Assuntos
Proteína Supressora de Tumor p53/metabolismo , Pontos de Checagem do Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Using genetically modified mouse models, we report here that p53 upregulated modulator of apoptosis (Puma) and Bcl-2 interacting mediator of cell death (Bim), two pro-apoptotic members of the B-cell lymphoma protein-2 (Bcl-2) family of proteins, cooperate in causing bone marrow and gastrointestinal tract toxicity in response to chemo and radiation therapy. Deletion of both Puma and Bim provides long-term survival without evidence of increased tumor susceptibility following a lethal challenge of carboplatin and ionizing radiation. Consistent with these in vivo findings, studies of primary mast cells demonstrated that the loss of Puma and Bim confers complete protection from cytokine starvation and DNA damage, similar to that observed for Bax/Bak double knockout cells. Biochemical analyses demonstrated an essential role for either Puma or Bim to activate Bax, thereby leading to mitochondrial outer membrane permeability, cytochrome c release and apoptosis. Treatment of cytokine-deprived cells with ABT-737, a BH3 mimetic, demonstrated that Puma is sufficient to activate Bax even in the absence of all other known direct activators, including Bim, Bid and p53. Collectively, our results identify Puma and Bim as key mediators of DNA damage-induced bone marrow failure and provide mechanistic insight into how BH3-only proteins trigger cell death.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Dano ao DNA , Hemoglobinúria Paroxística/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/metabolismo , Anemia Aplástica , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Compostos de Bifenilo/farmacologia , Doenças da Medula Óssea , Transtornos da Insuficiência da Medula Óssea , Sobrevivência Celular/genética , Citocromos c/genética , Citocromos c/metabolismo , Deleção de Genes , Hemoglobinúria Paroxística/genética , Hemoglobinúria Paroxística/patologia , Mastócitos/metabolismo , Mastócitos/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Nitrofenóis/farmacologia , Permeabilidade , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/genética , Sulfonamidas/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Most inherited TP53 mutations have been identified in individuals with a family cancer predisposition syndrome, in which the activity of p53 mutants is severely reduced. However, germline p53 mutants in children with 'sporadic' adrenocortical or choroid plexus tumors exhibit a wide range of functional activity. Here, we demonstrate the occurrence of a complex germline TP53 mutation in two unrelated families with different cancer phenotypes, neither fulfilling the classic criteria for Li-Fraumeni syndrome. The TP53 mutation consists of a duplication of 7 bp in exon 4, resulting in a frame shift and premature stop signal. Haplotype analysis indicated that the mutation arose independently in the two families. Analysis of the DNA secondary structure predicts the TP53 mutation occurred within a hairpin loop. Additional germline complex mutations occurring within the same region of exon 4 have been identified in the IARC database. Our findings suggest that certain TP53 regions are prone to intrinsic genetic alterations, possibly through defects in DNA replication or repair. Further, carriers of the same TP53 mutation can have diverse cancer profiles, illustrating the complexity of genetic counseling and risk prediction.
RESUMO
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that has an important role in immunity and inflammation by inducing cellular responses such as apoptosis. The transcription factor nuclear factor-kappaB (NF-kappaB) can paradoxically suppress and promote apoptosis in response to TNF-alpha. In this study, we found that p53 upregulated modulator of apoptosis (PUMA), a p53 downstream target and a BH3-only Bcl-2 family member, is directly regulated by NF-kappaB in response to TNF-alpha. TNF-alpha treatment led to increases in PUMA mRNA and protein levels in human colon cancer cells. The induction of PUMA was p53 independent, and mediated by the p65 component of NF-kappaB through a kappaB site in the PUMA promoter. The apoptotic effect of PUMA induction by TNF-alpha was unmasked by depleting the antiapoptotic protein Bcl-X(L). In mice, PUMA was also induced by TNF-alpha in an NF-kappaB-dependent manner. TNF-alpha-induced apoptosis in a variety of tissues and cell types, including small intestinal epithelial cells, hepatocytes, and thymocytes, was markedly reduced in PUMA-deficient mice. Collectively, these results demonstrated that PUMA is a direct target of NF-kappaB and mediates TNF-alpha-induced apoptosis in vitro and in vivo.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína bcl-X/metabolismoRESUMO
Paediatric choroid plexus carcinomas (CPC) and adrenocortical carcinomas (ACC) are exceedingly rare tumours, each occurring at an annual rate of 0.3 cases per million children or less. Although both tumour types are associated with Li-Fraumeni syndrome (LFS), the penetrance of germline TP53 mutations in CPC remains to be established. We report here a young boy without a family history of cancer who presented with CPC and subsequently ACC. Genetic testing revealed a novel de novo germline TP53 mutation (E285V). Neither tumour underwent loss of heterozygosity. Consistent with this observation, functional analyses demonstrated that E285V acts as a dominant negative mutant that is defective in regulating target gene expression, growth suppression and apoptosis. These results further strengthen the association between germline TP53 mutations and childhood CPC, even when occurring in the absence of familial tumour susceptibility.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Carcinoma/genética , Neoplasias do Plexo Corióideo/genética , Genes p53 , Mutação em Linhagem Germinativa , Segunda Neoplasia Primária/genética , Neoplasias do Córtex Suprarrenal/diagnóstico , Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/diagnóstico , Carcinoma Adrenocortical/metabolismo , Carcinoma/diagnóstico , Carcinoma/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias do Plexo Corióideo/diagnóstico , Neoplasias do Plexo Corióideo/metabolismo , Humanos , Lactente , Masculino , Segunda Neoplasia Primária/diagnóstico , Segunda Neoplasia Primária/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismoAssuntos
DNA/metabolismo , Ribossomos/metabolismo , Transdução de Sinais , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Humanos , Mutação , Regiões Promotoras Genéticas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: An inherited germline P53 mutation has been identified in cases of childhood adrenocortical carcinoma (ACT), a neoplasm with a high incidence in southern Brazil. The penetrance of ACT in carriers of the point mutation, which encodes an arginine-to-histidine substitution at codon 337 of TP53 (R337H), has not been determined. OBJECTIVE: To investigate the penetrance of childhood ACT in carriers of the R337H TP53 mutation. METHODS: The family histories of 30 kindreds of 41 southern Brazilian children with ACT were obtained. A PCR based assay was used to detect this P53 mutation in a large number of relatives of children with ACT. In all, 927 individuals were tested for the mutation, 232 from the non-carrier and 695 (including the 40 probands) from the carrier parental lines. RESULTS: 40 children with ACT carried the TP53 R337H mutation; the remaining child with ACT was not tested. There was no evidence of Li-Fraumeni syndrome in any of the kindreds; however, seven met the criteria for Li-Fraumeni-like syndrome. The carrier parental line was identified in each kindred. Of the 695 individuals tested in the carrier parental line, 240 (34.5%) were positive for the mutation, while none of the 232 individuals in the other parental line carried the mutation. The penetrance of ACT was 9.9% (95% confidence interval, 8.7% to 11.1%). CONCLUSIONS: The TP53 R337H mutation dramatically increases predisposition to childhood ACT but not to other cancers, and explains the increased frequency of ACT observed in this geographic region.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Penetrância , Proteína Supressora de Tumor p53/genética , Distribuição por Idade , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Masculino , Linhagem , Fatores de RiscoAssuntos
Apoptose/genética , Ciclo Celular/genética , Regulação da Expressão Gênica/genética , Genes Reguladores/genética , Estresse Fisiológico/genética , Proteína Supressora de Tumor p53/genética , Animais , Citocinas/genética , Citocinas/metabolismo , Humanos , Transdução de Sinais/genética , Estresse Fisiológico/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Malignant transformation occurs in cells that overexpress c-Myc or that inappropriately activate E2F-1. Transformation occurs after the selection of cells that have acquired resistance to apoptosis that is triggered by these oncogenes, and a key mediator of this cell death process is the p53 tumor suppressor. In IL-3-dependent immortal 32D.3 myeloid cells the ARF/p53 apoptotic pathway is inactivated, as these cells fail to express ARF. Nonetheless, both c-Myc and E2F-1 overexpression accelerated apoptosis when these cells were deprived of IL-3. Here we report that c-Myc or E2F-1 overexpression suppresses Bcl-2 protein and RNA levels, and that restoration of Bcl-2 protein effectively blocks the accelerated apoptosis that occurs when c-Myc- or E2F-1-overexpressing cells are deprived of IL-3. Blocking p53 activity with mutant p53 did not abrogate E2F-1-induced suppression of Bcl-2. Analysis of immortal myeloid cells engineered to overexpress c-Myc and E2F-1 DNA binding mutants revealed that DNA binding activity of these oncoproteins is required to suppress Bcl-2 expression. These results suggest that the targeting of Bcl-2 family members is an important mechanism of oncogene-induced apoptosis, and that this occurs independent of the ARF/p53 pathway.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Genes bcl-2 , Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Fatores de Transcrição/fisiologia , Animais , Apoptose/genética , Linhagem Celular Transformada , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , DNA/genética , DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Genes myc , Genes p53 , Interleucina-3/farmacologia , Camundongos , Mutação , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes de Fusão/fisiologia , Temperatura , Fatores de Transcrição/genética , Transfecção , Proteína Supressora de Tumor p14ARF/deficiência , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteína bcl-XRESUMO
The tumor suppressor p53 plays a central role in the protection against DNA damage and other forms of physiological stress primarily by inducing cell cycle arrest or apoptosis. Mutation of p53, which is the most frequent genetic alteration detected in human cancers, inactivates these growth regulatory functions and causes a loss of tumor suppressor activity. In some cases, mutation also confers tumor-promoting functions, such as the transcriptional activation of genes involved in cell proliferation, cell survival and angiogenesis. Consequently, cells expressing some forms of mutant p53 show enhanced tumorigenic potential with increased resistance to chemotherapy and radiation. Our current understanding of these activities is summarized in this review. By dissecting out mechanistic differences between wild-type and mutant p53 activities, it may be possible to develop therapeutics that restore tumor suppressor function to mutant p53 or that selectively inactivate mutant p53 tumor-promoting functions.
Assuntos
Divisão Celular/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Sequência de Aminoácidos , Animais , Divisão Celular/genética , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
BH3-only proteins function at a proximal point in a conserved cell death pathway by binding, through their BH3 domains, to other Bcl-2 family members and triggering mitochondrial events associated with apoptosis. Here, we describe a strongly pro-apoptotic BH3-only protein, designated Bbc3, whose expression increases in response to diverse apoptotic stimuli. bbc3 mRNA levels were induced by exposure to DNA-damaging agents and by wild-type p53, which mediates DNA damage-induced apoptosis. p53 transactivated bbc3 through consensus p53 binding sites within the bbc3 promoter region, indicating that bbc3 is a direct target of p53. Additionally, bbc3 mRNA was induced by p53-independent apoptotic stimuli, including dexamethasone treatment of thymocytes, and serum deprivation of tumor cells. Insulin-like growth factor-1 and epidermal growth factor, growth factors with broad anti-apoptotic activity, were each sufficient to suppress Bbc3 expression in serum-starved tumor cells. These results suggest that the transcriptional regulation of bbc3 contributes to the transduction of diverse cell death and survival signals.
Assuntos
Apoptose/genética , Regulação da Expressão Gênica/fisiologia , Proteínas/genética , Proteínas Proto-Oncogênicas , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Sítios de Ligação , Morte Celular , Sobrevivência Celular , Células Cultivadas , Sequência Consenso , Fibroblastos/citologia , Fibroblastos/fisiologia , Genes p53 , Humanos , Leucemia Mieloide , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Proteínas/química , Proteínas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
A single dose of Mpl ligand (Mpl-L) given immediately after lethal DNA-damaging regimens prevents the death of mice. However, the mechanism of this myeloprotection is unknown. The induction of p53-dependent apoptosis in response to DNA damage signals suggests that immediate administration of Mpl-L may inhibit p53-dependent apoptosis. This hypothesis was tested by administering a single injection of pegylated murine Megakaryocyte Growth and Development Factor (PEG-rmMGDF, a truncated recombinant Mpl-L) to p53(-/-) and wild-type mice immediately after carboplatin (80 mg/kg) and 7.5 Gy total body gamma-irradiation. PEG-rmMGDF was required to prevent the death of wild-type mice, whereas p53(-/-) mice survived with or without the exogenous cytokine. The degree of platelet depression and subsequent recovery was comparable in p53(-/-) mice to wild-type animals given PEG-rmMGDF. Hence, either Mpl-L administration or p53-deficiency protected multipotent hematopoietic progenitors and committed megakaryocyte precursors. The myelosuppressive regimen induced expression of p53 and the p53 target, p21(Cipl) in wild-type bone marrow, indicating that Mpl-L acts downstream of p53 to prevent apoptosis. Constitutive expression of the proapoptotic protein Bax, was not further increased. Bax(-/-) mice survived the lethal regimen only when given PEG-rmMGDF; however, these Bax(-/-) mice showed more rapid hematopoietic recovery than did identically-treated wild-type mice. Therefore, administration of Mpl-L immediately after myelosuppressive chemotherapy or preparatory regimens for autologous bone marrow transplantation should prevent p53-dependent apoptosis, decrease myelosuppression, and reduce the need for platelet transfusions.
Assuntos
Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2 , Trombopoetina/farmacologia , Proteína Supressora de Tumor p53/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Células da Medula Óssea/efeitos da radiação , Carboplatina/administração & dosagem , Carboplatina/intoxicação , Proteínas de Ciclo Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Proteínas de Ligação a DNA , Morte , Hematopoese/efeitos dos fármacos , Camundongos , Camundongos Knockout , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacologia , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Células-Tronco/efeitos da radiação , Trombopoetina/administração & dosagem , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Irradiação Corporal Total/efeitos adversos , Proteína X Associada a bcl-2RESUMO
The incidence of pediatric adrenal cortical carcinoma (ACC) in southern Brazil is 10-15 times higher than that of pediatric ACC worldwide. Because childhood ACC is associated with Li-Fraumeni syndrome, we examined the cancer history and p53 status of 36 Brazilian patients and their families. Remarkably, 35 of 36 patients had an identical germ-line point mutation of p53 encoding an R337H amino acid substitution. Differences within intragenic polymorphic markers demonstrated that at least some mutant alleles arose independently, thus eliminating a founder effect. In tumor cells, the wild-type allele was deleted, and mutant p53 protein accumulated within the nuclei. Although these features are consistent with Li-Fraumeni syndrome-associated adrenal tumors, there was no history of increased cancer incidence among family members. Therefore, this inherited R337H p53 mutation represents a low-penetrance p53 allele that contributes in a tissue-specific manner to the development of pediatric ACC.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Genes p53 , Mutação em Linhagem Germinativa , Mutação Puntual , Sequência de Bases , Núcleo Celular/metabolismo , Criança , Primers do DNA , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Linhagem , Proteína Supressora de Tumor p53/metabolismoRESUMO
The most frequently expressed drug resistance genes, MDR1 and MRP1, occur in human tumors with mutant p53. However, it was unknown if mutant p53 transcriptionally regulated both MDR1 and MRP1. We demonstrated that mutant p53 did not activate either the MRP1 promoter or the endogenous gene. In contrast, mutant p53 strongly up-regulated the MDR1 promoter and expression of the endogenous MDR1 gene. Notably, cells that expressed either a transcriptionally inactive mutant p53 or the empty vector showed no endogenous MDR1 up-regulation. Transcriptional activation of the MDR1 promoter by mutant p53 required an Ets binding site, and mutant p53 and Ets-1 synergistically activated MDR1 transcription. Biochemical analysis revealed that Ets-1 interacted exclusively with mutant p53s in vivo but not with wild-type p53. These findings are the first to demonstrate the induction of endogenous MDR1 by mutant p53 and provide insight into the mechanism.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Genes MDR/genética , Genes p53/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Sequência de Bases , Sítios de Ligação , Western Blotting , Caspases/metabolismo , Linhagem Celular , DNA/metabolismo , Citometria de Fluxo , Vetores Genéticos , Glutationa Transferase/metabolismo , Humanos , Luciferases/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Biossíntese de Proteínas , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas c-ets , Ribonucleases/metabolismo , Transcrição Gênica , Ativação Transcricional , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
The mdr1b gene is thought to be a "stress-responsive" gene, however it is unknown if this gene is regulated by p53 in the whole animal. Moreover, it is unknown if overexpression of mdr1b affects cell survival. The dependence of mdr1b upon p53 for upregulation was evaluated in p53 knockout mice. Wild-type (wt) or p53-/- mice were treated singly or in combination with gamma irradiation (IR) and/or the potent DNA damaging agent, diethylnitrosoamine (DEN). Both IR and DEN induced mdr1b in wild-type animals, but not in the p53-/- mice. IR also upregulated endogenous mdr1b in the H35 liver cell line, and the mdr1b promoter was activated by IR and activation correlated with p53 levels; moreover activation required an intact p53 binding site. Colony survival studies revealed that co-transfection of both mdr1b and p53 dramatically reduced colony numbers compared to cells transfected with either p53 or mdr1b alone and cells microinjected with both mdr1b and p53 had a more dramatic loss in viability compared to cells injected with either expression vector alone. Further studies using acridine orange and ethidium bromide to measure apoptosis revealed that mdr1b caused apoptosis and this was enhanced by p53, however the increased apoptosis required a functional p53 transactivation domain. These studies indicate that mdr1b is a downstream target of p53 in the whole animal and expression of mdr1b facilitates p53-mediated cell death.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/efeitos da radiação , Alquilantes/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Morte Celular/fisiologia , Células Cultivadas , Sequência Conservada , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dietilnitrosamina/farmacologia , Raios gama , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/efeitos da radiação , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Radiação Ionizante , Ratos , Homologia de Sequência do Ácido Nucleico , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Irradiação Corporal Total , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
E2F-1 induces p53 accumulation and E2F-1 and p53 form a physical complex, which affects the ability of E2F-1 to activate transcription. We mapped the domains on E2F-1 that interact with p53 and found two p53-binding domains. To understand the functional consequences of the E2F-1/p53 association on p53 activities we identified the domains of E2F-1 that were responsible for the accumulation of p53. Unexpectedly, we found that the E2F-1 transactivation domain was dispensable for p53 induction. By contrast, further deletion of the DP-1 interaction/'marked' box domain eliminated p53 accumulation. Radiolabeling pulse/chase analysis demonstrated that E2F-1 caused post-translational stabilization of p53. Although E2F-1 caused the stabilization of p53, E2F-1 expression impaired p53-dependent transactivation. Thus, the E2F-1 : p53 interaction may provide a checkpoint function to inactivate overactive E2F-1, but the association may also inactivate p53 transactivation to allow cell cycle progression.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação , Ciclo Celular , Células Cultivadas , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Camundongos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Proteína Supressora de Tumor p14ARFRESUMO
Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.
Assuntos
Regulação da Expressão Gênica , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos T/citologia , Animais , Apoptose , Relação CD4-CD8 , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Sobrevivência Celular , Citometria de Fluxo , Imunofluorescência , Deleção de Genes , Marcação In Situ das Extremidades Cortadas , Janus Quinase 3 , Camundongos , Camundongos Knockout , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/metabolismo , Timo/citologia , Timo/crescimento & desenvolvimento , Proteína X Associada a bcl-2RESUMO
One consequence of transforming growth factor beta (TGF-beta) treatment is inhibition of Cdk4 synthesis, and this is dependent on p53. Here, we show that the 5' untranslated region (UTR) of the cdk4 mRNA is both necessary and sufficient for wild-type p53-dependent TGF-beta-regulated translational inhibition of cdk4. Wild-type p53 bound selectively to the 5' UTR of the cdk4 mRNA and inhibited translation of RNAs that contain this region. RNA binding and translational control are two genetically separable functions of p53, as are specific and nonspecific RNA binding. Moreover, transactivation-defective mutants of p53 retain the ability to regulate cdk4 translation. Our findings suggest that p53 functions as a regulator of translation in response to TGF-beta in vivo.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas , RNA não Traduzido/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regiões 5' não Traduzidas , Animais , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Regulação para Baixo , Mutação , Proteína Supressora de Tumor p53/genéticaRESUMO
The p19(ARF) tumor suppressor antagonizes Mdm2 to induce p53-dependent cell cycle arrest. Individual TKO (triple knock out) mice nullizygous for ARF, p53, and Mdm2 develop multiple tumors at a frequency greater than those observed in animals lacking both p53 and Mdm2 or p53 alone, demonstrating that p19(ARF) can act independently of the Mdm2-p53 axis in tumor surveillance. Reintroduction of ARF into TKO mouse embryo fibroblasts (MEFs), but not into those lacking both p53 and ARF, arrested the cell division cycle in the G1 phase. Inhibition of the retinoblastoma protein had no effect on the ability of ARF to arrest TKO MEFs. Thus, in the absence of Mdm2, p19(ARF) interacts with other targets to inhibit cell proliferation.
Assuntos
Genes Supressores de Tumor , Proteínas Nucleares , Proteínas/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Células Cultivadas , Imunofluorescência , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Reação em Cadeia da Polimerase , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p14ARF , Proteína Supressora de Tumor p53/genéticaRESUMO
Activation of the p53 tumor suppressor leads to either a cell cycle arrest or to apoptosis and the factors that influence these responses are poorly understood. It is clear, however, that p53 regulates these processes by inducing a series of downstream target genes. One recently identified p53-target gene, EI24 (alias PIG8), induces apoptosis when ectopically expressed. To better understand the biological properties of EI24 and its potential relevance to disease, in particular cancer, we determined the chromosomal location and pattern of gene expression of EI24. EI24 is widely expressed in adult tissues and throughout mouse embryogenesis. The genomic locus of EI24 was mapped to the proximal region of mouse chromosome 9 and human chromosome 11q23-->q24, a region frequently altered in human cancers. These results suggest that EI24 may play an important role in the p53 tumor suppressor pathway.