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1.
Nat Cell Biol ; 3(6): 552-8, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11389439

RESUMO

Loss of function of the retinoblastoma protein, pRB, leads to lack of differentiation, hyperproliferation and apoptosis. Inactivation of pRB results in deregulated E2F activity, which in turn induces entry to S-phase and apoptosis. Induction of apoptosis by either the loss of pRB or the deregulation of E2F activity occurs via both p53-dependent and p53-independent mechanisms. The mechanism by which E2F induces apoptosis is still unclear. Here we show that E2F1 directly regulates the expression of Apaf-1, the gene for apoptosis protease-activating factor 1. These results provide a direct link between the deregulation of the pRB pathway and apoptosis. Furthermore, because the pRB pathway is functionally inactivated in most cancers, the identification of Apaf-1 as a transcriptional target for E2F might explain the increased sensitivity of tumour cells to chemotherapy. We also show that, independently of the pRB pathway, Apaf-1 is a direct transcriptional target of p53, suggesting that p53 might sensitize cells to apoptosis by increasing Apaf-1 levels.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Regiões Promotoras Genéticas/genética , Proteínas/genética , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Fator Apoptótico 1 Ativador de Proteases , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Embrião de Mamíferos/metabolismo , Humanos , Camundongos , Proteínas/metabolismo , Proteína do Retinoblastoma/metabolismo , Transcrição Gênica , Ativação Transcricional
2.
Trends Cell Biol ; 3(2): 43-6, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14731718

RESUMO

The retinoblastoma protein (pRB) is one of the best-studied tumour suppressor gene products. Its loss during the genesis of many human tumours, its inactivation by several DNA tumour virus oncoproteins, and its ability to inhibit cell growth when introduced into dividing cells all suggest that pRB negatively regulates some aspect of normal cell growth. The discovery that pRB associates with transcription factors such as E2F has provided the first model for pRB function. In this review, we discuss how pRB may regulate cell growth by repressing transcription of genes essential for cell proliferation.

3.
J Cell Biol ; 155(2): 207-16, 2001 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11591730

RESUMO

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53-DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.


Assuntos
Apoptose , Neurônios/metabolismo , Proteínas/genética , Ativação Transcricional , Proteína Supressora de Tumor p53/fisiologia , Animais , Fator Apoptótico 1 Ativador de Proteases , Sequência de Bases , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Camptotecina/farmacologia , Linhagem Celular , Células Cultivadas , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Proteínas/fisiologia , RNA Mensageiro/biossíntese
5.
Curr Opin Genet Dev ; 8(1): 28-35, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9529602

RESUMO

Experimental data generated in the past year have further emphasized the essential role for the E2F transcription factors in the regulation of cell proliferation. Genetic studies have shown that E2F activity is required for normal development in fruitflies, and the generation of E2F-1(-/-) mice has demonstrated that individual members of the E2F transcription factor family are likely to have distinct roles in mammalian development and homeostasis. Additional mechanisms regulating the activity of the E2F transcription factors have been reported, including subcellular localization and proteolysis of the E2Fs in the proteasomes. Novel target genes for the E2F transcription factors have been identified that link the E2Fs directly to the initiation of DNA replication.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Divisão Celular , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Previsões , Humanos , Camundongos , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética
6.
Oncogene ; 25(2): 230-9, 2006 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-16158053

RESUMO

ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases -420 to +58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.


Assuntos
Neoplasias da Mama/genética , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/metabolismo , Regulação da Expressão Gênica/genética , Histona Desacetilases/metabolismo , Proteínas rho de Ligação ao GTP/genética , Acetilação , Sítios de Ligação , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F2/antagonistas & inibidores , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Fator de Transcrição E2F4/antagonistas & inibidores , Fator de Transcrição E2F4/genética , Fator de Transcrição E2F6/antagonistas & inibidores , Fator de Transcrição E2F6/genética , Fator de Transcrição E2F6/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Genes Supressores de Tumor , Inibidores de Histona Desacetilases , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Luciferases/metabolismo , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/farmacologia , Elementos de Resposta , Proteína do Retinoblastoma/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
7.
Mol Cell Biol ; 13(12): 7267-77, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8246949

RESUMO

The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a related gene product, p107, in association with the cyclin A-cdk2 or the cyclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since overexpression of pRB or p107 in cells lacking a functional pRB leads to the repression of E2F activity. The products of the adenovirus E1A gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of E1A required for this function are also essential for binding to a number of cellular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regions of E1A, as well as in vivo expression of E1A proteins containing deletions of either conserved region 1 (CR1) or CR2, we find that CR2 of E1A can form stable complexes with E2F. E1A proteins containing both CR1 and CR2 also associate with E2F, although the presence of these proteins results in the release of free E2F from its complexes. In vitro reconstitution experiments indicate that E1A-E2F interactions are not direct and that pRB can serve to facilitate these interactions. Complexes containing E1A, p107, cyclin A, and E2F were identified in vivo, which indicates that E1A may associate with E2F through either p107 or pRB. Peptide competition experiments demonstrate that the pRB-binding domain of the human E2F-1 protein can compete with the CR1 but not CR2 domain of E1A for binding to pRB. These results indicate that E1A CR1 and E2F-1 may bind to the same or overlapping sites on pRB and that E1A CR2 binds to an independent region. On the basis of our results, we propose a two-step model for the release of E2F from pRB and p107 cellular proteins.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Sequência de Bases , Ligação Competitiva , Sequência Conservada , Ciclinas/metabolismo , Primers do DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Proteínas/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Proteína p107 Retinoblastoma-Like , Deleção de Sequência , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Células Tumorais Cultivadas/metabolismo
8.
Mol Cell Biol ; 13(10): 6501-8, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8413249

RESUMO

Loss of a functional retinoblastoma tumor suppressor gene product, pRB, is a key step in the development of many human tumors. pRB is a negative regulator of cell proliferation and appears to participate in control of entry into the S phase of the cell cycle. The recent demonstration that pRB binds to transcription factor E2F has provided a model for the mechanism of pRB-mediated growth regulation. Since adenovirus E1A proteins dissociate the pRB-E2F complexes and stimulate E2F-dependent transcription, it has been suggested that pRB inhibits E2F transactivation. Although some evidence for this hypothesis has been provided, it has not been possible to determine the mechanism of pRB-mediated inhibition of E2F transactivation. In this study, we constructed mutants of E2F-1 that do not bind to pRB yet retain the ability to transactivate the adenovirus E2 promoter through E2F DNA-binding sites. We demonstrated that transactivation mediated by the wild-type E2F-1 protein was inhibited by overexpression of wild-type pRB but not by a naturally occurring mutant of pRB. Transactivation mediated by mutants of E2F-1 which do not bind to pRB was not affected by overexpression of wild-type pRB. Furthermore, when the E2F-1 transactivation domain was fused to the GAL4 DNA-binding domain, pRB inhibited GAL4-E2F-1 transactivation through GAL4 sites. Expression of pRB did not inhibit transactivation mediated by GAL4-E2F-1 mutant constructs which were devoid of pRB binding. In conclusion, these data demonstrate that pRB inhibits E2F-dependent transactivation by direct protein-protein interaction.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteína do Retinoblastoma/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sítios de Ligação , DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Proteínas Fúngicas/metabolismo , Humanos , Mutagênese , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Células Tumorais Cultivadas
9.
Mol Cell Biol ; 16(3): 1047-57, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8622649

RESUMO

The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated ED2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rate fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G1 arrest mediated by the p16INK4 tumor suppressor protein, and E2F-1 was shown to override a G1 block mediated by a neutralizing antibody to cyclin D1. The p16INK4-induced G1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simulataneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G1 progession, is sufficient to induce S-phase entry, and overcomes a p16-mediated G1 block, and since E12F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p126-cyclin D-pRB pathway. Furthermore, our date suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Fase S , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/genética , Ciclo Celular/genética , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F3 , Fator de Transcrição E2F5 , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Ratos , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética
10.
Mol Cell Biol ; 14(12): 8241-9, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7969161

RESUMO

E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type 16 (HPV16) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Divisão Celular , Proteínas Oncogênicas Virais/fisiologia , Proteínas Repressoras , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Transformação Celular Neoplásica , Transformação Celular Viral , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Teste de Complementação Genética , Humanos , Técnicas In Vitro , Queratinócitos/citologia , Masculino , Camundongos , Papillomaviridae , Proteínas E7 de Papillomavirus , Proteína do Retinoblastoma/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Dedos de Zinco
11.
Mol Cell Biol ; 13(12): 7813-25, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8246996

RESUMO

E2F is a transcription factor that helps regulate the expression of a number of genes that are important in cell proliferation. Recently, several laboratories have isolated a cDNA clone that encodes an E2F-like protein, known as E2F-1. Subsequent characterization of this protein showed that it had the properties of E2F, but it was difficult to account for all of the suggested E2F activities through the function of this one protein. Using low-stringency hybridization, we have isolated cDNA clones that encode two additional E2F-like proteins, called E2F-2 and E2F-3. The chromosomal locations of the genes for E2F-2 and E2F-3 were mapped to 1p36 and 6q22, respectfully, confirming their independence from E2F-1. However, the E2F-2 and E2F-3 proteins are closely related to E2F-1. Both E2F-2 and E2F-3 bound to wild-type but not mutant E2F recognition sites, and they bound specifically to the retinoblastoma protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined action of a family of proteins.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , DNA/metabolismo , Primers do DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Fator de Transcrição E2F3 , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Ligação Proteica , Proteína 1 de Ligação ao Retinoblastoma , Homologia de Sequência de Aminoácidos , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Ativação Transcricional
12.
Mol Cell Biol ; 17(1): 407-15, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8972221

RESUMO

Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. We show here that cyclin E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein interaction. These observation suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene requires an active cdk2 kinase tethered to its promoter region.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ciclo Celular , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Proteínas Associadas aos Microtúbulos/fisiologia , Ativação Transcricional/fisiologia , Proteínas Supressoras de Tumor , Células 3T3 , Animais , Sítios de Ligação , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Fatores de Transcrição E2F , Inibidores Enzimáticos , Fase G1/fisiologia , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/análise , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão , Proteína 1 de Ligação ao Retinoblastoma , Proteína p107 Retinoblastoma-Like , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Transfecção
13.
Mol Cell Biol ; 17(9): 5508-20, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9271426

RESUMO

The E2F transcription factors are essential for regulating the correct timing of activation of several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130, and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G1 arrests mediated by the p16INK4A tumor suppressor protein or neutralizing antibodies to cyclin D1, E2F-4 and -5 do not. Using E2F-1 and E2F-4 as representatives of the two subgroups, we showed here, by constructing a set of chimeric proteins, that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well as the ability to efficiently transactivate an E2F-responsive promoter to E2F-4. We found that the E2F-1 amino terminus directs chimeric proteins to the nucleus. Surprisingly, a short nuclear localization signal derived from simian virus 40 large T antigen could perfectly substitute for the presence of the E2F-1 amino terminus in these assays. Thus, nuclearly localized E2F-4, when overexpressed, displayed biological activities similar to those of E2F-1. Furthermore, we showed that nuclear localization of endogenous E2F-4 is cell cycle regulated, with E2F-4 being nuclear in the G0 and early G1 phases and mainly cytoplasmic after the pRB family members have become phosphorylated. We propose a novel mechanism for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G0 until mid- to late G1 phase whereas E2F-1 is active in late G1 and S phases, until it is inactivated by cyclin A-dependent kinase in late S phase.


Assuntos
Proteínas de Ciclo Celular , Fase S , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F4 , Fator de Transcrição E2F5 , Inibidores Enzimáticos/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Inibidores de Proteínas Quinases , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Ativação Transcricional
14.
Mol Cell Biol ; 18(11): 6679-97, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9774682

RESUMO

The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Clonagem Molecular , Ciclina E/metabolismo , DNA/biossíntese , Pegada de DNA , Replicação do DNA/genética , Fatores de Transcrição E2F , Imunofluorescência , Humanos , Camundongos , Dados de Sequência Molecular , RNA/metabolismo , Proteína do Retinoblastoma/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Proteínas de Schizosaccharomyces pombe , Fator de Transcrição DP1
15.
Mol Cell Biol ; 19(9): 6379-95, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10454584

RESUMO

Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G(0)-G(1) phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteínas Tirosina Fosfatases/metabolismo , Fase S/fisiologia , Fatores de Transcrição/metabolismo , Fosfatases cdc25 , Animais , Apoptose , Sequência de Bases , Sítios de Ligação , Divisão Celular , Linhagem Celular , Ciclina E/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Fator de Transcrição E2F3 , Marcação de Genes , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Transcrição Gênica
16.
Mol Cell Biol ; 20(19): 7059-67, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10982822

RESUMO

Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105(Rb), and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105(Rb) complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105(Rb) complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.


Assuntos
Carcinoma de Células Escamosas/patologia , Proteínas de Transporte , Proteínas de Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Papillomaviridae/genética , Proteínas , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias do Colo do Útero/patologia , Proteínas Virais/biossíntese , Proteínas Virais/fisiologia , Fosfatases cdc25/genética , Sítios de Ligação , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/fisiologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Transformação Celular Neoplásica/genética , Transformação Celular Viral/genética , Sequência Consenso , Cisteína Endopeptidases/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F4 , Feminino , Genes do Retinoblastoma , Humanos , Substâncias Macromoleculares , Complexos Multienzimáticos/metabolismo , Proteínas de Neoplasias/genética , Papillomaviridae/fisiologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteína do Retinoblastoma/biossíntese , Proteína do Retinoblastoma/genética , Proteína 1 de Ligação ao Retinoblastoma , Proteína p130 Retinoblastoma-Like , Fator de Transcrição DP1 , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas/metabolismo , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/genética
17.
Mol Cell Biol ; 18(2): 1084-93, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9448006

RESUMO

PML is a nuclear protein with growth-suppressive properties originally identified in the context of the PML-retinoic acid receptor alpha (RAR alpha) fusion protein of acute promyelocytic leukemia. PML localizes within distinct nuclear structures, called nuclear bodies, which are disrupted by the expression of PML-RAR alpha. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RAR alpha expression. Both PML and PML-RAR alpha form complexes with the nonphosphorylated form of pRB in vivo, and they interact with the pocket region of pRB. The regions of PML and PML-RAR alpha involved in pRB binding differ; in fact, the B boxes and the C-terminal region of PML, the latter of which is not present in PML-RAR alpha, are essential for the formation of stable complexes with pRB. Functionally, PML abolishes activation of glucocorticoid receptor-regulated transcription by pRB, whereas PML-RAR alpha further increases it. Our results suggest that PML may be part of transcription-regulatory complexes and that the oncogenic potential of the PML-RAR alpha protein may derive from the alteration of PML-regulated transcription.


Assuntos
Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Proteínas de Fusão Oncogênica/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Divisão Celular , Humanos , Corpos de Inclusão/metabolismo , Substâncias Macromoleculares , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Proteína da Leucemia Promielocítica , Ligação Proteica , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor
18.
Oncogene ; 35(33): 4368-78, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-26804170

RESUMO

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.


Assuntos
Transformação Celular Neoplásica , Neoplasias do Colo/patologia , Proteínas Tirosina Quinases/fisiologia , Proteínas ras/fisiologia , Regiões 5' não Traduzidas/fisiologia , Tirosina Quinase da Agamaglobulinemia , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/genética
19.
Leukemia ; 30(6): 1388-98, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26876596

RESUMO

TEN-ELEVEN-TRANSLOCATION-2 (TET2) and DNA-METHYLTRANSFERASE-3A (DNMT3A), both encoding proteins involved in regulating DNA methylation, are mutated in hematological malignancies affecting both myeloid and lymphoid lineages. We previously reported an association of TET2 and DNMT3A mutations in progenitors of patients with angioimmunoblastic T-cell lymphomas (AITL). Here, we report on the cooperative effect of Tet2 inactivation and DNMT3A mutation affecting arginine 882 (DNMT3A(R882H)) using a murine bone marrow transplantation assay. Five out of eighteen primary recipients developed hematological malignancies with one mouse developing an AITL-like disease, two mice presenting acute myeloid leukemia (AML)-like and two others T-cell acute lymphoblastic leukemia (T-ALL)-like diseases within 6 months following transplantation. Serial transplantations of DNMT3A(R882H) Tet2(-/-) progenitors led to a differentiation bias toward the T-cell compartment, eventually leading to AITL-like disease in 9/12 serially transplanted recipients. Expression profiling suggested that DNMT3A(R882H) Tet2(-/-) T-ALLs resemble those of NOTCH1 mutant. Methylation analysis of DNMT3A(R882H) Tet2(-/-) T-ALLs showed a global increase in DNA methylation affecting tumor suppressor genes and local hypomethylation affecting genes involved in the Notch pathway. Our data confirm the transformation potential of DNMT3A(R882H) Tet2(-/-) progenitors and represent the first cooperative model in mice involving Tet2 inactivation driving lymphoid malignancies.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Transtornos Linfoproliferativos/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Animais , Diferenciação Celular , DNA Metiltransferase 3A , Dioxigenases , Genes Supressores de Tumor , Transtornos Linfoproliferativos/etiologia , Camundongos , Receptores Notch/genética
20.
Oncogene ; 17(2): 143-55, 1998 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-9674698

RESUMO

The E2F transcription factors are key targets for the retinoblastoma protein, pRB. By inactivation of E2Fs, pRB prevents progression to the S phase. To test proliferative functions of E2F, we generated transgenic mice expressing human E2F-1 and/or human DP-1. When the hydroxymethyl glutaryl coenzyme A reductase promoter was used to express DP-1, overexpression occurred in a variety of tissues and did not confer phenotypic changes. In contrast, expression of E2F-1 from the same promoter was obtained only in testicles, in which E2F-1 overexpression caused atrophy and sterility through a process involving increased apoptosis in the germinal epithelium. This effect was potentiated by simultaneous overexpression of DP-1. Testicular atrophy as a result of overexpression of E2F-1 and DP-1 is independent of functional p53, since p53-nullizygous transgenic mice overexpressing E2F-1 and DP-1 also suffered testicular atrophy.


Assuntos
Apoptose/genética , Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Atrofia , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteína 1 de Ligação ao Retinoblastoma , Testículo/patologia , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética
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