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 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.
Assuntos
Cromatina/fisiologia , DNA de Plantas/fisiologia , Arabidopsis , Centrômero/metabolismo , Centrômero/fisiologia , Cromatina/metabolismo , Metilação de DNA/fisiologia , DNA Antissenso/metabolismo , DNA Antissenso/fisiologia , DNA de Plantas/metabolismo , Eucromatina/metabolismo , Eucromatina/fisiologia , Genes de Plantas/fisiologia , Heterocromatina/metabolismo , Heterocromatina/fisiologia , Hibridização in Situ Fluorescente , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/fisiologiaRESUMO
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
The treatment of cancer using macromolecular therapeutics such as oligonucleotides or peptides requires efficient delivery systems capable of intracellular penetration and may also benefit from use of a combination of therapeutics with different mechanisms of action. With this possibility in mind, we constructed cationic liposome loaded with the proapoptotic peptide, d-(KLAKLAK)(2) and the Bcl-2 antisense oligodeoxynucleotide, G3139, and determined whether the combination of the proapoptotic macromolecules in a single cationic liposome can enhance antitumor efficacy. Advantage was taken of alternating charge interaction to entrap macromolecules of opposite charge. The polycationic peptide d-(KLAKLAK)(2) was first condensed with the polyanionic oligodeoxynucleotide G3139 to obtain overall negatively charged peptide/oligodeoxynucleotide complexes. The complexes were then entrapped into DOTAP/DOPE cationic liposomes (CL). This sequential charge interaction ensured efficient entrapment of d-(KLAKLAK)(2) and G3139 with a high loading efficiency (50%) and capacity (7.5 wt %). In vitro treatment of mouse melanoma B16(F10) with CL loaded with d-(KLAKLAK)(2)/G3139 led to significantly enhanced antitumor efficacy, mediated by stimulated induction of apoptotic (caspase 3/7) activity, when compared to CL loaded with G3139 alone. Intratumoral injection of CL loaded with d-(KLAKLAK)(2)/G3139 in B16(F10) mice xenograft also led to suppressed tumor growth associated with enhanced apoptotic activity. Thus, the combination of proapoptotic peptide d-(KLAKLAK)(2) and antisense oligonucleotide G3139 in a cationic liposome led to enhanced apoptotic/antitumor efficacy and may provide a promising tool for cancer treatment.
Assuntos
DNA Antissenso/química , DNA Antissenso/fisiologia , Terapia Genética/métodos , Lipossomos/química , Melanoma Experimental/terapia , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , DNA Antissenso/genética , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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
Protocadherins (PCDH), localized to synaptic junctions, contribute to the formation of neuronal networks during brain development; thus, it is speculated that protocadherins may play a role in evolution of neuronal complexity. While protocadherin genes are highly conserved in vertebrates, EST evidence from the locus suggests apparently species-specific cis-antisense transcripts. Novel cis-antisense transcripts, which partially overlap the PCDHalpha12 variable exon, PCDHbeta3 single-exon gene, and PCDHpsi5 unprocessed pseudogene in the human 5q31 PCDHalpha/beta/gamma gene cluster and which are coexpressed with sense-strand transcripts in fetal and adult brain, were identified computationally and validated by gene-specific strand-specific reverse transcriptase PCR (SSRTPCR) and sequencing. Absence of antisense transcripts arising from equivalent genomic locations in mouse indicates that the antisense transcripts originated in the primates after the primate-rodent divergence. Furthermore, not all expected orthologues of human sense and antisense PCDH transcripts were detected in rhesus macaque brain, implying that protocadherin expression patterns differ between primate species. RT followed by quantitative real-time PCR (QPCR) analysis of the three genes in the brain of all three species, and of the PCDHbeta15 gene paralogous to PCDHpsi5 in human and rhesus, revealed that the presence of antisense transcripts was significantly associated with lower sense expression levels across all orthologues. This inverse relationship, along with the pattern of sense and antisense coexpression in the brain, is consistent with a regulatory role for the primate-specific PCDH cis-antisense transcripts, which may represent recent evolutionary inventions modulating the activity of this conserved gene cluster.
Assuntos
Caderinas/genética , Cromossomos Humanos Par 5 , DNA Antissenso/fisiologia , Família Multigênica , Primatas , Precursores de Proteínas/genética , Animais , Humanos , Elementos Reguladores de Transcrição , Especificidade da Espécie , Transcrição GênicaRESUMO
P-glycoprotein, encoded by the multidrug resistance (MDR)-1 gene, expels various drugs from cells resulting in drug resistance. However, its functional relevance to lymphocytes and the regulatory mechanism remain unclear. Although MDR-1 is known to be induced by various cytotoxic stimuli, it is poorly understood whether the activation stimuli such as cytokines induce MDR-1 transcription. We investigated the transcriptional regulation of MDR-1 in lymphocytes by activation stimuli, particularly by interleukin (IL)-2. IL-2 induced translocation of YB-1, a specific transcriptional factor for MDR-1, from the cytoplasm into nucleus of lymphocytes in a dose-dependent manner and resulted in the sequential events; transcription of MDR-1, expression of P-glycoprotein on the cell surface, and excretion of the intracellular dexamethasone added in vitro. Transfection of YB-1 anti-sense oligonucleotides inhibited P-glycoprotein expression induced by IL-2. Cyclosporin A, a competitive inhibitor of P-glycoprotein, recovered intracellular dexamethasone levels in lymphocytes. We provide the first evidence that IL-2, a representative lymphocyte-activation stimulus, induces YB-1 activation followed by P-glycoprotein expression in lymphocytes. Our findings imply that lymphocytes activation by IL-2 in vivo, in the context of the pathogenesis of autoimmune diseases, results in P-glycoprotein-mediated multidrug resistance, and that P-glycoprotein could be an important target for the treatment of refractory autoimmune diseases.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Genes MDR , Interleucina-2/fisiologia , Linfócitos/metabolismo , Ativação Transcricional , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Sequência de Bases , Ciclosporina/farmacologia , DNA Antissenso/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Proteínas de Ligação a DNA/fisiologia , Dexametasona/farmacologia , Humanos , Ativação Linfocitária , Dados de Sequência Molecular , Monócitos/metabolismo , Proteínas Nucleares , Transporte Proteico , Regulação para Cima , Proteína 1 de Ligação a Y-BoxRESUMO
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
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
The plastid genome is transcribed by nucleus-encoded (NEP) and plastid-encoded (PEP) RNA polymerases. PEP is a prokaryotic-type enzyme whose activity is regulated by sigma-like transcription initiation factors that are nucleus-encoded. cDNAs coding for six different potential a-like factors have been cloned and sequenced recently. However, functional analyses of these factors are still limited. We have used an anti-sense approach in order to study the function of SIG1, SIG2 and SIG3. Only SIG2 anti-sense plants show a visible phenotype characterized by chlorophyll deficiency. Surprisingly, this phenotype is different from the phenotype of SIG2 knockout plants in that the chlorophyll deficiency is limited to cotyledons. In later developmental stages, the SIG2 anti-sense plants can overcome SIG2 mRNA under-expression by adjusting SIG2 protein levels to that of wild-type plants, suggesting that SIG2 expression is also regulated at the post-transcriptional level. The efficient recovery of the wild-type phenotype could also be supported by partial take-over of SIG2 function by one of the six other sigma factors. A good candidate for such substitution of SIG2 function represents SIG3. SIG3 is constitutively expressed during plant development and its specificity in promoter discrimination is less pronounced than that of SIG1 and SIG2. Finally, SIG3 protein is enhanced in SIG2 anti-sense plants when compared to wild-type plants. SIG2 is present as a soluble factor while SIG3 is partly attached to the plastid membranes. We suggest that membrane localization is necessary for efficient SIG3 function. Therefore, SIG3 cannot substitute for SIG2 function in early chloroplast biogenesis, when plastid membranes are not yet made up.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Plastídeos/metabolismo , Fator sigma/fisiologia , Fatores de Transcrição/fisiologia , Especificidade de Anticorpos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Cotilédone/genética , Cotilédone/metabolismo , DNA Antissenso/genética , DNA Antissenso/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Imuno-Histoquímica/métodos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Fator sigma/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
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
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 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 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
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
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
Breast cancer resistance protein (BCRP), an ABC half-transporter, is overexpressed in cancer cell lines selected with doxorubicin/verapamil, topotecan, or mitoxantrone. BCRP-overexpressing cells show cross-resistance to camptothecin derivatives such as irinotecan, SN-38 (the active metabolite of irinotecan), and topotecan. To test whether BCRP confers SN-38 resistance, we selected two SN-38 resistant sublines from PC-6 human small-cell lung cancer cells by SN-38, and then characterized these cells. Compared to PC-6 cells, the resistant sublines PC-6/SN2-5 and PC-6/SN2-5H were approximately 18- and 34-fold resistant, respectively. The intracellular SN-38 accumulation was reduced in the sublines, and BCRP mRNA was overexpressed in proportion to the degree of SN-38 resistance. These findings suggest that BCRP confers SN-38 resistance in the sublines. To confirm this hypothesis, PC-6/SN2-5 cells were transfected with antisense oligonucleotides complementary to portions of BCRP mRNA. The antisense oligonucleotides significantly suppressed BCRP mRNA expression, and enhanced SN-38 sensitivity in the subline. These data indicate that BCRP is directly involved with SN-38 resistance, by efflux transport of SN-38.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Neoplasias Pulmonares/genética , Proteínas de Neoplasias , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos Fitogênicos/metabolismo , Northern Blotting , Células CACO-2 , Camptotecina/análogos & derivados , Camptotecina/metabolismo , DNA Antissenso/genética , DNA Antissenso/fisiologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Irinotecano , Neoplasias Pulmonares/patologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair. The enzyme consists of two dissimilar subunits, M1 and M2. It is known that the M2 subunit plays a role in tumorgenicity and metastasis. In this study, we transfected human oropharyngeal KB cancer cells with human RR M1 and M2 antisense cDNA expressed by an inducible vector system. The transfectants were double-selected with hygromycin and G418. The clones, designated KB-M1AS, KB-M2AS and KB-CAT, represented transfectant clones that contained M1 antisense cDNA, M2 antisense cDNA, and a CAT reporter gene, respectively. In a colony-forming assay, colony formation for the KB-M2AS clone decreased approximately 50% when M2 antisense mRNA expression was induced by isopropylthiogalactose (IPTG). However, the KB-M1AS clone revealed no significant inhibition under IPTG induction. RR enzyme activity, as measured by 14CDP reduction assay, revealed a 30% decrease in the IPTG-induced KB-M2AS clone relative to non-IPTG-induced samples at 144 hours. As shown by Northern blot, expression of the M2 antisense mRNA showed peaks at 48 hours and 144 hours after induction by IPTG. M2 antisense mRNA expression induced by IPTG was 33-fold greater than the uninduced control at 144 hours. Western blot analysis showed that the M2 subunit protein level decreased in the KB-M2AS clone beginning at 72 hours after induction and continued to decrease to 50% of the uninduced control at 144 hours, then showed a slight recovery at 168 hours. In conclusion, M2 antisense mRNA expression by an inducible system can effectively decrease RR M2 protein expression, reduce enzyme activity, and inhibit growth. Furthermore, this approach can be employed in future antisense investigations.
Assuntos
DNA Antissenso/biossíntese , DNA Complementar/biossíntese , Inibidores do Crescimento/biossíntese , Ribonucleosídeo Difosfato Redutase/biossíntese , Western Blotting , Células Clonais , DNA Antissenso/fisiologia , DNA Complementar/fisiologia , Ativação Enzimática , Indução Enzimática , Inibidores do Crescimento/fisiologia , Humanos , Células KB , RNA Mensageiro/biossíntese , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Ribonucleosídeo Difosfato Redutase/fisiologia , Transfecção , Ensaio Tumoral de Célula-TroncoRESUMO
Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development. The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases. The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants. Immunoblot analyses performed with a monoclonal antibody to the E1alpha mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein. MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants. Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time. The potential role for AtPDHK gene manipulation in crop improvement is discussed.