RESUMO
Expression of the seven open reading frames (ORFs) of single-stranded DNA Curtoviruses such as Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV) is driven by a bi-directional promoter. To investigate this bi-directional promoter activity with respect to viral late gene expression, transgenic Arabidopsis plants expressing a GUS reporter gene under the control of either the BCTV or BSCTV bi-directional promoter were constructed. Transgenic plants harboring constructs showed higher expression levels when the promoter of the less virulent BCTV was used than when the promoter of the more virulent BSCTV was used. In transgenic seedlings, the reporter gene constructs were expressed primarily in actively dividing tissues such as root tips and apical meristems. As the transgenic plants matured, reporter gene expression diminished but viral infection of mature transgenic plants restored reporter gene expression, particularly in transgenic plants containing BCTV virion-sense gene promoter constructs. A 30 base pair conserved late element (CLE) motif was identified that was present three times in tandem in the BCTV promoter and once in that of BSCTV. Progressive deletion of these repeats from the BCTV promoter resulted in decreased reporter gene expression, but BSCTV promoters in which one or two extra copies of this motif were inserted did not exhibit increased late gene promoter activity. These results demonstrate that Curtovirus late gene expression by virion-sense promoters depends on the developmental stage of the host plant as well as on the number of CLE motifs present in the promoter.
Assuntos
Arabidopsis/virologia , Geminiviridae/genética , Plantas Geneticamente Modificadas/virologia , Regiões Promotoras Genéticas/fisiologia , Sequência de Bases , DNA Intergênico/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Ativação Transcricional/fisiologiaRESUMO
Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV), members of curtoviruses, encode seven open reading frames (ORFs) within a approximately 3 kb genome. One of these viral ORFs, C1, is known to play an important role in the early stage of viral infection in plants during initiation of viral DNA replication. We used promoter:: reporter (beta-glucuronidase) gene fusions in transgenic Ara-bidopsis to identify the putative promoter region of BCTV ORF C1. Unlike other geminiviruses, the intergenic region of BCTV was not sufficient to promote C1 expression in transgenic plants. When sequences extending into the coding region of C1 were tested, strong expression of the reporter protein was observed in vascular tissues of transgenic plants. This expression was not dependent on the presence of the intergenic regions or proximal 5' portions of the C1 coding region. Transgenic plants expressing a reporter gene under control of the putative complete C1 promoter were inoculated with virus to determine if any viral transcript affected C1 expression. Virus inoculated plants did not show any altered pattern or change in of reporter gene expression level. These results suggest that (1) important transcriptional activator elements for C1 expression reside in the 3' portion of C1 coding area itself, (2) C1 protein does not auto-regulate its own expression and (3) C1 expression of two curtoviruses is controlled differently compared to other geminiviruses.
Assuntos
Geminiviridae/genética , Regulação Viral da Expressão Gênica , Fases de Leitura Aberta/genética , Elementos Reguladores de Transcrição/genética , Proteínas Virais/genética , Arabidopsis/genética , Arabidopsis/virologia , DNA Intergênico/genética , Genes Reporter , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Deleção de SequênciaRESUMO
Induction of mRNA for BIK proapoptotic protein by doxorubicin or gamma-irradiation requires the DNA-binding transcription factor activity of p53. In MCF7 cells, pure antiestrogen fulvestrant also induces BIK mRNA and apoptosis. Here, we provide evidence that, in contrast to doxorubicin or gamma-irradiation, fulvestrant induction of BIK mRNA is not a direct effect of the transcriptional activity of p53, although p53 is necessary for this induction. It is known that p53 up-regulated modulator of apoptosis (PUMA) mRNA is induced directly by the transcriptional activity of p53. Whereas gamma-irradiation induced both BIK and PUMA mRNA, only BIK mRNA was induced by fulvestrant. Whereas both fulvestrant and doxorubicin induced BIK mRNA, only doxorubicin enhanced the DNA-binding activity of p53 and induced PUMA mRNA. Small interfering RNA (siRNA) suppression of p53 expression as well as overexpression of dominant-negative p53 effectively inhibited the fulvestrant induction of BIK mRNA, protein, and apoptosis. Transcriptional activity of a 2-kb BIK promoter, which contained an incomplete p53-binding sequence, was not affected by fulvestrant when tested by reporter assay. Fulvestrant neither affected the stability of the BIK mRNA transcripts. Interestingly, other human breast cancer cells, such as ZR75-1, constitutively expressed BIK mRNA even without fulvestrant. In these cells, however, BIK protein seemed to be rapidly degraded by proteasome, and siRNA suppression of BIK in ZR75-1 cells inhibited apoptosis induced by MG132 proteasome inhibitor. These results suggest that expression of BIK in human breast cancer cells is regulated at the mRNA level by a mechanism involving a nontranscriptional activity of p53 and by proteasomal degradation of BIK protein.
Assuntos
Antineoplásicos Hormonais/farmacologia , Proteínas Reguladoras de Apoptose/biossíntese , Neoplasias da Mama/genética , Estradiol/análogos & derivados , Proteínas de Membrana/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Estradiol/farmacologia , Fulvestranto , Raios gama , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Leupeptinas/farmacologia , Proteínas de Membrana/genética , Proteínas Mitocondriais , Regiões Promotoras Genéticas , Inibidores de Proteassoma , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/biossíntese , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
To obtain insights into similarities and differences in the biological actions of related drugs or toxic agents, their transcriptomal signature profiles (TSPs) have been examined in a large number of studies. However, many such reports did not provide proper justification for the dosage criteria of each agent. Using a well characterized cell culture model of estrogen-dependent proliferation of MCF7 human breast cancer cells, we demonstrate how different approaches to dosage standardization exert critical influences on TSPs, leading to different and even conflicting conclusions. Using quantitative cellular response (QCR)-based dosage criteria, TSPs were determined by Affymetrix microarray when cells were proliferating at comparable rates in the presence of various estrogens. We observed that TSPs of the xenoestrogens (e.g., genistein or bisphenol A) were clearly different from the TSP of 17beta-estradiol; namely, the former strongly enhanced expression of genes involved in mitochondrial oxidative phosphorylation, whereas the latter showed minimal effects. In contrast, TSPs for genistein and 17beta-estradiol were indistinguishable by using the marker gene expression-based dosage criteria, conditions in which there was comparable expression of the mRNA transcripts for the estrogen-inducible WISP2 gene. Our findings indicate that determination and interpretation of TSPs in pharmacogenomic and toxicogenomic studies that examine the transcriptomal actions of related agents by microarray require a clear rationale for the dosage standardization method to be used. We suggest that future studies involving TSP analyses use quantitative and objective dosage standardization methods, such as those with quantitative cellular response or marker gene expression-based dosage criteria.
Assuntos
Estrogênios/farmacologia , Padrões de Referência , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Estradiol/metabolismo , Estrogênios/metabolismo , Perfilação da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/metabolismo , Transcrição GênicaRESUMO
Evidence has been accumulating that some estrogen-dependent human breast cancers require estrogen for not only proliferation but also survival. To obtain insights into the molecular mechanisms of apoptosis of breast cancer cells subjected to estrogen starvation or exposed to antiestrogens, we characterized changes in the gene expression profile of MCF-7/BUS human breast cancer cells and revealed a strong induction of Bik, a member of the BH3-only proapoptotic proteins. The Bik mRNA transcript and protein were strongly induced by estrogen starvation or exposure to fulvestrant, a pure antiestrogen that competes with the natural estrogens for binding to the estrogen receptors. This Bik induction preceded apoptotic cell death, which was blocked by zVAD-fmk, a pancaspase inhibitor. Amounts of the Bcl-2-related proteins, such as Bcl-2, Bcl-XL, or Bax, showed only marginal changes in the presence or absence of estrogens or antiestrogens. Suppression of Bik expression by using the small interfering RNA effectively blocked the fulvestrant-induced breast cancer cell apoptosis. These results indicate that Bik is induced in MCF-7/BUS cells in the absence of estrogen signaling and plays a critical role in the antiestrogen-provoked breast cancer cell apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas Reguladoras de Apoptose , Sequência de Bases , Neoplasias da Mama , Linhagem Celular Tumoral , Primers do DNA , Feminino , Humanos , Cinética , Proteínas Mitocondriais , RNA Interferente Pequeno/genética , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed approximately 3-5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced approximately 1.5-2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, transforming growth factor alpha and stromal cell-derived factor 1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, depend on the E2 concentration, especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Expressão Gênica/efeitos dos fármacos , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CXCL12 , Quimiocinas CXC/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Ligantes , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Fator de Crescimento Transformador alfa/genéticaRESUMO
The CITED family proteins bind to CBP/p300 transcriptional integrators through their conserved C-terminal acidic domain and function as coactivators. The 21-kDa mouse Cited4 protein, a novel member of the CITED family, interacted with CBP/p300 as well as isoforms of the TFAP2 transcription factor, coactivating TFAP2-dependent transcription. The cited4 gene consisted of only a single exon located on chromosome 4 at 56.5-56.8 cM flanked by marker genes kcnq4 and scml1. Expression of Cited4 protein was strong and selective in embryonic hematopoietic tissues and endothelial cells. In adult animals, Cited4 showed strong milk cycle-dependent induction in pregnant and lactating mammary epithelial cells. Strong induction of Cited4 expression was also observed in SCp2 mouse mammary epithelial cells during their prolactin-dependent in vitro differentiation. These results implied possible roles for Cited4 in regulation of gene expression during development and differentiation of blood cells, endothelial cells, and mammary epithelial cells.