhibit H-2(b)D(d) as compared with H-2(b) NK cells, rendering Ly49A+ NK cells in D(d+) mice particularly reactive. Minor reductions of D(d) levels and/or increases of activating ligands on environmental cells may thus suffice to abrogate Ly49A-mediated NK cell inhibition. The mechanistic explanation for all these phenomena is likely the partial masking of Ly49A by D(d) on the same cell via a lateral binding site in the H-2D(d) molecule.
Asunto(s)
Antígenos Ly , Proteínas Portadoras/fisiología , Antígenos H-2/metabolismo , Células Asesinas Naturales/fisiología , Proteínas de la Membrana/fisiología , Animales , Antígeno de Histocompatibilidad H-2D , Lectinas Tipo C , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Subfamilia A de Receptores Similares a Lectina de Células NK , Receptores Similares a Lectina de Células NKRESUMEN
We have established H-2D(d)-transgenic (Tg) mice, in which H-2D(d) expression can be extinguished by Cre recombinase-mediated deletion of an essential portion of the transgene (Tg). NK cells adapted to the expression of the H-2D(d) Tg in H-2(b) mice and acquired reactivity to cells lacking H-2D(d), both in vivo and in vitro. H-2D(d)-Tg mice crossed to mice harboring an Mx-Cre Tg resulted in mosaic H-2D(d) expression. That abrogated NK cell reactivity to cells lacking D(d). In D(d) single Tg mice it is the Ly49A+ NK cell subset that reacts to cells lacking D(d), because the inhibitory Ly49A receptor is no longer engaged by its D(d) ligand. In contrast, Ly49A+ NK cells from D(d) x MxCre double Tg mice were unable to react to D(d)-negative cells. These Ly49A+ NK cells retained reactivity to target cells that were completely devoid of MHC class I molecules, suggesting that they were not anergic. Variegated D(d) expression thus impacts specifically missing D(d) but not globally missing class I reactivity by Ly49A+ NK cells. We propose that the absence of D(d) from some host cells results in the acquisition of only partial missing self-reactivity.
Asunto(s)
Antígenos Ly , Proteínas Portadoras/biosíntesis , Silenciador del Gen/inmunología , Antígenos H-2/genética , Integrasas/fisiología , Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/inmunología , Proteínas de la Membrana/biosíntesis , Transgenes/inmunología , Proteínas Virales/fisiología , Animales , Células Cultivadas , Cruzamientos Genéticos , Citomegalovirus/genética , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica/genética , Vectores Genéticos/síntesis química , Antígenos H-2/biosíntesis , Antígeno de Histocompatibilidad H-2D , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Integrasas/genética , Células Asesinas Naturales/enzimología , Lectinas Tipo C , Subgrupos Linfocitarios/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Subfamilia A de Receptores Similares a Lectina de Células NK , Receptores de Estrógenos/genética , Receptores Similares a Lectina de Células NK , Eliminación de Secuencia/inmunología , Proteínas Virales/genéticaRESUMEN
Cancer cell lines are widely used in many types of cancer research, including studies aimed at understanding DNA hypermethylation of gene promoters in cancer. Hypermethylation of promoters is capable of repressing the expression of tumor suppressor genes and may play a role in the development and/or progression of cancer. Although both primary malignancies and cancer cell lines exhibit this epigenetic phenomenon, there has been no direct comparison between them. In order to address this question, we have utilized restriction landmark genomic scanning to measure the hypermethylation phenotypes of cancer cell lines and compared these data with the same analysis performed on primary malignancies. In all cases, cancer cell lines exhibit significantly higher levels of CpG island hypermethylation than the primary malignancies they represent. Colon cancer cell lines are most similar to their respective tumors, with only a 5-fold increase in hypermethylation, while head and neck squamous cell carcinoma cell lines show a 93-fold increase in hypermethylation. Furthermore, >57% of the loci methylated in cell lines are never methylated in 114 primary malignancies studied. Seventy percent of loci hypermethylated in cell lines are hypermethylated in lines from more than one type of cancer. These data indicate that most CpG island hypermethylation observed in cancer cell lines is due to an intrinsic property of cell lines as opposed to the malignant tissue from which they originated.
Asunto(s)
Islas de CpG/genética , Metilación de ADN , ADN de Neoplasias/metabolismo , Neoplasias/genética , ADN de Neoplasias/genética , Electroforesis en Gel Bidimensional , Células HL-60 , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
The association of trans-acting T cell factors (TCFs) or lymphoid enhancer factor 1 (LEF-1) with their coactivator beta-catenin mediates transient transcriptional responses to extracellular Wnt signals. We show here that T cell maturation depends on the presence of the beta-catenin--binding domain in TCF-1. This domain is necessary to mediate the survival of immature CD4(+)CD8(+) double-positive (DP) thymocytes. Accelerated spontaneous thymocyte death in the absence of TCF-1 correlates with aberrantly low expression of the anti-apoptotic protein Bcl-x(L). Increasing anti-apoptotic effectors in thymocytes by the use of a Bcl-2 transgene rescued TCF-1-deficient DP thymocytes from apoptosis. Thus, TCF-1, upon association with beta-catenin, transiently ensures the survival of immature T cells, which enables them to generate and edit T cell receptor (TCR) alpha chains and attempt TCR-mediated positive selection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas del Citoesqueleto/inmunología , Proteínas de Unión al ADN/inmunología , Transactivadores , Factores de Transcripción/inmunología , Animales , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Diferenciación Celular/inmunología , Supervivencia Celular/inmunología , Factor Nuclear 1-alfa del Hepatocito , Factor de Unión 1 al Potenciador Linfoide , Ratones , Transducción de Señal/inmunología , Factor 1 de Transcripción de Linfocitos T , beta CateninaRESUMEN
Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.
Asunto(s)
Antígenos Ly , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Factores de Transcripción/fisiología , Regiones no Traducidas 5'/análisis , Regiones no Traducidas 5'/aislamiento & purificación , Animales , Secuencia de Bases , Sitios de Unión/genética , Sitios de Unión/inmunología , Proteínas Portadoras/genética , Células Cultivadas , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Factor Nuclear 1-alfa del Hepatocito , Antígenos de Histocompatibilidad Clase I/genética , Lectinas Tipo C , Factor de Unión 1 al Potenciador Linfoide , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Regiones Promotoras Genéticas/inmunología , Receptores Inmunológicos/genética , Receptores Similares a Lectina de Células NK , Factor 1 de Transcripción de Linfocitos T , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Aberrant DNA methylation is believed to be important in tumorigenesis by causing either transcriptional inactivation of genes or chromosomal instability. Several laboratories have identified promoter hypermethylation of tumor suppressor genes in acute myeloid leukemia (AML). However, these studies do not provide a global assessment of overall methylation changes and do not allow the identification of novel methylated sequences. Previously, nonrandom CpG island methylation was reported in 17 adult de novo AML diagnostic samples when compared with the corresponding remission samples by means of restriction landmark genomic scanning (RLGS). That study has been expanded on by an analysis of a larger set of CpG islands (1740 vs 1184), which now provides details of 33 cloned methylated loci, including 21 known genes or expressed sequence tags. Five of these cloned loci appear to be methylated only in AML and not in the 6 solid tumors studied in this study (more than 98 samples analyzed). Chromosomal location was available for 30 of the 33 loci, and 5 of these 30 (17%) are localized to chromosome 11, suggesting a trend toward overrepresentation of methylation events on this chromosome. These results provide evidence for widespread aberrant methylation in AML, with identification of novel methylation targets, epigenetic changes that appear unique to AML, and apparent preferential methylation on chromosome 11.
Asunto(s)
Cromosomas Humanos Par 11 , Metilación de ADN , Leucemia Mieloide Aguda/genética , Adulto , Southern Blotting , Mapeo Cromosómico , Clonación Molecular , Desoxirribonucleasas de Localización Especificada Tipo II , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Inducción de Remisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Notch proteins influence cell-fate decisions in many developing systems. Several gain-of-function studies have suggested a critical role for Notch 1 signaling in CD4-CD8 lineage commitment, maturation and survival in the thymus. However, we show here that tissue-specific inactivation of the gene encoding Notch 1 in immature (CD25+CD44-)T cell precursors does not affect subsequent thymocyte development. Neither steady-state numbers nor the rate of production of CD4+ and CD8+ mature thymocytes is perturbed in the absence of Notch 1. In addition, Notch 1-deficient thymocytes are normally sensitive to spontaneous or glucocorticoid-induced apoptosis. In contrast to earlier reports, these data formally exclude an essential role for Notch 1 in CD4-CD8 lineage commitment, maturation or survival.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular , Timo/inmunología , Factores de Transcripción , Proteínas Virales , Animales , Apoptosis/efectos de los fármacos , Antígenos CD4/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , División Celular , Linaje de la Célula , Células Cultivadas , Eliminación de Gen , Marcación de Gen , Glucocorticoides/farmacología , Integrasas/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Notch1 , Bazo/inmunología , Subgrupos de Linfocitos T/clasificación , Timo/citología , TransgenesRESUMEN
DNA methylation is the only known mechanism for an epigenetic genomic DNA modification that is capable of altering gene expression. A recent study reveals that the pattern of CpG island methylation is largely characteristic of tumor type, suggesting that distinct sets of genes are inactivated by methylation during development of each tumor type. We compared previously the methylation status between normal liver and liver tumors in SV40 T/t antigen transgenic mice (MT-D2 mice) using Restriction Landmark Genomic Scanning for Methylation (RLGS-M) and identified several loci/spots that appeared to be methylated frequently in liver tumors. One of these spots, B236, identified a locus on chromosome 12 (D12Ncvs7) syntenic with human 14q12-q21 that is frequently lost in certain human cancers. Shotgun sequencing of a bacterial artificial chro mosome clone containing this spot/locus was performed to identify genes within this region. The Genescan program predicted an open reading frame of a novel, intron-less gene adjacent to the B236 spot that encodes a putative 493-amino acid protein containing the SNAG repressor motif in the NH2-terminal region and five C2H2-type zinc finger motifs in the COOH-terminal half. This putative gene, methylated in liver tumor (mlt 1), is a novel member of the SNAG transcriptional repressor family with 43% amino acid identity to insulinoma-associated protein 1. An open reading frame encoding a protein quite similar to mouse mlt 1 (56% amino acid identity) was located in the syntenic region of the human genome, indi cating that mlt 1 is evolutionarily conserved in human. Northern blot analysis revealed that mlt 1 is normally expressed in brain, spleen, stom ach, and liver. However, mlt 1 expression was silenced in the liver tumors of MT-D2 mice. The putative promoter region of mlt 1 is unmethylated in normal tissues but methylated in all liver tumors from 11 MT-D2 mice We also found that mlt 1 was methylated and not expressed in N18TG-22 cells, a mouse neuroblastoma cell line. Treatment of N18TG-2 cells with a demethylating agent, 5-aza-deoxycytidine, resulted in an expression of mlt 1, indicating that the repression of mlt 1 is attributable to methylation Thus, mlt 1 is a novel target gene that is silenced by methylation during liver tumorigenesis initiated by SV40 T antigen.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Azacitidina/análogos & derivados , Metilación de ADN , Silenciador del Gen , Neoplasias Hepáticas Experimentales/genética , Proteínas Represoras/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Secuencia de Bases , Proteínas de Unión al ADN/genética , Decitabina , Femenino , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Neoplasias Hepáticas Experimentales/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Neuroblastoma/genética , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , ARN Mensajero/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genéticaRESUMEN
Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.
Asunto(s)
Antígenos Ly , Proteínas Portadoras/genética , Regulación de la Expresión Génica/inmunología , Leucemia Experimental/inmunología , Proteínas de la Membrana/genética , Receptores Inmunológicos/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transgenes/inmunología , Animales , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/metabolismo , Relación Dosis-Respuesta Inmunológica , Epítopos de Linfocito T/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidad H-2D , Epítopos Inmunodominantes/inmunología , Lectinas Tipo C , Leucemia Experimental/prevención & control , Leucemia Experimental/virología , Activación de Linfocitos/genética , Linfopenia/genética , Linfopenia/inmunología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Virus de la Leucemia Murina de Moloney/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK , Trasplante de Neoplasias , Receptores Inmunológicos/biosíntesis , Receptores Similares a Lectina de Células NK , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/trasplanteRESUMEN
NK cell function is negatively regulated by MHC class I-specific inhibitory receptors. Transduction of the inhibitory signal involves protein tyrosine phosphatases such as SHP-1 (SH2-containing protein tyrosine phosphatase-1). To investigate the role of SHP-1 for NK cell development and function, we generated mice expressing a catalytically inactive, dominant-negative mutant of SHP-1 (dnSHP-1). In this paper we show that expression of dnSHP-1 does not affect the generation of NK cells even though MHC receptor-mediated inhibition is partially impaired. Despite this defect, these NK cells do not kill syngeneic, normal target cells. In fact dnSHP-1-expressing NK cells are hyporesponsive toward MHC-deficient target cells, suggesting that non-MHC-specific NK cell activation is significantly reduced. In contrast, these NK cells mediate Ab-dependent cell-mediated cytotoxicity and prevent the engraftment with beta2-microglobulin-deficient bone marrow cells. A similar NK cell phenotype is observed in viable motheaten (mev) mice, which show reduced SHP-1 activity due to a mutation in the Shp-1 gene. In addition, NK cells in both mouse strains show a tendency to express more inhibitory MHC-specific Ly49 receptors. Our results demonstrate the importance of SHP-1 for the generation of functional NK cells, which are able to react efficiently to the absence of MHC class I molecules from normal target cells. Therefore, SHP-1 may play an as-yet-unrecognized role in some NK cell activation pathways. Alternatively, a reduced capacity to transduce SHP-1-dependent inhibitory signals during NK cell development may be compensated by the down-modulation of NK cell triggering pathways.
Asunto(s)
Antígenos Ly , Antígenos de Histocompatibilidad Clase I/genética , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/inmunología , Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/genética , Animales , Trasplante de Médula Ósea/inmunología , Catálisis , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Cruzamientos Genéticos , Citotoxicidad Inmunológica/genética , Activación Enzimática/genética , Activación Enzimática/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Inmunidad Innata/genética , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Lectinas Tipo C , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Mutantes , Ratones Transgénicos , Mutagénesis Insercional , Mutación Puntual , Proteína Fosfatasa 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/metabolismo , Receptores Similares a Lectina de Células NK , Proteínas Tirosina Fosfatasas con Dominio SH2 , Linfocitos T/citología , Células Tumorales Cultivadas , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genética , Dominios Homologos src/genética , Dominios Homologos src/inmunologíaRESUMEN
SV40 T/t antigen-induced liver tumors from transgenic mice were analyzed by Restriction Landmark Genomic Scanning (RLGS). Using NotI as the restriction landmark, RLGS targets CpG islands found in gene-rich regions of the genome. Since many RLGS landmarks are mapped, the candidate gene approach can be used to help determine which genes are altered in tumors. RLGS analysis revealed one tumor-specific amplification mapping close to CcnA2 (cyclin A2) and Fgf2 (fibroblast growth factor 2). Southern analysis confirmed that both oncogenes are amplified in this tumor and in a second, independent liver tumor. Whereas Fgf2 RNA is undetectable in tumors, CcnA2 RNA and cyclin A2 protein was overexpressed in 25 and 50% of tumors, respectively. Combining RLGS with the candidate gene approach indicates that cyclin A2 amplification and overexpression is a likely selected event in transgenic mouse liver tumors. Our results also indicate that our mouse model for liver tumorigenesis in mice accurately recapitulates events observed in human hepatocellular carcinoma.
Asunto(s)
Ciclina A/biosíntesis , Ciclina A/genética , Neoplasias Hepáticas Experimentales/genética , Factores de Edad , Animales , Northern Blotting , Southern Blotting , Mapeo Cromosómico , Islas de CpG , Ciclina A2 , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/genética , Marcadores Genéticos , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Ratones Transgénicos , Modelos Genéticos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S/genética , Regulación hacia ArribaRESUMEN
NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.
Asunto(s)
Antígenos Ly , Citotoxicidad Inmunológica , Antígenos H-2/fisiología , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Receptores Inmunológicos/fisiología , Animales , Médula Ósea/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citotoxicidad Inmunológica/genética , Antígenos H-2/metabolismo , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Células Asesinas Naturales/inmunología , Lectinas Tipo C , Ligandos , Activación de Linfocitos/genética , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Quimera por Radiación/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores Similares a Lectina de Células NK , Autotolerancia/genéticaAsunto(s)
Antígenos Ly , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/genética , Receptores Inmunológicos/genética , Animales , Células Clonales , Humanos , Lectinas Tipo C , Receptores Similares a Lectina de Células NK , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de TiempoRESUMEN
The Medical Advisory Service of the Health Insurance in the area of the Northern Rhine (MDK Nordrhein) has set up an internal concept for quality management since 1998. This concept includes the installation and performance of quality circles. Staff members were internally qualified as "presenters". They worked out principles for quality circles of the MDK North Rhine which were implemented as binding basic rules by the managing conference of the MDK. The principles will be presented in detail.
Asunto(s)
Participación en las Decisiones , Programas Nacionales de Salud , Alemania , Humanos , Garantía de la Calidad de Atención de Salud , Gestión de la Calidad TotalRESUMEN
CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.
Asunto(s)
Metilación de ADN , Fosfatos de Dinucleósidos/análisis , Neoplasias/genética , Adenocarcinoma/genética , Secuencia de Bases , Neoplasias Encefálicas/genética , Neoplasias de la Mama/genética , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Lobular/genética , Neoplasias del Colon/genética , Fosfatos de Dinucleósidos/genética , Femenino , Genoma Humano , Humanos , Masculino , Datos de Secuencia Molecular , Mapeo RestrictivoRESUMEN
Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect alterations in DNA methylation associated with murine SV40 T/t antigen-induced hepatocarcinogenesis. An altered locus/spot (S130) was cloned and found to correspond to sequences in the 5' flanking region and 5' portion of the cDNA for the murine mac25/insulin-like growth factor binding protein-7 (Igfbp-7) gene. IGFBPs are believed to be capable of binding insulin, Igf1, and Igf2 and modulating mitogenic effects. Previous studies have shown that Igf2 has an important role in promoting liver tumorigenesis. Quantitative PCR was used to access the methylation status of the NotI site just 5' to the coding region and the expression level of the mac25/igfbp-7 gene. The results indicated that the degree of methylation was inversely related to the expression level and is consistent with a role for DNA methylation in silencing mac25/Igfbp-7 gene expression and function for mac25/Igfbp-7 as a tumor suppressor gene.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Proteínas Portadoras/genética , Metilación de ADN , Regulación de la Expresión Génica , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Neoplasias Hepáticas/genética , Virus 40 de los Simios/genética , Animales , Secuencia de Bases , ADN Complementario , Biblioteca de Genes , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Muridae , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.
Asunto(s)
Antígenos Ly , Células Clonales/inmunología , Citotoxicidad Inmunológica , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Antígenos H-2/inmunología , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/genética , Autotolerancia , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Linfoma de Burkitt/patología , Línea Celular , Linaje de la Célula , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta Inmunológica , Proteínas Fluorescentes Verdes , Factor Nuclear 1-alfa del Hepatocito , Antígeno de Histocompatibilidad H-2D , Humanos , Células Jurkat , Células Asesinas Naturales/metabolismo , Lectinas Tipo C , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Recuento de Linfocitos , Subgrupos Linfocitarios , Factor de Unión 1 al Potenciador Linfoide , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subfamilia A de Receptores Similares a Lectina de Células NK , Regiones Promotoras Genéticas , Quimera por Radiación , Receptores Inmunológicos/biosíntesis , Receptores Similares a Lectina de Células NK , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factor 1 de Transcripción de Linfocitos T , Timoma/patología , Neoplasias del Timo/patología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.