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1.
Mol Cell Biol ; 24(7): 2890-904, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15024077

RESUMO

The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARalpha), and PLZF-RARalpha encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARalpha, or PLZF-RARalpha. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (gamma-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. beta-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.


Assuntos
Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra , Animais , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desmoplaquinas , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteína 1 Parceira de Translocação de RUNX1 , Fatores de Transcrição/genética , Transplante Isogênico , Proteínas Wnt , gama Catenina
2.
Mol Cell Biol ; 24(20): 8917-28, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15456866

RESUMO

Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and gamma-irradiation which was mediated by p53. cyclin A1-/- cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1-/- cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.


Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina A/metabolismo , Reparo do DNA , DNA/metabolismo , DNA/efeitos da radiação , Regulação da Expressão Gênica , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Quinases relacionadas a CDC2 e CDC28/genética , Células Cultivadas , Ciclina A/genética , Ciclina A1 , Quinase 2 Dependente de Ciclina , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Raios gama , Células-Tronco Hematopoéticas , Humanos , Autoantígeno Ku , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Regiões Promotoras Genéticas , Distribuição Aleatória , Recombinação Genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Raios Ultravioleta
3.
Oncogene ; 24(16): 2739-44, 2005 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-15829981

RESUMO

Cyclin A1 is an alternative A-type cyclin that is essential for spermatogenesis, but it is also expressed in hematopoietic progenitor cells and in acute myeloid leukemia. Its functions during cell cycle progression of somatic cells are incompletely understood. Here, we have analysed the cell cycle functions of cyclin A1 in transformed and nontransformed cells. Murine embryonic fibroblasts derived from cyclin A1-deficient mice were significantly impaired in their proliferative capacity. In accordance, cyclin A1-/- cells accumulated in G1 and G2/M phase while the percentage of S phase cells decreased. Also, lectin stimulated splenic lymphocytes from cyclin A1-/- mice proliferated slower than their wild-type counterparts. Forced cyclin A1 overexpression in NIH3T3 cells and in U937 leukemic cells either by transient transfection or by retroviral infection enhanced S phase entry. Consequently, siRNA mediated silencing of cyclin A1 in highly cyclin A1 expressing ML1 leukemic cells significantly slowed S phase entry, decreased proliferation and inhibited colony formation. Taken together, these analyses demonstrate that cyclin A1 contributes to G1 to S cell cycle progression in somatic cells. Cyclin A1 overexpression enhances S phase entry consistent with an oncogenic function. Finally, cyclin A1 might be a therapeutic target since its silencing inhibited leukemia cell growth.


Assuntos
Ciclina A/genética , Fase G1 , Fase S , Animais , Proliferação de Células , Transformação Celular Neoplásica , Ciclina A/metabolismo , Ciclina A1 , Feminino , Expressão Gênica , Humanos , Cinética , Leucemia/genética , Leucemia/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células NIH 3T3 , Oligorribonucleotídeos , Células U937
4.
Oncogene ; 23(57): 9162-72, 2004 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-15516979

RESUMO

Microarray analyses were performed to identify target genes that are shared by the acute myeloid leukemia (AML) translocation products PML-RARalpha, PLZF-RARalpha and AML1-ETO in inducibly transfected U937 cell lines. The cytoplasmic serine and threonine kinase MNK1 was identified as one of the target genes. At the protein level, MNK1 was significantly induced by each of the three fusion proteins. Protein half-life analyses showed that PML-RARalpha enhanced MNK1 protein stability in U937 cells and ATRA exposure decreased MNK1 half-life in NB4 cells. EIF4E, the main MNK1 substrate, plays a role in the pathogenesis of a variety of cancers. Upon MNK1 overexpression, eIF4E phosphorylation increased as a sign of functional activation. Interestingly, MNK1 protein expression decreased during myeloid differentiation. Inhibition of MNK1 activity by a specific inhibitor (CGP57380) enhanced differentiation of HL60 and 32D cells, further suggesting a role for MNK1 in the myeloid differentiation. In addition, kinase dead mutants of MNK1 significantly impaired proliferation of 32D cells. Immunohistochemistry of primary AML bone marrow biopsies showed strong cytoplasmic MNK1 expression in 25 of 99 AML specimens (25%). MNK1 expression was associated with high levels of c-myc expression. Taken together, we identified MNK1 as a target gene of several leukemogenic fusion proteins in AML. MNK1 plays a role in myeloid differentiation. These data suggest a role for MNK1 in the AML fusion protein-associated differentiation block.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética
5.
Int J Mol Med ; 11(3): 311-5, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12579332

RESUMO

The human cyclin A1 gene is highly expressed in pachytene spermatocytes and is essential for spermatogenesis. To analyze mechanisms of cyclin A1 gene expression in vivo, we cloned a 1.3 kb fragment of the promoter upstream of the cDNA of enhanced green fluorescent protein (EGFP). Four lines of transgenic mice were generated that carried the transgene. Cyclin A1 promoter activity in the organs of the transgenic mice was analyzed using fluorescence microscopy and flow cytometry. Expression of EGFP was seen in male germ cells of all four murine lines. Spermatogonia at the basal membrane expressed low levels of EGFP, but bright green fluorescence was present in spermatocytes entering meiosis. Interestingly, a further sharp increase in EGFP expression was found in spermatocytes approximately at the stage of the first meiotic division. EGFP levels stayed high thereafter and EGFP was present in mature spermatozoa. A portion of c-kit expressing cells in the testis also expressed EGFP indicating cyclin A1 promoter activity in a subpopulation of spermatogonia. These data suggest that cyclin A1 is active not only in pachytene spermatocytes but also in earlier phases of spermatogenesis.


Assuntos
Ciclina A/genética , Ciclina A/metabolismo , Espermatogênese , Animais , Ciclina A1 , Regulação da Expressão Gênica , Humanos , Masculino , Meiose , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-kit/metabolismo , Espermatócitos/metabolismo , Espermatogônias/metabolismo , Espermatozoides/metabolismo , Testículo/citologia
6.
J Basic Microbiol ; 44(5): 360-73, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15378530

RESUMO

The gene mak1FN coding for maltokinase from Actinoplanes missouriensis is located in a cluster similar to glycogen metabolism clusters identified in Streptomyces coelicolor. Sequence comparisons demonstrate that mak1-related genes coding for homologous proteins are present in many bacterial genomes including taxonomic distantly related groups such as Rhodospirillales or green sulfur bacteria. More than 50% of the aligned sequences are longer than the mak1 gene from A. missouriensis, and the N-terminal portion of these putative maltokinases exhibit high sequence homologies with trehalose synthases. A more detailed sequence comparison indicates a relationship of maltokinases to aminoglycoside phospho-transferases and protein kinases. Transformation of S. lividans with plasmid vectors containing either the mak1 gene from A. missouriensis or the pep2 gene from S. coelicolor resulted in recombinant strains, which produced measurable amounts of maltokinase activity. The proteins Pep2 and Mak1 were over expressed with Streptomyces lividans 66 as a heterologous host and further characterized. The possible physiological function of maltokinases is discussed.


Assuntos
Micromonosporaceae/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Streptomyces coelicolor/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Sequência Conservada , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Estabilidade Enzimática , Expressão Gênica , Genes Bacterianos , Glucosiltransferases/genética , Concentração de Íons de Hidrogênio , Canamicina Quinase/genética , Micromonosporaceae/genética , Dados de Sequência Molecular , Família Multigênica , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Filogenia , Proteínas Quinases/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhodospirillales/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência , Streptomyces coelicolor/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Temperatura
7.
J Biol Chem ; 279(8): 6959-66, 2004 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-14645373

RESUMO

Suppression of collagen synthesis is a major therapeutic goal in the treatment of fibrotic disorders. We show here that alpha-melanocyte-stimulating hormone (alpha-MSH), a neuropeptide well known for its pigment-inducing capacity, modulates collagen synthesis and deposition. Alpha-MSH in vitro suppresses the synthesis of collagen types I, III, and V and down-regulates the secretion of procollagen type I C-terminal peptide (PICP) in human dermal fibroblasts treated with the fibrogenic cytokine transforming growth factor-beta1 (TGF-beta1). Alpha-MSH did not interfere with TGF-beta1 signaling, because TGF-beta1-induced expression of collagen mRNA was not affected, implying a posttranscriptional mechanism. Human dermal fibroblasts in vitro express a high affinity binding site for MSH, which was identified by reverse transcription PCR and immunofluorescence analysis as the melanocortin-1 receptor (MC-1R). Immunohistochemical studies on normal adult human skin confirmed MC-1R expression in distinct dermal fibroblastic cells. The MC-1R on fibroblasts appears to be functionally relevant because alpha-MSH increased the amount of intracellular cAMP, and coincubation with a synthetic peptide corresponding to the human Agouti signaling protein abrogated the inhibition of TGF-beta1-induced PICP secretion by alpha-MSH. To assess the in vivo relevance of these findings, a mouse model was used in which dermal fibrosis was induced by repetitive intracutaneous injections with TGF-beta1. The inductive activity of TGF-beta1 on collagen deposition and the number of dermal cells immunoreactive for vimentin and alpha-smooth muscle actin was significantly suppressed by injection of alpha-MSH. Melanocortins such as alpha-MSH may therefore represent a novel class of modulators with potential usefulness for the treatment of fibrotic disorders.


Assuntos
Colágeno/química , Neuropeptídeos/química , alfa-MSH/química , Actinas/metabolismo , Proteína Agouti Sinalizadora , Animais , Sítios de Ligação , Células Cultivadas , Colágeno/metabolismo , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Fibroblastos/metabolismo , Fibrose/metabolismo , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Melanócitos/metabolismo , Camundongos , Microscopia de Fluorescência , Músculo Liso/metabolismo , Peptídeos/química , Ligação Proteica , Receptor Tipo 1 de Melanocortina/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1
8.
J Biol Chem ; 279(32): 33727-41, 2004 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-15159402

RESUMO

The CDK2-associated cyclin A1 is essential for spermatogenesis and contributes to leukemogenesis. The detailed molecular functions of cyclin A1 remain unclear, since the molecular networks involving cyclin A1-CDK2 have not been elucidated. Here, we identified novel cyclin A1/CDK2 interaction partners in a yeast triple-hybrid approach. Several novel proteins (INCA1, KARCA1, and PROCA1) as well as the known proteins GPS2 (G-protein pathway suppressor 2), Ku70, receptor for activated protein kinase C1/guanine nucleotide-binding protein beta-2-like-1, and mRNA-binding motif protein 4 were identified as interaction partners. These proteins link the cyclin A1-CDK2 complex to diverse cellular processes such as DNA repair, signaling, and splicing. Interactions were confirmed by GST pull-down assays and co-immunoprecipitation. We cloned and characterized the most frequently isolated unknown gene, which we named INCA1 (inhibitor of CDK interacting with cyclin A1). The nuclear INCA1 protein is evolutionarily conserved and lacks homology to any known gene. This novel protein and two other interacting partners served as substrates for the cyclin A1-CDK2 kinase complex. Cyclin A1 and all interaction partners were highly expressed in testis with varying degrees of tissue specificity. The highest expression levels were observed at different time points during testis maturation, whereas expression levels in germ cell cancers and infertile testes decreased. Taken together, we identified testicular interaction partners of the cyclin A1-CDK2 complex and studied their expression pattern in normal organs, testis development, and testicular malignancies. Thereby, we establish a new basis for future functional analyses of cyclin A1. We provide evidence that the cyclin A1-CDK2 complex plays a role in several signaling pathways important for cell cycle control and meiosis.


Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina A/metabolismo , Testículo/química , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Chlorocebus aethiops , Ciclina A1 , Quinase 2 Dependente de Ciclina , Reparo do DNA , Expressão Gênica , Biblioteca Gênica , Glutationa Transferase/genética , Humanos , Técnicas de Imunoadsorção , Infertilidade Masculina/metabolismo , Masculino , Meiose , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Splicing de RNA , RNA Mensageiro/análise , RNA Mensageiro/química , Proteínas Recombinantes de Fusão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência , Transdução de Sinais , Neoplasias Testiculares/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Transfecção
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