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
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
In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.
Assuntos
Anticorpos Antibacterianos/sangue , Flagelina/imunologia , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/imunologia , Administração Oral , Animais , Vacinas Bacterianas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas contra Salmonella/administração & dosagem , Vacinas Atenuadas/imunologiaRESUMO
In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.