RESUMEN

BACKGROUND: Mediator complex participates in transcriptional regulation by connecting regulatory DNA sequences to the RNA polymerase II initiation complex. Recently, we discovered through exome sequencing that as many as 70% of uterine leiomyomas harbour specific mutations in exon 2 of mediator complex subunit 12 (MED12). In this work, we examined the role of MED12 exon 2 mutations in other tumour types. METHODS: The frequency of MED12 exon 2 mutations was analysed in altogether 1158 tumours by direct sequencing. The tumour spectrum included mesenchymal tumours (extrauterine leiomyomas, endometrial polyps, lipomas, uterine leiomyosarcomas, other sarcomas, gastro-intestinal stromal tumours), hormone-dependent tumours (breast and ovarian cancers), haematological malignancies (acute myeloid leukaemias, acute lymphoid leukaemias, myeloproliferative neoplasms), and tumours associated with abnormal Wnt-signalling (colorectal cancers (CRC)). RESULTS: Five somatic alterations were observed: three in uterine leiomyosarcomas (3/41, 7%; Gly44Ser, Ala38_Leu39ins7, Glu35_Leu36delinsVal), and two in CRC (2/392, 0.5%; Gly44Cys, Ala67Val). CONCLUSION: Somatic MED12 exon 2 mutations were observed in uterine leiomyosarcomas, suggesting that a subgroup of these malignant tumours may develop from a leiomyoma precursor. Mutations in CRC samples indicate that MED12 may, albeit rarely, contribute to CRC tumorigenesis.

Asunto(s)

Neoplasias Colorrectales/genética , Leiomiosarcoma/genética , Complejo Mediador/genética , Neoplasias Uterinas/genética , Neoplasias Colorrectales/patología , Exoma , Exones , Femenino , Humanos , Leiomioma/genética , Leiomioma/patología , Leiomiosarcoma/patología , Mutación , Análisis de Secuencia de ADN/métodos , Neoplasias Uterinas/patología

RESUMEN

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily divergent glycoproteins. The secreted pregnancy-specific glycoproteins constitute a subgroup within the CEA family. They are predominantly expressed in trophoblast cells throughout placental development and are essential for a positive outcome of pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. The murine CEA gene family member CEA cell adhesion molecule 9 (Ceacam9) also exhibits a trophoblast-specific expression pattern. However, its mRNA is found only in certain populations of trophoblast giant cells during early stages of placental development. It is exceptionally well conserved in the rat (over 90% identity on the amino acid level) but is absent from humans. To determine its role during murine development, Ceacam9 was inactivated by homologous recombination. Ceacam9(-/-) mice on both BALB/c and 129/Sv backgrounds developed indistinguishably from heterozygous or wild-type littermates with respect to sex ratio, weight gain, and fertility. Furthermore, the placental morphology and the expression pattern of trophoblast marker genes in the placentae of Ceacam9(-/-) females exhibited no differences. Both backcross analyses and transfer of BALB/c Ceacam9(-/-) blastocysts into pseudopregnant C57BL/6 foster mothers indicated that Ceacam9 is not needed for the protection of the embryo in a semiallogeneic or allogeneic situation. Taken together, Ceacam9 is dispensable for murine placental and embryonic development despite being highly conserved within rodents.

Asunto(s)

Moléculas de Adhesión Celular/fisiología , Desarrollo Embrionario y Fetal/fisiología , Isoantígenos/inmunología , Placentación , Trofoblastos/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Cruzamientos Genéticos , Desarrollo Embrionario y Fetal/inmunología , Femenino , Fertilidad/genética , Proteínas Fetales/deficiencia , Proteínas Fetales/genética , Proteínas Fetales/fisiología , Marcación de Gen , Genotipo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fenotipo , Placenta/inmunología , Embarazo , Ratas , Organismos Libres de Patógenos Específicos

RESUMEN

In terrestrial mammals, hearing starts with the perception of acoustic pressure by the tympanic membrane. Vibrations in this membrane are then transduced into the inner ear by the ossicle chain of the middle ear, composed of the malleus, incus and stapes. The proper connection of the ossicle chain with the tympanic membrane, provided by the insertion of the manubrium of the malleus into the eardrum, is essential for the functionality of the hearing apparatus. We describe here the mechanisms regulating the development of the manubrium and its integration into the tympanic membrane. We show that the external acoustic meatus (EAM), which eventually forms the outer epithelium of the tympanic membrane, plays an essential role in this developmental process. Histological and expression analyses indicate that the manubrium develops close to the EAM with a similar temporal sequence. In addition, when the middle ear ossicles are allowed to develop in vitro under conditions that do not support further EAM development, the manubrium develops only up to the stage of its induction at the time of explantation. Moreover, genetically or teratogenically derived alterations in the EAM also have an effect on manubrial development. Finally, we show that the EAM is the source of two quite opposite activities, one that induces chondrogenesis and another that represses it. The combination of these two activities results in the proper positioning of the manubrium.

Asunto(s)

Inducción Embrionaria , Martillo/embriología , Membrana Timpánica/embriología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Condrogénesis , Oído Medio/embriología , Oído Medio/cirugía , Epitelio , Factor 4 de Crecimiento de Fibroblastos , Factor 9 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento , Proteínas del Grupo de Alta Movilidad/genética , Proteínas de Homeodominio/genética , Mandíbula/embriología , Mesodermo , Ratones , Ratones Mutantes , Modelos Biológicos , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas , Factor de Transcripción SOX9 , Trasplante de Tejidos , Factores de Transcripción/genética

RESUMEN

Foregut development produces a characteristic sequence of gastrointestinal and respiratory organs, but the signaling pathways that ensure this developmental order remain largely unknown. Here, mutations of activin receptors ActRIIA and ActRIIB are shown to disrupt the development of posterior foregut-derived organs, including the stomach, pancreas, and spleen. Foregut expression of genes including Shh and Isl1 is shifted in mutant mice. The endocrine pancreas is particularly sensitive to the type and extent of receptor inactivation. ActRIIA(+/-)B(+/-) animals lack axial defects, but have hypoplastic pancreatic islets, hypoinsulinemia, and impaired glucose tolerance. Thus, activin receptor-mediated signaling regulates axial patterning, cell differentiation, and function of foregut-derived organs.

Asunto(s)

Tipificación del Cuerpo/fisiología , Proteínas del Tejido Nervioso , Páncreas/embriología , Páncreas/fisiología , Receptores de Factores de Crecimiento/metabolismo , Transactivadores , Receptores de Activinas Tipo II , Animales , Diferenciación Celular , Sistema Digestivo/embriología , Fenómenos Fisiológicos del Sistema Digestivo , Prueba de Tolerancia a la Glucosa , Proteínas Hedgehog , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hiperplasia , Insulina/sangre , Proteínas con Homeodominio LIM , Masculino , Ratones , Ratones Mutantes , Páncreas/patología , Proteínas/genética , Proteínas/metabolismo , Receptores de Factores de Crecimiento/genética , Bazo/anomalías , Bazo/fisiología , Estómago/anomalías , Estómago/embriología , Factores de Transcripción

RESUMEN

The type II activin receptors, ActRIIA and ActRIIB, have been shown to play critical roles in axial patterning and organ development in mice. To investigate whether their function is required for mesoderm formation and gastrulation as implicated in Xenopus studies, we generated mice carrying both receptor mutations by interbreeding the ActRIIA and ActRIIB knockout mutants. We found that embryos homozygous for both receptor mutations were growth arrested at the egg cylinder stage and did not form mesoderm. Further analyses revealed that ActRIIA(-/-)ActRIIB(+/-) and about 15% of the ActRIIA(-/-) embryos failed to form an elongated primitive streak, resulting in severe disruption of mesoderm formation in the embryo proper. Interestingly, we observed similar gastrulation defects in ActRIIA(-/-)nodal(+/-) double mutants, which, if they developed beyond the gastrulation stage, displayed rostral head defects and cyclopia. These results provide genetic evidence that type II activin receptors are required for egg cylinder growth, primitive streak formation, and rostral head development in mice.

Asunto(s)

Tipificación del Cuerpo/fisiología , Desarrollo Embrionario y Fetal/fisiología , Receptores de Factores de Crecimiento/fisiología , Receptores de Activinas Tipo II , Animales , Secuencia de Bases , Tipificación del Cuerpo/genética , Cruzamientos Genéticos , Cartilla de ADN/genética , Desarrollo Embrionario y Fetal/genética , Femenino , Gástrula/citología , Cabeza/embriología , Hibridación in Situ , Mesodermo/citología , Ratones , Ratones Noqueados , Embarazo , Receptores de Factores de Crecimiento/genética , Transducción de Señal , Factor de Crecimiento Transformador beta/fisiología

RESUMEN

Messenger RNA expression of activin betaC subunit in the liver was compared with that for betaA subunit before and after 70% hepatectomy. mRNA for betaC was abundantly expressed in the liver but decreased at 12 h and later after 70% hepatectomy, whereas that for betaA was increased 12 h after the hepatectomy. We also compared the expression of mRNA for betaA and betaC in cultured rat hepatocytes. mRNA for betaC subunit was abundantly expressed in the beginning of the culture but was reduced gradually after stimulation with epidermal growth factor. In contrast, mRNA for betaA subunit was undetectable before stimulation and was increased 24 to 48 h after stimulation with the mitogen. These results indicate that expression of mRNA for betaC and betaA is regulated differently. The role of activin C may be different from that of activin A in the liver.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/genética , Inhibinas , Hígado/química , Péptidos/genética , Proteínas de Secreción Prostática , ARN Mensajero/análisis , Animales , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Hepatectomía , Humanos , Hígado/citología , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Masculino , ARN Mensajero/genética , Ratas , Ratas Wistar , Factores de Tiempo

RESUMEN

The mouse inhibin/activin beta C gene (Inhbc), a member of the transforming growth factor-beta (TGF-beta) superfamily, was cloned, mapped, and characterized. The gene spans approximately 14 kb, is composed of two exons, and maps to the distal region of mouse chromosome 10, which is syntenic to chromosome 12q13.1, where the human inhibin/activin beta C gene (INHBC) maps. The primary translation product is a preproprotein of 352 amino acids. The mature C-terminal domain of 116 amino acids shares 94% identity with its human homolog. Primer extension analysis shows that transcription starts approximately 130 bp upstream of the translation initiation site, and no TATA box was found in the promoter. Ribonuclease protection analyses reveal that mouse Inhbc is predominantly expressed in adult liver. Embryonic expression is detected beginning from Day 14.5 of gestation.

Asunto(s)

Mapeo Cromosómico , Regulación del Desarrollo de la Expresión Génica , Inhibinas/genética , Activinas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Genes/genética , Humanos , Hígado/química , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Especificidad de Órganos , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción Genética/genética

RESUMEN

We have characterized the genomic organization of a mouse type I activin receptor. Using the mouse tsk7L cDNA, 4 overlapping lambda clones containing the activin receptor IA (ActRIA) gene were isolated from a mouse 129 Sv genomic library. The mouse ActRIA gene is encoded by 10 exons and spans approximately 40 kb. The size of the introns was determined and the intron/exon boundaries were sequenced. Primer extension analysis of the 5' non-translated region using RNA from different organs or tissues revealed a strong transcription start site 68 nucleotides upstream of the ATG. Knowledge of the structure of the ActRIA gene is essential for the production of ActRIA deficient mice by homologous recombination.

Asunto(s)

Ratones/genética , Receptores de Factores de Crecimiento/genética , Receptores de Activinas , Animales , Secuencia de Bases , Encéfalo/metabolismo , Línea Celular , Clonación Molecular , ADN/química , Cartilla de ADN , Exones , Biblioteca Genómica , Humanos , Intrones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Especificidad de Órganos , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Mapeo Restrictivo , Transcripción Genética

RESUMEN

We have determined the precise chromosomal location, the exon structure, and the expression pattern of CGM2, a member of the carcinoembryonic antigen (CEA) gene family. CGM2 cDNA was amplified by reverse transcription-polymerase chain reaction (RT/PCR) from the colon adenocarcinoma cell line, LS174T. A defective exon is missing from this cDNA clone, leading to a novel domain organization for the human CEA family with two immunoglobulin-like domains. The derived C-terminal domain predicts that the CGM2 protein is membrane-bound through a glycosyl phosphatidylinositol anchor. RT/PCR analyses identified CGM2 transcripts in mucinous ovarian and colonic adenocarcinomas as well as in adjacent colonic tissue, but not in other tumors including leukocytes from six chronic myeloid leukemia patients. Thus, unlike several other family members, CGM2 is not expressed in granulocytes but reveals a more CEA-like expression pattern. Northern blot analyses identified a 2.5-kilobase CGM2 mRNA that is strongly down-regulated in colonic adenocarcinomas compared with adjacent colonic mucosa, suggesting a possible tumor suppressor function. In addition, a 3.2-kilobase transcript was observed in a number of colon tumors that is not detectable in normal colonic tissue. This mRNA species could represent a tumor-specific CGM2 splice variant.

Asunto(s)

Adenocarcinoma/genética , Antígenos de Neoplasias/genética , Antígeno Carcinoembrionario/genética , Moléculas de Adhesión Celular/genética , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cósmidos , ADN Complementario , Regulación hacia Abajo , Exones , Proteínas Ligadas a GPI , Humanos , Mucosa Intestinal/inmunología , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales Cultivadas

RESUMEN

We have made a null mutation in the mouse activin beta B gene by deleting the portion of the gene encoding the proteolytic cleavage site and the majority of the coding region for the mature processed protein. Mice homozygous for this mutation complete embryogenesis and are completely viable. Approximately 40% of the homozygous mutant animals are born with open eyes. Aside from the incompletely penetrant eye defects, histopathological analysis has not revealed any other abnormalities in homozygous mutant animals. Breeding tests have shown that both male and female homozygous mutant animals are fertile.

Asunto(s)

Activinas , Desarrollo Embrionario y Fetal/fisiología , Fertilidad/fisiología , Homocigoto , Mutación , Oligopéptidos , Péptidos/genética , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Alelos , Animales , Secuencia de Bases , Southern Blotting , Línea Celular , Embrión de Mamíferos/química , Embrión de Mamíferos/citología , Desarrollo Embrionario y Fetal/genética , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Péptidos/fisiología , Reacción en Cadena de la Polimerasa

RESUMEN

Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region.

Asunto(s)

Antígeno Carcinoembrionario/genética , Moléculas de Adhesión Celular , Regulación de la Expresión Génica , Genes , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Secuencia de Bases , Clonación Molecular/métodos , Cósmidos , Vectores Genéticos , Biblioteca Genómica , Glicoproteínas/genética , Humanos , Datos de Secuencia Molecular , Mapeo Nucleótido , Sondas de Oligonucleótidos , Especificidad de Órganos , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética

RESUMEN

Complementary DNA clones coding for both carcinoembryonic antigen (CEA), a well characterized colonic tumor marker, and nonspecific cross-reacting antigen (NCA), a related antigen, were expressed in Chinese hamster ovary (CHO) cells and L-cells (mouse fibroblasts). A genomic clone coding for CEA was also expressed in CHO cells. Positive clones were identified by fluorescence flow cytometry and enzyme-linked immunosorbent assay. Membrane location of the recombinant CEA and NCA was confirmed by indirect immunofluorescence labeling of the transfectants, followed by visualization under a fluorescence microscope. The apparent molecular weight of the expressed CEA and NCA were 180,000 and 96,000, respectively, for both cell lines, as determined by immunoblot analysis. The CEA and NCA expressed on CHO cells were sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the CEA and NCA proteins on L-cells were resistant to removal by PI-PLC. Unlike NCA, which contains three methionine residues, the only methionine in CEA is in the C-terminal hydrophobic domain. This domain in CEA was shown to be removed and replaced by a phosphatidylinositol glycan (PI-G) anchor (Hefta et al., Proc. Natl. Acad. Sci. USA, 85: 4648-4652, 1988). The recombinant CEA from both CHO cells and L-cells could be labeled with [3H]-ethanolamine (a component of the PI-G anchor) but not with [35S] methionine, whereas the recombinant NCA could be labeled with both [3H]ethanolamine and [35S]methionine. The labeling studies and PI-PLC treatment results are consistent with the CEA and NCA expressed on CHO cells possessing a PI-G anchor. The CEA expressed on the L-cell transfectants may contain a PI-G anchor which is resistant to cleavage by PI-PLC. In addition, the membrane-bound and secreted levels of CEA from the CHO and L-cell transfectants were determined.

Asunto(s)

Antígenos de Neoplasias/genética , Antígeno Carcinoembrionario/genética , Moléculas de Adhesión Celular , Glicoproteínas/genética , Animales , Antígenos de Neoplasias/análisis , Antígeno Carcinoembrionario/análisis , Línea Celular , Membrana Celular/inmunología , Cricetinae , Cricetulus , Femenino , Citometría de Flujo , Expresión Génica , Glicoproteínas/análisis , Humanos , Immunoblotting , Células L/inmunología , Ratones , Peso Molecular , Ovario , Mapeo Restrictivo , Transfección

RESUMEN

The pregnancy-specific glycoprotein (PSG) genes encode a group of proteins which are found in large amounts in placenta and maternal serum. In situ hybridization analyses of metaphase chromosomes reveal that all the human pregnancy-specific glycoprotein (PSG) genes are located on the long arm of chromosome 19 (19q13.2-13.3), overlapping the region containing the closely-related carcinoembryonic antigen (CEA) gene subgroup. Higher resolution analyses indicate that the PSG genes are closely linked within an 800kb SacII restriction endonuclease fragment. This has been confirmed through restriction endonuclease mapping and DNA sequence analyses of isolated genomic clones, which show that at least some of these genes are located in very close proximity. Further, these studies have helped to identify a new member of the PSG gene subfamily (PSG7). DNA/RNA hybridization analyses, using gene-specific oligonucleotide probes based on published sequences, showed that five from six PSG genes tested are coordinately transcribed in the placenta. Due to the close proximity of these genes and their coordinated expression pattern, common transcriptional regulatory elements may exist.

Asunto(s)

Cromosomas Humanos Par 19 , ADN/genética , Genes , Ligamiento Genético , Proteínas Gestacionales/genética , Secuencia de Bases , Antígeno Carcinoembrionario/genética , Línea Celular , Clonación Molecular , ADN/aislamiento & purificación , Femenino , Biblioteca Genómica , Humanos , Datos de Secuencia Molecular , Peso Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Placenta/metabolismo , Embarazo , ARN/genética , ARN/aislamiento & purificación , Mapeo Restrictivo

RESUMEN

Five members of the human CEA gene family [human pregnancy-specific beta 1-glycoprotein (PS beta G); hsCGM1, 2, 3 and 4] have been isolated and identified through sequencing the exons containing their N-terminal domains. Sequence comparisons with published data for CEA and related molecules reveal the existence of highly-conserved gene subgroups within the CEA family. Together with published data eleven CEA family members have so far been determined. Apart from the highly conserved coding sequences, these genes also show strong sequence conservation in their introns, indicating a duplication of whole gene units during the evolution of the CEA gene family.

Asunto(s)

Antígenos de Neoplasias , Antígeno Carcinoembrionario/genética , Moléculas de Adhesión Celular , Secuencia de Aminoácidos , Animales , Antígenos , Bacteriófago lambda/genética , Secuencia de Bases , Cósmidos , Sondas de ADN , Enzimas de Restricción del ADN , ADN Recombinante , Exones , Glicoproteínas/genética , Humanos , Intrones , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Neoplasias Experimentales/análisis , Hibridación de Ácido Nucleico , Glicoproteínas beta 1 Específicas del Embarazo/genética , ARN Mensajero/análisis

RESUMEN

Immunocompromised children with acute leukemias and solid tumors are at high risk of fatal varicella infection. Reviewing a total of 242 patients at risk we have found that zoster immune plasma from reconvalescent patients (ZIP) and commercially available specific varicella/zoster immune globulin (VZIG) both prevent fatal disease. In addition, Acyclovir was effective against VZV-infections in this group of patients. We summarize our present policy of prophylaxis and treatment of immunocompromised children with neoplastic disease who have been exposed to VZV.

Asunto(s)

Aciclovir/uso terapéutico , Herpes Zóster/terapia , Inmunización Pasiva/métodos , Neoplasias/complicaciones , Enfermedad Aguda , Niño , Preescolar , Enfermedad de Hodgkin/complicaciones , Humanos , Leucemia/complicaciones , Pronóstico