RESUMEN
Classic Hodgkin lymphoma (cHL) is characterized by multinucleated cells called Reed-Sternberg (RS) cells and genetic complexity. Although CD30 also characterizes cHL cells, its biological roles are not fully understood. In this report, we examined the link between CD30 and these characteristics of cHL cells. CD30 stimulation increased multinucleated cells resembling RS cells. We found chromatin bridges, a cause of mitotic errors, among the nuclei of multinucleated cells. CD30 stimulation induced DNA double-strand breaks (DSBs) and chromosomal imbalances. RNA sequencing showed significant changes in the gene expression by CD30 stimulation. We found that CD30 stimulation increased intracellular reactive oxygen species (ROS), which induced DSBs and multinucleated cells with chromatin bridges. The PI3K pathway was responsible for CD30-mediated generation of multinucleated cells by ROS. These results suggest that CD30 involves generation of RS cell-like multinucleated cells and chromosomal instability through induction of DSBs by ROS, which subsequently induces chromatin bridges and mitotic error. The results link CD30 not only to the morphological features of cHL cells, but also to the genetic complexity, both of which are characteristic of cHL cells.
Asunto(s)
Enfermedad de Hodgkin , Células de Reed-Sternberg , Humanos , Células de Reed-Sternberg/metabolismo , Células de Reed-Sternberg/patología , Enfermedad de Hodgkin/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Línea Celular , Inestabilidad Cromosómica/genética , Cromatina/genética , Cromatina/metabolismo , Antígeno Ki-1/genética , Antígeno Ki-1/metabolismoRESUMEN
BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 (PTCH1) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1 in an individual with NBCCS. METHODS AND RESULTS: A de novo germline mutation of PTCH1 (c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%-15.6% depending on the tissue) identical to the one found in iPSC clones. CONCLUSIONS: This is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation.
Asunto(s)
Síndrome del Nevo Basocelular/genética , Mutación del Sistema de Lectura , Células Madre Pluripotentes Inducidas/fisiología , Mosaicismo , Receptor Patched-1/genética , Neoplasias Cutáneas/genética , Adulto , Alelos , Células Cultivadas , Femenino , HumanosRESUMEN
A recent report indicated involvement of CD30 in progression of human leukemia virus type 1 (HTLV-1) infection, but the exact roles of CD30 in this process remain unclear. This study was conducted to determine the role of CD30 by stimulating CD30 expressed on HTLV-1-infected cell lines with CD30 ligand and observing its effects. CD30 stimulation increased multinucleated cells and inhibited proliferation of HTLV-1-infected cells. This inhibition was recovered by interruption of CD30 stimulation. Chromatin bridges found in multinucleated cells suggested DNA damage. CD30 stimulation triggered DNA double-strand breaks (DSBs) and chromosomal imbalances. CD30 stimulation induced reactive oxygen species (ROS), which induced DSBs. Generation of ROS and multinucleated cells by CD30 was dependent on phosphoinositide 3-kinase. RNA sequencing showed that CD30 stimulation produced significant changes in gene expression profiles, including upregulation of programmed death ligand 1 (PD-L1). Tax, which has also been shown to induce multinucleation and chromosomal instability, failed to induce CD30. These results suggest that induction of CD30, independent of Tax, triggers morphological abnormalities, chromosomal instability, and alteration of gene expression in HTLV-1-infected cells.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano , Humanos , Virus Linfotrópico T Tipo 1 Humano/genética , Fosfatidilinositol 3-Quinasas , Especies Reactivas de Oxígeno , Línea Celular , Inestabilidad CromosómicaRESUMEN
Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1. The PTCH1 gene has five alternatively used first exons resulting in the translation of three isoforms of the PTCH1 protein; that is, PTCHL, PTCHM and PTCHS. However, the biological significance of each isoform is unclear. Here we show an individual with NBCCS carrying a nonsense mutation in PTCH1 exon2, c.387G>A (p.W129X). As the mutation lay upstream of the ATG codon used for PTCHS translation, the mutant allele still expressed RNA isoforms that encode PTCHS. These results clearly demonstrate that a selective haploinsufficiency of longer isoforms of the PTCH1 protein, PTCHL and PTCHM, but not PTCHS is sufficient to cause NBCCS. Although mice selectively deficient in PTCHS isoforms are currently unavailable, this study sheds light on the complex in vivo roles of PTCH1 isoforms.
Asunto(s)
Síndrome del Nevo Basocelular/genética , Haploinsuficiencia , Receptores de Superficie Celular/genética , Síndrome del Nevo Basocelular/metabolismo , Síndrome del Nevo Basocelular/patología , Niño , Codón sin Sentido/genética , Exones , Células HEK293 , Humanos , Masculino , Receptores Patched , Receptor Patched-1 , Biosíntesis de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Gorlin syndrome is a rare autosomal dominant hereditary disease with a high incidence of tumors such as basal cell carcinoma and medulloblastoma. Disease-specific induced pluripotent stem cells (iPSCs) and an animal model have been used to analyze disease pathogenesis. In this study, we generated iPSCs derived from fibroblasts of four patients with Gorlin syndrome (Gln-iPSCs) with heterozygous mutations of the PTCH1 gene. Gln-iPSCs from the four patients developed into medulloblastoma, a manifestation of Gorlin syndrome, in 100% (four out of four), of teratomas after implantation into immunodeficient mice, but none (0/584) of the other iPSC-teratomas did so. One of the medulloblastomas showed loss of heterozygosity in the PTCH1 gene while the benign teratoma, i.e. the non-medulloblastoma portion, did not, indicating a close clinical correlation between tumorigenesis in Gorlin syndrome patients and Gln-iPSCs.
Asunto(s)
Síndrome del Nevo Basocelular/genética , Neoplasias Cerebelosas/genética , Pérdida de Heterocigocidad/genética , Meduloblastoma/genética , Receptor Patched-1/genética , Adolescente , Adulto , Niño , Femenino , Humanos , Células Madre Pluripotentes Inducidas , MasculinoRESUMEN
Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 (PTCH1) gene. To date, we have detected 73 mutations in PTCH1 and ten of them (14 %) were suspected splicing mutations. Eight out of the ten mutations were localized near the splice donor site. Five mutations were localized within the invariant GT-AG splice site, whereas the other five mutations occurred outside the invariant GT-AG site including the last exonic nucleotide. When the transcripts were examined, all mutations resulted in aberrant splicing, including exon skipping or the activation of cryptic splice sites. This is the first extensive report of NBCCS focusing on splice site mutations, and it highlights the importance of analyzing transcripts especially for mutations lying outside the GT-AG splicing consensus site. In addition, the splice site score calculated by Splice-Site Analyzer Tool provided by Tel Aviv University helped predict aberrant splice patterns in most of the cases.
Asunto(s)
Síndrome del Nevo Basocelular/genética , Mutación , Receptor Patched-1/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Sitios de Empalme de ARN , Empalme del ARNRESUMEN
Porcine T-cell receptor alpha (TCRalpha)-chain cDNA clones were isolated from libraries made from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes (PBL) of a 5-month-old Clawn strain pig. Among 109 cDNA clones with the Jalpha-gene segment, 44 different Jalpha-gene segments were found out of the 61 Jalpha-gene segments previously identified in the porcine germline sequence. Among the 103 complete TCRalpha-chain cDNA clones with the rearranged Valpha- and Jalpha-gene segments, 33 different Valpha-gene segments were identified, which randomly rearranged to Jalpha-gene segments indicating lack of any specific combinations between Valpha- and Jalpha-gene segments with only one exception of the same set of Jalpha-gene segments in duplicate clones. Among the cDNA clones from PBL of an individual 5-month-old Clawn strain pig, a broad distribution of the Jalpha-gene segment usage was observed over the entire Jalpha-gene cluster. The Jalpha-gene segment usage in an individual 1-month-old thymus from a LW strain pig also gave a pattern consistent with the 5-month-old pig. These distributions of the Jalpha-gene segment usage were similar to the previously reported patterns for human T-cells and those of adult murine T-cells. Among the porcine cDNA clones isolated, TCRalpha-chain CDR3 length ranged from 4 to 14 amino acids with the average being 9.35 amino acids. Present report provides groundwork for further studies on porcine TCRalpha-chain expression.
Asunto(s)
Regiones Determinantes de Complementariedad/genética , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Variación Genética , Humanos , Cadenas J de Inmunoglobulina/genética , Linfocitos/inmunología , Ratones , Datos de Secuencia Molecular , Especificidad de la Especie , Sus scrofa , Porcinos , Timo/inmunologíaRESUMEN
We analyzed mutant mice showing behavioral defects such as severe tremor, up-and-down and side-to-side wriggling of neck without coordination, and found that the gene causing the defects was located between 46 and 60.55 centimorgans (cM) on the mouse chromosome 6. In this region, nucleotide transition of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) gene was found, which caused a glutamic acid to change into lysine. Since PMCA2 is expressed in the cerebellum and plays an important role to maintain the homeostasis of the intracellular Ca2+ as a Ca2+ pump, the behavioral defect can be ascribed to the impairment of Ca2+ regulation in neurons of the cerebellum. To confirm the defect of Ca2+ homeostasis in the mutant mice, we measured high K+-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in the cerebellar neurons. Contrary to our expectation, the extent of the [Ca2+]i increase in all the regions tested in the cerebellar slice was far smaller than that of the wild type mice, while the resting [Ca2+]i remained almost unaltered. The rate of rise in [Ca2+]i during high K+-induced depolarization was significantly reduced, and the extrusion rate of increased [Ca2+]i was also reduced. These results suggested that voltage-gated Ca2+ channels were down-regulated in the mutant mice in order to regulate [Ca2+]i toward the normal homeostasis. The behavioral defects may be ascribed to the down-regulated Ca2+ homeostasis since dynamic changes in [Ca2+]i are important for various neuronal functions.
Asunto(s)
Señalización del Calcio/genética , ATPasas Transportadoras de Calcio/deficiencia , ATPasas Transportadoras de Calcio/genética , Calcio/metabolismo , Membrana Celular/metabolismo , Cerebelo/metabolismo , Proteínas de Homeodominio , Neuronas/metabolismo , Mutación Puntual/fisiología , Proteínas Protozoarias , Animales , Conducta Animal/fisiología , Proteínas de Transporte de Catión , Cerebelo/crecimiento & desarrollo , Cerebelo/fisiopatología , Cromosomas/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Genotipo , Masculino , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Nucleótidos/genética , Fenotipo , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Células de Purkinje/metabolismo , Células de Purkinje/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
OBJECTIVE: The aim of this study was to investigate the molecular pathogenesis of keratocystic odontogenic tumors (KCOTs) that developed in nevoid basal cell carcinoma syndrome (NBCCS) patients. STUDY DESIGN: We analyzed germline and somatic mutations of the PTCH1 and its related genes, SMO and SUFU in 10 KCOTs that developed in 8 unrelated NBCCS patients. Methylation status of the PTCH1 promoter was also investigated by bisulfite sequencing. RESULTS: Somatic mutations of PTCH1 were detected in 3 KCOTs. Two out of 3 somatic mutations were either identified as a polymorphism or located on the same allele as the germline mutation. Neither abnormal methylation of the PTCH1 promoter, loss of PTCH1, nor somatic mutation of SMO or SUFU was detected in any of the samples. CONCLUSIONS: Our results suggest that the tumorigenesis of most KCOTs associated with NBCCS cannot be explained by the classical 2-hit theory.
Asunto(s)
Síndrome del Nevo Basocelular/complicaciones , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Tumores Odontogénicos/complicaciones , Receptores de Superficie Celular/genética , Adolescente , Adulto , Anciano , Síndrome del Nevo Basocelular/genética , Preescolar , Análisis Mutacional de ADN , Femenino , Humanos , Pérdida de Heterocigocidad , Masculino , Tumores Odontogénicos/genética , Adulto JovenRESUMEN
Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1, encoding a receptor for the secreted protein, sonic hedgehog. Recently, a Chinese family with NBCCS carrying a missense mutation in PTCH2, a close homolog of PTCH1, was reported. However, the pathological significance of missense mutations should be discussed cautiously. Here, we report a 13-year-old girl diagnosed with NBCCS based on multiple keratocystic odontogenic tumors and rib anomalies carrying a frameshift mutation in the PTCH2 gene (c.1172_1173delCT). Considering the deleterious nature of the frameshift mutation, our study further confirmed a causative role for the PTCH2 mutation in NBCCS. The absence of typical phenotypes in this case such as palmar/plantar pits, macrocephaly, falx calcification, hypertelorism and coarse face, together with previously reported cases, suggested that individuals with NBCCS carrying a PTCH2 mutation may have a milder phenotype than those with a PTCH1 mutation.
Asunto(s)
Síndrome del Nevo Basocelular/genética , Biomarcadores de Tumor/sangre , Mutación del Sistema de Lectura/genética , Tumores Odontogénicos/genética , Receptores de Superficie Celular/genética , Adolescente , Secuencia de Aminoácidos , Síndrome del Nevo Basocelular/sangre , Síndrome del Nevo Basocelular/patología , Secuencia de Bases , Estudios de Casos y Controles , ADN de Neoplasias/genética , Femenino , Humanos , Datos de Secuencia Molecular , Tumores Odontogénicos/sangre , Tumores Odontogénicos/patología , Receptores Patched , Receptor Patched-1 , Receptor Patched-2 , Reacción en Cadena de la Polimerasa , Pronóstico , Receptores de Superficie Celular/sangreRESUMEN
Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.