RESUMEN
Laminins are structural components of basement membranes that regulate and control many cellular functions. Changes in basement membrane composition play significant roles in etiology of diseases. Inflammatory bowel diseases are conditions that lead to defects in the mucosal barrier which includes the basement membrane underlying the epithelium. This review will summarize the main findings related to the involvement of laminins and of the laminin-binding receptors in inflammatory conditions such as Crohn's disease and ulcerative colitis. We will review the current literature devoted to studies in humans (immunolocalisation, genetic factors, microarray data), as well as experimental cell models that show that laminins contribute to the inflammation process probably linked to the deregulation of proinflammatory cytokines.
Asunto(s)
Enfermedades Inflamatorias del Intestino/etiología , Laminina/fisiología , Animales , Citocinas/fisiología , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Perfilación de la Expresión Génica , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Laminina/genética , Laminina/metabolismo , Análisis por Micromatrices , Modelos Biológicos , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Receptores de Laminina/fisiologíaRESUMEN
Tenascin-C is an adhesion-modulatory extracellular matrix molecule that is highly expressed in tumors. To investigate the effect of tenascin-C on tumor cells, we analyzed its antiadhesive nature and effect on tumor cell proliferation in a fibronectin context. Glioblastoma and breast carcinoma cell adhesion was compromised by a mixed fibronectin/tenascin-C substratum, which concomitantly caused increased tumor-cell proliferation. We identified the antiadhesive mechanism as a specific interference of tenascin-C with cell binding to the HepII/syndecan-4 site in fibronectin through direct binding of tenascin-C to the 13th fibronectin type III repeat (FNIII13). Cell adhesion and proliferation levels were restored by the addition of FNIII13. Overexpression of syndecan-4, but not syndecan-1 or -2, reverted the cell adhesion defect of tenascin-C. We characterized FNIII13 as the binding site for syndecan-4. Thus we describe a novel mechanism by which tenascin-C impairs the adhesive function of fibronectin through binding to FNIII13, thereby inhibiting the coreceptor function of syndecan-4 in fibronectin-induced integrin signaling.
Asunto(s)
Fibronectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanos/metabolismo , Tenascina/farmacología , Animales , Sitios de Unión , Células CHO , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Pollos , Cricetinae , Humanos , Fragmentos de Péptidos/metabolismo , Receptores de Fibronectina/biosíntesis , Sindecano-4 , Células Tumorales CultivadasRESUMEN
In adenovirus type 12 (Ad12)-induced tumor cells, in Ad12-transformed cells and in continuously passaged cell lines from these sources, the viral DNA is integrated in multiple copies, usually at a single chromosomal location. In different tumors or cell lines, the sites of integration of Ad12 DNA are all different. Rare exceptions exist. In most instances, the integrated viral DNA resides very stably in the host cell genomes. However, upon continuous serial passage of such cell lines, the integrated viral DNA can be destabilized and lost. In two instances, i.e. in the Ad12-induced hamster tumor cell lines H1111(1) and CLAC1, we have investigated the loss of integrated viral DNA in detail. After extended serial passage, these two cell lines seemed to be devoid of Ad12 DNA sequences, as detectable by Southern blot hybridization, but continued to induce tumors after reinjection into hamsters. Cells from these two cell lines were now recloned three times, and DNAs from cultures derived from several individual clones were reinvestigated for the presence of several parts of the viral genome by the polymerase chain reaction (PCR). Some of the clones still carried parts of the Ad12 genome. However, several clones were isolated that proved free of all parts of the viral genome, except for minute segments from the right terminus of the Ad12 genome. Apparently, the loss of integrated viral DNA from these cell lines proceeded as a continuous, gradual, multistep process whose pattern could differ from cell clone to cell clone, once destabilization had been initiated. The mechanism of destabilization is not understood. Cell populations of 2 x 10(6) to 3 x 10(7), and as low as 10(2), cells from the clones, that contained only minimal remnants from the right viral DNA terminus, were reinjected into newborn or 13-20 day-old weanling Syrian hamsters (Mesocricetus auratus). Tumors developed within 5-17 days after injection. Tumor cell clones also grew in soft agar. The injection of primary hamster skin fibroblasts never elicited tumor formation. The tumor cells induced by this reinjection proved repeatedly free of Ad12 DNA both by Southern blot hybridization and by PCR, except for those cell and tumor clones that contained small segments of the right terminal E4 region of the Ad12 genome. The tumor cells, however, retained their oncogenic phenotype. The results raise questions about the cell clone-specific excision patterns of integrated foreign DNA from the recipient genome and the possibility of a hit-and-run mechanism of adenoviral oncogenesis.
Asunto(s)
Adenovirus Humanos/genética , Virus ADN Tumorales/genética , ADN de Neoplasias/análisis , Genoma Viral , Células Tumorales Cultivadas/virología , Animales , Animales Recién Nacidos , Southern Blotting , Cricetinae , ADN Viral/análisis , Mesocricetus , Neoplasias Experimentales/genética , Neoplasias Experimentales/virología , Fenotipo , Reacción en Cadena de la Polimerasa , Células Tumorales Cultivadas/citologíaRESUMEN
Adherent cells assemble fibronectin into a fibrillar matrix on their apical surface. The fibril formation is initiated by fibronectin binding to the integrins alpha5 beta1 and alphav beta3, and is completed by a process that includes fibronectin self-assembly. We found that a 76- amino acid fragment of fibronectin (III1-C) that forms one of the self-assembly sites caused disassembly of preformed fibronectin matrix without affecting cell adhesion. Treating attached fibroblasts or endothelial cells with III1-C inhibited cell migration and proliferation. Rho-dependent stress fiber formation and Rho-dependent focal contact protein phosphorylation were also inhibited, whereas Cdc42 was activated, leading to actin polymerization into filopodia. ACK (activated Cdc42-binding kinase) and p38 MAPK (mitogen-activated protein kinase), two downstream effectors of Cdc42, were activated, whereas PAK (p21-activated kinase) and JNK/SAPK (c-Jun NH2-terminal kinase/ stress-activated protein kinase) were inhibited. III1-C treatment also modulated activation of JNK and ERK (extracellular signal-regulated kinases) in response to growth factors, and reduced the activity of the cyclin E-cdk2 complex. These results indicate that the absence of fibronectin matrix causes activation of Cdc42, and that fibronectin matrix is required for Rho activation and cell cycle progression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Matriz Extracelular/fisiología , Fibronectinas/fisiología , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Actinas/efectos de los fármacos , Actinas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ciclo Celular/efectos de los fármacos , Citoesqueleto/fisiología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Fibroblastos/citología , Fibroblastos/fisiología , Fibronectinas/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Laminina/farmacología , Laminina/fisiología , Proteína Quinasa 1 Activada por Mitógenos , Fragmentos de Péptidos/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Venas Umbilicales , Vitronectina/farmacología , Vitronectina/fisiología , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Loss of attachment to an extracellular matrix substrate arrests the growth of untransformed cells in the G1 phase. This anchorage-dependent cell cycle arrest is linked to increased expression of the p21Cip1 (p21) and p27Kip1 (p27) cyclin-dependent kinase inhibitors. The result is a loss of cdk2-associated kinase activity, especially that of cyclin E-cdk2. The levels of p21 and p27 are also upregulated in unattached transformed cells, but cyclin E-cdk2 activity remains high, and the cells are able to grow in an anchorage-independent manner. Increased expression of cyclin E and cdk2 appears to be partially responsible for the maintenance of cyclin E-cdk2 activity in transformed cells. To explore further the regulation of cyclin E-cdk2 in transformed cells, we have analysed the subcellular distribution of cyclin-cdk complexes and their inhibitors in normal human fibroblasts, their transformed counterparts, and in various human tumor cell lines. In substrate-attached normal fibroblasts, cyclin E and cdk2 were exclusively in the nuclear fraction, associated with one another. When normal fibroblasts were detached and held in suspension, cyclin E-cdk2 complexes remained nuclear, but were now found associated with the p21 and p27 cdk inhibitors and lacked histone H1 phosphorylating activity. In contrast, the transformed fibroblasts and tumor cells, which are anchorage-independent, had more than half of their cyclin E, cdk2, p21 and p27 in the cytoplasmic fraction, both in attached and suspended cultures. The cytoplasmic p21 and p27 were bound to cyclin E-cdk2, as well as to complexes containing cyclin A and cyclin D. The nuclear cyclin E-cdk2 complexes from the transformed cells grown in suspension contained only low levels of p21 and p27 and had histone H1 kinase activity. Thus, at least three mechanisms contribute to keeping cyclin E-cdk2 complexes active in suspended anchorage-independent cells: cyclin E and cdk2 are upregulated, as reported previously, cdk inhibitors are sequestered away from the nucleus by cytoplasmic cyclin-cdk complexes, and the binding of the inhibitors to nuclear cyclin E-cdk2 complexes is impaired.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Citoplasma/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Supresoras de Tumor , Animales , Línea Celular Transformada , Núcleo Celular/metabolismo , Transformación Celular Neoplásica , Células Cultivadas , Ciclina A/metabolismo , Ciclina D , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Fibroblastos/metabolismo , Humanos , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
Most nonmalignant cells are anchorage-dependent; they require substrate attachment for growth and, in some instances, survival. This requirement is lost on oncogenic transformation. The cyclin E-CDK2 complex, which is required for the G1-S transition of the cell cycle, was activated in late G1 phase in attached human fibroblasts, but not in fibroblasts maintained in suspension. In transformed fibroblasts the complex was active regardless of attachment. The lack of cyclin E-CDK2 activity in suspended cells appeared to result from increased expression of CDK2 inhibitors and a concomitant decrease in phosphorylation of CDK2 on threonine-160. Suppression of cyclin E-CDK2 activity may thus underlie the anchorage dependence of cell growth.
Asunto(s)
Quinasas CDC2-CDC28 , Adhesión Celular , Proteínas de Ciclo Celular , Transformación Celular Neoplásica , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Fase G1 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor , Línea Celular , Línea Celular Transformada , Núcleo Celular/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
We have studied the stability of integrated adenovirus type 12 (Ad12) DNA sequences and the relation of foreign DNA persistence to the state of methylation of this DNA. In the Ad12-transformed hamster cell line T637, multiple copies of Ad12 DNA are chromosomally integrated. Some of these integrated viral genomes are rearranged in that internal parts of the viral DNA have become juxtaposed to its left terminus. Fluorescent in situ hybridization analyses demonstrate that the Ad12 DNA in cell line T637 and in some of its revertants is located at one site on one of the hamster chromosomes. Major portions of the integrated viral genomes in cell line T637 have become extensively de novo methylated in specific patterns. Most of the rearranged Ad12 DNA sequences in the T637 genome are un- or hypomethylated. In the morphological revertants of the Ad12-transformed hamster cell line T637, the majority of the integrated Ad12 genomes has been lost. Surprisingly, we have found that the un- or hypomethylated rearranged viral sequences have been selectively lost, in contrast to some of the methylated sequences that are stably retained.
Asunto(s)
Adenovirus Humanos/genética , ADN Viral/metabolismo , Genes Virales , Integración Viral , Animales , Sitios de Unión , Línea Celular Transformada , Cricetinae , Reordenamiento Génico , Humanos , MetilaciónRESUMEN
We have studied the integration of adenovirus type 12 (Ad12) DNA in transformed and hamster tumor cells over many years. Upon infection of hamster cells with Ad12, viral DNA has been found in association with hamster chromosomes, possibly in part integrated into the host genome. Ad12 DNA integration is not sequence specific. Transcriptionally active sites of the host genome show a preponderance for foreign DNA insertion. We are pursuing the mechanism of Ad12 DNA integrative recombination in a cell-free system prepared from hamster cell nuclear extracts. In a number of Ad12-transformed hamster cell lines or in cell lines carrying foreign DNA, we have located the inserted Ad12 DNA copies on hamster chromosomes by fluorescent in situ hybridization (FISH). Among the consequences of Ad12 DNA integration, we have studied the de novo methylation of the integrated foreign (Ad12) DNA and increases in DNA methylation in several cellular genes and DNA segments in Ad12-transformed and hamster tumor cells. Several lines of evidence argue for the notion that parameters in addition to nucleotide sequence, in particular site of integration and/or the chromatin configuration of the integrated DNA, are important in generating de novo methylation patterns. The de novo methylation of integrated foreign DNA can be interpreted as an old cellular defense mechanism against the activity of foreign genes in an established genome. Pursuing this concept, we have asked for the most likely portal of entry of foreign DNA, supposedly the gastrointestinal tract in most animals. This hypothesis has been tested by feeding mice linearized or circular, double-stranded bacteriophage M13mp18 DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenovirus Humanos/genética , Transfección/métodos , Integración Viral , Animales , Bacteriófago M13 , Línea Celular , Transformación Celular Neoplásica , Sistema Libre de Células , Cricetinae , ADN Viral/sangre , ADN Viral/genética , ADN Viral/metabolismo , Hibridación Fluorescente in Situ , Riñón , Mamíferos , RatonesRESUMEN
The de novo methylation of foreign DNA integrated into the mammalian genome is a fundamental process whose mechanism has not yet been elucidated. We have studied de novo methylation in adenovirus type 12 (Ad12) genomes inserted into the genomes of Ad12-induced hamster tumor cells. De novo methylation of Ad12 DNA, which is not methylated in the virion, is initiated in two paracentrally located regions and spreads from there across the integrated Ad12 genomes. (i) After extensive cultivation of cloned Ad12-induced hamster tumor cell lines, the same segments in integrated Ad12 DNA in different cell lines become methylated or remain unmethylated, depending on their positions in the viral genome. (ii) When Ad12 DNA or Ad12 DNA fragments are transfected into hamster cells and permanent cell lines are established by selection for the cotransfected neomycin phosphotransferase gene, patterns of de novo methylation in terminally or internally located segments of Ad12 DNA are different from those in Ad12-induced tumor cell lines. (iii) A detailed study on the topology of the integrated viral genomes in the Ad12-transformed hamster cell lines T637 and A2497-3 and in the Ad12-induced hamster tumors T191, T1111(1), and T181 has been performed. Some of the integrated viral genomes are inserted into the cellular genome in an orientation colinear with the virion genome; others have been rearranged. An originally internally located Ad12 DNA segment has become transposed to the left-terminal sequences of the viral genome in several cell lines and tumors. In the complete Ad12 genomes, the internally located PstI-D fragment becomes extensively methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences. When this DNA segment has been juxtaposed to the left-terminal, hypomethylated fragment of Ad12 DNA in rearranged genomes, the PstI-D fragment remains unmethylated. We therefore reason that the initiation of de novo methylation in integrated Ad12 DNA cannot be directed exclusively by the nucleotide sequence. Other parameters, such as site of integration, conformation of integrates, mode of cell selection, or chromatin structure related to transcriptional activity, may play decisive roles.
Asunto(s)
Adenoviridae/genética , ADN Viral/metabolismo , Integración Viral , Animales , Secuencia de Bases , Línea Celular , Cricetinae , Reordenamiento Génico , Genoma Viral , Humanos , Metilación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Transcripción GenéticaRESUMEN
We investigated whether, upon the integration of multiple copies of adenovirus type 12 (Ad12) DNA into an established mammalian (hamster) genome, the pattern of foreign DNA insertion would remain stable or change with consecutive passages of cells in culture. By the injection of purified Ad12 into newborn hamsters, tumors were induced, cells from these tumors were cultivated, and five independent cell lines, HT5, H201/2, H201/3, H271, and H281, were established. These cell lines carried different copy numbers of Ad12 DNA per cell in an integrated form and differed in morphology. Cell line HT5 had been passed twice through hamsters as tumor cells and was subsequently passaged in culture. Patterns of Ad12 DNA integration were determined by restriction cleavage of the nuclear DNA with BamHI, EcoRI, HindIII, MspI, or PstI followed by Southern blot hybridization using 32P-labeled Ad12 DNA or its cloned terminal DNA fragments as hybridization probes. In this way, the off-size fragments, which represented the sites of linkage between Ad12 and cellular DNAs, were determined. At early passage levels in culture, the integration sites of Ad12 DNA in the hamster genome, as characterized by the positions of off-size fragments in agarose or polyacrylamide gel electrophoresis, were different in the five different tumor cell lines. Upon repeated passage, however, the off-size fragment patterns generated by the five restriction endonucleases became very similar in the five tumor cell lines. This surprising result indicates that under cell culture conditions, Ad12-transformed tumor cell lines that carry the foreign (Ad12) genome in selective, probably very similar sites of the cellular genome evolve.
Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Viral/genética , Cricetinae/genética , ADN Viral/genética , Mastadenovirus/genética , Integración Viral/genética , Animales , Animales Recién Nacidos , Línea Celular Transformada/citología , ADN Viral/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Genoma , Pase SeriadoRESUMEN
The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Müller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells.
Asunto(s)
Adenoviridae/genética , ADN Viral/metabolismo , Animales , Autorradiografía , Southern Blotting , Línea Celular Transformada , Cricetinae , Desoxirribonucleasa HpaII , Desoxirribonucleasas de Localización Especificada Tipo II , Electroforesis en Gel de Agar , Mesocricetus , Metilación , Hibridación de Ácido Nucleico , Mapeo Restrictivo , Células Tumorales CultivadasRESUMEN
Patterns of DNA methylation in complex genomes like those of mammalian cells have been viewed as indicators of different levels of genetic activities. It is as yet unknown how these complicated patterns are generated and maintained during cell replication. There is evidence from many different biological systems that the sequence-specific methylation of promoters in higher eukaryotes is one of the important factors in controlling gene activity at a long-term level. In general, the fifth nucleotide 5-methyldeoxycytidine can be considered as a modulator of protein-DNA interactions. The degree and direction of this modulation has to be assessed experimentally in each individual instance. The establishment of de novo patterns of DNA methylation is characterized by the gradual non-random spreading of DNA methylation by an essentially unknown mechanism. In this review, some of the general concepts of DNA methylation in mammalian systems are presented, and research currently performed in the authors' laboratory has been summarized.