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1.
Oncotarget ; 7(39): 63924-63936, 2016 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-27590506

RESUMO

Epidermal growth factor receptor (EGFR) is commonly overexpressed in breast cancer and is associated with poor clinical outcomes; however, an increasing number of patients have shown a poor effective response to EGFR tyrosine kinase inhibitors (EGFR-TKI). Here, we found that AXL expression was positively correlated with poor progression in breast cancer patients. Suppression of AXL by an anti-tumor protein, E1A, enhanced EGFR-TKI (gefitinib, erlotinib and lapatinib) sensitization, resulting in significant inhibition of tumor growth in breast cancer cells. Additionally, AXL overexpression dramatically impaired E1A-mediated EGFR-TKI sensitization. These findings show that downregulation of AXL expression by E1A contributes to sensitization to EGFR-TKI in breast cancer, suggesting that combinatorial therapy of AXL inhibitors or E1A gene therapy with EGFR-TKI may be a potential therapeutic strategy for treatment of breast cancer patients.


Assuntos
Proteínas E1A de Adenovirus/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Progressão da Doença , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Cloridrato de Erlotinib/administração & dosagem , Feminino , Gefitinibe , Terapia Genética , Humanos , Estimativa de Kaplan-Meier , Lapatinib , Camundongos , Camundongos SCID , Transplante de Neoplasias , Prognóstico , Quinazolinas/administração & dosagem , Receptor Tirosina Quinase Axl
2.
Sci Rep ; 6: 19415, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26762895

RESUMO

Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming.


Assuntos
Reprogramação Celular , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Ativação Transcricional , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/farmacologia , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Camundongos , Fatores de Transcrição SOXB1/farmacologia
3.
Arch Immunol Ther Exp (Warsz) ; 62(3): 195-204, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24504082

RESUMO

The adenovirus type 5 E1A protein (E1A) plays a critical role in anti-cancer gene therapy and has been tested in clinical trials. The expression of E1A significantly reduces tumorigenesis, promotes cell death, and inhibits cancer cell mobility. Chemosensitization is one of the anti-tumor effects of E1A, increasing in vitro and in vivo sensitization of anti-cancer drugs, including cisplatin, gemcitabine, etoposide, doxorubicin, paclitaxel, and tumor necrosis factor-related apoptosis-inducing ligand and histone deacetylase inhibitors in different types of cancer cells. E1A also demonstrates anti-metastasis activity through various molecular mechanisms such as the repression of protease expression, suppression of HER2/neu and downregulation of microRNA (miR-520h). Moreover, E1A has been reported to reprogram transcription in tumor cells and stabilize tumor suppressors such as PP2A/C, p21 and p53. Because E1A plays a potentially significant role in anti-tumor therapy, there exists an urgent need to study the anti-cancer activities of E1A. This paper presents a review of our current understanding of the tumor-suppressive functions and molecular regulation of E1A, as well as the potential clinical applications of E1A.


Assuntos
Proteínas E1A de Adenovirus/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas E1A de Adenovirus/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Humanos , Receptor ErbB-2/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 31(8): 1392-5, 2011 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-21868306

RESUMO

OBJECTIVE: To study the effect of binding activities of the NH(2) terminus of E1A to the proteins regulating cell growth on ras-induced cell senescence and explore the mechanism of E1A-mediated escape from ras-induced senescence by E1A in human fibroblast. METHODS: In primary human fibroblasts, the proteins regulating cell growth in association with E1A NH(2) terminus, including the Rb family proteins, p300/CBP, and p400, were inactivated or interfered. The effect of alterations in the binding activities of these proteins on cell senescence bypass mediated by E1A was evaluated by cell growth curve. RESULTS: The Inactivation of Rb family proteins alone was not sufficient to rescue ras-induced cell senescence, whereas inactivation of both the Rb proteins and p300/CBP blocked ras-induced senescence of human fibroblasts. CONCLUSION: Rb and p300/CBP binding activities are both required for E1A to bypass ras-induced senescence in human fibroblasts.


Assuntos
Proteínas E1A de Adenovirus/farmacologia , Senescência Celular/efeitos dos fármacos , Fibroblastos/citologia , Proteínas ras/antagonistas & inibidores , Humanos , Cultura Primária de Células , Proteína do Retinoblastoma/metabolismo , Pele/citologia , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas ras/farmacologia
5.
Zhonghua Jie He He Hu Xi Za Zhi ; 33(1): 51-5, 2010 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-20368026

RESUMO

OBJECTIVE: the relationship between latent adenovirus infection and airway inflammation had not been well documented. The aim of this study was to illustrate the roles of adenovirus E1A protein on the transactivation of NF-kappaB, AP-1 in response to inflammatory stimuli and the effect of N-Acetylcysteine (NAC) upon the transactivation of NF-kappaB and AP-1 in cells stably expressing E1A protein. METHODS: rat alveolar epithelial cells stably expressing adenoviral E1A or control plasmid were developed. For isolation of nuclear extracts, 5 x 10(5) cells were plated and grown overnight in 60 mm dishes. Experiments were repeated 3 times. The cell model of stably expressing adenoviral E1A was stimulated by LPS or TNF-alpha and treated with NAC, a precursor for cysteine. The NF-kappaB and AP-1 transcriptional activity were measured by LUC report system. The expression of NF-kappaB and AP-1 were measured by Western blot. Differences between groups were assessed for significance by Student' t test, and multiple comparisons were made by one-way ANOVA. RESULTS: the luciferase activity derived by NF-kappaB element was (9 698 +/- 98) RLU in untreated E1A-positive clones and (101 195 +/- 234), and (170 385 +/- 443) RLU in LPS and TNF-alpha-stimulated cells, which were significantly higher than that of the control group 2 077 +/- 107, 67 846 +/- 332, 95 743 +/- 211 respectively. The luciferase activity derived by AP-1 element was 9 034 +/- 78 RLU in untreated E1A-positive clones and 26 343 +/- 398 and 31 731 +/- 332 RLU in LPS and TNF-alpha-stimulated cells, which were significantly higher than that of the control group 2 845 +/- 93, 10 772 +/- 432, 11 005 +/- 556 respectively. The densitometry of the NF-kappaB expression in E1A-positive clones were 79.3 +/- 4.6 and 80.3 +/- 3.8 respectively without treatment and were 81.8 +/- 3.9 - 89.9 +/- 1.6 and 94.1 +/- 1.9 to 99.8 +/- 1.6 respectively under LPS or TNF-alpha stimulation, which were significantly higher than that of the control group (68.3 +/- 3.8, 69.4 +/- 4.3 respectively) without stimulation and 70.1 +/- 2.8 to 80.8 +/- 3.6, 73.4 +/- 4.9 to 83.2 +/- 6.7 respectively under stimulation. The level of AP-1 expression did not show difference upon treatment with LPS or TNF-alpha in either cell clones. The densitometry of the NF-kappaB expression in E1A-positive clones were 3.2 +/- 0.1 and 3.3 +/- 0.1 respectively under LPS and TNF-alpha-stimulation and 1.98 +/- 0.2 and 1.9 +/- 0.2 respectively upon treatment for LPS and TNF-alpha with NAC pre-incubation. CONCLUSIONS: these results indicate that E1A protein upregulated NF-kappaB transcription activity induced by LPS and TNF-alpha in rat alveolar epithelial cells and this effect could be repressed by NAC. The mechanisms underlying transactivation of NF-kappaB involved by E1A may be related to oxidative stress.


Assuntos
Acetilcisteína/farmacologia , Proteínas E1A de Adenovirus/farmacologia , Antioxidantes/farmacologia , NF-kappa B/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular , Células Epiteliais , Alvéolos Pulmonares/citologia , Ratos , Ativação Transcricional
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 24(1): 20-2, 2008 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-18177611

RESUMO

AIM: To study the effect of peptide seals specific to CD59 on the expression of apoptosis-related survivin, caspase-3 and bax in HeLa cells and investigate the mechanism of peptide seals specific to CD59 in inducing apoptosis of HeLa cells. METHODS: The peptide seals specific to CD59 were put into HeLa cells and HeLa cells with CD59-transfected gene, respectively. After 24 hours of interaction, the inhibitory ratio of cell proliferation was measured by MTT and the expression of survivin, caspase-3 and bax was detected by immunohistochemistry. RESULTS: The inhibitory ratio of HeLa cells with CD59-transfected gene + peptide seal group was higher than that of HeLa cells + peptide seal group. There was a significant difference in depressing the proliferation between two groups (P < 0.01). Compared with HeLa cells + peptide seal group, the survivin expression of HeLa cells with CD59-transfected gene + peptide seal group was decreased (P < 0.05) while caspase-3 expression was increased (P < 0.05). There was no significant difference in the expression of bax in four groups. CONCLUSION: The peptide seal specific to CD59 can down-regulate the expression of survivin, activate caspase-3 and enhance the apoptosis of HeLa cells.


Assuntos
Apoptose/fisiologia , Antígenos CD59/farmacologia , Caspase 3/fisiologia , Proliferação de Células/efeitos dos fármacos , Proteína X Associada a bcl-2/fisiologia , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/farmacologia , Antígenos CD59/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Vetores Genéticos , Células HeLa , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , RNA Mensageiro/farmacologia
7.
Respiration ; 72(6): 629-35, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16355004

RESUMO

BACKGROUND: An imbalance between neutrophil protease and surrounding antiprotease levels has been shown to be important in the pathogenesis of chronic obstructive pulmonary disease (COPD). Adenoviral E1A DNA and protein are frequently detected in the lungs of COPD patients. As secretory leukoprotease inhibitor (SLPI) and elafin/skin-derived antileukoproteinase (SKALP) are locally produced in the lung and inhibit neutrophil elastase activity, we hypothesized that adenoviral E1A might affect the production of these antiproteases. OBJECTIVES: To examine the effect of E1A on SLPI and elafin/SKALP secretion in A549 (alveolar epithelial) cells and primary human bronchial epithelial (HBE) cells. METHODS: SLPI and elafin/SKALP were quantitated from cell culture supernatants using an ELISA. SLPI mRNA expression was examined by Northern blotting, and SLPI promoter activity was measured using a reporter gene assay. RESULTS: E1A significantly suppressed SLPI and elafin/SKALP secretion by A549 cells upon interleukin (IL)-1beta stimulation. E1A also suppressed SLPI and elafin/SKALP secretion by HBE cells. SLPI mRNA expression in A549 cells was suppressed by E1A regardless of IL-1beta stimulation. IL-1beta-induced SLPI promoter activity was suppressed by E1A gene transfection into A549 cells. CONCLUSIONS: Our findings of adenoviral E1A-mediated suppression of SLPI and elafin/SKALP secretion suggest that E1A may be involved in the enhancement of alveolar damage and play a role in the COPD process.


Assuntos
Proteínas E1A de Adenovirus/fisiologia , Brônquios/metabolismo , Proteínas/metabolismo , Alvéolos Pulmonares/metabolismo , Doença Pulmonar Obstrutiva Crônica/virologia , Adenoviridae/genética , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/farmacologia , Infecções por Adenovirus Humanos/virologia , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Secretadas Inibidoras de Proteinases , Proteínas/antagonistas & inibidores , Doença Pulmonar Obstrutiva Crônica/metabolismo , Inibidor Secretado de Peptidases Leucocitárias , Latência Viral
8.
J Endocrinol ; 187(2): 283-92, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16293776

RESUMO

Transcriptional coactivators are essential mediators of signal amplification in the regulation of gene expression in response to hormones and extracellular signals. We previously identified Bridge-1 (PSMD9) as a PDZ-domain coregulator that augments insulin gene transcription via interactions with the basic helix-loop-helix transcription factors E12 and E47, and that increases transcriptional activation by the homeodomain transcription factor PDX-1. In these studies, we find that transcriptional activation by Bridge-1 can be regulated via interactions with the histone acetyltransferase and nuclear receptor coactivator p300. In transfection assays, transcriptional activation by Bridge-1 is increased by the inhibition of endogenous histone deacetylase activity with trichostatin A, indicating that the transcriptional activation function of Bridge-1 can be regulated by histone modifications. The exogenous expression of p300 enhances the transcriptional activation by Bridge-1 in a dose-dependent manner. In contrast, the sequestration of p300 by the overexpression of the adenoviral protein E1A, but not by an E1A mutant protein that is unable to interact with p300, suppresses the transcriptional activation by Bridge-1. We demonstrate that p300 and Bridge-1 proteins interact in immunopre-cipitation and glutathione-S-transferase (GST) pull-down assays. Bridge-1 interacts directly with multiple regions within p300 that encompass C/H1 or C/H2 cysteine- and histidine-rich protein interaction domains and the histone acetyltransferase domain. Deletion or point mutagenesis of the Bridge-1 PDZ domain substantially reduces transcriptional activation by Bridge-1 and interrupts interactions with p300. We propose that p300 interactions with Bridge-1 can augment the transcriptional activation of regulatory target genes by Bridge-1.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Insulina/metabolismo , Proteínas/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas E1A de Adenovirus/farmacologia , Animais , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Deleção de Genes , Glutationa Transferase/metabolismo , Sequências Hélice-Alça-Hélice , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Imunoprecipitação , Mutagênese Sítio-Dirigida , Mutação Puntual , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína , Proteínas/genética , Transcrição Gênica , Leveduras , Fatores de Transcrição de p300-CBP/farmacologia
9.
Front Biosci ; 10: 1396-414, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15769634

RESUMO

The E1A gene of human adenovirus (Ad) serotypes 2 and 5 induces susceptibility of cells from several species, including human, to lysis by natural killer cells, activated macrophages and a variety of other immunologic and nonimmune cellular injuries. This E1A activity is the rationale behind some treatment strategies using combined adenoviral vector infection and chemotherapy for cancer. This review will consider the evolution of the studies that have resulted in the current understanding of the cellular mechanisms of E1A-induced tumor cell cytolytic susceptibility and sensitization to apoptotic injury. The translation of in vitro observations to experimental models testing E1A-induced tumor rejection in the context of the cellular immune response and E1A-induced sensitization of human tumor cells to therapeutic injuries will be discussed. Review of available information on the molecular mechanisms of E1A-induced cellular sensitivity to immune and nonimmune injuries will be used as a basis for consideration of possible future directions of this research.


Assuntos
Proteínas E1A de Adenovirus/farmacologia , Apoptose/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Proteínas E1A de Adenovirus/genética , Animais , Apoptose/imunologia , Testes de Carcinogenicidade , Humanos , Oncogenes/imunologia , Células Tumorais Cultivadas
10.
Biochem J ; 386(Pt 1): 29-34, 2005 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-15588252

RESUMO

Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.


Assuntos
Proteínas de Ligação a DNA/química , Transativadores/química , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/farmacologia , Animais , Sítios de Ligação , Células Cultivadas , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Genes Reporter , Humanos , Mamíferos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Prolina/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Serina/química , Proteína Smad3 , Relação Estrutura-Atividade , Transativadores/metabolismo , Transativadores/fisiologia , Ativação Transcricional , Transfecção , Fator de Crescimento Transformador beta/metabolismo
11.
Cancer Lett ; 216(1): 1-8, 2004 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-15500943

RESUMO

E1AF is an ets-oncogene family transcription factor. E1AF was shown to upregulate multiple matrix metalloproteinase (MMP) genes and contribute to the malignant phenotype of cancer cells by inducing invasive and metastatic activities. E1AF is upregulated by hepatocyte growth factor (HGF) stimulation, which indicates that E1AF would participate in cell motility by HGF/scatter factor. On the other hand, E1AF upregulates p21waf1/cip1 to induce cell cycle arrest when cells are exposed to stress. EWS/ETS fusions are frequently observed in Ewing's sarcoma, and we have revealed that EWS/ETS chimeric protein activates telomerase activity by upregulating hTERT. However, substitution ets binding site (EBS) mutants did not affect the responsiveness to EWS/E1AF. DNA-IP assay showed that the complexes contained EWS/E1AF bound to the hTERT promoter, which suggested that EWS/ETS functions as a co-activator for TERT transcription. Our findings that EWS/ETS acts as a transcriptional co-factor may imply that the transcription pathway is regulated by the interaction of transcription factors.


Assuntos
Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/farmacologia , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/genética , Neoplasias/fisiopatologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/farmacologia , Transcrição Gênica , Ciclo Celular , Humanos , Metaloproteinases da Matriz/biossíntese , Fenótipo , Proteínas Proto-Oncogênicas c-ets , Regulação para Cima
12.
Mol Immunol ; 41(12): 1241-6, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15482860

RESUMO

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by macrophages in response to intracellular pathogens and provides an obligatory signal for the differentiation of T-helper-1 cells. p300 is an important histone acetyltransferase (HAT) and has been implicated in the regulation of gene expression. Whether p300 plays a role in the IL-12 expression has not been investigated before. In this study, we analyzed the roles of p300 in the regulation of human IL-12 p40. By using RT-PCR and a series of co-transfection studies, we found that p300 had a stimulating effect on the expression of endogenous IL-12 p40 mRNA and on the activity of IL-12 p40 promoter. We also showed that the HAT activity of p300 was essential to its function in regulating IL-12 promoter, since the mutant p300 with the HAT domain deleted did not have such a stimulation effect. In addition, the adenovirus E1A protein suppressed the p300 function, whereas the mutant E1A lacking the p300 interaction domain did not. Furthermore, p300 was able to reinforce the c-Rel-mediated activation of IL-12 p40. Results presented in this paper implicate that p300 is involved in the transcriptional regulation of IL-12 p40, and IL-12 p40 is one of the target genes of p300.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Interleucina-12/genética , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Ativação Transcricional , Acetiltransferases/genética , Acetiltransferases/fisiologia , Proteínas E1A de Adenovirus/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Histona Acetiltransferases , Humanos , Subunidade p40 da Interleucina-12 , Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/fisiologia , Fatores de Transcrição , Transfecção , Fatores de Transcrição de p300-CBP
13.
Cell Cycle ; 3(7): 931-40, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15190207

RESUMO

p21(Waf1/Cip1/Sdi1) is the primary mediator of cell cycle arrest in response to different forms of stress and in the programs of senescence and differentiation. p21 interacts with many regulatory proteins and has broad effects on cellular gene expression. p21 was previously shown to stimulate NFkappaB transcriptional activity through its effect on the p300/CBP transcription cofactor family. p21 expression in human cells increases mRNA levels of different genes, some of which have been implicated in carcinogenesis and age-related diseases. Here we report that p21 expression stimulates promoters of six p21-responsive human genes and the cytomegalovirus promoter, as well as an artificial promoter containing NFkappaB response elements. The IkappaBalpha super-repressor blocked the effect of p21 on all but one of the promoters, and the response to p21 was abrogated by the mutagenesis of an NFkappaB element. p21 inducibility of all the tested promoters and of the endogenous p21-responsive genes was strongly inhibited by adenoviral E1A protein and its deletion mutants that bind p300/CBP but not p21 or Rb. Sulindac and some other non-steroidal anti-inflammatory drugs that inhibit NFkappaB decrease the effects of p21 on the responsive promoters and endogenous genes. These findings suggest the feasibility of developing agents that will counteract p21-mediated induction of disease-associated genes.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica/genética , NF-kappa B/metabolismo , Ativação Transcricional/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas I-kappa B/farmacologia , Mutagênese Sítio-Dirigida , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Regiões Promotoras Genéticas/genética , Sulindaco/farmacologia , Ativação Transcricional/efeitos dos fármacos
14.
J Neurochem ; 89(1): 15-23, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15030385

RESUMO

Cholecystokinin (CCK) is one of the most abundant peptide transmitters in the mammalian brain. Despite the physiological significance of CCK expression in long-term memory and psychiatric disorders, little is known about the factors that regulate the expression of CCK peptides. Here, we report that KCl and forskolin synergistically increase CCK gene transcription via protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) signalling pathways, activating cAMP response element-binding protein (CREB) associated with the CRE(- 80) element of the CCK promoter. Whereas, CREB Ser133 phosphorylation was essential for transcriptional activation, the synergistic stimulation was not correlated to the level of Ser133 phosphorylation, indicating that recruitment and/or activation of additional downstream factors were required for maximal stimulation. Transcriptional activation was reduced by co-expression of adenovirus 12S E1A, that inhibits binding of CREB-binding protein (CBP) to CREB. Moreover GAL4-CREB-DIEDML, which mediates the phosphorylation-independent binding of CBP, and the C-terminal domain of CBP was synergistically activated by forskolin and KCl. Taken together the results imply that neuronal CCK gene transcription is regulated by the cumulative action of calcium and cAMP via stimulation of the PKA and ERK signalling pathways and that synergy is accomplished by the coordinate activation of CREB and CBP.


Assuntos
Colecistocinina/genética , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Cloreto de Potássio/farmacologia , Transativadores/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/farmacologia , Animais , Proteína de Ligação a CREB , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Nucleares/efeitos dos fármacos , Células PC12 , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , RNA Mensageiro/metabolismo , Ratos , Elementos de Resposta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transativadores/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transfecção , Regulação para Cima/efeitos dos fármacos
15.
Ai Zheng ; 23(2): 146-9, 2004 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-14960232

RESUMO

BACKGROUND & OBJECTIVE: It has been well demonstrated that E1A, as a tumor suppression gene, is capable of inhibiting the growth and metastasis of different tumors, and reversing the malignant phenotype. Particularly, the gene possesses the ability to greatly enhance the drug-sensitivity of tumor cells to several antitumor agents, and also increase the radio-sensitivity. However, the associated genes through which E1A can exert its antitumor functions still remain unknown. The aim of this study was to isolate E1A anticancer-related genes,which were differentially expressed in drug-sensitive tumor cells using suppression subtractive hybridization (SSH). METHODS: To construct SSH library of human lymph node metastasis tumor cells (LN686) using the mRNA from LN686 cells treated by E1A protein and the parental LN686 cells as tester and driver, respectively. Positive clones in the library were selected randomly, and dot blot was used for the analysis of expression pattern of the differentially expressed-gene fragments. The sequences of cDNA fragments were analyzed and compared with that in GenBank. The mRNA levels of the novel genes in tester and driver were determined by semi-quantitative RT-PCR analysis. RESULTS: The SSH library contained about 7000 positive clones. Random analysis of 384 clones with PCR demonstrated that 362 clones contained inserted fragments. The consequence of dot blot demonstrated that these genes were over-expressed in the tester compared to the driver significantly. The 362 clones were sequenced and BLAST analysis was conducted, 10 clones are shown to be novel ESTs, and were registered in GenBank. The mRNA levels of the seven novel genes were over-expressed in LN686 cells treated by E1A protein compared to those of parental LN686 cells by semi-quantitative RT-PCR analysis, and the difference of mRNA expression was approximately 3-8 times. CONCLUSION: Ten novel gene fragments were isolated by the SSH technology, and it provided the basis for further cloning their full- length genes and studying their functions.


Assuntos
Proteínas E1A de Adenovirus/farmacologia , DNA Complementar/isolamento & purificação , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase
16.
J Biol Chem ; 279(9): 7832-9, 2004 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-14681221

RESUMO

Tight regulation of luteinizing hormone-beta subunit (LHbeta) expression is critical for differentiation and maturation of mammalian sexual organs and reproductive function. Two transcription factors, steroidogenic factor-1 (SF-1) and early growth response-1 (Egr-1), play a central role in activating LHbeta promoter, and the synergy between these two factors is essential in mediating gonadotropin-releasing hormone stimulation of LHbeta promoter. Here we demonstrate that the transcriptional co-activator p300 regulates this synergy. Overexpression of p300 results in strong stimulation of LHbeta promoter but only in the presence of both SF-1 and Egr-1, and not in the presence of other Egr proteins. Mutation of the binding sites for either SF-1 or Egr-1 completely abolishes the synergy between these two factors, as well as the influence of p300. Importantly, LHbeta promoter is precipitated using p300 antibodies in a chromatin immunoprecipitation assay with LbetaT2 gonadotropes, and this effect is enhanced by gonadotropin-releasing hormone. The influence of p300 on LHbeta promoter is potentiated by steroid receptor co-activator, as well as by E1A proteins, and attenuated by Smad nuclear interacting protein 1. Taken together, these results suggest that p300 is recruited to LHbeta promoter where it coordinates the functional synergy between SF-1 and Egr-1.


Assuntos
Proteínas de Ligação a DNA/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Hormônio Luteinizante Subunidade beta/genética , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/farmacologia , Proteínas E1A de Adenovirus/farmacologia , Animais , Sítios de Ligação , Proteínas de Transporte/farmacologia , Linhagem Celular , DNA/química , DNA/genética , DNA/metabolismo , Sinergismo Farmacológico , Fatores de Transcrição Fushi Tarazu , Expressão Gênica , Proteínas de Homeodomínio , Técnicas de Imunoadsorção , Mutagênese , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Proteínas de Ligação a RNA , Receptores Citoplasmáticos e Nucleares , Fator Esteroidogênico 1 , Transativadores/genética , Transfecção
17.
Curr Top Microbiol Immunol ; 272: 365-97, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12747556

RESUMO

Expression of the adenovirus oncoprotein E1A 12S induces the heterotrimeric transcription factor, NF-Y. NF-Y binds to the two CCAAT motifs upstream of the transcriptional start site of the human cdc2 promoter and is required for activation of the promoter by E1A 12S in cycling cells. The observations that a number of eukaryotic cell cycle regulatory genes also contain the CCAAT motifs and NF-Y binds to them support the notion that E1A 12S could play an important role in deregulated expression of these genes through activation of NF-Y gene in cycling cells.


Assuntos
Proteínas E1A de Adenovirus/farmacologia , Proteína Quinase CDC2/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Adenovírus Humanos/química , Humanos , Fatores de Transcrição/metabolismo
18.
Biochem Biophys Res Commun ; 305(4): 1099-104, 2003 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-12767945

RESUMO

The adenovirus-5 E1A gene encodes two main proteins of 289 and 243 amino acid residues from 13S and 12S mRNA, respectively. The E1A gene products function as transcriptional regulators and have anti-tumor activities. Despite the fact that E1A gene therapy has been tested in clinical trials, the molecular mechanism by which it suppresses tumor cell growth is still not completely understood. Here, we show that E1A increases the expression of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1), which inhibits cell growth. We further show that 13S E1A, but not 12S E1A, can transactivate the p21 promoter through Sp1 sites. Interestingly, the E1A-induced transactivation occurs only in cancer cells, not in normal cells. This study provides new insight into the links between E1A and the CDK inhibitor and may have important clinical implications.


Assuntos
Proteínas E1A de Adenovirus/farmacologia , Ciclinas/genética , Neoplasias/genética , Ativação Transcricional , Sítios de Ligação , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Humanos , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Células Tumorais Cultivadas
19.
J Immunol ; 170(8): 4139-47, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12682245

RESUMO

The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-kappaB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence. To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions. An E1a-sensitive coactivator was found to be required for expression. In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-kappaB and Sp1. Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-kappaB p65. Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation. These data and the NF-kappaB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.


Assuntos
Comunicação Celular/imunologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Histonas/metabolismo , NF-kappa B/fisiologia , Regiões Promotoras Genéticas/imunologia , Fator de Transcrição Sp1/fisiologia , Células 3T3 , Acetilação , Acetiltransferases/metabolismo , Acetiltransferases/farmacologia , Proteínas E1A de Adenovirus/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Proteína de Ligação a CREB , Comunicação Celular/genética , Proteínas de Ciclo Celular/farmacologia , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/biossíntese , Cromatina/genética , Cromatina/metabolismo , Células Clonais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/imunologia , Histona Acetiltransferases , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Proteínas Nucleares/farmacologia , Processamento de Proteína Pós-Traducional/imunologia , Sequências Reguladoras de Ácido Nucleico/imunologia , Proteínas Repressoras/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição Sp1/metabolismo , Transativadores/farmacologia , Fator de Transcrição RelA , Fatores de Transcrição , Ativação Transcricional , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Transcrição de p300-CBP
20.
J Biol Chem ; 278(17): 14897-905, 2003 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-12594215

RESUMO

The retinoblastoma protein (pRB) and its homologues, p107 and p130, prevent cell cycle progression from G(0)/G(1) to S phase by forming complexes with E2F transcription factors. Upon phosphorylation by G(1) cyclin-cyclin-dependent kinase (Cdk) complexes such as cyclin D1-Cdk4/6 and cyclin E-Cdk2, they lose the ability to bind E2F, and cells are thereby allowed to progress into S phase. Functional loss of one or more of the pRB family members, as a result of genetic mutation or deregulated phosphorylation, is considered to be an essential prerequisite for cellular transformation. In this study, we found that pRB family proteins have the ability to stimulate cyclin D1 transcription by activation of the NF-kappaB transcription factor. The cyclin D1-inducing activity of pRB is abolished by adenovirus E1A oncoprotein but not by the deletion of the A-box, the B-box, or the C-terminal region of the pocket, indicating that multiple pocket sequences are independently involved in cyclin D1 activation. Intriguingly, tumor-derived pRB pocket mutants retain the cyclin D1-inducing activity. Our results reveal a novel role of pRB family proteins as potential activators of NF-kappaB and inducers of G(1) cyclin. Certain pRB pocket mutants may give rise to a cellular situation in which deregulated E2F and cyclin D1 cooperatively promote abnormal cell proliferation.


Assuntos
Ciclina D1/biossíntese , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas , Proteína do Retinoblastoma/fisiologia , Proteínas E1A de Adenovirus/farmacologia , Divisão Celular , Ciclina G , Ciclina G1 , Ciclinas/biossíntese , Humanos , Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-Like , Transfecção , Células Tumorais Cultivadas
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