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1.
Braz J Med Biol Res ; 56: e12897, 2023.
Article in English | MEDLINE | ID: mdl-37909496

ABSTRACT

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.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Telomerase , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Telomerase/metabolism , Focal Adhesions/metabolism , Lung Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Cell Death , Cell Proliferation , Cell Line , Cell Line, Tumor
2.
Braz. j. med. biol. res ; 56: e12897, 2023. graf
Article in English | LILACS-Express | LILACS | ID: biblio-1520468

ABSTRACT

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.

3.
Braz. j. med. biol. res ; 48(5): 382-391, 05/2015. tab, graf
Article in English | LILACS | ID: lil-744376

ABSTRACT

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.


Subject(s)
Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Alzheimer Disease , Apolipoproteins E/genetics , Brain/pathology , Cholesterol Ester Transfer Proteins/genetics , Polymorphism, Genetic/genetics , Age Distribution , Atrophy , Alzheimer Disease/epidemiology , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Follow-Up Studies , Genotype , Genetic Predisposition to Disease/epidemiology , Genetic Predisposition to Disease/genetics , Magnetic Resonance Imaging , Risk Factors
4.
Braz J Med Biol Res ; 48(5): 382-91, 2015 May.
Article in English | MEDLINE | ID: mdl-25760027

ABSTRACT

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.


Subject(s)
Antimetabolites/metabolism , Bromodeoxyuridine/metabolism , Cell Cycle/genetics , Gene Silencing/physiology , Genes, erbB-1/genetics , Micronuclei, Chromosome-Defective , Animals , Cattle , Cell Cycle/drug effects , Cell Line, Tumor , DNA Replication , G1 Phase , Gene Amplification/physiology , Humans , In Situ Hybridization, Fluorescence , Micronuclei, Chromosome-Defective/chemically induced , Microscopy, Confocal , Mitosis Modulators/pharmacology , Mitotic Index/statistics & numerical data , S Phase , Vincristine/pharmacology
5.
Qual Manag Health Care ; 23(2): 99-118, 2014.
Article in English | MEDLINE | ID: mdl-24710186

ABSTRACT

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.


Subject(s)
Diabetes Mellitus, Type 2/therapy , Quality Improvement/organization & administration , Aged , Female , Humans , Italy , Male , Middle Aged , Models, Organizational , Organizational Case Studies , Quality Indicators, Health Care , Quality of Health Care/standards
6.
Braz. j. med. biol. res ; 45(8): 721-729, Aug. 2012. ilus, tab
Article in English | LILACS | ID: lil-643658

ABSTRACT

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.


Subject(s)
Animals , Rats , Carcinoma, Hepatocellular/pathology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cyclic AMP/pharmacology , Liver Neoplasms/pathology , Tretinoin/pharmacology , Cell Line, Tumor , Carcinoma, Hepatocellular/metabolism , Drug Combinations , Enzyme-Linked Immunosorbent Assay , Immunoblotting , Liver Neoplasms/metabolism , Microscopy, Confocal , Mitotic Index , Polymerase Chain Reaction
7.
Genet Mol Res ; 11(2): 1475-85, 2012 May 21.
Article in English | MEDLINE | ID: mdl-22653597

ABSTRACT

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.


Subject(s)
Genome/genetics , Animals , Ants/classification , Ants/genetics , DNA Transposable Elements/genetics , Phylogeny
8.
Braz J Med Biol Res ; 45(8): 721-9, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22618858

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular/pathology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cyclic AMP/pharmacology , Liver Neoplasms/pathology , Tretinoin/pharmacology , Animals , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Drug Combinations , Enzyme-Linked Immunosorbent Assay , Immunoblotting , Liver Neoplasms/metabolism , Microscopy, Confocal , Mitotic Index , Polymerase Chain Reaction , Rats
9.
Genet Mol Res ; 9(2): 849-57, 2010 May 04.
Article in English | MEDLINE | ID: mdl-20449818

ABSTRACT

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.


Subject(s)
DNA Transposable Elements/genetics , DNA-Binding Proteins/genetics , Diptera/genetics , Transposases/genetics , Animals , Base Sequence , Chromosomes/genetics , In Situ Hybridization , Microscopy, Confocal , Molecular Sequence Data , Phylogeny
10.
Dentomaxillofac Radiol ; 37(7): 398-403, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18812602

ABSTRACT

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.


Subject(s)
Keratinocytes/radiation effects , Mouth Mucosa/radiation effects , Radiography, Panoramic/adverse effects , Adult , Cell Nucleus/radiation effects , Chromosomes/radiation effects , DNA Damage , Female , Humans , Male , Micronucleus Tests , Mouth Mucosa/cytology , Surveys and Questionnaires , X-Rays/adverse effects , Young Adult
11.
Insect Mol Biol ; 15(2): 109-18, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16640721

ABSTRACT

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.


Subject(s)
Diptera/genetics , Expressed Sequence Tags , Amino Acid Sequence , Animals , Codon , DNA, Complementary , Diptera/metabolism , Gene Expression Regulation, Developmental , Insecta/genetics , Larva/growth & development , Larva/metabolism , Molecular Sequence Data , Polymorphism, Single Nucleotide , RNA, Messenger , Salivary Glands/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
12.
Eur J Histochem ; 48(3): 267-72, 2004.
Article in English | MEDLINE | ID: mdl-15590417

ABSTRACT

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.


Subject(s)
Down Syndrome/genetics , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Muscle Proteins/metabolism , Animals , CHO Cells , COS Cells , Cell Line , Chlorocebus aethiops , Cricetinae , Endoplasmic Reticulum/ultrastructure , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/genetics , Humans , Immunohistochemistry , Membrane Proteins/genetics , Membrane Proteins/ultrastructure , Molecular Chaperones , Muscle Proteins/genetics , Muscle Proteins/ultrastructure , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion
13.
Mutat Res ; 562(1-2): 111-7, 2004 Aug 08.
Article in English | MEDLINE | ID: mdl-15279834

ABSTRACT

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.


Subject(s)
Mouth Mucosa/radiation effects , Radiography, Panoramic , Female , Humans , Male , Mouth Mucosa/ultrastructure , Mutagenicity Tests
14.
Endocrinology ; 144(10): 4298-305, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12959980

ABSTRACT

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.


Subject(s)
Intracellular Membranes/physiology , Mutation/physiology , Oncogene Proteins/genetics , Protein Tyrosine Phosphatases/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Signal Transduction/physiology , Animals , Cell Differentiation/physiology , Cell Line , Cell Survival/physiology , Glial Cell Line-Derived Neurotrophic Factor , Intracellular Signaling Peptides and Proteins , Nerve Growth Factors/metabolism , Oncogene Proteins/metabolism , PC12 Cells/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Proto-Oncogene Proteins c-ret , Rats , Receptor Protein-Tyrosine Kinases/metabolism
17.
Br J Cancer ; 86(6): 917-23, 2002 03 18.
Article in English | MEDLINE | ID: mdl-11953824

ABSTRACT

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.


Subject(s)
Histone Deacetylases/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae , Telomerase/genetics , Thyroid Neoplasms/genetics , Trans-Activators/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Gene Expression , Gene Library , Humans , Molecular Sequence Data , Sirtuin 1 , Sirtuin 2 , Sirtuins , Tumor Cells, Cultured
18.
Oncogene ; 20(48): 6973-82, 2001 Oct 25.
Article in English | MEDLINE | ID: mdl-11704822

ABSTRACT

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.


Subject(s)
Carcinoma, Papillary/pathology , Drosophila Proteins , Oncogene Proteins, Fusion/physiology , Signal Transduction/physiology , Stress Fibers/physiology , Thyroid Gland/cytology , Thyroid Neoplasms/pathology , rho GTP-Binding Proteins/physiology , 3T3 Cells , Actins/metabolism , Adenocarcinoma/pathology , Animals , Apoptosis , Breast Neoplasms/pathology , Cell Line , Cell Line, Transformed , Cell Survival , DNA Replication , Dimerization , Female , Humans , MAP Kinase Signaling System , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Multiple Endocrine Neoplasia Type 2a/genetics , Multiple Endocrine Neoplasia Type 2a/metabolism , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , Organ Specificity , Phenotype , Protein-Tyrosine Kinases , Proto-Oncogene Proteins , Proto-Oncogene Proteins c-ret , Rats , Receptor Protein-Tyrosine Kinases , Recombinant Fusion Proteins/physiology , Transfection , Tumor Cells, Cultured
19.
Histochem Cell Biol ; 115(5): 403-11, 2001 May.
Article in English | MEDLINE | ID: mdl-11449888

ABSTRACT

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.


Subject(s)
Cell Nucleus/pathology , Cytoskeleton/pathology , Lung Neoplasms/pathology , Actin Cytoskeleton/metabolism , Actins/metabolism , Actins/ultrastructure , Animals , Cell Nucleus/ultrastructure , Cytoskeleton/ultrastructure , Humans , Keratins/metabolism , Lung Neoplasms/ultrastructure , Microscopy, Confocal , Rats , Tubulin/metabolism , Tubulin/ultrastructure , Tumor Cells, Cultured , Vimentin/metabolism
20.
Gene ; 253(1): 107-15, 2000 Jul 25.
Article in English | MEDLINE | ID: mdl-10925207

ABSTRACT

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.


Subject(s)
Proto-Oncogene Proteins/genetics , Transcription, Genetic , X Chromosome/genetics , Base Sequence , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , DNA/chemistry , DNA/genetics , Exons , Genes/genetics , Guanine Nucleotide Exchange Factors , Humans , Introns , Promoter Regions, Genetic , Proto-Oncogene Mas , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Tumor Cells, Cultured
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