RESUMO
G-quadruplexes (G4) are structures formed at the ends of telomeres rich in guanines and stabilized by molecules that bind to specific sites. TMPyP4 and thymoquinone (TQ) are small molecules that bind to G4 and have drawn attention because of their role as telomerase inhibitors. The aim of this study was to evaluate the effects of telomerase inhibitors on cellular proliferation, senescence, and death. Two cell lines, LC-HK2 (non-small cell lung cancer - NSCLC) and RPE-1 (hTERT-immortalized), were treated with TMPyP4 (5 µM) and TQ (10 µM). Both inhibitors decreased telomerase activity. TMPyP4 increased the percentage of cells with membrane damage associated with cell death and decreased the frequency of cells in the S-phase. TMPyP4 reduced cell adhesion ability and modified the pattern of focal adhesion. TQ acted in a concentration-dependent manner, increasing the frequency of senescent cells and inducing cell cycle arrest in G1 phase. Thus, the present results showed that TMPyP4 and TQ, although acting as telomerase inhibitors, had a broader effect on other signaling pathways and processes in cells, differing from each other. However, they act both on malignant and immortalized cells, and further studies are needed before their anti-cancer potential can be considered.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Telomerase , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Telomerase/metabolismo , Adesões Focais/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Morte Celular , Proliferação de Células , Linhagem Celular , Linhagem Celular TumoralRESUMO
G‐quadruplexes (G4) are structures formed at the ends of telomeres rich in guanines and stabilized by molecules that bind to specific sites. TMPyP4 and thymoquinone (TQ) are small molecules that bind to G4 and have drawn attention because of their role as telomerase inhibitors. The aim of this study was to evaluate the effects of telomerase inhibitors on cellular proliferation, senescence, and death. Two cell lines, LC‐HK2 (non-small cell lung cancer - NSCLC) and RPE‐1 (hTERT-immortalized), were treated with TMPyP4 (5 μM) and TQ (10 μM). Both inhibitors decreased telomerase activity. TMPyP4 increased the percentage of cells with membrane damage associated with cell death and decreased the frequency of cells in the S‐phase. TMPyP4 reduced cell adhesion ability and modified the pattern of focal adhesion. TQ acted in a concentration-dependent manner, increasing the frequency of senescent cells and inducing cell cycle arrest in G1 phase. Thus, the present results showed that TMPyP4 and TQ, although acting as telomerase inhibitors, had a broader effect on other signaling pathways and processes in cells, differing from each other. However, they act both on malignant and immortalized cells, and further studies are needed before their anti-cancer potential can be considered.
RESUMO
Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene that encodes epidermal growth factor receptor (EGFR). ErbB1 amplification and mutation are associated with tumor aggressiveness and low response to therapy. The aim of the present study was to design a schedule to synchronize the cell cycle of A549 cell line (a non-small cell lung cancer) and to analyze the possible association between the micronuclei (MNs) and the extrusion of ErbB1 gene extra-copies. After double blocking, by the process of fetal bovine serum deprivation and vincristine treatment, MNs formation was monitored with 5-bromo-2-deoxyuridine (BrdU) incorporation, which is an S-phase marker. Statistical analyses allowed us to infer that MNs may arise both in mitosis as well as in interphase. The MNs were able to replicate their DNA and this process seemed to be non-synchronous with the main cell nuclei. The presence of ErbB1 gene in the MNs was evaluated by fluorescent in situ hybridization (FISH). ErbB1 sequences were detected in the MNs, but a relation between the MNs formation and extrusion of amplified ErbB1could not be established. The present study sought to elucidate the meaning of MNs formation and its association with the elimination of oncogenes or other amplified sequences from the tumor cells.
Assuntos
Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Alzheimer , Apolipoproteínas E/genética , Encéfalo/patologia , Proteínas de Transferência de Ésteres de Colesterol/genética , Polimorfismo Genético/genética , Distribuição por Idade , Atrofia , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Seguimentos , Genótipo , Predisposição Genética para Doença/epidemiologia , Predisposição Genética para Doença/genética , Imageamento por Ressonância Magnética , Fatores de RiscoRESUMO
Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene that encodes epidermal growth factor receptor (EGFR). ErbB1 amplification and mutation are associated with tumor aggressiveness and low response to therapy. The aim of the present study was to design a schedule to synchronize the cell cycle of A549 cell line (a non-small cell lung cancer) and to analyze the possible association between the micronuclei (MNs) and the extrusion of ErbB1 gene extra-copies. After double blocking, by the process of fetal bovine serum deprivation and vincristine treatment, MNs formation was monitored with 5-bromo-2-deoxyuridine (BrdU) incorporation, which is an S-phase marker. Statistical analyses allowed us to infer that MNs may arise both in mitosis as well as in interphase. The MNs were able to replicate their DNA and this process seemed to be non-synchronous with the main cell nuclei. The presence of ErbB1 gene in the MNs was evaluated by fluorescent in situ hybridization (FISH). ErbB1 sequences were detected in the MNs, but a relation between the MNs formation and extrusion of amplified ErbB1 could not be established. The present study sought to elucidate the meaning of MNs formation and its association with the elimination of oncogenes or other amplified sequences from the tumor cells.
Assuntos
Antimetabólitos/metabolismo , Bromodesoxiuridina/metabolismo , Ciclo Celular/genética , Inativação Gênica/fisiologia , Genes erbB-1/genética , Micronúcleos com Defeito Cromossômico , Animais , Bovinos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Replicação do DNA , Fase G1 , Amplificação de Genes/fisiologia , Humanos , Hibridização in Situ Fluorescente , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Microscopia Confocal , Moduladores de Mitose/farmacologia , Índice Mitótico/estatística & dados numéricos , Fase S , Vincristina/farmacologiaRESUMO
PURPOSE: The study aimed to establish whether the organization for the management of type 2 diabetes mellitus at 9 diabetic units (DUs), in 5 neighboring local health authorities (LHAs), was able to (a) comply with the organizational model prescribed by specific regional standards; (b) ensure adequate clinical management of diabetic patients; (c) assess whether the relationship between primary care physicians (PCPs) and diabetologists (SDs) was instrumental to the needs of patients; (d) optimize specialist treatment at the DUs; (e) optimize drug management; and (f) check whether organizational changes led to variations in clinical results. METHODS: This 6-stage study analyzed procedures, precoded actions, and recordable processes. Stage (1) Defining clinical and organizational endpoints; (2) Drafting flowcharts to describe the actions and work procedures implemented within each LHA; (3) Comparing the flowcharts with the data obtained from related literature; (4) Establishing a protocol shared with PCPs for the management and treatment of patients with type 2 diabetes; (5) Changing the procedures at the DUs; and (6) Evaluating the results. The data were assessed before and after establishing a shared protocol for SDs and PCPs (year 2009 vs 2011). RESULTS: The study shows inconsistencies in the organization of work in the 5 LHAs; however, collaboration with PCPs has guaranteed: (a) unchanged hemoglobin A1C values before and after applying the protocol; (b) a percentage increase in the number of patients with type 2 diabetes who were identified thanks to these protocols; (c) an increase in the use of biguanides compared to the preprotocol period; and (d) no change in the number of patients hospitalized because of acute complications from type 2 diabetes mellitus. CONCLUSIONS: This study confirms how adequate collaboration between SDs and PCPs keeps the risk of complications stable. Nevertheless, shared protocols and clearly defined roles are required.
Assuntos
Diabetes Mellitus Tipo 2/terapia , Melhoria de Qualidade/organização & administração , Idoso , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Modelos Organizacionais , Estudos de Casos Organizacionais , Indicadores de Qualidade em Assistência à Saúde , Qualidade da Assistência à Saúde/normasRESUMO
Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation (E-cadherin, connexin 26 (Cx26), and Cx32). RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.
Assuntos
Animais , Ratos , Carcinoma Hepatocelular/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/farmacologia , Neoplasias Hepáticas/patologia , Tretinoína/farmacologia , Linhagem Celular Tumoral , Carcinoma Hepatocelular/metabolismo , Combinação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Neoplasias Hepáticas/metabolismo , Microscopia Confocal , Índice Mitótico , Reação em Cadeia da PolimeraseRESUMO
Mobile elements are widely present in eukaryotic genomes. They are repeated DNA segments that are able to move from one locus to another within the genome. They are divided into two main categories, depending on their mechanism of transposition, involving RNA (class I) or DNA (class II) molecules. The mariner-like elements are class II transposons. They encode their own transposase, which is necessary and sufficient for transposition in the absence of host factors. They are flanked by a short inverted terminal repeat and a TA dinucleotide target site, which is duplicated upon insertion. The transposase consists of two domains, an N-terminal inverted terminal repeat binding domain and a C-terminal catalytic domain. We identified a transposable element with molecular characteristics of a mariner-like element in Atta sexdens rubropilosa genome. Identification started from a PCR with degenerate primers and queen genomic DNA templates, with which it was possible to amplify a fragment with mariner transposable-element homology. Phylogenetic analysis demonstrated that this element belongs to the mauritiana subfamily of mariner-like elements and it was named Asmar1. We found that Asmar1 is homologous to a transposon described from another ant, Messor bouvieri. The predicted transposase sequence demonstrated that Asmar1 has a truncated transposase ORF. This study is part of a molecular characterization of mobile elements in the Atta spp genome. Our finding of mariner-like elements in all castes of this ant could be useful to help understand the dynamics of mariner-like element distribution in the Hymenoptera.
Assuntos
Genoma/genética , Animais , Formigas/classificação , Formigas/genética , Elementos de DNA Transponíveis/genética , FilogeniaRESUMO
Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3ß) and liver differentiation (E-cadherin, connexin 26 (Cx26), and Cx32). RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3ß (inactive form) expression while the expression of Cx43, Tyr216-GSK-3ß (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.
Assuntos
Carcinoma Hepatocelular/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/farmacologia , Neoplasias Hepáticas/patologia , Tretinoína/farmacologia , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Combinação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Neoplasias Hepáticas/metabolismo , Microscopia Confocal , Índice Mitótico , Reação em Cadeia da Polimerase , RatosRESUMO
Mariner-like elements are widely present in diverse organisms. These elements constitute a large fraction of the eukaryotic genome; they transpose by a "cut-and-paste" mechanism with their own transposase protein. We found two groups of mobile elements in the genus Rhynchosciara. PCR using primers designed from R. americana transposons (Ramar1 and Ramar2) were the starting point for this comparative study. Genomic DNA templates of four species: R. hollaenderi, R. millerii, R. baschanti, and Rhynchosciara sp were used and genomic sequences were amplified, sequenced and the molecular structures of the elements characterized as being putative mariner-like elements. The first group included the putative full-length elements. The second group was composed of defective mariner elements that contain a deletion overlapping most of the internal region of the transposase open reading frame. They were named Rmar1 (type 1) and Rmar2 (type 2), respectively. Many conserved amino acid blocks were identified, as well as a specific D,D(34)D signature motif that was defective in some elements. Based on predicted transposase sequences, these elements encode truncated proteins and are phylogenetically very close to mariner-like elements of the mauritiana subfamily. The inverted terminal repeat sequences that flanked the mariner-like elements are responsible for their mobility. These inverted terminal repeat sequences were identified by inverse PCR.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Dípteros/genética , Transposases/genética , Animais , Sequência de Bases , Cromossomos/genética , Hibridização In Situ , Microscopia Confocal , Dados de Sequência Molecular , FilogeniaRESUMO
OBJECTIVES: The aim of this study was to evaluate the genotoxic effects of X-rays on epithelial gingival cells during panoramic dental radiography using a differentiated protocol for the micronucleus test. METHODS: 40 healthy individuals who underwent this procedure for diagnostic purposes on request from their dentists agreed to participate in this study. All of them answered a questionnaire before the examination. Epithelial gingival cells were obtained from the keratinized mucosa of the upper dental arcade by gentle scraping with a cervical brush immediately before exposure and 10 days later. Cytological preparations were stained according to the Feulgen-Rossenbeck reaction, counterstained with fast green 1% for 1 min and analysed under a light microscope. Micronuclei, nuclear projections (broken eggs) and degenerative nuclear alterations (pyknosis, karyolysis, karyorrhexis and condensed chromatin) were scored. RESULTS: The frequency of micronuclei was significantly higher after exposure (P < 0.05), as were the frequencies of nuclear alterations indicative of apoptosis (P < 0.001). CONCLUSIONS: These results indicate that X-ray radiation emitted during panoramic dental radiography induces a genotoxic effect on epithelial gingival cells that increases the frequency of chromosomal damage and nuclear alterations indicative of apoptosis.
Assuntos
Queratinócitos/efeitos da radiação , Mucosa Bucal/efeitos da radiação , Radiografia Panorâmica/efeitos adversos , Adulto , Núcleo Celular/efeitos da radiação , Cromossomos/efeitos da radiação , Dano ao DNA , Feminino , Humanos , Masculino , Testes para Micronúcleos , Mucosa Bucal/citologia , Inquéritos e Questionários , Raios X/efeitos adversos , Adulto JovemRESUMO
The diptera Rhynchosciara americana (sciaridae) is an important model organism in polyteny and gene amplification research, but up to now a limited amount of data regarding DNA sequences and molecular aspects of this species is available. Considering the importance of going further on the DNA puffs biological meaning, we proposed to generate EST sequences from a DNA library constructed from salivary glands. After their categorization in gene ontology terms, they were used to construct an 'electronic Northern' that represents a general view of the salivary gland metabolic status in an important phase of larval development: the spinning of communal cocoon. In this phase occurs the last polytene DNA replication cycle concomitantly with the specific loci amplification related to protein secretion.
Assuntos
Dípteros/genética , Etiquetas de Sequências Expressas , Sequência de Aminoácidos , Animais , Códon , DNA Complementar , Dípteros/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Insetos/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , RNA Mensageiro , Glândulas Salivares/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
We used immunocytochemical and fluorescence assays to investigate the subcellular location of the protein encoded by Down syndrome critical region gene 2 (DSCR2) in transfected cells. It was previously suggested that DSCR2 is located in the plasma membrane as an integral membrane protein. Interestingly, we observed this protein in the endoplasmic reticulum (ER) of cells. We also studied whether the truncated forms of DSCR2 showed different subcellular distributions. Our observations indicate that DSCR2 probably is not inserted into the membrane of the endoplasmic reticulum since the fragments lacking the predicted transmembrane (TM) helices remained associated with the ER. Our analyses suggest that, although DSCR2 is associated with the endoplasmic reticulum, it is not an integral membrane protein and it is maintained on the cytoplasmic side of the ER by indirect interaction with the ER membrane or with another protein.
Assuntos
Síndrome de Down/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Animais , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Retículo Endoplasmático/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Chaperonas Moleculares , Proteínas Musculares/genética , Proteínas Musculares/ultraestrutura , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de SequênciaRESUMO
The genotoxic effects of X-ray emitted during dental panoramic radiography were evaluated in exfoliated cells from oral epithelium through a differentiated protocol of the micronucleus test. Thirty-one healthy individuals agreed to participate in this study and were submitted to this procedure for diagnosis purpose after being requested by the dentist. All of them answered a questionnaire before the examination. Cells were obtained from both sides of the cheek by gentle scrapping with a cervical brush, immediately before the exposure and after 10 days. Cytological preparations were stained according to Feulgen-Rossenbeck reaction and analyzed under light and laser scanning confocal microscopies. Micronuclei, nuclear projections (buds and broken eggs) and degenerative nuclear alterations (condensed chromatin, karyolysis and karyorrhexis) were scored. The frequencies of micronuclei, karyolysis and pycnosis were similar before and after exposure (P > 0.90), whereas the condensation of the chromatin and the karyorrhexis increased significantly after exposure (P < 0.0001). In contrast, both bud and broken egg frequencies were significantly higher before the examination (P < 0.005), suggesting that these structures are associated to the normal epithelium differentiation. The results suggest that the X-ray exposure during panoramic dental radiography induces a cytotoxic effect by increasing apoptosis. We also believe that the score of other nuclear alterations in addition to the micronucleus improves the sensitivity of genotoxic effects detection.
Assuntos
Mucosa Bucal/efeitos da radiação , Radiografia Panorâmica , Feminino , Humanos , Masculino , Mucosa Bucal/ultraestrutura , Testes de MutagenicidadeRESUMO
The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for RetM918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors RetC634Y and RetM918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.
Assuntos
Membranas Intracelulares/fisiologia , Mutação/fisiologia , Proteínas Oncogênicas/genética , Proteínas Tirosina Fosfatases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Peptídeos e Proteínas de Sinalização Intracelular , Fatores de Crescimento Neural/metabolismo , Proteínas Oncogênicas/metabolismo , Células PC12/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
We used subtractive library screening to identify the changes that occur in gene expression during thyroid cell neoplastic transformation. Complementary DNA from normal thyroid cells (HTC 2) was subtracted from a complementary DNA library constructed from a human thyroid papillary carcinoma cell line. The library was screened for genes upregulated in human thyroid papillary carcinoma cell line cells, and several cDNA clones were isolated. One of these clones has a sirtuin core and high homology with the human silent information regulator protein family. This clone, designated "SIR-T8", was overexpressed in human thyroid carcinoma cell lines and tissues, but not in adenomas. The human SIR-T8 protein has a molecular weight of 39 kDa and is primarily located in the cytoplasm under the nuclear membrane. The SIR-T8 gene is located on chromosome 17q25-1.
Assuntos
Histona Desacetilases/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Telomerase/genética , Neoplasias da Glândula Tireoide/genética , Transativadores/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Expressão Gênica , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Sirtuína 1 , Sirtuína 2 , Sirtuínas , Células Tumorais CultivadasRESUMO
Thyroid papillary carcinomas are characterized by RET/PTC rearrangements that cause the tyrosine kinase domain of the RET receptor to fuse with N-terminal sequences encoded by heterologous genes. This results in the aberrant expression of a ligand-independent and constitutively active RET kinase. We analysed actin reorganization induced by the RET/PTC1 oncogene in PC Cl 3 rat thyroid epithelial cells. Differently from oncogenes Src, Ras and Raf, RET/PTC1 caused actin filaments to form prominent stress fibers. Moreover, stress fibers were identified in human thyroid papillary carcinoma cell lines harboring RET/PTC1 rearrangements but not in thyroid carcinoma cells negative for RET/PTC rearrangements. RET/MEN 2A, a constitutively active but unrearranged membrane-bound RET oncoprotein, did not induce stress fibers in PC Cl 3 cells. Induction of stress fibers by RET/PTC1 was restricted to thyroid cells; it did not occur in NIH3T3 fibroblasts or MCF7 mammary cells. RET/PTC1-mediated stress fiber formation depended on Rho but not Rac small GTPase activity. In addition, inhibition of Rho, but not of Rac, caused apoptosis of RET/PTC1-expressing thyroid cells. We conclude that Rho is implicated in the actin reorganization and cell survival mediated by the chimeric RET/PTC1 oncogene in thyroid epithelial cells, both phenotypes being cell type- and oncogene type-specific.
Assuntos
Carcinoma Papilar/patologia , Proteínas de Drosophila , Proteínas de Fusão Oncogênica/fisiologia , Transdução de Sinais/fisiologia , Fibras de Estresse/fisiologia , Glândula Tireoide/citologia , Neoplasias da Glândula Tireoide/patologia , Proteínas rho de Ligação ao GTP/fisiologia , Células 3T3 , Actinas/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Transformada , Sobrevivência Celular , Replicação do DNA , Dimerização , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Especificidade de Órgãos , Fenótipo , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
Tumor cells generally present various types of nuclear alterations, which can be associated with genetic instability. The origin and mechanism of formation of the nuclear alterations are largely unknown, with the micronucleus being the most well studied alteration. The purpose of this study was to characterize the cytoskeleton filaments and to analyze the possible association between nuclear alterations and the cytoskeleton in the human lung carcinoma cells HK2 and A549. The cytoskeleton analysis was performed by using antibodies against lamin B, vimentin, cytokeratin-8, and alpha-tubulin and the secondary antibody labeled with FITC. The analysis of the actin filament was made with phalloidin-TRITC. The analyses of cytoskeleton were performed from optical sections obtained by confocal laser scanning microscopy. Filaments of the cytoskeleton of tumor cells present some differences in their distribution pattern and their expression when compared with the filaments of normal cells. The HK2 cells presented actin fibers arranged either concentrically or in clusters and tubulin filaments arranged radially, while in the A549 cells the distribution pattern was similar to that of normal cells. The lamin B filaments were the most important to identify nuclear alterations. These alterations in cytoskeleton distribution could not be associated with nuclear alterations.
Assuntos
Núcleo Celular/patologia , Citoesqueleto/patologia , Neoplasias Pulmonares/patologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Humanos , Queratinas/metabolismo , Neoplasias Pulmonares/ultraestrutura , Microscopia Confocal , Ratos , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura , Células Tumorais Cultivadas , Vimentina/metabolismoRESUMO
The dbl oncogene is generated by substitution of the 5' portion of its normal counterpart with an unrelated human sequence. To analyze the genomic structure and transcriptional regulation of the dbl proto-oncogene, we have isolated human genomic clones containing the entire human proto-dbl gene, localized in Xq26. Restriction mapping of a 600kb YAC clone (yWXD311) placed proto-dbl about 50kb telomeric to the coagulation Factor IX gene. The genomic DNA fragment containing the 5' end of proto-dbl was subcloned into plasmid vectors and the nucleotide sequences of exon 1, the flanking intronic region and genomic DNA 5' of the first codon were determined. Sequence analysis of 85119bp from the region revealed the genomic structure of proto-dbl. It contains 25 exons coding for a 4.7kb transcript including large 5'- and 3'- (1218bp and 701bp, respectively) untranslated regions (UTRs). RNase protection and primer extension assays on RNA from medullary thyroid carcinoma (TT) cells, which normally express dbl, revealed a transcription start site 1218bp upstream of the ATG of the first exon. A 1.6kb genomic 5' of the translation start sites drives the expression of a CAT-reporter in transient transfections in the TT cell line, though lacking TATA or CAAT boxes.
Assuntos
Proteínas Proto-Oncogênicas/genética , Transcrição Gênica , Cromossomo X/genética , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , DNA/química , DNA/genética , Éxons , Genes/genética , Fatores de Troca do Nucleotídeo Guanina , Humanos , Íntrons , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
Oncogenic variants of the receptor tyrosine kinase, Ret, cause formation of tumors of neuroendocrine derivation in the multiple endocrine neoplasia type 2 and, thus, likely interfere with antiproliferative and/or differentiative extracellular signals. Here we took advantage of two rat pheochromocytoma-derived cell lines (PC12/MEN2A and PC12/MEN2B) to investigate whether Ret-induced nerve growth factor (NGF) unresponsiveness might involve impairment of ERK signaling. In fact, these cells, stably transfected with distinct forms of the active ret oncogene, fail to block proliferation, even upon NGF stimulation. In these cells we show the presence of both chronic ERKs activity and high expression levels of MKP-3, an ERK-specific phosphatase. Despite the presence of MKP-3, ERK activity can be further stimulated by NGF, but it fails to translocate into the nucleus and consequently to induce immediate-early gene transcription. Because of the presence of MKP-3, our results suggest the existence of a negative regulatory feedback acting on ERKs as a mechanism responsible for the abrogation of NGF-induced terminal differentiation. Indeed, MKP-3 seems to be implicated in the persistence of ERKs in cell cytoplasm. This interpretation is further supported by the observation that in ret-transfected cells, forced expression of an active form of MEK-1 may overcome this block; it restores transcription from the c-fos promoter, induces translocation of ERKs into the nucleus, and inhibits cell proliferation.