RESUMO
PURPOSE: Laryngeal carcinomas always resist to radiotherapy. Hypoxia is an important factor in radioresistance of laryngeal carcinoma. Glucose transporter-1 (GLUT-1) is considered to be a possible intrinsic marker of hypoxia in malignant tumors. We speculated that the inhibition of GLUT-1 expression might improve the radiosensitivity of laryngeal carcinoma. METHODS: We assessed the effect of GLUT-1 expression on radioresistance of laryngeal carcinoma and the effect of GLUT-1 expressions by antisense oligodeoxynucleotides (AS-ODNs) on the radiosensitivity of laryngeal carcinoma in vitro and in vivo. RESULTS: After transfection of GLUT-1 AS-ODNs: MTS assay showed the survival rates of radiation groups were reduced with the prolongation of culture time (p<0.05); Cell survival rates were significantly reduced along with the increasing of radiation dose (p<0.05). There was significant difference in the expression of GLUT-1mRNA and protein in the same X-ray dose between before and after X-ray radiation (p<0.05). In vivo, the expressions of GLUT-1 mRNA and protein after 8Gy radiation plus transfection of GLUT-1 AS-ODNs were significant decreased compared to 8Gy radiation alone (p<0.001). CONCLUSION: Radioresistance of laryngeal carcinoma may be associated with increased expression of GLUT-1 mRNA and protein. GLUT-1 AS-ODNs may enhance the radiosensitivity of laryngeal carcinoma mainly by inhibiting the expression of GLUT-1.
Assuntos
Transportador de Glucose Tipo 1/genética , Neoplasias Laríngeas/radioterapia , Neoplasias Laríngeas/terapia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA Antissenso/genética , DNA Antissenso/fisiologia , Citometria de Fluxo , Humanos , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The mechanisms that control neural stem and progenitor cell survival are unknown. In several pathological conditions, death receptor (DR) ligands and inflammatory cytokines exert a deleterious effect on neurons, whereas primitive neural cells migrate and survive in the site of lesion. Here, we show that even in the presence of inflammatory cytokines, DRs are unable to generate death signals in primitive neural cells. Neural stem and progenitor cells did not express caspase 8, the presence of which is required for initiating the caspase cascade. However, exogenous or cytokine-mediated expression of caspase 8 was not sufficient to restore their DR sensitivity. Searching for molecules potentially able to block DR death-inducing signaling complex (DISC), we found that primitive neural cells expressed high levels of the death effector domain-containing protein PED (also known as PEA-15). PED localized in the DISC and prevented caspase 8 recruitment and activation. Moreover, lentiviral-mediated delivery of PED antisense DNA resulted in dramatic down-regulation of the endogenous gene expression and sensitization of primitive neural cells to apoptosis mediated by inflammatory cytokines and DRs. Thus, absence of caspase 8 and high expression of PED constitute two levels of protection from apoptosis induced by DRs and inflammatory cytokines in neural stem and progenitor cells.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Regulação da Expressão Gênica/fisiologia , Células-Tronco Multipotentes/fisiologia , Neurônios/fisiologia , Fosfoproteínas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Reguladoras de Apoptose , Caspase 8 , Células Cultivadas , Primers do DNA , DNA Antissenso/fisiologia , Citometria de Fluxo , Técnicas de Transferência de Genes , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia de Fluorescência , Células-Tronco Multipotentes/metabolismo , Fosfoproteínas/fisiologia , Ribonucleases/metabolismoRESUMO
The mammalian genome contains a large layer of hidden biological information. High-throughput methods have provided new insights into the regulatory networks that orchestrate the "when, where and how" of gene expression, revealing a complex interplay between proteins, regulatory RNAs, and chemical and structural alterations of the genome itself. Naturally occurring antisense transcription has been considered as an important feature in creating transcriptional and hence cellular and organismal complexity. Here, we review the current understanding of the extent, functions and significance of antisense transcription. We critically discuss results from genome-wide studies and documented examples of individual antisense transcripts. So far, the regulatory potential of gene overlaps has been demonstrated only in a few selected cases of experimentally characterized antisense transcripts. Facing the large-scale antisense transcription observed in eukaryotic genomes, it still remains an open challenge to distinguish transcriptional noise from biological function of gene overlapping patterns.
Assuntos
DNA Antissenso/fisiologia , Transcrição Gênica , Animais , Cromatina/genética , Evolução Molecular , Regulação da Expressão Gênica , Genoma , Impressão Genômica , Proteínas de Homeodomínio/genética , Modelos Genéticos , RNA/química , Edição de RNA , Interferência de RNA , Inativação do Cromossomo XRESUMO
An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated. The antisense tomato plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern blot analysis confirmed that the expression of LeFAD3 was inhibited in the tomato genome. Levels of 18:3 decreased and correspondingly levels of 18:2 increased in total lipids of leaves and roots. After heat stress, the fresh weight of the aerial parts of antisense transgenic plants was higher than that of the wild type (WT) plants. The membrane system ultrastructure of chloroplasts in leaf cells and all of the subcellular organelles in the root tips of transgenic plants remained more intact than those of WT. Relative electric conductivity increased less in transgenic plants than in WT. Under heat stress, the maximal photochemical efficiency of photosystem II (Fv/Fm) and the O(2) evolution rate decreased more in WT than in transgenic plants. These results suggested that the depletion of LeFAD3 increased the saturation of fatty acids and alleviated high temperature stress.
Assuntos
DNA Antissenso/fisiologia , Retículo Endoplasmático/enzimologia , Ácidos Graxos Dessaturases/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Solanum lycopersicum/enzimologia , Northern Blotting , DNA Antissenso/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Temperatura Alta , Solanum lycopersicum/genética , Microscopia Eletrônica de Transmissão , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/fisiologia , Plantas Geneticamente Modificadas/genética , Rhizobium/genética , TemperaturaRESUMO
A gene that encodes systemin, a mobile 18-amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato leaves, has been isolated from tomato, Lycopersicon esculentum. Induction of proteinase inhibitors in plants is a response to insect or pathogen attacks. The gene has 10 introns and 11 exons, ten of which are organized as five homologous pairs with an unrelated sequence in the eleventh, encoding systemin. Systemin is proteolytically processed from a 200-amino acid precursor protein, prosystemin. Prosystemin messenger RNA was found in all organs of the plant except the roots and was systemically wound-inducible in leaves. Tomato plants transformed with an antisense prosystemin complementary DNA exhibited greatly suppressed systemic wound induction of proteinase Inhibitor I and II synthesis in leaves.
Assuntos
Regulação da Expressão Gênica , Peptídeos/genética , Proteínas de Plantas/genética , Plantas Comestíveis/genética , Sequência de Aminoácidos , Northern Blotting , DNA Antissenso/fisiologia , Medicago sativa , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Biossíntese Peptídica , Plantas Tóxicas , RNA Mensageiro/análise , Solanum tuberosum , Nicotiana , Transformação GenéticaRESUMO
The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence supports the conclusion that the nuclear DNA ligase III protein plays an essential role in both base excision repair and homologous recombination. However, the role of DNA ligase III protein in mitochondrial genome dynamics has been obscure. Human tumor-derived HT1080 cells were transfected with an antisense DNA ligase III expression vector and clones with diminished levels of DNA ligase III activity identified. Mitochondrial protein extracts prepared from these clones had decreased levels of DNA ligase III relative to extracts from cells transfected with a control vector. Analysis of these clones revealed that the DNA ligase III antisense mRNA-expressing cells had reduced mtDNA content compared to control cells. In addition, the residual mtDNA present in these cells had numerous single-strand nicks that were not detected in mtDNA from control cells. Cells expressing antisense ligase III also had diminished capacity to restore their mtDNA to pre-irradiation levels following exposure to gamma-irradiation. An antisense-mediated reduction in cellular DNA ligase IV had no effect on the copy number or integrity of mtDNA. This observation, coupled with other evidence, suggests that DNA ligase IV is not present in the mitochondria and does not play a role in maintaining mtDNA integrity. We conclude that DNA ligase III is essential for the proper maintenance of mtDNA in cultured mammalian somatic cells.
Assuntos
DNA Ligases/genética , DNA Antissenso/fisiologia , DNA Mitocondrial/genética , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , DNA Ligases/farmacologia , DNA Antissenso/genética , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Transporte de Elétrons , Regulação Enzimológica da Expressão Gênica , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxigênio/farmacocinética , Plasmídeos/genética , Proteínas de Ligação a Poli-ADP-Ribose , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/efeitos da radiação , Transfecção , Células Tumorais Cultivadas , Proteínas de XenopusRESUMO
Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.
Assuntos
DNA Antissenso/química , DNA/química , Processamento Alternativo , Animais , Bovinos , DNA/genética , DNA Antissenso/genética , DNA Antissenso/fisiologia , Sangue Fetal/fisiologia , Globinas/genética , Células HeLa , Humanos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/genética , RNA Complementar/química , RNA Complementar/genética , RNA Complementar/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TransfecçãoRESUMO
Murine melanoma B16-F1 cells of low metastatic potential were transfected with the human gene for the prepro form of urokinase in an SV40 expression vector (plasmid pSV2-uPA), and cells expressing high amounts of the human urokinase gene product were selected for by an enzyme-linked immunosorbent assay specific for human high molecular weight urokinase. Southern analysis showed one of the cell lines (clone 7) had incorporated 150 copies of the pSV2-uPA plasmid into its genomic DNA. The human urokinase synthesized by the pSV2-uPA-transfected murine B16 cells was found to be glycosylated and did not bind to the murine cell surface urokinase receptor sites. In an in vivo assay that measures metastasis from a primary tumor (spontaneous metastatic assay), clone 7 cells showed an increased ability to metastasize (12 of 12 mice showed metastatic tumors), while control cells showed a lower ability to metastasize (only 2 of 11 mice showed metastatic tumors). In a second in vivo assay, which measures only the steps of the metastatic migration process during which tumor cells extravasate from the blood and then grow into pulmonary tumors (lung colonization assay), a significant multifold increase in the ability to form lung tumors was shown by the high human urokinase-secreting B16-F1 cells. In B16-F10 cells incorporating an antisense sequence to preprourokinase (plasmid pSV1-ASuPA-265) and secreting significantly decreased amounts of murine urokinase, a corresponding significant decrease in lung colonization was observed. These results provide direct experimental support for a role of secreted (non-surface-bound) urokinase in the colonization steps of the metastatic process. Furthermore, the data indicate that the higher lung colonization ability of the B16-F10 line than of the B16-F1 line is primarily based on the quantitative differences in their abilities to produce urokinase.
Assuntos
Fibrinolíticos , Metástase Neoplásica/fisiopatologia , Ativadores de Plasminogênio/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Northern Blotting , Southern Blotting , Movimento Celular/fisiologia , Células Cultivadas , Cromatografia de Afinidade , DNA Antissenso/fisiologia , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Técnicas In Vitro , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Ativadores de Plasminogênio/metabolismo , RNA/análise , Vírus 40 dos Símios , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
In the Burkitt's lymphoma (BL) cell line BL67 the first exon of the c-myc gene is fused to the mu-switch region of the immunoglobulin heavy-chain gene (IgH). BL67 cells express IgH/c-myc hybrid RNAs which are initiated in the immunoglobulin locus, transcribed across the chromosomal breakpoint into the first exon of c-myc and spliced using the physiological splice donor and acceptor sites of the c-myc gene. We have isolated cDNAs of these hybrid RNAs and characterized the start points in the Ig heavy-chain gene. Two promoters were identified in the mu-switch region of BL67 cells which give rise to antisense transcription of the mu-gene. These promoters are also active in other BL cell lines, in B cells without Ig translocation and in a T-cell line. Both promoters co-localize with DNAase I-hypersensitive sites, HNF and HSW, in the mu-switch region. The structures of IgH/c-myc hybrid RNAs and of the corresponding promoters are described.
Assuntos
Linfoma de Burkitt/genética , DNA Antissenso/fisiologia , Genes myc/genética , Região de Troca de Imunoglobulinas/genética , Cadeias mu de Imunoglobulina/genética , Sequência de Bases , Clonagem Molecular , Desoxirribonuclease I , Éxons , Expressão Gênica/genética , Biblioteca Gênica , Humanos , Íntrons , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Translocação Genética/genética , Células Tumorais CultivadasRESUMO
We recently demonstrated that suppressing 1alpha,25-(OH)2-D3 by increasing dietary calcium decreases adipocyte intracellular Ca2+ ([Ca2+]i), stimulates lipolysis, and inhibits lipogenesis. High calcium diets also increase core temperature and white adipose tissue uncoupling protein 2 (UCP2) expression in aP2-agouti transgenic mice. Accordingly, we have evaluated the role of 1alpha,25-(OH)2-D3 in regulating human adipocyte UCP2 expression. Treatment of human adipocytes for 48 h with 1 nM 1alpha,25-(OH)2-D3 inhibited UCP2 mRNA and protein levels by 50% (P<0.002) and completely blocked isoproterenol- or fatty acid-stimulated two- to threefold increases in UCP2 expression. However, a specific agonist for the membrane vitamin D receptor (mVDR), 1alpha,25-dihydroxylumisterol3, was unable to inhibit basal, isoproterenol-stimulated, or fatty acid-stimulated UCP2 expression, whereas a specific mVDR antagonist,1beta,25-dihydroxyvitamin D3, was unable to prevent the 1alpha,25-(OH)2-D3 inhibition of UCP2 expression. In contrast, nuclear vitamin D receptor (nVDR) knockout via antisense oligodeoxynucleotide (ODN) prevented the inhibitory effect of 1alpha,25-(OH)2-D3 on adipocyte UCP2 expression and protein levels. These data indicate that 1a,25-(OH)2-D3 exerts an inhibitory effect on adipocyte UCP2 expression via the nVDR. Thus, suppression of 1alpha,25-(OH)2-D3 and consequent up-regulation of UCP2 may contribute to our previous observation of increased thermogenesis in mice fed with high calcium diets.
Assuntos
Adipócitos/efeitos dos fármacos , Calcitriol/farmacologia , Ergosterol/análogos & derivados , Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Proteínas/genética , Adipócitos/citologia , Adipócitos/metabolismo , Northern Blotting , Western Blotting , Calcitriol/análogos & derivados , DNA Antissenso/genética , DNA Antissenso/fisiologia , Ergosterol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Canais Iônicos , Proteínas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Calcitriol/antagonistas & inibidores , Receptores de Calcitriol/genética , Receptores de Calcitriol/fisiologia , Proteína Desacopladora 2RESUMO
Nine thousand and eighty-eight base pairs of the chicken Hoxa 11 gene, including 8470 bases 5' of the translation start site were sequenced, and the characteristics of the upstream sequence investigated. Consistent with previous findings that middle repetitive elements are rare in the HoxA cluster, no repetitive elements were found other than simple oligonucleotide repeats. Multiple and pairwise alignments of the chicken upstream sequence with its human and mouse orthologs revealed multiple regions of 80% or higher homology across species. For the chicken, these regions were separated by sequences with no significant homology to human, mouse, or in most cases any other Genbank sequences. Selective clustering of transcription factor binding motifs was found to occur within the conserved homologous regions, suggesting evolutionary conservation of critical regulatory sequences. Of particular interest, seven conserved Cdx binding sites were found in the Hoxa 11 promoter, suggesting regulation by a non-clustered Caudal homeobox gene. Previous analysis of the mouse and human Hoxa 11 genes found a conserved antisense transcript, of unknown function. The chicken Hoxa 11 antisense strand included a conserved open reading frame capable of encoding 168 amino acids. Comparison of this region in mouse and chicken showed seven insertion/deletions, with each a multiple of three bases, thereby preserving open reading frame.
Assuntos
Elementos de DNA Transponíveis , DNA Antissenso/fisiologia , Proteínas de Homeodomínio/genética , Proteínas Oncogênicas/genética , Filogenia , Animais , Sequência de Bases , Sítios de Ligação , Galinhas , Sequência Conservada , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
OBJECTIVE: To clone the full length of renal cell carcinoma (RCC) related novel gene GYLZ-RCC18 and study its function. METHODS: SMART RACE technology was used to clone the full length of GYLZ-RCC18. RT-PCR was used to detect its expression in renal cell carcinoma tissue at different stages and grades. We transfected the antisense oligonucleotide of GYLZ-RCC18 to renal cell carcinoma cell line, GRC-1, and analyzed proliferation activity, growth rate, apoptosis, and mortality changes. RESULTS: The full length of GYLZ-RCC18 (GenBank accession number: BE825133) cDNA was about 3.5 kb. GYLZ-RCC18 had a higher expression in higher grades and stages of renal cell carcinoma than in lower ones. The expression of GYLZ-RCC18 in renal cell carcinoma was much higher than in normal kidney. After the transfection of GYLZ-RCC18 antisense oligonucleotide, the mortality of GRC-1 increased significantly, while proliferative activity and growth rate were substantially inhibited at the same time. The antisense oligonucleotide induced apoptosis of GRC-1 through the entire observation time. CONCLUSION: GYLZ-RCC18 is an important novel gene related to renal cell carcinoma. Overexpression of this gene results in higher growth and proliferative activity and has an antiapoptosis effect on renal cell carcinoma cells. Transfection of the antisense oligonucleotide may inhibit the generation and development of renal cell carcinoma.
Assuntos
Carcinoma de Células Renais/genética , Genes Neoplásicos/genética , Neoplasias Renais/genética , Proteínas de Neoplasias/genética , Apoptose/genética , Apoptose/fisiologia , Carcinoma de Células Renais/patologia , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Clonagem Molecular , DNA Antissenso/genética , DNA Antissenso/fisiologia , DNA Complementar/química , DNA Complementar/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/patologia , Oligonucleotídeos/genética , Análise de Sequência de DNA , Transfecção , Células Tumorais CultivadasRESUMO
OBJECTIVE: To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth muscle cells (SMCs) adhesion and migration stimulated by fibronectin. METHODS: Adhesion and migration of cultured SMCs were stimulated by different concentrations of fibronectin (FN), FAK and its phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation. SMCs adhesion and migration were also measured by morphological enumeration and modified Boyden Chambers, respectively. RESULTS: FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20 microg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5 - 60 microg/ml FN groups was reduced by 17.89% - 27.67%. Cell migration stimulated by FN at 10, 20, 40 and 60 microg/ml were reduced by 23.26%, 21.63%, 19.31% and 17.88%, respectively (P < 0.05). CONCLUSIONS: FAK phosphorylation and FAK-mediated signal transduction play important roles in SMCs adhesion and migration stimulated by ECM. The process can be inhibited effectively by FAK antisense ODNs.
Assuntos
Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fibronectinas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Animais , Células Cultivadas , DNA Antissenso/genética , DNA Antissenso/fisiologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Músculo Liso Vascular/citologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Ratos , TransfecçãoRESUMO
OBJECTIVE: To investigate the role of cyclooxygenase-2 (COX-2) in tumorigenesis of gastric cancer, and the introduction of into human gastric cancer cells to suppress COX-2 expression. METHODS: The high COX-2 expressing human gastric cancer cell line SGC 7901 was stably transfected with the COX-2 antisense recombinant vector and plain vector (named as 7901-AS and 7901-P cells). The COX-2 expression levels in transfected cells were detected by immunocytochemistry and dot blotting methods. Proliferation and tumorigenic ability of transfected (7901-AS, 7901-P) and control (SGC 7901) cells were evaluated in vitro with MTT assay and in vivo with nude mice. RESULTS: Antisense treatment for COX-2 gene significantly reduced the expression level of COX-2 protein and mRNA and led inhibition of proliferation in 7901-AS cells. The tumor graft of 7901-AS in nude mice 30 days after implantation had less volume and weight than that of SGC7901 and 7901-P [(486.67 +/- 15.28) mg vs (826.67 +/- 77.67) mg and (776.67 +/- 300.06) mg, P < 0.01]. CONCLUSIONS: Overexpression of COX-2 in human gastric cancer cell line SGC 7901 was related to the malignant phenotype of cancer cells. Inhibition of COX-2 expression by antisense technique could reverse malignant phenotypes of gastric cancer cells.
Assuntos
DNA Antissenso/genética , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Neoplasias Gástricas/patologia , Animais , Divisão Celular/genética , Divisão Celular/fisiologia , Ciclo-Oxigenase 2 , DNA Antissenso/fisiologia , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Isoenzimas/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Fenótipo , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/genética , Células Tumorais CultivadasRESUMO
OBJECTIVE: To investigate the effects of antisense Smad(4) on the biological characteristics of the fat-storing cell line CFSC. METHODS: Fat-storing cells of line CFSC from rat with liver fibrosis were cultured and transfected with 50 MOI of recombinant adenoviral vector carrying antisense Smad(4) (AdvATSmad(4)) or the control empty adenovirus (Adv0), both produced by 293 packaging cells, respectively. Two, four, and six days after the transfection the cultured cells were collected to undergo trypan blue staining and cell counting. The growth curves were drawn. The presence of antisense Smad(4) was detected by RT-PCR and Western blotting. (3)H-TdR was added into the culture media to be co-cultured for 6 hours. Then the cells were collected to examine the (3)H-TdR incorporation rate. RT-PCR and immunohistochemistry were used to examine the expression of COL1A1 and type I collagen, kinds of extracellular matrix (ECM). RESULTS: Compared with the control CFSC and the Adv0-transfected CFSC cells, the cell growth curve, (3)H-TdR incorporation rate, proline incorporation rate, expression of Smad(4), and expression of extracellular matrix were markedly decreased in the AdvATSmad(4)-transfected CFSCs. CONCLUSION: The antisense Smad(4) gene inhibits the expression of Smad(4) mRNA and protein, proline incorporation and cell growth, thus down-regulating the production of ECM. Antisense Smad(4) gene may be used as a choice of gene therapy for liver fibrosis.
Assuntos
DNA Antissenso/fisiologia , Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Adenoviridae/genética , Animais , Western Blotting , Divisão Celular , Linhagem Celular , Colágeno Tipo I/metabolismo , DNA Antissenso/genética , Proteínas de Ligação a DNA/genética , Matriz Extracelular/metabolismo , Vetores Genéticos/genética , Fígado/metabolismo , Fígado/patologia , Prolina/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad4 , Transativadores/genética , TransfecçãoRESUMO
Antisense DNA has been given a big attention as future therapeutic reagents. However, there are several shortcomings including biological instability and low membrane permeability. In order to overcome these shortcomings, the first generation of oligonucleotides has been synthesized. The efficacy of these analogues in vitro or in vivo seems to be promising. In this reviewing article, we mainly describe the intracellular delivery of oligonucleotides from the pharmaceutical point of view.
Assuntos
DNA Antissenso , Animais , DNA Antissenso/metabolismo , DNA Antissenso/farmacologia , DNA Antissenso/fisiologiaRESUMO
The human wild-type Rb gene cDNA has been cis- or trans inserted into the retrovirus vector DOL, resulting in a sense-expression vector DOLRB and an antisense-expression vector DOLRBAS of Rb gene. By eletroporation transfection techniques, the vector DOLRB has been introduced into the human breast carcinoma cell line MDAMB468 and human hepatocellular carcinoma cell line SMMC7721 both of which have an inactivated Rb gene and the vector DOLBAS, into normal human embryonic lung fibroblasts HEL cells. With the expression of Rb protein, the growth rate of the MDAMB468 cells is decreased by about 50%, their colony formation ability in soft agar is repressed completely, and their tumorigenicity in nude mice is repressed partially. Meanwhile, the cell population of G1 phase of Rb+ MDAMB468 cells is increased markedly. About 75% of transfected SMMC7721 cells have been killed by Rb gene product. For HEL cells, with the transient expression of antisense Rb gene, the Rb protein synthesis is reduced and the growth rate of those cells increased, but no colonies of HEL cells are formed in soft agar.
Assuntos
Genes do Retinoblastoma/fisiologia , Proteína do Retinoblastoma/biossíntese , Animais , Neoplasias da Mama/genética , Carcinoma Hepatocelular/genética , DNA Antissenso/fisiologia , Genes do Retinoblastoma/genética , Humanos , Neoplasias Hepáticas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
We demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV, MMLV, or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment, bound to the antisense oligonucleotide, by reverse transcriptase associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization, by binding to the AMV- and MMLV-RTs, rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription. Furthermore, the RNase H activity of HIV-RT was only slightly affected by the phosphorothioate oligonucleotide. The anti-HIV activities of phosphorothioate- or 5'-linked lipid-oligonucleotides are also described and some of the problems that still need to be solved are pointed out.
Assuntos
DNA Antissenso/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Vírus da Mieloblastose Aviária/genética , Sequência de Bases , HIV/genética , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/genética , DNA Polimerase Dirigida por RNA/fisiologiaRESUMO
The accessibility of DNA to regulatory proteins is a major property of the chromatin environment that favours or hinders transcription. Recent studies in flies reported that H3K9me2-marked heterochromatin is accessible while H3K27me3-marked chromatin forms extensive domains of low accessibility. Here we show that plants regulate DNA accessibility differently. H3K9me2-marked heterochromatin is the least accessible in the Arabidopsis thaliana genome, and H3K27me3-marked chromatin also has low accessibility. We see that very long genes without H3K9me2 or H3K27me3 are often inaccessible and generated significantly lower amounts of antisense transcripts than other genes, suggesting that reduced accessibility is associated with reduced recognition of alternative promoters. Low accessibility of H3K9me2-marked heterochromatin and long genes depend on cytosine methylation, explaining why chromatin accessibility differs between plants and flies. Together, we conclude that restriction of DNA accessibility is a local property of chromatin and not necessarily a consequence of microscopically visible compaction.