RESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been associated with diabetes in several epidemiological studies. However, the diabetogenic action of TCDD on pancreatic cells is unclear. Here, we investigated the direct toxic effects of TCDD on a rat insulin-secreting beta cell line. We found that TCDD enhances exocytosis of MTT formazan and lysosomal proteins such as beta-hexosaminindase and Lamp-1. This TCDD-induced exocytosis was abrogated by T-type calcium channel blockers (mibefradil, flunarizine) but not by an aryl hydrocarbon receptor antagonist (alpha-naphtoflavone). Indeed, cytosolic calcium levels were increased by TCDD. Furthermore, TCDD stimulated insulin secretion, which was inhibited by flunarizine. Taken together, our results suggest that TCDD-induced calcium influx via T-type channels regulates vesicular trafficking, such as lysosomal and secretory granule exocytosis, and that TCDD might exert adverse effects on beta cells by continuous insulin release followed by beta cell exhaustion. This could contribute to the link between TCDD exposure and the risk of developing diabetes.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Cálcio/metabolismo , Poluentes Ambientais/toxicidade , Exocitose/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Ratos , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Via Secretória/efeitos dos fármacosRESUMO
Increased levels and non-telomeric roles have been reported for shelterin proteins, including RAP1 in cancers. Herein using Rap1 null mice, we provide the genetic evidence that mammalian Rap1 plays a major role in hematopoietic stem cell survival, oncogenesis and response to chemotherapy. Strikingly, this function of RAP1 is independent of its association with the telomere or with its known partner TRF2. We show that RAP1 interacts with many members of the DNA damage response (DDR) pathway. RAP1 depleted cells show reduced interaction between XRCC4/DNA Ligase IV and DNA-PK, and are impaired in DNA Ligase IV recruitment to damaged chromatin for efficient repair. Consistent with its role in DNA damage repair, RAP1 loss decreases double-strand break repair via NHEJ in vivo, and consequently reduces B cell class switch recombination. Finally, we discover that RAP1 levels are predictive of the success of chemotherapy in breast and colon cancer.
Assuntos
Antineoplásicos/farmacologia , Carcinogênese/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/metabolismo , Fluoruracila/farmacologia , Raios gama , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Camundongos Knockout , Mutagênicos/toxicidade , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Complexo Shelterina , Análise de SobrevidaRESUMO
Here, we show that H-ras(V12) causes the p53-knockout mouse astrocytes (p53-/- astrocytes) to be transformed into brain cancer stem-like cells. H-ras(V12) triggers the p53-/- astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras(V12) also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras(V12)-overexpressing p53-/- astrocytes (p53-/-ast-H-ras(V12)) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.
Assuntos
Astrócitos/patologia , Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Proteína Oncogênica p21(ras)/metabolismo , Animais , Astrócitos/metabolismo , Biomarcadores/análise , Neoplasias Encefálicas/metabolismo , Transformação Celular Neoplásica/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/química , Neurônios/patologia , Proteína Oncogênica p21(ras)/análise , Proteína Oncogênica p21(ras)/genética , Fosfatidilinositol 3-Quinases/metabolismo , Tubulina (Proteína)/análise , Proteína Supressora de Tumor p53/genéticaRESUMO
After successful clinical application, arginine deiminase (ADI) has been proposed to be a new cancer therapeutic. In the present study, we examined the effect of ADI in combination with ionizing radiation (IR) on MCF-7 cell growth and clonogenic cell death. Cell growth was inhibited by IR in a dose-dependent manner and ADI enhanced the radiosensitivity. ADI itself did not suppress the growth of MCF-7 cells due to the high level of expression of argininosuccinate synthetase (ASS), which convert citrulline, a product of arginine degradation by ADI, to arginine. Previously, it was suggested that ammonia, another product of arginine degradation by ADI, is the main cause of the growth inhibition of irradiated hepatoma cells contaminated with ADI-expressing mycoplasma [van Rijn et al. (2003)]. However, we found that ammonia is not the only factor that enhances radiosensitivity, as enhancement was also observed in the absence of ammonia. In order to identify the enhancing effect, levels of ASS and proteins related to the cell cycle were examined. ASS was unchanged by ADI plus IR, but p21 (a CDK inhibitor) was upregulated and c-Myc downregulated. These findings indicate that changes in the expressions of cell cycle proteins are involved in the enhancement of radiosensitivity by ADI. We suggest that ADI is a potential adjunct to cancer therapy.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Hidrolases/farmacologia , Mycoplasma/enzimologia , Tolerância a Radiação/efeitos dos fármacos , Amônia/farmacologia , Argininossuccinato Sintase/genética , Argininossuccinato Sintase/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , Tolerância a Radiação/efeitos da radiação , Radiação IonizanteRESUMO
Clusterin, a secretory glycoprotein, has been shown to be up-regulated in the reactive astrocytes in response to brain injury and neurodegenerative diseases, but its function has not been clearly elucidated. In this study, we investigate whether clusterin has growth-stimulatory activity in astrocytes. Suppression of clusterin with antisense oligonucleotide induced growth arrest, whereas transient overexpression of clusterin by cDNA transfection or exogenous treatment with purified clusterin promoted proliferation of the primary astrocytes in culture. This clusterin-stimulated proliferation was abrogated by PD98059, an inhibitor of mitogen-activated protein kinase kinase. These results suggest that clusterin might play an important role in astrogliosis by stimulating the proliferation of astrocytes through activation of the extracellular signal-regulated kinase 1/2 signaling pathway.
Assuntos
Astrócitos/fisiologia , Proliferação de Células , Clusterina/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Western Blotting/métodos , Contagem de Células/métodos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Clusterina/química , Clusterina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Citometria de Fluxo/métodos , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Ratos , Ratos Sprague-Dawley , Timidina/metabolismo , Transfecção , Trítio/metabolismoRESUMO
BACKGROUND: Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. METHODS: We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. RESULTS: USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. CONCLUSION: USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Proteases Específicas de Ubiquitina/metabolismo , Animais , Quinase 1 do Ponto de Checagem , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Proteínas Quinases/metabolismo , Células Tumorais Cultivadas , Proteases Específicas de Ubiquitina/antagonistas & inibidoresRESUMO
Glioblastoma (GBM) is the most common and aggressive primary brain tumor. To better understand how GBM evolves, we analyzed longitudinal genomic and transcriptomic data from 114 patients. The analysis shows a highly branched evolutionary pattern in which 63% of patients experience expression-based subtype changes. The branching pattern, together with estimates of evolutionary rate, suggests that relapse-associated clones typically existed years before diagnosis. Fifteen percent of tumors present hypermutation at relapse in highly expressed genes, with a clear mutational signature. We find that 11% of recurrence tumors harbor mutations in LTBP4, which encodes a protein binding to TGF-ß. Silencing LTBP4 in GBM cells leads to suppression of TGF-ß activity and decreased cell proliferation. In recurrent GBM with wild-type IDH1, high LTBP4 expression is associated with worse prognosis, highlighting the TGF-ß pathway as a potential therapeutic target in GBM.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Evolução Clonal/genética , Dacarbazina/análogos & derivados , Glioblastoma/patologia , Mutação/genética , Recidiva Local de Neoplasia/patologia , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Proliferação de Células , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Genômica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Isocitrato Desidrogenase/genética , Proteínas de Ligação a TGF-beta Latente/genética , Estudos Longitudinais , Gradação de Tumores , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Taxa de Sobrevida , Temozolomida , Transcriptoma , Fator de Crescimento Transformador beta/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Glioblastoma multiforme (GBM) displays cellular hierarchies harboring a subpopulation of stem-like cells (GSCs). Enhancer of Zeste Homolog 2 (EZH2), the lysine methyltransferase of Polycomb repressive complex 2, mediates transcriptional repression of prodifferentiation genes in both normal and neoplastic stem cells. An oncogenic role of EZH2 as a transcriptional silencer is well established; however, additional functions of EZH2 are incompletely understood. Here, we show that EZH2 binds to and methylates STAT3, leading to enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. The EZH2-STAT3 interaction preferentially occurs in GSCs relative to non-stem bulk tumor cells, and it requires a specific phosphorylation of EZH2. Inhibition of EZH2 reverses the silencing of Polycomb target genes and diminishes STAT3 activity, suggesting therapeutic strategies.
Assuntos
Glioblastoma/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Fator de Transcrição STAT3/metabolismo , Animais , Transformação Celular Neoplásica , Proteína Potenciadora do Homólogo 2 de Zeste , Inativação Gênica , Glioblastoma/patologia , Humanos , Metilação , Camundongos , Fosforilação , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
We previously reported that clusterin enhances astrocyte proliferation and extracellular signal-regulated kinase (ERK) activity. It, however, remains largely unknown how clusterin promotes cell growth. Here, we investigate the signaling pathway and related molecules underlying astrocyte proliferation by clusterin. Exogenous clusterin stimulates Ras-dependent Raf-1/mitogen-activated protein kinase kinase (MEK)/ERK activation. Clusterin-induced astrocyte proliferation and ERK1/2 phosphorylation were abrogated by either AG1478 (an inhibitor of epidermal growth factor receptor, EGFR) or EGFR small interfering RNA. Furthermore, clusterin treatment provoked tyrosine phosphorylation of EGFR (pY(1173)), which was also blocked by AG1478. These results suggest that clusterin requires EGFR activation to deliver its mitogenic signal through the Ras/Raf-1/MEK/ERK signaling cascade in astrocytes.
Assuntos
Astrócitos/fisiologia , Proliferação de Células/efeitos dos fármacos , Clusterina/farmacologia , Receptores ErbB/metabolismo , Animais , Células Cultivadas , Clusterina/fisiologia , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Mitógenos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/efeitos dos fármacos , Quinazolinas , RNA Interferente Pequeno/farmacologia , Ratos , Transdução de Sinais , Tirfostinas/farmacologiaRESUMO
Clusterin has been known as a chaperone-like molecule capable of interacting with various proteins. In this study, we show that clusterin interacts with the microtubule-destabilizing stathmin family protein SCLIP by GST pull-down and co-immunoprecipitation assays. Interestingly, SCLIP interacts with 80 kDa mature form of clusterin in the cytosolic fraction of PC12 cells permeabilized by low concentration of a weak nonionic detergent digitonin, but not with intracellular variants of clusterin known as binding isoforms of Ku70 or TGF-beta receptors. Both clusterin and SCLIP are co-localized at the perinuclear region and growth cone of PC12 cells. In addition, we show that the minimal domains for the interaction are mapped to the C-terminal valine-rich region (367-447) of clusterin and the N-terminal palmitoylation and membrane attachment site (1-34) of SCLIP. Finally, we demonstrate that ectopic expression of clusterin in PC12 cells elongates neurite-formation triggered by NGF and induces spontaneous neurite outgrowth even in the absence of NGF. Taken together, these results suggest that the clusterin interacts with SCLIP and the interaction may act as an important modulator during neuronal differentiation.
Assuntos
Diferenciação Celular/fisiologia , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Crescimento Neural/metabolismo , Neuritos/fisiologia , Animais , Citoplasma/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Células PC12 , Estrutura Terciária de Proteína , Ratos , Canais de Cátion TRPMRESUMO
Since arginine deiminase (ADI; EC 3.5.3.6) inhibits cell proliferation by arresting cells in the G1 phase, we tested its synergistic effect on cell death induced by dexamethasone (DEX), which also induces apoptosis by G1 cell cycle arrest. ADI inhibited cell proliferation and induced apoptosis in human leukemic CEM cells in a dose-dependent manner. Simultaneous treatment with ADI and DEX showed synergistic effects on DNA fragmentation and LDH release. In addition, ADI exerted its anti-proliferative activity against DEX-resistant CEM cells. ADI suppressed expression of c-myc, a potential key regulator of cell proliferation and apoptosis, and increased expression of p27Kip1 cyclin-dependent kinase inhibitor. These results suggest that ADI efficiently increases the anti-cancer effect of DEX on human leukemic CEM cells through G1 cell cycle arrest involving downregulation of c-myc and upregulation of p27Kip1.