RESUMO
Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in Myeloid Neoplams (MNs) exhibit DNA hypermethylation via 2-hydroxyglutarate (2HG) over-production. Clinical impact of azacitidine (AZA) remains inconsistent in IDH1/2-mutated MNs and the potential of serum 2HG as a suitable marker of response to AZA is unknown. To address these questions, we retrospectively analyzed 93 MNs patients (78 AML, 11 MDS, 4 CMML) with IDH1/2 mutations treated with AZA. After a median of 5 cycles of AZA, overall response rate was 28% (including 15% complete remission) and median OS was 12.3 months (significantly shorter in AML compared to MDS/CMML patients). In multivariate analysis of AML patients, DNMT3A mutation was associated with shorter OS while IDH1/2 mutation subtypes had no independent impact. No difference was observed in serum 2HG levels upon AZA treatment between responding and refractory patients suggesting that serum 2HG cannot be used as a surrogate marker of AZA response.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Azacitidina/uso terapêutico , Humanos , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Estudos RetrospectivosRESUMO
Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that convert isocitrate to alpha-ketoglutarate (alphaKG). Somatic point mutations in IDH1/2 that are found in rare distinct subsets of cancers confer a gain of function in cancer cells which results in the accumulation and secretion in vast excess of the oncometabolite D-2-hydroxyglutarate (D-2HG). Overproduction of D-2HG interferes with cellular metabolism and epigenetic regulation, contributing to oncogenesis. High levels of D-2HG inhibit alphaKG-dependent dioxygenases including histone, DNA and RNA demethylases, resulting in histone, DNA and RNA hypermethylation and cell differentiation blockade. In addition, D-2HG is a biomarker suitable for the detection of IDH1/2 mutations at diagnosis, and is also predictive of clinical response. The U.S. Food and Drug Administration (FDA) approved ivosidenib, a mutant-IDH1 enzyme inhibitor, for patients with relapsed or refractory IDH1-mutated acute myeloid leukemia (AML) in 2018, and also as front-line therapy for newly diagnosed elderly patients 75 years or older or who are ineligible to receive intensive chemotherapy in 2019. Ivosidenib represents a novel drug class for targeted therapy in AML.
Assuntos
Glicina/análogos & derivados , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Piridinas/uso terapêutico , Adulto , Idoso , Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética , Glicina/uso terapêutico , Humanos , MutaçãoAssuntos
Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/patologia , Isocitrato Desidrogenase/genética , Linfócitos/patologia , Mutação/genética , Células Mieloides/patologia , Animais , Células Cultivadas , Neoplasias Hematológicas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos/metabolismo , Camundongos , Células Mieloides/metabolismo , Retroviridae/genéticaAssuntos
Leucemia Megacarioblástica Aguda/patologia , Megacariócitos/citologia , Megacariócitos/metabolismo , Fator de Resposta Sérica/fisiologia , Transativadores/fisiologia , Animais , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Integrases/metabolismo , Camundongos , Camundongos Knockout , Trombocitopenia/etiologia , Trombocitopenia/patologiaRESUMO
Tel-Abl and Tel-Jak2 are fusion proteins associated with human haematologic neoplasms. They possess constitutive tyrosine kinase activity and activate common downstream signalling pathways like Stat-5, PI3-K/Akt, Ras/MapK and NF-kappaB. In this study, we showed the specific requirement of Src family members for the Tel-Abl-mediated cell growth, activation of Stat5, PI3-K/Akt and Ras/MapK while dispensable for Tel-Jak2. Hck was found strongly phosphorylated in Tel-Abl-expressing Ba/F3 cells and sensitive to imatinib mesylate treatment, providing evidence that Hck is a target of Tel-Abl tyrosine kinase activity. Overexpression of a kinase dead form of Hck inhibits the proliferation of Ba/F3 cells expressing Tel-Abl as the phosphorylation of Akt and Erk1/2. These results argue for an important role of Hck in Tel-Abl oncogenic signalling.
Assuntos
Transformação Celular Neoplásica , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-hck/metabolismo , Benzamidas , Linhagem Celular , Humanos , Mesilato de Imatinib , Fosforilação , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Pirimidinas/farmacologiaRESUMO
Abnormal activation of tyrosine kinases and of signaling pathways they control plays a critical role in the neoplastic process of human hematopoietic malignancy. The nuclear factor-kappaB (NF-kappaB) pathway is one of the signalings activated by the TEL-Jak2 and TEL-Abl oncoproteins and required for their antiapoptotic activity. To define the signal relay responsible for this activation, we used mouse embryonic fibroblast (MEF) cells and observed that TEL-Jak2- and TEL-Abl-mediated NF-kappaB induction was abolished in cells lacking the IkappaB kinase (IKK)alpha but not in IKKbeta(-/-) cells. Similar observations were performed with oncogenic forms of the FMS-like tyrosine kinase 3 (Flt-3) involved in the pathogenesis of one-third of acute myeloid leukemias. Rescue of TEL-Jak2-mediated NF-kappaB activation was obtained with a kinase-proficient form of IKKalpha in IKKalpha(-/-) MEF. Hematopoietic cells transformed by TEL-Jak2 and TEL-Abl showed sustained IKKalpha activity without promotion of NF-kappaB2/p100 processing, generally associated to IKKalpha functions. Furthermore, IAP1, IAP2 and XIAP, which are central regulators of the NF-kappaB-mediated survival pathway, were highly expressed in cells transformed by these oncoproteins. Our results indicate that these oncogenic tyrosine kinases preferentially use an IKKalpha-dependent mechanism to induce a persistent NF-kappaB activity and allow the production of antiapoptotic effectors that participate to their leukemogenic properties.
Assuntos
Transformação Celular Neoplásica/metabolismo , Quinase I-kappa B/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , NF-kappa B/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática/fisiologia , Humanos , Immunoblotting , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologia , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
FISH identified a cryptic t(5;14)(q35;q32) in T acute lymphoblastic leukemia (ALL), whereas it was not observed in B ALL samples. This translocation is present in five out of 23 (22%) children and adolescents with T ALL tested. RanBP17, a gene coding for a member of the importin beta protein family, and Hox11Like2, an orphan homeobox gene were mapped close to the chromosome 5 breakpoints and CTIP2, which is highly expressed during normal T cell differentiation, was localized in the vicinity of the chromosome 14 breakpoints. The Hox11L2 gene was found to be transcriptionally activated as a result of the translocation, probably under the influence of CTIP2 transcriptional regulation elements. These data establish the t(5;14)(q35;q32) as a major abnormality, and Hox11 family member activation as an important pathway in T ALL leukemogenesis.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 5 , Proteínas de Homeodomínio/genética , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas Oncogênicas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Adolescente , Adulto , Sequência de Aminoácidos , Estudos de Casos e Controles , Transformação Celular Neoplásica , Criança , Pré-Escolar , Quebra Cromossômica , Análise Citogenética , Feminino , Humanos , Leucemia-Linfoma de Células T do Adulto/etiologia , Masculino , Dados de Sequência Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Proteínas Proto-Oncogênicas , Alinhamento de Sequência , Proteína ran de Ligação ao GTP/genéticaRESUMO
Signal Transducer and Activator of Transcription (STATs) are important mediators of cytokine and growth factor-induced signal transduction. STAT5A and STAT5B have been shown to play a role in survival and proliferation of hematopoietic cells both in vitro and in vivo and to contribute to the growth and viability of cells transformed by the TEL-JAK2 oncoprotein. In this study, we investigated the molecular mechanisms by which constitutively active STAT5 proteins induce cell proliferation and survival of Ba/F3 cell lines expressing either dominant positive STAT5A or STAT5B variants or TEL-JAK2 or TEL-ABL fusion proteins. Our results showed that active STAT5 constitutively interacted with p85, the regulatory subunit of the PI 3-kinase. A constitutive activity of the PI 3-kinase/Akt pathway was observed in these cells and required for their cell cycle progression. In contrast, while activity of the PI 3-kinase/Akt pathway was required for survival of Ba/F3 cells expressing the constitutively active forms of STAT5A or STAT5B, it was dispensable for cells transformed by TEL-JAK2 or TEL-ABL fusion proteins, suggesting that additional survival pathways take place in these transformed cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas do Leite , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Transativadores/fisiologia , Animais , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Eritropoetina/farmacologia , Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/fisiologia , Células-Tronco Hematopoéticas/enzimologia , Humanos , Camundongos , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fator de Transcrição STAT5 , Transativadores/genética , Transativadores/metabolismo , Transformação Genética , Proteínas Supressoras de TumorRESUMO
Constitutively active tyrosine kinases are frequently expressed in various types of human leukemias as the result of chromosomal translocations. The TEL-Jak2 fusion oncoprotein possesses transforming properties in both animal and cellular models, that are tightly dependent on Stat5 activation. In the IL-3-independent TEL-Jak2-transformed Ba/F3 cells, activation of the PI-3K/Akt pathway appears essential to cell proliferation. Here we report a sustained activation of NF-kappaB factors in Ba/F3 cells, which inhibition dramatically impairs cell viability, indicating that NF-kappaB signaling exerts a major role in the anti-apoptotic activities of TEL-Jak2 oncoprotein.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas I-kappa B , Leucemia/metabolismo , NF-kappa B/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Serina-Treonina Quinases , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Interleucina-3/farmacologia , Leucemia/etiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Proteínas de Fusão Oncogênica/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The leukemia-associated TEL-Jak2 fusion protein possesses a constitutive tyrosine kinase activity and transforming properties in hematopoietic cell lines and animal models. In the murine pro-B Ba/F3 cell line, this fusion constitutively activates the Signal Transducer and Activator of Transcription 5 (Stat5) factors and, as a consequence, induces the sustained expression of various Stat5-target genes including the Cytokine Inducible SH2-containing protein (Cis) gene, which codes for a member of the Suppressor of Cytokine Signaling (Socs) protein family. In TEL-Jak2-transformed Ba/F3 cells, we also observed the upregulation of the Socs1 gene, whose product has been reported to negatively regulate the Jak kinase activity. In transient transfection experiments, Socs1 physically interacts with TEL-Jak2 and interferes with the TEL-Jak2-induced phosphorylation and activation of Stat5 factors, probably through the Socs1-induced proteasome-mediated degradation of the fusion protein. Interestingly, TEL-Jak2-expressing Ba/F3 cells were found to be resistant to the anti-proliferative activities of gamma interferon (IFN-gamma) seemingly as a consequence of Socs1 constitutive expression. These results indicate that the Socs1-dependent cytokine feedback loop, although active, is bypassed by the TEL-Jak2 fusion, but may play a role in the leukemogenic process by altering the cytokine responses of the leukemic cells. Our results also suggest that Socs1 plays a role in shutting down the signaling from the normally activated Jak2 kinase by inducing its proteasome-dependent degradation.
Assuntos
Proteínas de Transporte/biossíntese , Citocinas/biossíntese , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Repressoras/biossíntese , Animais , Linfócitos B/metabolismo , Linhagem Celular Transformada , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Janus Quinase 2 , Camundongos , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Receptores de Interferon/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina , Ubiquitinas/metabolismo , Receptor de Interferon gamaRESUMO
The TEL/ETV6 gene is located at 12p13 and is frequently involved in chromosomal translocations in human malignancies usually resulting in the expression of fusion proteins between the amino terminal part of TEL, and either unrelated transcription factors or protein tyrosine kinases. We report here a novel gene named TELB which is located on human chromosomal band 6p21 and encodes a protein highly related to TEL. TELB is widely expressed in different tissues and, similarly to TEL encodes a sequence-specific transcriptional repressor.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Genes , Leucemia/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Linfoma de Burkitt/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 6/ultraestrutura , Subunidade alfa 2 de Fator de Ligação ao Core , DNA Complementar/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/biossíntese , Drosophila melanogaster/genética , Éxons/genética , Etiquetas de Sequências Expressas , Proteínas do Olho/genética , Regulação Leucêmica da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Família Multigênica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-ets , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de Transcrição/biossíntese , Transcrição Gênica , Transfecção , Translocação Genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
We previously reported a fusion between TEL and JAK2 in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2-induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EmuSRalpha enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRbeta locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells. (Blood. 2000;95:3891-3899)
Assuntos
Leucemia de Células T/genética , Proteínas de Fusão Oncogênica/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , DNA Complementar , Elementos Facilitadores Genéticos , Humanos , Leucemia de Células T/sangue , Leucemia de Células T/imunologia , Leucemia de Células T/patologia , Contagem de Leucócitos , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Timo/imunologia , Timo/patologia , Transcrição GênicaRESUMO
The involvement of the cytokine signaling pathway in oncogenesis has long been postulated. Recently, rearrangements of the gene encoding the tyrosine Janus kinase 2 (JAK2) have been reported in human leukemias indicating a direct JAK-signal transduction and activator of transcription (STAT)-mediated leukemic process. The leukemia-associated TEL-JAK2 fusion protein is formed by the oligomerization domain of the translocated ets leukemia (TEL) protein fused to the catalytic domain of JAK2. TEL-mediated oligomerization results in a constitutive tyrosine kinase activity that, in turn, is able to confer growth factor independence to the murine hematopoietic interleukin-3 (IL-3)-dependent Ba/F3 cell line. Results of the present study indicate that fusion proteins containing the oligomerization domain of TEL and the tyrosine kinase domains of Jak1, Jak2, JAK3, or TYK2 share similar properties and are able to efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. Electrophoretic mobility shift assays demonstrated Stat5 as the only activated Stat factor in TEL-Jak2- and TEL-Jak1-expressing cells, whereas other Stats, namely Stat1 and Stat3, could be detected in TEL-JAK3-, TEL-TYK2-, and also in TEL-ABL-expressing Ba/F3 cells. High levels of expression of the Stat5-target genes pim-1, osm, and Cis were observed in all the cytokine-independent cell lines. Furthermore, the expression of a dominant negative form of Stat5A markedly interfered with the growth factor independence process mediated by TEL-Jak2 in Ba/F3 cells. Because the BCR-ABL and TEL-PDGFbetaR oncoproteins also activate Stat5, activation of this factor should be a crucial step in activated tyrosine kinase-mediated leukemogenesis. (Blood. 2000;95:2076-2083)
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Animais , Northern Blotting , Domínio Catalítico , Divisão Celular , Linhagem Celular , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/química , Ativação Enzimática , Humanos , Interleucina-3/metabolismo , Leucemia/enzimologia , Leucemia/metabolismo , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets , Fatores de Tempo , Fatores de Transcrição/química , Transfecção , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Chromosomal translocations involving the human 12p13 band frequently affect the TEL gene, usually resulting in gene fusion between TEL and genes encoding proteins of various types. The most frequent 12p13 translocation is the t(12;21)(p13;q22), which recombines TEL with the AML1 gene on chromosome 21 and is frequently associated with deletion of the untranslocated TEL allele. Using antisera against different parts of TEL and against the AML1 proteins, we undertook Western blot and immunofluorescence analyses of leukemic samples with and without 12p13 abnormalities. In t(12;21) samples, TEL-AML1 was detected as several protein species in the nuclei, whereas the AML1-TEL protein, was inconsistently expressed. AML1 was found to be expressed but no normal TEL proteins were detected. A survey of the TEL proteins in a panel of human leukemic samples without t(12;21) revealed a variation in the ratio of TEL protein isoforms. We also analysed a leukemic cell line bearing a t(12;22)(p13;q11) that was found to affect the 5' untranslated (UT) region of TEL and to be associated with inactivation of the untranslocated TEL allele. No MN1-TEL fusion could be detected upon RT-PCR analysis, in contrast to the previously investigated t(12;22). Strikingly, extremely low levels of apparently normal TEL proteins, expressed from the translocated allele, were detected by Western blot analysis. These results suggest that the level of TEL expression can be important for leukemogenesis.
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 21 , Proteínas de Ligação a DNA/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Adulto , Animais , Criança , Pré-Escolar , Subunidade alfa 2 de Fator de Ligação ao Core , Células HL-60 , Células HeLa , Humanos , Isomerismo , Leucemia Mielomonocítica Aguda/patologia , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-ets , Coelhos , Frações Subcelulares , Translocação Genética , Células Tumorais Cultivadas , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
We and others have recently shown that loss of the mitochondrial membrane potential (Deltapsi) precedes apoptosis and chemical-hypoxia-induced necrosis and is prevented by Bcl-2. In this report, we examine the biochemical mechanism used by Bcl-2 to prevent Deltapsi loss, as determined with mitochondria isolated from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 had no effect on the respiration rate of isolated mitochondria, it prevented both Deltapsi loss and the permeability transition (PT) induced by various reagents, including Ca2+, H2O2, and tert-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintained Deltapsi, suggesting that the functional target of Bcl-2 is regulation of Deltapsi but not PT. Bcl-2 also maintained Deltapsi in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Deltapsi. Although treatment with SF6847 in the absence of Ca2+ caused massive H+ influx in control mitochondria, the presence of Bcl-2 induced H+ efflux after transient H+ influx. In this case, Bcl-2 did not enhance K+ efflux. Furthermore, Bcl-2 enhanced H+ efflux but not K+ flux after treatment of mitochondria with Ca2+ or tert-butyl hydroperoxide. These results suggest that Bcl-2 maintains Deltapsi by enhancing H+ efflux in the presence of Deltapsi-loss-inducing stimuli.
Assuntos
Mitocôndrias Hepáticas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Apoptose , Transporte Biológico Ativo , Cálcio/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/fisiologia , Potenciais da Membrana , Camundongos , Fosforilação Oxidativa , Consumo de Oxigênio , Peróxidos/farmacologia , Ratos , terc-Butil HidroperóxidoRESUMO
Recombinant human p53 isolated either from E. coli or from insect cells is poorly active for binding to DNA but it can be dramatically stimulated by phosphorylation, antibody binding to the carboxy-terminal negative regulatory domain, short peptides derived from this negative regulatory domain or short single strands of DNA. We report here that Xenopus p53 has a very similar behavior. Using a new set of monoclonal antibodies directed either to the amino- or the carboxy-terminus of Xenopus p53, we demonstrate that the frog protein can be activated by specific carboxy-terminus monoclonal antibodies in order to bind to human p53 DNA response element. In addition, we report that such activation of both humans and frogs protein can also be achieved by small peptides derived from the carboxy-terminus of both p53. Although, the sequence of this region is not conserved in the various p53 species, the presence of conserved basic residues indicates that such activation is charge-dependent. This is confirmed by the finding that small poly-lysine peptides can activate both human and Xenopus p53. In vivo expression of Xenopus p53 indicates that this protein is able to transactivate a wide variety of human p53 response elements as long as the experiments are performed at 32 degrees C since activity at 37 degrees C, a temperature well above the natural temperature of Xenopus, is lost. Finally, we demonstrate that human mdm2 is able to down regulate the transcriptional activity of Xenopus p53.
Assuntos
Proteínas de Ligação a DNA/química , Proteína Supressora de Tumor p53/química , Xenopus laevis/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Janus family of tyrosine kinases (JAK) plays an essential role in development and in coupling cytokine receptors to downstream intracellular signaling events. A t(9;12)(p24;p13) chromosomal translocation in a T cell childhood acute lymphoblastic leukemia patient was characterized and shown to fuse the 3' portion of JAK2 to the 5' region of TEL, a gene encoding a member of the ETS transcription factor family. The TEL-JAK2 fusion protein includes the catalytic domain of JAK2 and the TEL-specific oligomerization domain. TEL-induced oligomerization of TEL-JAK2 resulted in the constitutive activation of its tyrosine kinase activity and conferred cytokine-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line.
Assuntos
Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteínas do Leite , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Biopolímeros , Divisão Celular , Linhagem Celular , Pré-Escolar , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Interleucina-3/fisiologia , Janus Quinase 2 , Leucemia-Linfoma de Células T do Adulto/genética , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Fosforilação , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-ets , Fator de Transcrição STAT5 , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Translocação Genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Erythropoietin (Epo) is known to control the erythroid developmental program through various biologic activities including maintenance of viability, cell proliferation, and/or cell maturation. In vitro experiments showed massive apoptosis in cultures of Epo-deprived colony-forming unit-erythroid (CFU-E) progenitors, demonstrating the Epo requirement of late-stage erythroid progenitors for survival. Based on these data, a model has been proposed whereby from CFU-E to proerythroblast stages, Epo acts rather as a survival factor than a proliferating factor. To investigate the relationship between Epo dependence and apoptotic mechanisms, we generated transgenic mice expressing the antiapoptotic human bcl-2 gene product in erythroid progenitors. Transgenic animals developed without any evidence of erythropoietic disorders. In vitro studies showed that overexpression of bcl-2 in erythroid progenitors delayed, but did not prevent the loss of CFU-E from Epo-deprivation. By measuring burst-forming unit-erythroid (BFU-E) and CFU-E-derived colonies, an enhanced sensitivity to low levels of Epo was demonstrated in adult bone marrow of transgenic mice with respect to nontransgenic animals. No spontaneous erythroid colonies were, however, observed in vitro in the absence of the cytokine, indicating that overexpression of bcl-2 is not sufficient to induce by itself a complete erythroid differentiation. Taken together, our data indicate that targeted erythroid overexpression of bcl-2 fails to alter the normal erythropoietic development in vivo and that erythroid progenitors remain strictly dependent on Epo for their survival.