RESUMO
UNLABELLED: Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella upon primary infection and zoster upon reactivation from latency in sensory ganglion neurons. The replication of herpesviruses requires manipulation of cell signaling pathways. Notably, CREB, a factor involved in the regulation of several cellular processes, is activated upon infection of T cells with VZV. Here, we report that VZV infection also induced CREB phosphorylation in fibroblasts and that XX-650-23, a newly identified inhibitor of the phosphorylated-CREB (pCREB) interaction with p300/CBP, restricted cell-cell spread of VZV in vitro CREB phosphorylation did not require the viral open reading frame 47 (ORF47) and ORF66 kinases encoded by VZV. Evaluating the biological relevance of these observations during VZV infection of human skin xenografts in the SCID mouse model of VZV pathogenesis showed both that pCREB was upregulated in infected skin and that treatment with XX-650-23 reduced infectious-virus production and limited lesion formation compared to treatment with a vehicle control. Thus, processes of CREB activation and p300/CBP binding are important for VZV skin infection and may be targeted for antiviral drug development. IMPORTANCE: Varicella-zoster virus (VZV) is a common pathogen that causes chicken pox and shingles. As with all herpesviruses, the infection is acquired for life, and the virus can periodically reactivate from latency. Although VZV infection is usually benign with few or no deleterious consequences, infection can be life threatening in immunocompromised patients. Otherwise healthy elderly individuals who develop zoster as a consequence of viral reactivation are at risk for postherpetic neuralgia (PHN), a painful and long-lasting complication. Current vaccines use a live attenuated virus that is usually safe but cannot be given to many immunodeficient patients and retains the capacity to establish latency and reactivate, causing zoster. Antiviral drugs are effective against severe VZV infections but have little impact on PHN. A better understanding of virus-host cell interactions is relevant for developing improved therapies to safely interfere with cellular processes that are crucial for VZV pathogenesis.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína p300 Associada a E1A/metabolismo , Herpesvirus Humano 3/fisiologia , Interações Hospedeiro-Patógeno , Fragmentos de Peptídeos/metabolismo , Sialoglicoproteínas/metabolismo , Replicação Viral , Animais , Modelos Animais de Doenças , Herpes Zoster/patologia , Herpes Zoster/virologia , Humanos , Camundongos SCID , Fosforilação , Processamento de Proteína Pós-Traducional , Pele/patologiaRESUMO
T-cell Prolymphocytic Leukemia (T-PLL) is a rare entity, and to date has never been reported in children. Here, we describe the first pediatric case of T-PLL in a 16-year old male and review his clinical course through treatment. He underwent therapy with alemtuzumab and pentostatin, which was successful in inducing initial remission. He then underwent an allogeneic matched sibling stem cell transplant following a myeloablative conditioning regimen and remains disease-free 1.5 years after diagnosis.
Assuntos
Leucemia Prolinfocítica de Células T/terapia , Adolescente , Transplante de Células-Tronco Hematopoéticas , Humanos , Masculino , Transplante HomólogoRESUMO
The use of image-guided percutaneous core needle biopsy (PCNB) to obtain tissue diagnosis of musculoskeletal lesions has become the standard of care in adult patients with a success rate of over 80%. Previous reports indicate a similar success rate in diagnosing pediatric solid tumors. In this large study, we analyzed >10 years of data in which PCNB was used for tissue diagnosis of musculoskeletal lesions in children; we evaluated the histopathologic accuracy, anesthetic requirements, and complications of these procedures. In 122 children, tissue diagnosis was successfully obtained in 82% of cases, and there were 0 complications associated with the procedure. There was a significantly higher PCNB diagnostic success rate in malignant lesions (93%). These data suggest that the use of PCNB is a safe and effective means of diagnosing musculoskeletal lesions in children.
Assuntos
Biópsia Guiada por Imagem , Doenças Musculoesqueléticas/diagnóstico , Recidiva Local de Neoplasia/diagnóstico , Adolescente , Adulto , Biópsia por Agulha , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Masculino , Doenças Musculoesqueléticas/cirurgia , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Estudos Retrospectivos , Adulto JovemRESUMO
Fewer than 40 cases of primary myelofibrosis have been reported in children; hematopoietic stem cell transplantation is the only available curative therapy for this disease. Here, we describe the case of a female infant diagnosed with primary myelofibrosis at the age of 6 months; she underwent successful matched unrelated bone marrow transplantation with complete resolution of disease. We discuss some unique characteristics of primary myelofibrosis in children and review outcome data for children with this disease.
Assuntos
Transplante de Medula Óssea , Mielofibrose Primária/terapia , Feminino , Humanos , Lactente , Mielofibrose Primária/diagnóstico , Resultado do TratamentoRESUMO
The HS-1 associated protein X-1 (HAX-1) is a ubiquitously expressed protein that protects cardiomyocytes from programmed cell death. Here we identify HAX-1 as a regulator of contractility and calcium cycling in the heart. HAX-1 overexpression reduced sarcoplasmic reticulum Ca-ATPase (SERCA2) pump activity in isolated cardiomyocytes and in vivo, leading to depressed myocyte calcium kinetics and mechanics. Conversely, downregulation of HAX-1 enhanced calcium cycling and contractility. The inhibitory effects of HAX-1 were abolished upon phosphorylation of phospholamban, which plays a fundamental role in controlling basal contractility and constitutes a key downstream effector of the beta-adrenergic signaling cascade. Mechanistically, HAX-1 promoted formation of phospholamban monomers, the active/inhibitory units of the calcium pump. Indeed, ablation of PLN rescued HAX-1 inhibition of contractility in vivo. Thus, HAX-1 represents a regulatory mechanism in cardiac calcium cycling and its responses to sympathetic stimulation, implicating its importance in calcium homeostasis and cell survival.
Assuntos
Apoptose , Testes de Função Cardíaca , Coração/fisiologia , Proteínas/metabolismo , Envelhecimento/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/metabolismo , Regulação para Baixo , Transferência Ressonante de Energia de Fluorescência , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos , Ligação Proteica , Ratos , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , TransgenesRESUMO
Alveolar soft part sarcoma (ASPS) is a very rare soft tissue sarcoma which arises primarily in children and young adults. Despite its unique histology and well-characterized genetic translocation, many questions remain regarding the pathogenesis and treatment of this tumor type. Though collective clinical experience with this tumor type spans more than 60 years, there has been little progress made in treating this uncommon but frequently fatal disease. This paper focuses on the available data regarding its molecular pathogenesis and insights into targeted therapeutics as well as the results of clinical trials performed to date to hopefully improve the outcome of patients with this rare malignancy.
RESUMO
Ischemic heart disease, which remains the leading cause of morbidity and mortality in the Western world, is invariably characterized by impaired cardiac function and disturbed Ca(2+) homeostasis. Because enhanced inhibitor-1 (I-1) activity has been suggested to preserve Ca(2+) cycling, we sought to define whether increases in I-1 activity in the adult heart may ameliorate contractile dysfunction and cellular injury in the face of an ischemic insult. To this end, we generated an inducible transgenic mouse model that enabled temporally controlled expression of active I-1 (T35D). Active I-1 expression in the adult heart elicited significant enhancement of contractile function, associated with preferential phospholamban phosphorylation and enhanced sarcoplasmic reticulum Ca(2+)-transport. Further phosphoproteomic analysis revealed alterations in proteins associated with energy production and protein synthesis, possibly to support the increased metabolic demands of the hyperdynamic hearts. Importantly, on ischemia/reperfusion-induced injury, active I-1 expression augmented contractile function and recovery. Further examination revealed that the infarct region and apoptotic as well as necrotic injuries were significantly attenuated by enhanced I-1 activity. These cardioprotective effects were associated with suppression of the endoplasmic reticulum stress response. The present findings indicate that increased I-1 activity in the adult heart enhances Ca(2+) cycling and improves mechanical recovery, as well as cell survival after an ischemic insult, suggesting that active I-1 may represent a potential therapeutic strategy in myocardial infarction.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Contração Miocárdica , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Função Ventricular Esquerda , Animais , Apoptose , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Transgênicos , Mutação , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Necrose , Fosforilação , Proteômica , Recuperação de Função Fisiológica , Retículo Sarcoplasmático/metabolismo , Estresse Fisiológico , Fatores de Tempo , Regulação para CimaRESUMO
Aberrant beta-adrenergic signaling and depressed calcium homeostasis, associated with an imbalance of protein kinase A and phosphatase-1 activities, are hallmarks of heart failure. Phosphatase-1 is restrained by its endogenous inhibitor, protein phosphatase inhibitor-1 (PPI-1). We assessed 352 normal subjects, along with 959 patients with heart failure and identified a polymorphism in PPI-1 (G147D) exclusively in black subjects. To determine whether the G147D variant could affect cardiac function, we infected adult cardiomyocytes with adenoviruses expressing D147 or wild-type (G147) PPI-1. Under basal conditions, there were no significant differences in fractional shortening or contraction or relaxation rates. However, the enhancement of contractile parameters after isoproterenol stimulation was significantly blunted in D147 compared with G147 and control myocytes. Similar findings were observed in calcium kinetics. The attenuated beta-agonist response was associated with decreased (50%) phosphorylation of phospholamban (PLN) at serine 16, whereas phosphorylation of troponin I and ryanodine receptor was unaltered. These findings suggest that the human G147D PPI-1 can attenuate responses of cardiomyocytes to beta-adrenergic agonists by decreasing PLN phosphorylation and therefore may contribute to deteriorated function in heart failure.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Contração Miocárdica/genética , Miócitos Cardíacos/fisiologia , Polimorfismo Genético , Negro ou Afro-Americano/genética , Estudos de Casos e Controles , Insuficiência Cardíaca , Humanos , Isoproterenol/farmacologia , Mutação de Sentido Incorreto , Contração Miocárdica/efeitos dos fármacos , Fosforilação , TransfecçãoRESUMO
BACKGROUND: Phospholamban (PLN) is an inhibitor of the Ca2+ affinity of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2). The amino acid sequence of PLN is highly conserved, and although all species contain asparagine (Asn), human PLN is unique in containing lysine (Lys) at amino acid 27. METHODS AND RESULTS: Human PLN was introduced in the null background. Expression of human PLN, at similar levels to mouse wild-type PLN, resulted in significant decreases in the affinity of SERCA2 for Ca2+, attributed to unique spatial conformation of this PLN form and increases in its monomeric active unit compared with mouse PLN. The increased inhibition by human PLN was associated with attenuated cardiac contractility in the intact-animal, organ, and cardiomyocyte levels and with depressed calcium kinetics. These inhibitory effects could not be fully reversed even on maximal isoproterenol stimulation. There were no alterations in the expression levels of SERCA2, calsequestrin, ryanodine receptor, and FKBP12, although the sodium/calcium exchanger and the L-type Ca2+ channel expression levels were upregulated. The depressed function resulted in increased heart/body weight ratios and phosphorylation levels of Akt, p38, and Erk1/2. CONCLUSIONS: Human PLN may play a more inhibitory role than that of other species in Ca2+ cycling. Expression of human PLN in the mouse is compensated by alterations in Ca2+-handling proteins and cardiac remodeling in an effort to normalize cardiac contractility. Thus, the unique amino acid sequence of human PLN may be critical in maintaining a high cardiac reserve, which is of paramount importance in the regulation of human cardiac function.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Cardiomegalia/etiologia , Sequência de Aminoácidos , Animais , Arginina , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Cinética , Lisina , Camundongos , Camundongos Knockout , Camundongos Transgênicos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Especificidade da EspécieRESUMO
Activation of the sympathetic nervous system is a common compensatory feature in heart failure, but sustained beta-adrenergic activation induces cardiomyocyte death, leading to cardiac remodeling and dysfunction. In mouse cardiomyocytes, we recently reported that prolonged exposure to beta-agonists is associated with transient increases in expression and phosphorylation of a small heat-shock protein, Hsp20. To determine the functional significance of Hsp20, we overexpressed this protein and its constitutively phosphorylated (S16D) or nonphosphorylated (S16A) mutant in adult rat cardiomyocytes. Hsp20 protected cardiomyocytes from apoptosis triggered by activation of the cAMP-PKA pathway, as indicated by decreases in the number of pyknotic nuclei, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling, and DNA laddering, which were associated with inhibition of caspase-3 activity. These protective effects were further increased by the constitutively phosphorylated Hsp20 mutant (S16D), which conferred full protection from apoptosis. In contrast, the nonphosphorylatable mutant (S16A) exhibited no antiapoptotic properties. Immunostaining studies and immunoprecipitations with Hsp20 or actin antibodies demonstrated that Hsp20 translocated to cytoskeleton and associated with actin on isoproterenol stimulation. These findings suggest that Hsp20 and its phosphorylation at Ser16 may provide cardioprotection against beta-agonist-induced apoptosis. Thus, Hsp20 may represent a novel therapeutic target in the treatment of heart failure.
Assuntos
Agonistas Adrenérgicos beta/toxicidade , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Isoproterenol/farmacologia , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Animais , Caspase 3 , Caspases/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP20 , Proteínas de Choque Térmico/química , Masculino , Camundongos , Miócitos Cardíacos/citologia , Fosfoproteínas/química , Fosforilação , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ~114,000 candidate compounds from Stanford University's small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Luciferases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Cálcio/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Proteoma/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional , ProteômicaRESUMO
OBJECTIVES: B-lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21) is a relatively uncommon manifestation of acute leukemia and limited predominantly to the pediatric population. Case-specific information regarding flow cytometric, morphologic, and laboratory findings of this subtype of leukemia is currently lacking. METHODS: We searched the databases of three large institutions for lymphoblastic leukemia with iAMP21 from 2005 through 2012 and analyzed the clinicopathologic features. RESULTS: We identified 17 cases with five or more RUNX1 signals on interphase nuclei, 14 of which were consistent with the Children's Oncology Group (COG) definition for iAMP21namely, the presence of three or more RUNX1 signals on one marker chromosome. These cases showed a statistically significant lower peripheral WBC count and older age at diagnosis compared with all pediatric cases of B-ALL. We also identified three cases with increased RUNX1 signals scattered on multiple marker chromosomes that did not meet the COG definition of iAMP21 but showed similar 21q instability and older age at presentation. CONCLUSIONS: Our findings not only demonstrate that B-ALL with iAMP21 is truly a distinct clinicopathologic entity but also suggest that a subset of cases of B-ALL with iAMP21 can show variable cytogenetic features.
Assuntos
Cromossomos Humanos Par 21/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adolescente , Criança , Pré-Escolar , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Análise Citogenética , Feminino , Citometria de Fluxo , Amplificação de Genes , Humanos , Masculino , Adulto JovemRESUMO
Acute myeloid leukemia (AML) is one of the most common leukemias with a 20% 5-year event-free survival in adults and 50% overall survival in children, despite aggressive chemotherapy treatment and bone marrow transplantation. The incidence and mortality rates for acute leukemia have only slightly decreased over the last 20 years, and therefore greater understanding of the molecular mechanisms associated with leukemic progression is needed. To this end, a number of transcription factors that appear to play a central role in leukemogenesis are being investigated; among them is the cAMP response element binding protein (CREB). CREB is a transcription factor that can regulate downstream targets involving in various cellular functions including cell proliferation, survival, and differentiation. In several studies, the majority of bone marrow samples from patients with acute lymphoid and myeloid leukemia demonstrate CREB overexpression. Moreover, CREB overexpression is associated with a poor outcome in AML patients. This review summarizes the role of CREB in leukemogenesis.
Assuntos
Proteína de Ligação a CREB/fisiologia , Leucemia Mieloide Aguda/fisiopatologia , Proteína de Ligação a CREB/genética , Progressão da Doença , Hematopoese/fisiologia , Humanos , MicroRNAs/genética , FosforilaçãoRESUMO
Central to discovering novel approaches to treating leukemias and lymphomas is a clear understanding of the signaling networks which lead to unchecked cell cycle progression, proliferation, and survival. Cyclic-adenosine monophosphate (cAMP) responsive element-binding protein (CREB) represents a critical integrator of numerous signals from cytoplasmic kinase cascades, and is directly involved in controlling the transcription of genes critical for normal cellular proliferation and survival. Several lines of evidence implicate CREB as a proto-oncogene, as a number of translocations involving CREB and dysregulation of expression are both associated with oncogenesis. Thus, CREB represents a potential therapeutic target in leukemia. Here, we review CREB function and regulation in normal and aberrant hematopoiesis.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Neoplasias Hematológicas/fisiopatologia , Hematopoese/fisiologia , Transdução de Sinais/fisiologia , Animais , Sobrevivência Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Hematopoese/genética , Humanos , Fosforilação , Proto-Oncogene Mas , Transdução de Sinais/genéticaRESUMO
The cyclic-AMP response element-binding protein (CREB) is a nuclear transcription factor activated by phosphorylation at Ser133 by multiple serine/threonine (Ser/Thr) kinases. Upon phosphorylation, CREB binds the transcriptional co-activator, CBP (CREB-binding protein), to initiate CREB-dependent gene transcription. CREB is a critical regulator of cell differentiation, proliferation and survival in the nervous system. Recent studies have shown that CREB is involved tumor initiation, progression and metastasis, supporting its role as a proto-oncogene. Overexpression and over-activation of CREB were observed in cancer tissues from patients with prostate cancer, breast cancer, non-small-cell lung cancer and acute leukemia while down-regulation of CREB in several distinct cancer cell lines resulted in inhibition of cell proliferation and induction of apoptosis, suggesting that CREB may be a promising target for cancer therapy. Although CREB, as a transcription factor, is a challenging target for small molecules, various small molecules have been discovered to inhibit CREB phosphorylation, CREB-DNA, or CREB-CBP interaction. These results suggest that CREB is a suitable transcription factor for drug targeting and therefore targeting CREB could represent a novel strategy for cancer therapy.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Proto-Oncogene MasRESUMO
Phospholamban has been suggested to be a key regulator of cardiac sarcoplasmic reticulum (SR) Ca cycling and contractility and a potential therapeutic target in restoring the depressed Ca cycling in failing hearts. Our understanding of the function of phospholamban stems primarily from studies in genetically altered mouse models. To evaluate the significance of this protein in larger mammalian species, which exhibit Ca cycling properties similar to humans, we overexpressed phospholamban in adult rabbit cardiomyocytes. Adenoviral-mediated gene transfer, at high multiplicities of infection, resulted in an insignificant 1.22-fold overexpression of phospholamban. There were no effects on twitch Ca-transient amplitude or decay under basal or isoproterenol-stimulated conditions. Furthermore, the SR Ca load and Na/Ca exchanger function were not altered. These apparent differences between phospholamban overexpression in rabbit compared with previous findings in the mouse may be due to a significantly higher (1.5-fold) endogenous phospholamban-to-sarco(endo)plasmic reticulum Ca-ATPase (SERCA) 2a ratio and potential functional saturation of SERCA2a by phospholamban in rabbit cardiomyocytes. The findings suggest that important species-dependent differences in phospholamban regulation of SERCA2a occur. In larger mammals, a higher fraction of SERCA2a pumps are regulated by phospholamban, and this may influence therapeutic strategies to enhance cardiac contractility and functional cardiac reserve.
Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Adenoviridae/genética , Agonistas Adrenérgicos beta/farmacologia , Animais , Transporte Biológico , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Vetores Genéticos , Ventrículos do Coração/metabolismo , Isoproterenol/farmacologia , Camundongos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Transdução Genética , Regulação para CimaRESUMO
BACKGROUND: Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers. METHODOLOGY/PRINCIPAL FINDINGS: Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice. CONCLUSIONS/SIGNIFICANCE: These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.
Assuntos
Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/citologia , Neuroblastoma/metabolismo , Vírus Oncolíticos/metabolismo , Antígeno AC133 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Antígenos CD/biossíntese , Linhagem Celular Tumoral , Linhagem da Célula , Chlorocebus aethiops , Glicoproteínas/biossíntese , Humanos , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Neoplasias/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Nestina , Peptídeos , Células-Tronco/metabolismo , Transcrição Gênica , Células VeroRESUMO
The depressed function of failing hearts has been partially attributed to increased protein phosphatase-1 through its impaired regulation by inhibitor-1. Phosphorylation of inhibitor-1 at Thr35 by PKA results in potent inhibition of protein phosphatase-1 activity, while phosphorylation at Ser67 or Thr75 by PKC attenuates the inhibitory activity. To examine the functional role of dual-site (Ser67, Thr75) phosphorylation of inhibitor-1 by PKC, the constitutively phosphorylated Ser67 (S67D) and/or Thr75 (T75D) human inhibitor-1 forms were expressed in adult cardiomyocytes. Expression of either single or double phosphorylated inhibitor-1 was associated with similar decreases in cardiac contractility, indicating that maximal inhibition can be elicited by each of these sites alone and that their inhibitory effects are not additive. Notably, activation of the cAMP pathway could only partially reverse the depressed contractile parameters. Accordingly, protein phosphatase-1 activity remained elevated, phosphorylation of phospholamban at Ser16 was decreased, and the EC(50) values of the sarcoplasmic reticulum calcium transport system were higher compared with controls. Thus phosphorylation of Ser67 and/or Thr75 in inhibitor-1 may mitigate the stimulatory effects of the cAMP pathway, resulting in compromised cardiac function.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Sítios de Ligação , Células Cultivadas , Ativação Enzimática , Masculino , Fosforilação , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Serina/metabolismo , Treonina/metabolismoRESUMO
Depressed cardiac Ca cycling by the sarcoplasmic reticulum (SR) has been associated with attenuated contractility, which can progress to heart failure. The histidine-rich Ca-binding protein (HRC) is an SR component that binds to triadin and may affect Ca release through the ryanodine receptor. HRC overexpression in transgenic mouse hearts was associated with decreased rates of SR Ca uptake and delayed relaxation, which progressed to hypertrophy with aging. The present study shows that HRC may mediate part of its regulatory effects by binding directly to sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2) in cardiac muscle, which is confirmed by coimmunostaining observed under confocal microscopy. This interaction involves the histidine- and glutamic acid-rich domain of HRC (320-460 aa) and the part of the NH(2)-terminal cation transporter domain of SERCA2 (74-90 aa) that projects into the SR lumen. The SERCA2-binding domain is upstream from the triadin-binding region in human HRC (609-699 aa). Specific binding between HRC and SERCA was verified by coimmunoprecipitation and pull-down assays using human and mouse cardiac homogenates and by blot overlays using glutathione S-transferase and maltose-binding protein recombinant proteins. Importantly, increases in Ca concentration were associated with a significant reduction of HRC binding to SERCA2, whereas they had opposite effects on the HRC-triadin interaction in cardiac homogenates. Collectively, our data suggest that HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart.