RESUMO
Ovarian epithelial carcinoma (OEC) is the most frequent ovarian tumor, characterized by a high mortality in advanced stages where conventional therapies are not effective. Based on the role of the immune system in the progression of this disease, immunotherapy using checkpoint blockade has been considered as a therapeutic alternative. Nevertheless, its results do not match up to the positive results in entities like melanoma and other malignancies, suggesting the need to find other therapies to be used alone or in combination. Dendritic cell- (DC-) based vaccines have shown promising results in several types of cancer, such as melanoma, prostate, and lung cancers, due to the essential role played by DCs in the activation of specific T cells, thus using other ways of activating the immune response than immune checkpoint blockade. During the last decade, we have used DC-based vaccines loaded with an allogeneic heat shock-conditioned melanoma cell lysate in the treatment of advanced stage patients in a series of clinical trials. In these studies, 60% of treated patients showed immunological responses which correlated positively with improved survival. Considering the relevance of ovarian cancer and the promising results of our DC-based vaccine, we show here that heat shock-conditioned cell lysates derived from ovarian epithelial carcinoma cell lines have the potential to induce the phenotypic and functional maturation of human DC, which in turn, is able to induce an efficient CD4+ and CD8+ T cell-mediated immune responses against ovarian cancer cell lines in vitro. In summary, OEC heat shock-conditioned cell lysate-loaded DCs may be considered for future combined immunotherapy approaches against ovarian tumors.
Assuntos
Carcinoma Epitelial do Ovário/imunologia , Células Dendríticas/imunologia , Resposta ao Choque Térmico , Neoplasias Ovarianas/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Feminino , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/imunologia , Humanos , Imunoterapia , Interferon gama/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/terapia , Linfócitos T/metabolismoRESUMO
Osteosarcoma is the most common primary bone tumor during childhood and adolescence. Several reports have presented data on serum biomarkers for osteosarcoma, but few reports have analyzed circulating microRNAs (miRNAs). In this study, we used next generation miRNA sequencing to examine miRNAs isolated from microvesicle-depleted extracellular vesicles (EVs) derived from six different human osteosarcoma or osteoblastic cell lines with different degrees of metastatic potential (i.e., SAOS2, MG63, HOS, 143B, U2OS and hFOB1.19). EVs from each cell line contain on average ~300 miRNAs, and ~70 of these miRNAs are present at very high levels (i.e., >1000 reads per million). The most prominent miRNAs are miR-21-5p, miR-143-3p, miR-148a-3p and 181a-5p, which are enriched between 3 and 100 fold and relatively abundant in EVs derived from metastatic SAOS2 cells compared to non-metastatic MG63 cells. Gene ontology analysis of predicted targets reveals that miRNAs present in EVs may regulate the metastatic potential of osteosarcoma cell lines by potentially inhibiting a network of genes (e.g., MAPK1, NRAS, FRS2, PRCKE, BCL2 and QKI) involved in apoptosis and/or cell adhesion. Our data indicate that osteosarcoma cell lines may selectively package miRNAs as molecular cargo of EVs that could function as paracrine agents to modulate the tumor micro-environment.
Assuntos
Neoplasias Ósseas/genética , Vesículas Extracelulares/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Osteossarcoma/genética , Apoptose , Adesão Celular , Linhagem Celular Tumoral , Redes Reguladoras de Genes , Humanos , Metástase Neoplásica , Análise de Sequência de RNA/métodosRESUMO
Renal transplantation (RTx) is an effective therapy to improve clinical outcomes in pediatric patients with terminal chronic kidney disease. However, chronic immunosuppression with glucocorticoids (GCs) reduces bone growth and BMD. The mechanisms causing GC-induced growth impairment have not been fully clarified. Fibroblast growth factor 23 (FGF23) is a peptide hormone that regulates phosphate homeostasis and bone growth. In pathological conditions, FGF23 excess or abnormal FGF receptors (FGFR) activity leads to bone growth impairment. Experimental data indicate that FGF23 expression is induced by chronic GC exposure. Therefore, we hypothesize that GCs impair bone growth by increasing FGF23 expression, which has direct effects on bone growth plate. In a post hoc analysis of a multicentric randomized clinical trial of prepubertal RTx children treated with early GC withdrawal or chronic GC treatment, we observed that GC withdrawal was associated with improvement in longitudinal growth and BMD, and lower plasma FGF23 levels as compared with a chronic GC group. In prepubertal rats, GC-induced bone growth retardation correlated with increased plasma FGF23 and bone FGF23 expression. Additionally, GC treatment decreased FGFR1 expression whereas it increased FGFR3 expression in mouse tibia explants. The GC-induced bone growth impairment in tibiae explants was prevented by blockade of FGF23 receptors using either a pan-FGFR antagonist (PD173074), a C-terminal FGF23 peptide (FGF23180-205) which blocks the binding of FGF23 to the FGFR-Klotho complex or a specific FGFR3 antagonist (P3). Finally, local administration of PD173074 into the tibia growth plate ameliorated cartilage growth impairment in GC-treated rats. These results show that GC treatment partially reduces longitudinal bone growth via upregulation of FGF23 and FGFR3 expression, thus suggesting that the FGF23/Klotho/FGFR3 axis at the growth plate could be a potential therapeutic target for the management of GC-induced growth impairment in children.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Osso e Ossos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glucocorticoides/administração & dosagem , Transplante de Rim , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/patologia , Criança , Feminino , Fator de Crescimento de Fibroblastos 23 , Seguimentos , Glucocorticoides/efeitos adversos , Humanos , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Falência Renal Crônica/cirurgia , Proteínas Klotho , Masculino , Proteínas de Membrana , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Osteosarcomas are bone tumors that frequently metastasize to the lung. Aberrant expression of the transcription factor, runt-related transcription factor 2 (RUNX2), is a key pathological feature in osteosarcoma and associated with loss of p53 and miR-34 expression. Elevated RUNX2 may transcriptionally activate genes mediating tumor progression and metastasis, including the RUNX2 target gene osteopontin (OPN/SPP1). This gene encodes a secreted matricellular protein produced by osteoblasts to regulate bone matrix remodeling and tissue calcification. Here we investigated whether and how the RUNX2/OPN axis regulates lung metastasis of osteosarcoma. Importantly, RUNX2 depletion attenuates lung metastasis of osteosarcoma cells in vivo. Using next-generation RNA-sequencing, protein-based assays, as well as the loss- and gain-of-function approaches in selected osteosarcoma cell lines, we show that osteopontin messenger RNA levels closely correlate with RUNX2 expression and that RUNX2 controls the levels of secreted osteopontin. Elevated osteopontin levels promote heterotypic cell-cell adhesion of osteosarcoma cells to human pulmonary microvascular endothelial cells, but not in the presence of neutralizing antibodies. Collectively, these findings indicate that the RUNX2/OPN axis regulates the ability of osteosarcoma cells to attach to pulmonary endothelial cells as a key step in metastasis of osteosarcoma cells to the lung.
Assuntos
Neoplasias Ósseas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Invasividade Neoplásica , Osteopontina/metabolismo , Osteossarcoma/metabolismo , Animais , Neoplasias Ósseas/patologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Xenoenxertos , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Osteossarcoma/secundárioRESUMO
The in vitro process of chondrogenic differentiation of mesenchymal stem cells (MSCs) induces a pre-apoptotic hypertrophic phenotype, guided by the active status of the WNT/ßcatenin pathway. To achieve a stable chondrocyte phenotype for cartilage tissue engineering, it is necessary to gain a better understanding of specific genes that regulate the cartilage tissue phenotype. RNA sequencing (RNA-seq) analysis of tissue samples from bone, cartilage, growth plate and muscle show that Dickkopf-1 (DKK1), a natural WNT canonical signaling inhibitor, is expressed in cartilage tissue. This observation reinforces the concept that inhibition of the WNT/ßcatenin pathway is critical for preventing avoid chondrocyte hypertrophy in vitro. We used two doses of DKK1 in a pellet cell culture system to inhibit the terminal differentiation of chondrocytes derived from bone marrow mesenchymal stem cells (MSCs). Bone marrow MSCs were cultured in chondrogenic induction medium with 50 and 200â¯ng/ml of DKK1 for 21â¯days. The highest doses of DKK1 reduce ßcatenin expression and nuclear localization at day 21, concomitant with reduced expression and activity of hypertrophy markers collagen type X (COL10A1) and alkaline phosphatase (ALPL), thus decreasing the pre-hypertrophic chondrocyte population. Furthermore, DKK1 stimulated expression of collagen type II (COL2A1) and glycosaminoglycans (GAGs), which represent healthy articular cartilage markers. We conclude that exogenous DKK1 impedes chondrocyte progression into a prehypertrophic stage and stimulates expression of healthy articular cartilage markers by blocking the WNT/ßcatenin pathway. Hence, DKK1 may promote a mature healthy articular cartilage phenotype and facilitate cartilage tissue engineering for joint repair.
Assuntos
Biomarcadores/análise , Células da Medula Óssea/patologia , Condrócitos/patologia , Condrogênese , Hipertrofia/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/patologia , Adulto , Apoptose , Células da Medula Óssea/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Feminino , Humanos , Hipertrofia/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual , Adulto JovemRESUMO
Osteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone-related genes, including those encoding extracellular matrix proteins or factors that control cell-cell, and cell-matrix interactions. Cell-cell communication in the many skeletal pericellular micro-niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by "A Disintegrin And Metalloproteinase" (ADAM) proteins. These cell membrane-associated metalloproteinases support proteolytic release ("shedding") of protein ectodomains residing at the cell surface. We analyzed microarray and RNA-sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα-signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (-0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic-nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2-Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.
Assuntos
Proteína ADAM17/genética , Proteína Morfogenética Óssea 2/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Retroalimentação Fisiológica , Osteoblastos/metabolismo , Osteogênese/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Sítios de Ligação , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoblastos/citologia , Comunicação Parácrina/genética , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
SALL2 is a poorly characterized transcription factor that belongs to the Spalt-like family involved in development. Mutations on SALL2 have been associated with ocular coloboma and cancer. In cancers, SALL2 is deregulated and is proposed as a tumor suppressor in ovarian cancer. SALL2 has been implicated in stemness, cell death, proliferation, and quiescence. However, mechanisms underlying roles of SALL2 related to cancer remain largely unknown. Here, we investigated the role of SALL2 in cell proliferation using mouse embryo fibroblasts (MEFs) derived from Sall2-/- mice. Compared to Sall2+/+ MEFs, Sall2-/- MEFs exhibit enhanced cell proliferation and faster postmitotic progression through G1 and S phases. Accordingly, Sall2-/- MEFs exhibit higher mRNA and protein levels of cyclins D1 and E1. Chromatin immunoprecipitation and promoter reporter assays showed that SALL2 binds and represses CCND1 and CCNE1 promoters, identifying a novel mechanism by which SALL2 may control cell cycle. In addition, the analysis of tissues from Sall2+/+ and Sall2-/- mice confirmed the inverse correlation between expression of SALL2 and G1-S cyclins. Consistent with an antiproliferative function of SALL2, immortalized Sall2-/- MEFs showed enhanced growth rate, foci formation, and anchorage-independent growth, confirming tumor suppressor properties for SALL2. Finally, cancer data analyses show negative correlations between SALL2 and G1-S cyclins' mRNA levels in several cancers. Altogether, our results demonstrated that SALL2 is a negative regulator of cell proliferation, an effect mediated in part by repression of G1-S cyclins' expression. Our results have implications for the understanding and significance of SALL2 role under physiological and pathological conditions.
Assuntos
Ciclo Celular/genética , Ciclina D1/genética , Ciclina E/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteínas Repressoras/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/patologia , Ciclina D1/metabolismo , Ciclina E/metabolismo , Proteínas de Ligação a DNA , Fibroblastos/metabolismo , Fase G1 , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos Knockout , Modelos Biológicos , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase S , Fatores de Transcrição , Transcrição GênicaRESUMO
Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/ß-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/ß-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/ß-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of ß-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, ß-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of ß-catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3ß inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of ß-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/ß-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662-3674, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Neoplasias Ósseas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Proteínas de Neoplasias/biossíntese , Osteossarcoma/metabolismo , Via de Sinalização Wnt , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Osteossarcoma/genética , Osteossarcoma/patologiaRESUMO
Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro-environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno-blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC-MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome-free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential. J. Cell. Biochem. 118: 351-360, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Exossomos/metabolismo , Proteínas de Neoplasias/metabolismo , Osteossarcoma/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that "bookmark" the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis.
Assuntos
Linhagem da Célula/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Padrões de Herança/genética , Mitose/genética , Osteoblastos/citologia , Osteogênese/genética , Biossíntese de Proteínas , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fase G1 , Regulação da Expressão Gênica , Humanos , Interfase , Camundongos , Osteoblastos/metabolismo , Osteocalcina/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G(1) phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G(1)/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G(1)/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias Ósseas/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Fase G1/genética , Humanos , Osteossarcoma/patologia , Fase S/genéticaRESUMO
ß-Catenin is crucial in the canonical Wnt signaling pathway. This pathway is up-regulated by CK2 which is associated with an enhanced expression of the antiapoptotic protein survivin, although the underlying molecular mechanism is unknown. AKT/PKB kinase phosphorylates and promotes ß-catenin transcriptional activity, whereas CK2 hyperactivates AKT by phosphorylation at Ser129; however, the role of this phosphorylation on ß-catenin transcriptional activity and cell survival is unclear. We studied in HEK-293T cells, the effect of CK2-dependent hyperactivation of AKT on cell viability, as well as analyzed ß-catenin subcellular localization and transcriptional activity and survivin expression. CK2α overexpression led to an augmented ß-catenin-dependent transcription and protein levels of survivin, and consequently an enhanced resistance to apoptosis. However, CK2α-enhancing effects were reversed when an AKT mutant deficient in Ser129 phosphorylation by CK2 was co-expressed. Therefore, our results strongly suggest that CK2α-specific enhancement of ß-catenin transcriptional activity as well as cell survival may depend on AKT hyperactivation by CK2.
Assuntos
Caseína Quinase II/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Apoptose , Núcleo Celular/metabolismo , Sobrevivência Celular , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Regulação para CimaRESUMO
ß-Catenin is a key protein in the canonical Wnt signaling pathway and in many cancers alterations in transcriptional activity of its components are observed. This pathway is up-regulated by the protein kinase CK2, but the underlying mechanism of this change is unknown. It has been demonstrated that CK2 hyperactivates AKT/PKB by phosphorylation at Ser129, and AKT phosphorylates ß-catenin at Ser552, which in turn, promotes its nuclear localization and transcriptional activity. However, the consequences of CK2-dependent hyperactivation of AKT on ß-catenin activity and cell viability have not been evaluated. We assessed this regulatory process by manipulating the activity of CK2 and AKT through overexpression of wild-type, constitutively active and dominant negative forms of these proteins as well as analyzing ß-catenin-dependent transcriptional activity, survivin expression and viability in HEK-293T cells. We observed that CK2α overexpression up-regulated the ß-catenin transcriptional activity, which correlated to an increased nuclear localization of ß-catenin as well as survivin expression. Importantly, these effects were strongly reversed when an AKT-S129A mutant was co-expressed in the same cells, followed by a significant decrease in cell viability but no changes in ß-catenin stability. Taken together, the data suggest that the CK2α-dependent up-regulation of ß-catenin activity requires phosphorylation of AKT in human embryonic kidney cells.
Assuntos
Caseína Quinase II/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcrição Gênica , beta Catenina/metabolismo , Caseína Quinase II/genética , Sobrevivência Celular , Células HEK293 , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Mutação , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Serina , Survivina , Transfecção , Regulação para Cima , beta Catenina/genéticaRESUMO
Toxoplasma gondii is one of the most successful protozoan parasites given its ability to manipulate the immune system and establish a chronic infection. It is a parasite with a significant impact on human health, mainly in immunocompromised patients. In Europe and North America, only a few clonal genotypes (I, II and III) seem to be responsible for the vast majority of Toxoplasma infections. Surface antigen 2 gene (SAG2) has been extensively used for genotyping T. gondii isolates. The analysis of this locus reveals that in Northern hemisphere, human disease causing isolates are mainly type II, whereas T. gondii isolated from different animals are both type II and III. Since the immune response depends on parasite genotype, it seems relevant to characterize parasites producing human toxoplasmosis in different geographical areas. The growing information about the prevalent T. gondii genotypes in South America mostly refers to domestic animals. This is the first report of genetic characterization of T. gondii isolates from clinical samples in Chile, South America. All the samples analyzed corresponded to SAG2 type I isolates, and they differ from classic SAG2 type I by genetic polymorphisms. This study contributes to the scarce available information on T. gondii at South America, and reinforces an emerging concept suggesting that SAG2 type I, rather than II, parasites are a frequent cause of clinical toxoplasmosis in this continent.
Assuntos
Antígenos de Protozoários/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose/genética , Toxoplasmose/parasitologia , Cromossomos/genética , Eletroforese em Gel de Campo Pulsado , Loci Gênicos/genética , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Análise de Sequência de DNA , Toxoplasma/isolamento & purificaçãoRESUMO
Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbfbeta heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G(1). Runx2 levels are modulated during the cell cycle, which are maximal in G(1) and minimal beyond the G(1)/S phase transition (S, G(2), and M phases). It is not known whether Cbfbeta gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbfbeta are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbfbeta gene expression during cell cycle progression in MC3T3-E1 osteoblasts, as well as ROS17/2.8 and SaOS-2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3-E1 cells arrested in late G(1) (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbfbeta protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3-E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbfbeta levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G(1) and S in MC3T3-E1 cells, but not in ROS17/2.8 and SaOS-2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma.
Assuntos
Ciclo Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Osteossarcoma/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/genética , Inibidores de Cisteína Proteinase , Fase G1/fisiologia , Expressão Gênica/genética , Humanos , Leupeptinas/farmacologia , Camundongos , Mitose/fisiologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteossarcoma/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Ratos , Ubiquitinação/efeitos dos fármacosRESUMO
Basic aspects of the generation, structure and function of Echinococcus granulosus protoscoleces are unknown. We review the work done on the structure and ultrastructure of the E. granulosus protoscolex and provide new data together with a comprehensive view of this form of the parasite. The surface, as observed by scanning electron microscopy, tightly correlates with five cellular territories characterized in the interior using light and transmission electron microscopy as well as a histochemical technique. Three of these territories are surrounded by a basal lamina that is also present in the internal side of the tegument, suggesting a complex internal organization. These cellular territories correlate with the expression of specific genes and the regionalization of DNA synthesis in protoscoleces. Additionally, a proposal to explain movements of the body of this form of the parasite in relation to the neck or to the germinal layer of the hydatid cyst is provided.
Assuntos
Echinococcus granulosus/citologia , Echinococcus granulosus/ultraestrutura , Animais , Equinococose/parasitologia , Histocitoquímica , Fígado/parasitologia , Pulmão/parasitologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Ovinos , Doenças dos Ovinos/parasitologiaRESUMO
The Trypanosoma cruzi karyotype shows an extensive chromosomal size polymorphism. Absence of condensed mitotic chromosomes and chromatin fragility are characteristic features of T. cruzi which would allow DNA breaks and chromosomal rearrangements during cell proliferation. We have investigated by pulsed field gel electrophoresis (PFGE) eventual changes in chromosomal size during exponential and stationary phases of T. cruzi epimastigotes in culture, in G0 trypomastigotes and throughout the cell cycle in synchronized epimastigotes. T. cruzi molecular karyotype was stable throughout the cell cycle and during differentiation. Thus, the chromosomal size polymorphism previously reported in T. cruzi contrasts with the stability of the molecular karyotype observed here and suggests that chromosomal rearrangements leading to changes in chromosomal size are scarce events during the clonal propagation of this parasite.
Assuntos
Ciclo Celular/genética , Cromossomos/genética , Cromossomos/fisiologia , Instabilidade Genômica , Trypanosoma cruzi/citologia , Trypanosoma cruzi/genética , Animais , Diferenciação Celular , Proliferação de Células , Replicação do DNA , DNA de Protozoário/biossíntese , Genoma de Protozoário/genética , CariotipagemRESUMO
Longitudinal bone growth occurs by a process called endochondral ossification that includes chondrocyte proliferation, differentiation, and apoptosis. Recent studies have suggested a regulatory role for intracellular Ca(2+) (Ca(i) (2+)) in this process. Indirect studies, using Ca(2+) channel blockers and measurement of Ca(i) (2+), have provided evidence for the existence of Ca(2+) channels in growth plate chondrocytes. Furthermore, voltage-gated Ca(2+) channels (VGCC), and specifically L- and T-type VGCCs, have been recently described in murine embryonic growth plates. Our aim was to assess the effect of L-type Ca(2+) channel blockers on endochondral ossification in an organ culture. We used cultures of fetal rat metatarsal rudiments at 20 days post gestational age, with the addition of the L-type Ca(2+) channel blockers verapamil (10-100 microM) or diltiazem (10-200 microM) to the culture medium. Longitudinal bone growth, chondrocyte differentiation (number of hypertrophic chondrocytes), and cell proliferation (incorporation of tritiated thymidine) were measured. Verapamil dose-dependently decreased growth, the number of hypertrophic chondrocytes, and cell proliferation, at concentrations of 10-100 microM. Growth and the number of hypertrophic chondrocytes decreased significantly with diltiazem at 50-100 microM, and proliferation decreased significantly at concentrations of 10-200 microM. Additionally, there was no increase in apoptosis over physiological levels with either drug. We confirmed the presence of L-type VGCCs in rat rudiments using immunohistochemistry, and showed that the antagonists did not alter the pattern of VGCC expression. In conclusion, our data suggest that L-type Ca(2+) channel activity in growth plate chondrocytes is necessary for normal longitudinal growth, participating in chondrocyte proliferation and differentiation.
Assuntos
Osso e Ossos , Canais de Cálcio Tipo L/metabolismo , Condrócitos/metabolismo , Lâmina de Crescimento/citologia , Osteogênese/fisiologia , Animais , Apoptose/fisiologia , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Diltiazem/farmacologia , Osteogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Verapamil/farmacologiaRESUMO
In higher eukaryotes, histone gene expression is coupled to DNA replication during the S-phase of the cell cycle. This coupling is primarily controlled at the transcriptional level. Considering the basal phylogenic position of platyhelminthes in the bilateria phylum, we have cloned a partial sequence of the histone H4 gene of Mesocestoides corti and studied its expression during the post larval development of this endoparasitic platyhelminth. In in vitro trypsin-induced tetrathyridia development to segmented adult worm, we found that histone H4 is expressed concomitantly with DNA synthesis throughout all stages of development. DNA synthesis and histone H4 mRNA levels were sharply increased at 24 h after inducing development. Afterwards, tetrathyridia grew in length from days 4 to 12 of development as proliferative cells gradually increased in number. Consequently, during this period of development histone H4 mRNA levels were upregulated. Taken together these results suggest that a replication-dependent expression pattern of histone H4 occurs in ancient bilateria, such as platyhelminthes, as previously observed in higher eukaryotes.
Assuntos
Replicação do DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/biossíntese , Histonas/genética , Mesocestoides/genética , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Masculino , Mesocestoides/embriologia , Mesocestoides/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.