RESUMO
Squamous cell carcinomas (SCCs) account for the majority of cancer mortalities. Although TP63 is an established lineage-survival oncogene in SCCs, therapeutic strategies have not been developed to target TP63 or it's downstream effectors. In this study we demonstrate that TP63 directly regulates NRG1 expression in human SCC cell lines and that NRG1 is a critical component of the TP63 transcriptional program. Notably, we show that squamous tumors are dependent NRG1 signaling in vivo, in both genetically engineered mouse models and human xenograft models, and demonstrate that inhibition of NRG1 induces keratinization and terminal squamous differentiation of tumor cells, blocking proliferation and inhibiting tumor growth. Together, our findings identify a lineage-specific function of NRG1 in SCCs of diverse anatomic origin.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Neuregulina-1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos Nus , Receptor ErbB-3/metabolismoRESUMO
Neuregulin 1 (NRG1) is required for development of the central and peripheral nervous system and regulates neurotransmission in the adult. NRG1 and the gene encoding its receptor, ERBB4, are risk genes for schizophrenia, although how alterations in these genes disrupt their function has not been fully established. Studies of knockout and transgenic mice have yielded conflicting results, with both gain and loss of function resulting in similar behavioral and electrophysiological phenotypes. Here, we used high affinity antibodies to NRG1 and ErbB4 to perturb the function of the endogenous proteins in adult mice. Treatment with NRG1 antibodies that block receptor binding caused behavioral alterations associated with schizophrenia, including, hyper-locomotion and impaired pre-pulse inhibition of startle (PPI). Electrophysiological analysis of brain slices from anti-NRG1 treated mice revealed reduced synaptic transmission and enhanced paired-pulse facilitation. In contrast, mice treated with more potent ErbB4 function blocking antibodies did not display behavioral alterations, suggesting a receptor independent mechanism of the anti-NRG1-induced phenotypes. We demonstrate that anti-NRG1 causes accumulation of the full-length transmembrane protein and increases phospho-cofilin levels, which has previously been linked to impaired synaptic transmission, indicating enhancement of non-canonical NRG1 signaling could mediate the CNS effects.
Assuntos
Sistema Nervoso Central/fisiologia , Eletrofisiologia/métodos , Neuregulina-1/metabolismo , Esquizofrenia/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Anticorpos Bloqueadores/administração & dosagem , Modelos Animais de Doenças , Predisposição Genética para Doença , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuregulina-1/genética , Neuregulina-1/imunologia , Estabilidade Proteica , Receptor ErbB-4/genética , Receptor ErbB-4/imunologia , Receptor ErbB-4/metabolismo , Risco , Esquizofrenia/genética , Transdução de Sinais , Transmissão SinápticaRESUMO
The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteína 2 de Ligação ao Retinoblastoma/antagonistas & inibidores , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Relação Estrutura-AtividadeRESUMO
Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, (3)H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.
Assuntos
Antígeno CTLA-4/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , ADP-Ribosil Ciclase 1/metabolismo , Apoptose , Linfócitos B/patologia , Antígeno CTLA-4/deficiência , Antígeno CTLA-4/genética , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo/genética , Inativação Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Prognóstico , RNA Interferente Pequeno/genética , Microambiente TumoralRESUMO
Although standard chemotherapies are commonly used to treat most types of solid tumors, such treatment often results in inadequate response to, or relapse after, therapy. This is particularly relevant for lung cancer because most patients are diagnosed with advanced-stage disease and are treated with frontline chemotherapy. By studying the residual tumor cells that remain after chemotherapy in several in vivo non-small cell lung cancer models, we found that these cells have increased levels of human epidermal growth factor receptor (HER) signaling due, in part, to the enrichment of a preexisting NRG1(HI) subpopulation. Neuregulin 1 (NRG1) signaling in these models can be mediated by either the HER3 or HER4 receptor, resulting in the differential activation of downstream effectors. Inhibition of NRG1 signaling inhibits primary tumor growth and enhances the magnitude and duration of the response to chemotherapy. Moreover, we show that inhibition of ligand-mediated Her4 signaling impedes disease relapse in cases where NRG1 inhibition is insufficient. These findings demonstrate that ligand-dependent Her4 signaling plays an important role in disease relapse.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neuregulina-1/antagonistas & inibidores , Transdução de Sinais , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Bloqueadores/uso terapêutico , Comunicação Autócrina/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Humanos , Ligantes , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/metabolismo , Neoplasia Residual/patologia , Neuregulina-1/metabolismo , Receptor ErbB-4 , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although chemotherapy is used to treat most advanced solid tumors, recurrent disease is still the major cause of cancer-related mortality. Cancer stem cells (CSCs) have been the focus of intense research in recent years because they provide a possible explanation for disease relapse. However, the precise role of CSCs in recurrent disease remains poorly understood and surprisingly little attention has been focused on studying the cells responsible for re-initiating tumor growth within the original host after chemotherapy treatment. We utilized both xenograft and genetically engineered mouse models of non-small cell lung cancer (NSCLC) to characterize the residual tumor cells that survive chemotherapy treatment and go on to cause tumor regrowth, which we refer to as tumor re-initiating cells (TRICs). We set out to determine whether TRICs display characteristics of CSCs, and whether assays used to define CSCs also provide an accurate readout of a cell's ability to cause tumor recurrence. We did not find consistent enrichment of CSC marker positive cells or enhanced tumor initiating potential in TRICs. However, TRICs from all models do appear to be in EMT, a state that has been linked to chemoresistance in numerous types of cancer. Thus, the standard CSC assays may not accurately reflect a cell's ability to drive disease recurrence.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Células-Tronco Neoplásicas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas with a median patient survival of only 5-7 years. The failure of existing therapies is mainly due to disease relapse when therapy-resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy-resistant MCL are needed. Here, we developed an in vivo model of therapy-resistant MCL by transplanting a patient-derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy-resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real-time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy-resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI-antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy-resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL-specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor-specific immunotherapy are effective and have excellent potential for clinical application to provide long-term, disease-free survival in MCL patients.
Assuntos
Proteínas Hedgehog/metabolismo , Linfoma de Célula do Manto/tratamento farmacológico , Transdução de Sinais , Animais , Antineoplásicos/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Mantle cell lymphoma (MCL) is an aggressive B-cell neoplasm with few patients achieving long-term survival with current treatment regimens. High-dose therapy is effective in reducing the tumor burden; however, patients eventually relapse due to minimal residual disease. Having demonstrated efficacy in other malignancies, the effectiveness of dendritic cell-based immunotherapy for minimal residual MCL was examined. We demonstrated that dendritic cells (DC) primed with MCL antigens stimulated the activation of MCL-specific T cells that recognized and destroyed both MCL cell lines and primary MCL in vitro. In addition, in vivo studies demonstrated that adoptively transferred MCL-specific T cells were able to significantly inhibit tumor growth in mice with minimal residual MCL. Subsequently, when combined with CHOP chemotherapy, adoptive T-cell therapy was able to significantly extend the survival of the mice by further reducing the tumor burden. These results clearly show that MCL-specific cellular immunotherapy is effective in treating minimal residual MCL, paving the way for future clinical studies.
Assuntos
Imunoterapia Adotiva/métodos , Neoplasias Renais/terapia , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/terapia , Linfoma de Célula do Manto/terapia , Linfócitos T/transplante , Animais , Antígenos de Neoplasias/imunologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Terapia Combinada , Ciclofosfamida/uso terapêutico , Células Dendríticas/imunologia , Doxorrubicina/uso terapêutico , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/imunologia , Neoplasias Renais/mortalidade , Neoplasias Renais/secundário , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Ativação Linfocitária/imunologia , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/mortalidade , Linfoma de Célula do Manto/patologia , Camundongos , Camundongos Endogâmicos NOD , Neoplasia Residual , Prednisona/uso terapêutico , Linfócitos T/imunologia , Resultado do Tratamento , Vincristina/uso terapêuticoRESUMO
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell-induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell-induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective down-regulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target.
Assuntos
Proteínas Hedgehog/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Células Estromais/citologia , Células Estromais/fisiologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologia , Proteína GLI1 em Dedos de ZincoRESUMO
Mantle cell lymphoma (MCL) has one of the worst clinical outcomes among the B-cell lymphomas, with a median survival of only 3 to 4 years. Therefore, a better understanding of the underlying mechanisms that regulate MCL proliferation/survival is needed to develop an effective therapy. Because sonic hedgehog (Shh)-GLI signaling has been shown to be important in the proliferation and survival of several cancers, and no such information is available for MCL, this study was undertaken. Our results show that the molecules associated with Shh-GLI signaling, such as PTCH and SMO receptors, and GLI1 and GLI2 target transcription factors were expressed in the human MCL cell lines and primary MCL cells from patients. Perturbation of this signaling in the presence of exogenous Shh/cyclopamine significantly (P < 0.001) influenced the proliferation of JVM2 MCL cells. Furthermore, down-regulation of GLI transcription factors using antisense oligonucleotides not only resulted in significantly (P < 0.001) decreased proliferation of the MCL cells but also significantly (P < 0.05) increased their susceptibility to chemotherapeutic drug, doxorubicin. Also, down-regulation of GLI decreased cyclin D1 and BCL2 transcript levels, which suggests that these key molecules might be regulated by GLI in MCL. Thus, our results indicate a significant role for Shh-GLI signaling in the proliferation of MCL, and molecular targeting of GLI is a potential therapeutic approach to improve the treatment for MCL.
Assuntos
Proteínas Hedgehog/metabolismo , Linfoma de Célula do Manto/terapia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Hedgehog/genética , Humanos , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Modelos Biológicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
A partial mechanism by which a conformationally-biased, response-selective agonist of complement component C5a, Tyr-Ser-Phe-Lys-Pro-Met-Pro-Leu-D-Ala-Arg or YSFKPMPLaR (EP54), acts as a molecular adjuvant is presented by showing the manner in which this peptide engages human dendritic cells (DC). Confocal microscopy was used to show that fluorescent-labeled EP54 (0.2 microM) and fluorescent-labeled B and T cell epitopes attached to EP54 (i.e., EP54-containing vaccines, 0.2 microM) were internalized by human DCs well within 30 min of exposure. After 24 h of exposure, EP54 and the B and T cell epitopes of the EP54-containing vaccines (20 microM) were presented on the DC surface in the context of HLA-ABC and HLA-DR determinants. Also, exposure of DCs to EP54 (50 microg/ml) induced the activation of genes specific for the Th1 cytokines IL-6, IL-12, INFgamma, and TNFalpha as well as the Th2 cytokine IL-4. Internalization, HLA expression, and cytokine gene activation were not observed in the presence of the inactive, scrambled EP54 constructs arguing that these effects of EP54 are mediated predominately via C5a receptors on the DC surface.
Assuntos
Apresentação de Antígeno , Complemento C5a/agonistas , Células Dendríticas/imunologia , Fragmentos de Peptídeos/imunologia , Adjuvantes Imunológicos , Complemento C5a/imunologia , Complemento C5a/farmacologia , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Fragmentos de Peptídeos/farmacologiaRESUMO
Tumors of the nervous system, including neuroblastoma and glioblastoma, are difficult to treat with current therapies. Despite the advances in cancer therapeutics, the outcomes in these patients remain poor and, therefore, new modalities are required. Recent literature demonstrates that cytotoxic effector cells can effectively kill tumors of the nervous system. In addition, we have previously shown that umbilical cord blood (UCB) contains precursors of antitumor cytotoxic effector cells. Therefore, to evaluate the antitumor potential of UCB-derived effector cells, studies were designed to compare the in vitro and in vivo antitumor effects of UCB- and peripheral blood (PB)-derived antigen-nonspecific and antigen-specific effector cells against tumors of the nervous system. Mononuclear cells (MNCs) from UCB were used to generate both interleukin-2 (IL-2)-activated killer (LAK) cells and tumor-specific cytotoxic T lymphocytes (CTLs). UCB-derived LAK cells showed a significant in vitro cytotoxicity against IMR-32, SK-NMC, and U-87 human neuroblastoma and glioblastoma, respectively. In addition, the CTLs generated using dendritic cells primed with IMR-32 tumor cell lysate showed a selective cytotoxicity in vitro against IMR-32 cells, but not against U-87 or MDA-231 cells. Furthermore, treatment of SCID mice bearing IMR-32 neuroblastoma with tumor-specific CTLs resulted in a significant (p < 0.01) inhibition of tumor growth and increased overall survival. Thus, these results demonstrate the potential of UCB-derived effector cells against human neuroblastoma and warrant further preclinical studies.
Assuntos
Sangue Fetal/citologia , Sangue Fetal/imunologia , Imunoterapia/métodos , Neuroblastoma/imunologia , Neuroblastoma/terapia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica/imunologia , Sangue Fetal/transplante , Glioblastoma/imunologia , Glioblastoma/terapia , Humanos , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Ativadas por Linfocina/transplante , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Camundongos , Camundongos SCID , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/transplanteRESUMO
PURPOSE: In B-cell chronic lymphocytic leukemia (CLL), high CD38 expression has been associated with unfavorable clinical course, advanced disease, resistance to therapy, shorter time to first treatment, and shorter survival. However, the genes associated with CLL patient subgroups with high and low CD38 expression and their potential role in disease progression is not known. EXPERIMENTAL DESIGN: To identify the genes associated with the clinical disparity in CLL patients with high versus low CD38 expression, transcriptional profiles were obtained from CLL cells from 39 different patients using oligonucleotide microarray. Gene expression was also compared between CLL cells and B cells from healthy individuals. RESULTS: Gene expression analysis identified 76 differentially expressed genes in CD38 high versus low groups. Out of these genes, HEM1, CTLA4, and MNDA were selected for further studies and their differential expression was confirmed by real-time PCR. HEM1 overexpression was associated with poor outcome, whereas the overexpression of CTLA4 and MNDA was associated with good outcome. Down-regulation of HEM1 expression in patient CLL cells resulted in a significant increase in their susceptibility to fludarabine-mediated killing. In addition, when gene expression patterns in CD38 high and low CLL cells were compared with normal B-cell profiles, ATM expression was found to be significantly lower in CD38 high compared with CD38 low CLL as confirmed by real-time reverse transcription-PCR. CONCLUSIONS: These results identify the possible genes that may be involved in cell proliferation and survival and, thus, determining the clinical behavior of CLL patients expressing high or low CD38.
Assuntos
ADP-Ribosil Ciclase 1/genética , Regulação Leucêmica da Expressão Gênica , Genes Neoplásicos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Antígenos CD/genética , Antígenos de Diferenciação/genética , Antígenos de Diferenciação Mielomonocítica/genética , Proteínas Mutadas de Ataxia Telangiectasia , Antígeno CTLA-4 , Proteínas de Ciclo Celular/genética , Proliferação de Células , Proteínas de Ligação a DNA/genética , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Membrana/genética , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
A therapeutic strategy that would mitigate the events leading to hyperplasia and facilitate re-endothelialization of an injured artery after balloon angioplasty could be effective for a long-term patency of the artery. It is hypothesized that erythropoietin (EPO), which has both anti-inflammatory and antiapoptotic properties, will prevent hyperplasia, and its ability to proliferate and mobilize endothelial progenitor cells will re-endothelialize the injured artery. To test this hypothesis, EPO (5000 IU/kg) in solution was injected intraperitoneally 6 hours before vascular injury and then on every alternate day for a week or as a single dose (5000 IU/kg) in a sustained release gel formulation 1 week before the vascular injury. Morphometric analysis revealed nearly continuous re-endothelialization of the injured artery in EPO solution-treated animals (90% vs less than 20% in saline control); however, the treatment also caused excessive neointima formation (intima/media ratio, 2.10 +/- 0.09 vs 1.60 +/- 0.02 saline control, n = 5, P < .001). The EPO gel also induced similar excessive neointima formation. Immunohistochemical analysis of the injured arteries from the animals treated with EPO solution demonstrated a significant angiogenic response in adventitia and media, thus explaining the formation of excessive neointima. Although the results are in contrast to expectation, they explain a greater degree of stenosis seen in hemodialysis access fistulas in patients who are on EPO therapy for anemic condition. The results also caution the use of EPO, particularly in patients who are at a risk of vascular injury or are suffering from an atherosclerotic condition.
Assuntos
Lesões das Artérias Carótidas/tratamento farmacológico , Eritropoetina/farmacologia , Túnica Íntima/efeitos dos fármacos , Animais , Artérias Carótidas/patologia , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Preparações de Ação Retardada , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Eritropoetina/administração & dosagem , Eritropoetina/efeitos adversos , Géis , Hiperplasia/induzido quimicamente , Imuno-Histoquímica , Injeções Intraperitoneais , Neovascularização Patológica/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Túnica Íntima/patologiaRESUMO
In B-cell chronic lymphocytic leukemia (CLL), Rai stage, immunoglobulin gene mutational status, chromosomal abnormalities, CD38 and ZAP-70 expression were used as prognostic markers. In this study, to understand the molecular basis of chromosomal abnormalities leading to tumor progression, 90 CLL patients were grouped into poor prognosis (with 11q deletion and trisomy 12) and good prognosis (with normal karyotype and 13q deletion) and their clinical outcome was assessed. Gene expression profiles of 35 CLL samples with poor outcome (11q deletion, n=9; trisomy 12, n=5) and good outcome (13q deletion, n=13; normal karyotype, n=8) were analyzed using oligonucleotide microarray. Significance analysis of microarray (SAM) identified 27 differentially expressed genes between these two subgroups with significant overexpression of ATF5 and underexpression of CDC16, PCDH8, SLAM, MNDA and ATF2 in CLL patients with poor outcome. ATF5 gene expression in CLL was further studied because of its role in the regulation of cell cycle progression/differentiation and apoptosis. The overexpression of ATF5 was confirmed by real-time PCR using 39 CLL samples from the poor and good outcome groups. ATF5 was significantly (p<0.001) overexpressed in the poor outcome group. Furthermore, ATF5 expression was significantly higher in the 11q deletion as well as trisomy 12 group alone compared to the 13q deletion and normal karyotype groups. ATF5 overexpression was also associated with significantly (p=0.04) shorter time to treatment. Similarly, expression of five underexpressed genes also correlated with longer time to treatment. Thus, this report demonstrates that ATF5 may be one of the key genes involved in increased proliferation and survival in 11q deletion or trisomy 12, whereas CD16, CD86, SLAM, MNDA and ATF2 may be involved in the decreased proliferation of CLL cells with 13q deletion or normal karyotype.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 12/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Trissomia/genética , Fatores Ativadores da Transcrição/metabolismo , Análise por Conglomerados , Análise Citogenética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Tempo , Resultado do TratamentoRESUMO
The maintenance and differentiation of retinal progenitors take place in the context of the microenvironment in which they reside at a given time during retinal histogenesis. To understand the nature of the microenvironment in the developing retina, we have examined the influence of activities present during the early stage of retinal histogenesis on enriched retinal progenitors, using the neurosphere model. Early and late retinal progenitors, enriched as neurospheres from embryonic day 14 (E14) and E18 rat retina, respectively, were cultured in embryonic day 3 (E3) chick retinal conditioned medium, simulating the microenvironment present during early retinal histogenesis. Examination of the differentiation and proliferation of retinal progenitors revealed that the early microenvironment contains at least three regulatory activities, which are partitioned in different size fractions of the conditioned medium with different heat sensitivity. First, it is characterized by activities, present in heat stable <30 kDa fraction, that promote the differentiation of retinal ganglion cells (RGCs), the early born neurons. Second, it contains activities, present in heat-sensitive >30 kDa fraction, that regulate the number of early born neurons and maintain the pool of retinal progenitors. Third, it possesses activities, present in heat-sensitive <30 kDa fraction, that prevent the premature differentiation of early retinal progenitors into the late born neurons. Thus, our observations demonstrate the regulatory influence of microenvironment on the maintenance and differentiation of retinal progenitors and establish neurospheres as a viable model system for the examination of such influences.
Assuntos
Retina/embriologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica/métodos , Modelos Animais , Células Fotorreceptoras de Vertebrados/citologia , Ratos , Ratos Sprague-Dawley , Retina/citologia , Células Ganglionares da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
B-cell chronic lymphocytic leukemia (B-CLL) is the most common B-cell leukemia among older populations in Western countries. The clinical course of B-CLL is heterogeneous: in some patients the disease course is indolent, in others it is aggressive. The B-CLL subgroups with chromosome 11q23 deletion have been associated with aggressive disease course involving ATM deletion, extensive bulky lymphadenopathy (BLA), and inferior clinical outcome. Using real-time reverse transcriptase-polymerase chain reaction, we found that ATM was consistently underexpressed in B-CLL patients with BLA, irrespective of 11q23 deletion status. In addition, B-CLL patients who presented with BLA had a significantly shorter time to treatment (2 months) than did patients without BLA (74 months). Moreover, gene expression analysis in B-CLL patients with and without BLA revealed differences in expression for genes involved in apoptosis, cell cycle, and cell adhesion. These results indicate an association between BLA and reduced expression of ATM, suggesting a role for ATM in disease progression in B-CLL.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Doenças Linfáticas/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Proteínas Mutadas de Ataxia Telangiectasia , Adesão Celular/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Doenças Linfáticas/diagnóstico , Doenças Linfáticas/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
The retina in adult mammals, unlike those in lower vertebrates such as fish and amphibians, is not known to support neurogenesis. However, when injured, the adult mammalian retina displays neurogenic changes, raising the possibility that neurogenic potential may be evolutionarily conserved and could be exploited for regenerative therapy. Here, we show that Müller cells, when retrospectively enriched from the normal retina, like their radial glial counterparts in the central nervous system (CNS), display cardinal features of neural stem cells (NSCs), i.e., they self-renew and generate all three basic cell types of the CNS. In addition, they possess the potential to generate retinal neurons, both in vitro and in vivo. We also provide direct evidence, by transplanting prospectively enriched injury-activated Müller cells into normal eye, that Müller cells have neurogenic potential and can generate retinal neurons, confirming a hypothesis, first proposed in lower vertebrates. This potential is likely due to the NSC nature of Müller cells that remains dormant under the constraint of non-neurogenic environment of the adult normal retina. Additionally, we demonstrate that the mechanism of activating the dormant stem cell properties in Müller cells involves Wnt and Notch pathways. Together, these results identify Müller cells as latent NSCs in the mammalian retina and hence, may serve as a potential target for cellular manipulation for treating retinal degeneration.
Assuntos
Mamíferos/embriologia , Células-Tronco Multipotentes/citologia , Neuroglia/citologia , Neurônios/citologia , Receptores Notch/metabolismo , Retina/citologia , Proteínas Wnt/metabolismo , Animais , Transplante de Células , Citometria de Fluxo , Ratos , Retina/transplanteRESUMO
Many fungi are known to secrete lectins, but their functional roles are not clearly understood. Sclerotium rolfsii, a soilborne plant pathogenic fungus capable of forming fruiting bodies called sclerotial bodies, secrete a cell wall-associated Thomsen-Friedenreich antigen-specific lectin. To understand the functional role of this lectin, we examined its occurrence and expression during development of the fungus. Furthermore, putative endogenous receptors of the lectin were examined to substantiate the functional role of the lectin. Immunolocalization studies using FITC-labeled lectin antibodies revealed discrete distribution of lectin sites at the branching points of the developing mycelia and uniformly occurring lectin sites on the mature sclerotial bodies. During development of the fungus the lectin is expressed in small amounts on the vegetative mycelia and reaching very high levels in mature sclerotial bodies with a sudden spurt in secretion at the maturation stage. Capping of the lectin sites on the sclerotial bodies by lectin antibodies or haptens inhibit strongly the germination of these bodies, indicating functional significance of the lectin. At the maturation stage the lectin interacts with the cell wall-associated putative endogenous receptor leading to the aggregation of mycelium to form sclerotial bodies. The lectin-receptor complex probably acts as signaling molecule in the germination process of sclerotial bodies. Using biotinylated lectin, the receptors were identified by determining the specific lectin binding to lipid components, extracted from sclerotial bodies, and separated on thin-layer chromatograms. Preliminary characterization studies indicated that the receptors are glycosphingolipids and resemble inositolphosphoceramides. These findings together demonstrate the importance of lectin-receptor interactions to explain hitherto speculated functional role of the lectins and also the glycosphingolipids of fungi.