RESUMEN

Protein tyrosine kinase 7 (PTK7) is an evolutionarily conserved transmembrane receptor with important roles in embryonic development and disease. Originally identified as a gene upregulated in colon cancer, it was later shown to regulate planar cell polarity (PCP) and directional cell movement. PTK7 is a Wnt co-receptor; however, its role in Wnt signaling remains controversial. Here, we find evidence that places PTK7 at the intersection of canonical and non-canonical Wnt signaling pathways. In presence of canonical Wnt ligands PTK7 is subject to caveolin-mediated endocytosis, while it is unaffected by non-canonical Wnt ligands. PTK7 endocytosis is dependent on the presence of the PTK7 co-receptor Fz7 (also known as Fzd7) and results in lysosomal degradation of PTK7. As we previously observed that PTK7 activates non-canonical PCP Wnt signaling but inhibits canonical Wnt signaling, our data suggest a mutual inhibition of canonical and PTK7 Wnt signaling. PTK7 likely suppresses canonical Wnt signaling by binding canonical Wnt ligands thereby preventing their interaction with Wnt receptors that would otherwise support canonical Wnt signaling. Conversely, if canonical Wnt proteins interact with the PTK7 receptor, they induce its internalization and degradation.

Asunto(s)

Caveolina 1/genética , Moléculas de Adhesión Celular/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Wnt/genética , Vía de Señalización Wnt , Proteína Wnt3A/genética , Animales , Caveolina 1/metabolismo , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular , Clatrina/genética , Clatrina/metabolismo , Embrión no Mamífero , Endocitosis , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ligandos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Células MCF-7 , Unión Proteica , Estabilidad Proteica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt3A/metabolismo , Xenopus laevis , beta Catenina/genética , beta Catenina/metabolismo

RESUMEN

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.

Asunto(s)

Movimiento Celular , Cresta Neural/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Cresta Neural/citología , Cresta Neural/embriología , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Vía de Señalización Wnt , Xenopus , Proteínas de Xenopus/genética

RESUMEN

In the polychaete Platynereis dumerilii exactly four primordial germ cells (PGCs) arise in early development and are subject to a transient mitotic arrest until the animals enter gametogenesis. In order to unravel the mechanisms controlling the number of PGCs in Platynereis, we tested whether the steroid 17ß-estradiol (E2) is able to induce PGC proliferation, as it had been described in other species. Our data provide strong support for such a mechanism, showing that E2 significantly increases the occurrence of larvae with supernumerary PGCs in Platynereis in a dose dependent manner. E2 responsiveness is restricted to early developmental stages, when the PGCs are specified. During these stages, embryos exhibit high expression levels of the estradiol receptor (ER). The ER transcript localizes to the yolk-free cytoplasm of unfertilized eggs and segregates into the micromeres during cleavage stages. Nuclear ER protein is found asymmetrically distributed between daughter cells. Neither transcript nor protein is detectable in PGCs at larval stages. Addition of the specific estradiol receptor inhibitor ICI-182,780 (ICI) abolishes the proliferative effect of E2, suggesting that it is mediated by ER signaling. Our study reports for the first time an ER mediated proliferative effect of E2 on PGCs in an invertebrate organism.

Asunto(s)

Embrión no Mamífero/efectos de los fármacos , Estradiol/farmacología , Estrógenos/farmacología , Células Germinativas/efectos de los fármacos , Receptores de Estradiol/metabolismo , Animales , Núcleo Celular/metabolismo , Proliferación Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Fulvestrant , Células Germinativas/citología , Células Germinativas/metabolismo , Técnicas para Inmunoenzimas , Hibridación in Situ , Poliquetos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Estradiol/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos

RESUMEN

Wnt signalling is an evolutionarily conserved pathway that directs cell-fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co-precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non-canonical Wnt signalling by turning off the canonical signalling branch.

Asunto(s)

Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Drosophila , Inmunoprecipitación , Modelos Biológicos , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Xenopus , Proteínas de Xenopus/metabolismo

RESUMEN

Therapy-associated myelodysplastic syndromes and acute myeloid leukaemia (t-AML/MDS) following high dose chemotherapy are significant problems, with a cumulative incidence of 20% or more in myeloablative treatment regimen. Retrospective findings indicated that t-AML/MDS associated genetic aberrations can be observed directly after exposure to chemotherapy and can precede t-AML by several months. To determine the incidence of post-therapeutic aberrations and their predictive value, we prospectively investigated 316 samples of 95 patients with non-Hodgkin lymphoma (NHL) who were treated with intermediate and high dose chemotherapy (Arm A and B of the megaCHOEP (cyclophosphamide, doxorubicin, etoposide, vincristine, prednisolone) trial of the German High Grade NHL study group). Molecular aberrations (RUNX1/RUNX1T1, PML-RARA, CBFB-MYH11, MLL-MLLT1, BCR-ABL1) were observed in 33.3% (Arm A) and 55.4% (Arm B) of patients and in 14.9% and 28.7% of respective samples. Cytogenetic analysis of 53 NHL patients after high dose therapy showed frequent chromosomal breakage. Clonal aberrations were found in three patients. None of these patients developed a t-AML/MDS during a 3-year clinical follow up period. We concluded that the high incidence of genetic aberrations reflected a dose-dependent, transient therapy-induced genetic damage which is not predictive of a t-AML/MDS.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Aberraciones Cromosómicas/efectos de los fármacos , Linfoma no Hodgkin/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ciclofosfamida/uso terapéutico , Relación Dosis-Respuesta a Droga , Doxorrubicina/uso terapéutico , Etopósido/uso terapéutico , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/inducido químicamente , Linfoma no Hodgkin/genética , Persona de Mediana Edad , Prednisolona/uso terapéutico , Pronóstico , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Translocación Genética , Vincristina/uso terapéutico

RESUMEN

AML-associated MLL-PTD contribute to leukemogenesis by a gain of function and confer an unfavorable prognosis. Like other leukemia associated aberrations they are also present in healthy adults. To delineate the leukemogenic mechanism we tracked down MLL-PTD in normal hematopoiesis and investigated cord blood samples. MLL-PTD were observed in 56/60 (93%) of all cord bloods. In contrast to AML, the transcript frequency in cord blood was four log scales lower as determined by real-time PCR. The CD34+ progenitor cell, CD33+ myeloid, CD19+ B-lymphoid and CD3+ T-lymphoid subfractions were positive. The ubiquitous presence of MLL-PTD in cord blood implicates a lifelong exposure, not an accumulation during lifetime. Since also present in the stem cell subfraction, these factors seem not to be major determinants in MLL-PTD leukemogenesis.

Asunto(s)

Duplicación de Gen , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Adulto , Antígenos CD/metabolismo , Linfocitos B/metabolismo , Linfocitos B/patología , Femenino , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , N-Metiltransferasa de Histona-Lisina , Humanos , Recién Nacido , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/patología , Masculino , Linfocitos T/metabolismo , Linfocitos T/patología

RESUMEN

Chronic myeloid leukemia (CML) is characterized by the presence of a t(9;22)(q34;q11.2), which leads to the well-known BCR-ABL1 fusion protein. We describe a patient who was diagnosed clinically with a typical CML but on cytogenetic analysis was found to have a t(9;22)(p24;q11.2). Chromosomal fluorescence in situ hybridization showed that the BCR gene locus spanned the breakpoint at band 22q11.2 but that the ABL1 gene was not rearranged. By means of a candidate gene approach, the JAK2 gene, at 9p24, was identified as the fusion partner of BCR in this case. The BCR-JAK2 fusion protein contains the coiled-coil dimerization domain of BCR and the protein tyrosine kinase domain (JH1) of JAK2. The patient's disease did not respond to Imatinib, and this unresponsiveness was most likely a result of the BCR-JAK2 fusion protein.

Asunto(s)

Cromosomas Humanos Par 22/genética , Cromosomas Humanos Par 9/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcr/genética , Proteínas Proto-Oncogénicas/genética , Translocación Genética/genética , Antineoplásicos/uso terapéutico , Benzamidas , Femenino , Humanos , Mesilato de Imatinib , Hibridación Fluorescente in Situ , Janus Quinasa 2 , Cariotipificación , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Persona de Mediana Edad , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico

RESUMEN

FLT3 is a receptor tyrosine kinase involved in the proliferation and differentiation of hematopoietic stem cells. FLT3 internal tandem duplications (FLT3/ITDs) are reported in acute myeloid leukemia (AML) and predict poor clinical outcome. We found FLT3/ITDs in 11.5% of 234 children with de novo AML. FLT3/ITD-positive patients were significantly older and had higher percentages of normal cytogenetic findings or French-American-British (FAB) classification M1/M2 and lower percentages of 11q23 abnormalities or FAB M5. FLT3/ITD-positive patients had lower remission induction rates (70% vs 88%; P =.01) and lower 5-year probability rates of event-free survival (pEF) (29% vs 46%; P =.0046) and overall survival (32% vs 58%; P =.037). Patients with high ratios (higher than the median) between mutant and wild-type FLT3 had significantly worse 2-year EFS rates than FLT3/ITD-negative patients (pEFS 20% vs 61%; P =.037), whereas patients with ratios lower than the median did not (pEFS 44% vs 61%; P =.26). FLT3/ITD was the strongest independent predictor for pEFS, with an increase in relative risk for an event of 1.92 (P =.01). Using an MTT (methyl-thiazol-tetrazolium)-based assay, we studied cellular drug resistance on 15 FLT3/ITD-positive and 125 FLT3/ITD-negative AML samples, but we found no differences in cellular drug resistance that could explain the poor outcomes in FLT3/ITD-positive patients. We conclude that FLT3/ITD is less common in pediatric than in adult AML. FLT3/ITD is a strong and independent adverse prognostic factor, and high ratios between mutant and WT-FLT3 further compromise prognosis. However, poor outcomes in FLT3/ITD-positive patients could not be attributed to increased in vitro cellular drug resistance.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Enfermedad Aguda , Adolescente , Antibióticos Antineoplásicos/uso terapéutico , Niño , Preescolar , Daunorrubicina/uso terapéutico , Femenino , Humanos , Idarrubicina/uso terapéutico , Lactante , Recién Nacido , Leucemia Mieloide Aguda/epidemiología , Masculino , Pronóstico , Factores de Riesgo , Secuencias Repetidas en Tándem , Tirosina Quinasa 3 Similar a fms

RESUMEN

ETV6, a member of the Ets family of transcription factors, is frequently rearranged to various translocation partners in human leukaemias. We previously described a CD3+/TCRalpha/beta+ mature T-cell acute lymphoblastic leukaemia (T-ALL) cell line, MT-ALL, carrying a t(1;10;12)(q25; p13;p13) with cytokine-inducible lineage switch into the myeloid lineage. Using reverse transcription polymerase chain reaction with primers complementary to ETV6 and ABL2, two ETV6-ABL2 fusion transcripts were identified in MT-ALL which resulted from alternative splicing of an ABL2 exon. The fusion transcripts code for putative ETV6-ABL2 fusion proteins containing the pointed domain of ETV6 and almost the complete ABL2 protein, including the SH2, SH3 domains and the protein tyrosine kinase domain (PTK). Identical ETV6-ABL2 fusion transcripts have been reported in an acute myeloid leukaemia (AML) M3 cell line, carrying both a t(15;17)(q22;q21) and a t(1;12)(q25;p13) with unusual inducible differentiation to eosinophils, and in a patient with AML-M4eo. Interestingly, the non-rearranged allele of ETV6 in the MT-ALL cell line carries an arginine to histidine (R399H) mutation which affects a conserved amino acid in the ets DNA binding domain.

Asunto(s)

Proteínas de Unión al ADN/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas de Fusión Oncogénica/genética , Mutación Puntual , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Fusión Artificial Génica , Secuencia de Bases , Humanos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-ets , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína ETS de Variante de Translocación 6