RESUMEN
Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD, we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation, we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3, a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.
Asunto(s)
Trastorno Bipolar/patología , Expresión Génica/fisiología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/fisiología , ARN Mensajero/metabolismo , Receptores CXCR4/genética , Diferenciación Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Variaciones en el Número de Copia de ADN/genética , Salud de la Familia , Femenino , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Potenciales de la Membrana/fisiología , Polimorfismo de Nucleótido Simple , Receptores CXCR4/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/fisiologíaRESUMEN
The family of immune associated nucleotide binding proteins (Ian) is a distinct family of GTP-binding proteins conserved in plants, mice, rats and humans that are associated with immune functions, suggesting involvement in conserved defense mechanisms. Recently, the rat Ian4 (rIan4) was cloned and it appears to be identical to the gene Iddm1/lyp responsible for severe lymphopenia and the development of insulin-dependent diabetes in the BB-DP rat. Here we describe the characterization of a new human member of the Ian family: hIan5. hIan5 is highly homologous to rIan4, has a predicted molecular weight of 35 kDa and contains distinct G motifs of GTP-binding proteins (G-1 to G-4) in the N-terminus. Human Ian5 is anchored to the mitochondria by the hydrophobic COOH-terminal domain. Human Ian5 is highly expressed in lymph node and spleen. Different blood fractions show high hIan5 expression in CD4- and CD8-positive T cells and monocytes, but not in B lymphocytes. In contrast, in B-CLL (chronic lymphocytic leukemia) and mantle cell lymphoma samples, hIan5 mRNA was upregulated. The current data underline the role of hIan5 in T-lymphocyte development and function, and for the first time suggest that upregulation of Ian proteins is associated with B-cell malignancy, possibly by inhibiting apoptosis.
Asunto(s)
Cromosomas Humanos Par 7/genética , Proteínas de Unión al GTP/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Fraccionamiento Celular , Proteínas de Unión al GTP/genética , Humanos , Ganglios Linfáticos/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Familia de Multigenes/genética , Plásmidos/genética , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Bazo/metabolismo , Linfocitos T/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The mixed lineage leukemia, MLL, gene is frequently rearranged in patients with secondary leukemia following treatment with DNA topoisomerase II inhibitors. By FISH and Southern blot analyses we identified a rearrangement in the MLL gene due to a novel t(3;11)(q28;q23) chromosomal translocation in a patient who developed AML-M5 3 years after treatment for a follicular lymphoma. Through inverse PCR, the LPP (lipoma preferred partner) gene on 3q28 was identified as the MLL fusion partner. LPP contains substantial identity to the focal adhesion protein, zyxin, and is frequently fused to HMGIC in lipomas. The breakpoint occurred in intron 8 of MLL and LPP. Two in-frame MLL-LPP transcripts, which fuse MLL exon 8 to LPP exon 9, were detected by RT-PCR, although the smaller of these contained a deletion of 120 bp from the MLL sequence. The predicted MLL-LPP fusion protein includes the A/T hook motifs and methyltransferase domain of MLL joined to the two last LIM domains of LPP. A reciprocal LPP-MLL transcript, predicted to include the proline-rich and leucine zipper motifs, and the first LIM domain of LPP were also detected by RT-PCR. In summary, LPP is a newly identified MLL fusion partner in secondary leukemia resulting from topoisomerase inhibitors. The MLL-LPP and LPP-MLL predicted proteins contain many of the features present in other MLL rearrangements.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Leucemia Monocítica Aguda/genética , Linfoma Folicular/genética , Neoplasias Primarias Secundarias/genética , Proteínas de Fusión Oncogénica/genética , Proto-Oncogenes , Factores de Transcripción , Translocación Genética/genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Rotura Cromosómica/genética , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 3/genética , Clonación Molecular , Resultado Fatal , Femenino , N-Metiltransferasa de Histona-Lisina , Humanos , Cariotipificación , Proteínas con Dominio LIM , Leucemia Monocítica Aguda/inducido químicamente , Linfoma Folicular/tratamiento farmacológico , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide , Neoplasias Primarias Secundarias/inducido químicamente , ARN Mensajero/genéticaRESUMEN
Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.
Asunto(s)
Proteínas de Unión al GTP/genética , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Cruzamientos Genéticos , ADN Complementario/química , ADN Complementario/genética , Femenino , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/fisiología , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Haplotipos , Humanos , Membranas Intracelulares/metabolismo , Células K562 , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , ARN/genética , ARN/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
The phenotype of hematopoietic cells transformed by the BCR/ABL oncoprotein of the Philadelphia chromosome is characterized by growth factor-independent proliferation, reduced susceptibility to apoptosis, and altered adhesion and motility. The mechanisms underlying this phenotype are not fully understood, but there is evidence that some of the properties of BCR/ABL-expressing cells are dependent on the activation of downstream effector molecules such as RAS, PI-3k, and bcl-2. We show here that the small GTP-binding protein Rac is activated by BCR/ABL in a tyrosine kinase-dependent manner. Upon transfection with a vector carrying the dominant-negative N17Rac, BCR/ABL-expressing myeloid precursor 32Dcl3 cells retained the resistance to growth factor deprivation-induced apoptosis but showed a decrease in proliferative potential in the absence of interleukin-3 (IL-3) and markedly reduced invasive properties. Moreover, compared with BCR/ABL-expressing cells, fewer BCR/ABL plus N17Rac double transfectants were capable of homing to bone marrow and spleen. Consistent with these findings, survival of SCID mice injected with the BCR/ABL plus N17Rac double transfectants was markedly prolonged as compared with that of mice injected with BCR/ABL-expressing cells. Together, these data support the important role of a Rac-dependent pathway(s) controlling motility in BCR/ABL-mediated leukemogenesis.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Proteínas de Unión al GTP/metabolismo , Leucemia Experimental/etiología , Leucemia Experimental/genética , Animales , Secuencia de Bases , División Celular , Línea Celular Transformada , Movimiento Celular , Supervivencia Celular , Cartilla de ADN/genética , Proteínas de Fusión bcr-abl/metabolismo , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Experimental/metabolismo , Ratones , Ratones SCID , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Fenotipo , Proteínas de Unión al GTP racRESUMEN
The Bcr-Abl fusion protein plays a crucial role in the initiation and maintenance of chronic myelogenous leukemia (CML). However, additional events are necessary for the transition from the chronic phase to the terminal phase of the disease. To identify genes involved in the disease progression, we constructed a subtractive library from enriched K562 cell mRNA. We obtained 1084 cDNA clones. After a specific hybridization of these clones with a cDNA probe from either chronic phase or K562 cells, 43 clones which present a differential hybridization level have been selected. Among them, several clones corresponded to ribosomal protein genes showing an increased transcription level during the blast crisis. We observed variations in the expression of a cellular adhesion molecule, a laminin-binding protein. An increased transcription level of the MAZ gene has been shown in the terminal phase of the disease. This gene encodes a protein that regulates the transcription of myc.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Transcripción Genética , Crisis Blástica , Proteínas Portadoras/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Cartilla de ADN , Proteínas de Unión al ADN , Progresión de la Enfermedad , Biblioteca de Genes , Humanos , Laminina/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Recuento de Leucocitos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-myc/biosíntesis , ARN Mensajero/biosíntesis , Proteínas Ribosómicas/biosíntesis , Factores de Transcripción/biosíntesis , Células Tumorales CultivadasRESUMEN
In vitro amplification of the Bcr-Abl cDNA has been widely used to assess for the presence of minimal residual disease in patients with chronic myelogenous leukaemia (CML) presenting with complete clinical and cytogenetic remission. However, the level of sensitivity achieved in different laboratories remains largely unknown. Moreover, the results are usually expressed as positive or negative, making a precise follow-up of the patients difficult. In an attempt to overcome these limitations, we devised a quantitative method to measure the amount of Bcr-Abl mRNA in clinical samples. This methodology involves a single reverse transcription step, followed by separate amplifications of Bcr-Abl and total Abl mRNA. These two amplifications are performed in the presence of different dilutions of a same internal standard. This standard consists of Bcr-Abl sequences with an eight bases deletion in exon 2 of Abl. One of the primers used in each separate reaction is labelled with fluorescein. Following amplification, PCR products derived from cellular RNA and those from the internal standard are separated and their relative fluorescence is determined using a laser fluorescent DNA sequencer (ALF, Pharmacia). The number of Bcr-Abl and total Abl mRNA molecules initially present in each sample is then calculated. The accuracy and reproducibility of this method was assessed by studying serial dilutions of K562 RNA into normal RNA. Blood samples from 10 patients in cytogenetic remission under interferon therapy were studied. Only one sample was found negative while the others contained between 0.05 and 17 hybrid Bcr-Abl mRNA molecules per 1000 molecules of Abl mRNA. These results suggest that a variable number of malignant cells are present in the majority of CML patients in cytogenetic remission following interferon therapy.