RESUMO
The expression of mucosa-associated lymphoid tissue 1 (MALT1) that activates nuclear factor (NF)-κB in lymphocyte lineages is rapidly inactivated in oral carcinoma cells at the invasive front and the patients with worst prognosis. However, its mechanism to accelerate carcinoma progression remains unknown, and this study was carried out to examine the role in invasion. HSC2 oral carcinoma cells stably expressing wild-type MALT1 (wtMALT1) reduced the invasion of basement membrane matrices and collagen gels, and the dominant-negative form (∆MALT1)-expressing cells aggressively invaded into collagen gels. MALT1 decelerated proliferation and migration of cells and downregulated expression of matrix metalloproteinase 2 and 9, which were confirmed by short interfering RNA transfections. Reporter assays and immunoblot analysis showed that MALT1 does not affect the NF-κB pathway but inhibits ERK/MAPK activation. This was confirmed by endogenous MALT1 expression in oral carcinoma cell lines. Orthotopic implantation of ∆MALT1-expressing HSC2 cells in mice grew rapid expansive and invasive tongue tumors in contrast to an absence of tumor formation by wtMALT1-expressing cells. These results demonstrate that MALT1 suppresses oral carcinoma invasion by inhibiting proliferation, migration, and extracellular matrix degradation and that the ERK/MAPK pathway is a target of MALT1 and further suggests a role as a suppressor of carcinoma progression.
Assuntos
Caspases/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Proteínas de Neoplasias/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Progressão da Doença , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Immunoblotting , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Bucais/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/metabolismo , Interferência de RNA , RNA Interferente PequenoRESUMO
DNA degradation is one of the biochemical hallmarks detected in apoptotic cells, and several nucleases have been reported to function cooperatively in this process. It has also been suggested that different sets of nucleases are activated by different stimuli, and induce distinct patterns of DNA degradation. Here we report that apoptosis-enhancing nuclease (AEN) is a novel direct target gene of p53. AEN is induced by p53 with various DNA damage, and its expression is regulated by the phosphorylation status of p53. We demonstrate that AEN is a typical exonuclease with conserved exonuclease domains Exo I-III, and it targets both single- and double-stranded DNA and RNA. AEN induces apoptosis by itself, and the conserved domains are essential for both AEN nuclease activity and its apoptosis-inducing ability. AEN possesses nuclear and nucleolar localization signals, and it translocates from the nucleolus to nucleoplasm upon apoptosis induction. We also show the dislocation of nucleophosmin in conjunction with the translocation of AEN to the nucleoplasm, indicating the ability of AEN in nucleolus disruption. In addition, AEN is shown to be required for efficient DNA fragmentation in p53-dependent apoptosis. These results suggest that AEN is an important downstream mediator of p53 in apoptosis induction.
Assuntos
Apoptose , Dano ao DNA , Exodesoxirribonucleases/metabolismo , Genes p53 , Proteína Supressora de Tumor p53/metabolismo , Fragmentação do DNA , Exodesoxirribonucleases/genética , Exonucleases/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Cinética , Mutação , Neoplasias/genética , FosforilaçãoRESUMO
Tumor suppressor p53 protein is the transcription factor responsible for various genes including DNA repair, growth arrest, apoptosis and antiangiogenesis. Recently, we showed that clathrin heavy chain (CHC), which was originally identified as a cytosolic protein regulating endocytosis, is present in nuclei and functions as a coactivator for p53. Here, we determined the detailed p53-binding site of CHC and a CHC deletion mutant containing this region (CHC833-1406) behaved as a monomer in cells. Monomeric CHC833-1406 still had a higher ability to transactivate p53 than wild-type CHC although this CHC mutant no longer had endocytic function. Moreover, similar to wild-type CHC, monomeric CHC enhances p53-mediated transcription through the recruitment of histone acetyltransferase p300. Immunofluorescent microscopic analysis exhibited that CHC833-1406 is predominantly localized in nuclei, suggesting that there may be a certain regulatory domain for nuclear export in the C-terminus of CHC. Thus, the trimerization domain of CHC is not necessary for the transactivation of p53 target genes and these data provide further evidence that nuclear CHC plays a role distinct from clathrin-mediated endocytosis.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Cadeias Pesadas de Clatrina/fisiologia , Transcrição Gênica , Ativação Transcricional , Proteína Supressora de Tumor p53/fisiologia , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/farmacologia , Dimerização , Endocitose/efeitos dos fármacos , Humanos , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Análise de Sequência de Proteína , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
How secondary palate formation is affected in the cleft lip genotype remains poorly understood. The purpose of this study was to analyze regional patterns of cell proliferation in CL/Fr mouse embryos with or without cleft lip. Pairs of palatal shelves were dissected at E13.5 from CL/Fr normal embryos (CL/Fr-N), CL/Fr embryos with bilateral cleft lip (CL/Fr-BCL), and a control strain of C57BL embryos (C57BL). The explants were examined histologically after 48 hrs of organ culture. Cell kinetics for proliferation in the palatal shelves was examined at E13.5 by the bromodeoxyuridine method in vivo. The CL/Fr-BCL palates fused as well as the CL/Fr-N palates in vitro. There were inter-group differences in the absolute number of BrdU-positive cells and the ratio of positive/(positive+negative) cells in the palate's mesenchyme (C57BL > CL/Fr-N > CL/Fr-BCL) and epithelium (C57BL > CL/Fr-N = CL/Fr-BCL). These findings indicate that a cleft palate follows reduced cell proliferation of secondary palatal mesenchyme in CL/Fr mice.
Assuntos
Fenda Labial/embriologia , Fissura Palatina/embriologia , Palato/embriologia , Animais , Bromodesoxiuridina/metabolismo , Proliferação de Células , Fenda Labial/complicações , Fissura Palatina/etiologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Técnicas de Cultura de ÓrgãosRESUMO
The effect of pressured environment from 1 to 24 ATA (Atmosphere Absolute) on total power spectrum (TP) of physiological tremor, whose value was the sum of power spectra for frequency range in 0.5-50 Hz, was investigated. The main effects obtained were as follows. (1) In the case of 3 ATA, TP during the pressure holding period denoted a similar value as the value in 1 ATA. In the latter half of the period, the TP increased. When the pressured environment is 4 ATA, in which the partial pressure of the nitrogen gas was 3.6 ATA, the TP decreased during the pressure holding period compared with the value in 1 ATA. Nitrogen narcosis was recognized at the partial pressure of 3.6 ATA, thus the effect denoted a decrease of TP. (2) In the cases of 16 and 19 ATA using heliox gas, during the pressure holding period, TP decreased compared with the value in 1 ATA, but in the case of 24 ATA the value increased. The partial pressure of helium gas of 23.6 ATA indicated a high pressure nervous syndrome, therefore, the influence of high pressure on TP was recognized as one of the causes of the increase of TP. (3) The influence of inhibitors of the autonomic nervous system on TP during the pressure holding period of 3 ATA was recognized. After intake of the inhibitor for the parasympathetic nerve (atropine) during the pressure period, TP increased, while for the intake of the inhibitor for the sympathetic nerve (propanol), TP decreased.
Assuntos
Pressão Atmosférica , Narcose por Gás Inerte/fisiopatologia , Tremor/fisiopatologia , Sistema Nervoso Autônomo/fisiologia , Dedos/fisiologia , HumanosRESUMO
We address here the effects of increasing fluencies of UV-radiation on stability, modifications, activity and HDM2-interactions of endogenous p53 tumor suppressor and on cellular damage response of human diploid fibroblasts. Low amounts of UVB/C-radiation induced a transient cell cycle arrest of the cells which correlated with rapid but transient increase in p53 levels. In contrast, high UV-fluency caused cell apoptosis and a slower but sustained increase in p53. Regulation of p53 target genes was highly dependent on the radiation dose used. Whereas low doses induced p21/Cip1/Waf1 and HDM2, high doses induced only GADD45 and BAX increasing the BAX:BCL-2 ratio. The levels of HDM2, a negative regulator of p53, increased only by the low dose of UVC and p53-HDM2 association was promoted. In the absence of HDM2-induction after the high dose of UV-radiation p53-HDM2-interaction was promoted, but HDM2 failed to downregulate p53. p53 site-specific modifications (Ser15, Ser33, Ser37, Lys382) varied kinetically and were dependent on the fluency of the radiation used. Maximal phosphorylation of p53 on Ser15 and Ser33 correlated with increased levels of HDM2-free p53. The results suggest that regulation of p53 and HDM2 by UV-radiation is highly dose-dependent and contributes to the outcome of the cellular response.
Assuntos
Regulação da Expressão Gênica , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Raios Ultravioleta , Northern Blotting , Bromodesoxiuridina/metabolismo , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Serina/química , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2 , Proteínas GADD45RESUMO
Ameloblastic fibro-odontoma is the unique entity of epithelial-ectomesenchymal odontogenic tumors, which is characterized by enamel formation in addition to dentine. We examined immunohistochemically a case of this tumor in which enamel having prism structures was developed in the absence of odontoblast differentiation but was in contact with mesenchymal matrices. Histological examination showed diverse morphological features of epithelial tumor cells, e.g., cuboidal cells comprising tooth bud-like projections, ameloblast- and stellate reticulum-like cells, and residual cells in forms of extended cords or islands of odontogenic epithelium. Immunostaining with anti-amelogenin sera proved that the intracellular production of amelogenins was initiated at the tooth bud-like stage. The secreted amelogenins were detected almost exclusively in the induced enamel and dentinoid areas, as well as in the core region of cementicle-like spheres deposited in the encapsulating stroma. The results obtained indicate that the odontogenic tumor epithelia and its products, i.e., amelogenins, participate in multifaceted aspects of dental hard tissue formation that takes place during oncogenesis.
Assuntos
Proteínas do Esmalte Dentário/metabolismo , Esmalte Dentário/patologia , Dentina/patologia , Odontoma/patologia , Adulto , Ameloblastos/metabolismo , Ameloblastos/patologia , Amelogenina , Diferenciação Celular , Corantes , Cemento Dentário/metabolismo , Cemento Dentário/patologia , Esmalte Dentário/metabolismo , Dentina/metabolismo , Ectoderma/patologia , Células Epiteliais/patologia , Epitélio/patologia , Humanos , Imuno-Histoquímica , Masculino , Neoplasias Mandibulares/metabolismo , Neoplasias Mandibulares/patologia , Mesoderma/patologia , Odontoblastos/patologia , Odontoma/metabolismo , Germe de Dente/metabolismo , Germe de Dente/patologiaRESUMO
Nonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates imperfect mRNAs that contain premature translation termination codons (PTCs) and code for nonfunctional or potentially harmful polypeptides. We show that a novel phosphatidylinositol 3-kinase-related protein kinase, hSMG-1, is a human ortholog of a product of Caenorhabditis elegans smg-1, one of seven smg genes involved in NMD. hSMG-1 phosphorylates hUPF1/SMG-2 in vivo and in vitro at specific serine residues in SQ motifs. hSMG-1 can associate with hUPF1/SMG-2 and other components of the surveillance complex. In particular, overexpression of a kinase-deficient point mutant of hSMG-1, hSMG-1-DA, results in a marked suppression of the PTC-dependent beta-globin mRNA degradation; whereas that of wild-type hSMG-1 enhances it. We also show that inhibitors of hSMG-1 induce the accumulation of truncated p53 proteins in human cancer cell lines with p53 PTC mutation. Taken together, we conclude that hSMG-1 plays a critical role in NMD through the direct phosphorylation of hUPF1/SMG-2 in the evolutionally conserved mRNA surveillance complex.
Assuntos
Fosfatidilinositol 3-Quinases/química , Sequência de Aminoácidos , Animais , Northern Blotting , Caenorhabditis elegans , Linhagem Celular , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Globinas/metabolismo , Células HeLa , Humanos , Metaloendopeptidases , Modelos Genéticos , Dados de Sequência Molecular , Família Multigênica , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Filogenia , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Isoformas de Proteínas , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , TransfecçãoRESUMO
Osmotic shock induced transient stabilization of p53, possibly due to increased degradation of Mdm2. Stabilized p53 was activated by p38(MAPK), resulting in G(1) arrest through induction of p21(WAF1). Among the postulated phosphorylation sites involved in p53 stabilization or activation (Ser(15), Ser(20), Ser(33), and Ser(46)), only Ser(33) was phosphorylated. Furthermore, interaction of p53 with the transcriptional coactivator p300 was induced, and Lys(382) of p53 was acetylated. Although inhibition of p38(MAPK) did not prevent nuclear accumulation of p53, phosphorylation of Ser(33) was markedly suppressed by SB203580, a specific inhibitor of p38(MAPK). Under these conditions, acetylation of Lys(382) and induction of p21(WAF1) were also inhibited, and cells with elevated levels of p53 showed normal cell cycle progression. Activated p38(MAPK) phosphorylated endogenous p53 at Ser(33) in living cells. In stable transformants expressing dominant negative MKK6, an upstream protein kinase of p38(MAPK), p53 stabilization was induced normally following osmotic shock, but phosphorylation of Ser(33), acetylation of Lys(382), and induction of p21(WAF1) were almost completely inhibited. These results suggest that phosphorylation at Ser(33) by p38(MAPK) is critical for activation of p53 following osmotic shock. Phosphorylation of neither Ser(15) nor Ser(20) was needed in this activation.
Assuntos
Fase G1 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pressão Osmótica , Fosforilação , Serina/química , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Animais , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Regulação para Baixo , Proteína p300 Associada a E1A , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Genes Dominantes , Genes p53 , Humanos , Imidazóis/farmacologia , Luciferases/metabolismo , Lisina/química , MAP Quinase Quinase 6 , Camundongos , Camundongos Knockout , Proteínas Nucleares , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transativadores , Transcrição Gênica , Ativação Transcricional , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Overexpression of the cyclin D1 (CCND1) gene, encoding a downstream effector of mitogenic signals that plays a central role in G1 phase progression, is often found in cancerous cells. In sporadic breast cancer (BC), this is one of the most frequent and early genetic lesions identified so far, found in more than 50% of the tumors. Inhibitors of the mevalonate/protein prenylation pathway belong to a new family of cancer therapeutic agents that act by blocking intracellular mitogenic signal transduction pathways, thereby preventing expansion of pre-cancerous foci and inhibiting growth of transformed cells. It is not known at present whether constitutively high intracellular levels of cyclin D1 might interfere with the cytostatic actions of mevalonate/protein prenylation inhibitors. This possibility was investigated here by assessing the cell cycle effects of Simvastatin, a non-toxic upstream inhibitor of the mevalonate pathway, on human BC MCF-7 cells expressing either normal or enhanced levels of cyclin D1 from of a stably transfected, tet-inducible expression vector. Results show that constitutive overexpression of this protein, such as that found in sporadic BCs, does not influence the growth inhibitory effects of Simvastatin in vitro. In addition, D1-overexpressing embryo fibroblasts were also found to be responsive to the cell cycle effects of mevalonate/protein prenylation pathway blockade, further suggesting that high intracellular levels of cyclin D1 do not prevent the cytostatic actions of compounds targeting this metabolic pathway.
Assuntos
Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Proteínas de Neoplasias/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Sinvastatina/uso terapêutico , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Humanos , Ácido Mevalônico , RatosRESUMO
Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte , Proteínas de Choque Térmico , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose , Northern Blotting , Dano ao DNA , Doxorrubicina/farmacologia , Raios gama , Regulação da Expressão Gênica , Genes Reporter , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oligonucleotídeos Antissenso/metabolismo , Fosforilação , Ligação Proteica , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Raios UltravioletaRESUMO
We propose a new role of retinoblastoma protein as a cell growth activator in its phosphorylated form. The hyper-phosphorylated retinoblastoma protein generated by the action of cdk2/cyclin E strongly stimulated the activity of DNA polymerase alpha, but did not stimulate DNA polymerases delta, epsilon, or primase. But, cdk4/cyclin D-phosphorylated retinoblastoma protein showed little stimulation. Hyper-phosphorylated retinoblastoma protein interacted with the catalytic subunit of DNA polymerase alpha, and stabilised DNA polymerase alpha from heat inactivation at 45 degrees C. These results suggest that in G1 phase, hypo-phosphorylated retinoblastoma protein suppresses the progression of cell cycle as a transcription inhibitor, but that after phosphorylation by cdk2/cyclin E at the G1/S boundary, hyper-phosphorylated retinoblastoma protein acts as a cell-cycle promoter by optimising the DNA polymerase alpha reaction.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , DNA Polimerase I/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteína do Retinoblastoma/metabolismo , Animais , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , DNA Polimerase I/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Temperatura Alta , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/fisiologia , Spodoptera/genética , TransfecçãoRESUMO
Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP) and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20), only DDP treatment induced p53 phosphorylation at serine 15 (Ser15). Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM) cell lines, the role of ATM in drug-induced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Proteínas de Ligação a DNA , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares , Paclitaxel/farmacologia , Células Tumorais Cultivadas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Androstadienos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Neoplasias do Colo/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Proteína Quinase Ativada por DNA , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Transcrição Gênica , Células Tumorais Cultivadas/efeitos dos fármacos , WortmaninaRESUMO
Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.
Assuntos
Apoptose/genética , Apoptose/fisiologia , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Proteínas Nucleares , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Transformação Celular Neoplásica , Dano ao DNA , Genes p53 , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Modelos Biológicos , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Ativação Transcricional , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
p53 is required for the induction of a G(1) and/or G(2) irreversible arrest after gamma irradiation (IR), whereas blocked DNA replication causes a p53-independent S-phase arrest. We have examined the response to p53 when DNA synthesis is blocked by hydroxyurea (HU) or aphidicolin or when DNA is damaged by gamma IR. Similarly to gamma IR, blocked DNA synthesis induces high levels of phosphorylated nuclear p53. Surprisingly, several (but not all) p53 transcriptional targets that are rapidly induced by gamma IR are weakly or not induced when DNA replication is blocked. Moreover, the p53 response to gamma IR is inhibited by pretreatment of cells with HU or aphidicolin, suggesting that blocked DNA replication prevents p53 from being fully active as a transcription factor. HU-induced stabilization of p53 neither requires functional ATM (ataxia telangiectasia mutated), nor interferes with the gamma IR-dependent activation of the ATM kinase. Thus, stalled replication forks activate kinases that modify and stabilize p53, yet act downstream of ATM to impair p53 transcriptional activity. The ramifications of this novel regulation of p53 are discussed.
Assuntos
Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular , Neoplasias Colorretais , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA , Fase G1 , Fase G2 , Raios gama , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
The present studies were undertaken to investigate the sexual dimorphism of porcine amelogenins and to gain information as to whether excesses of male amelogenins, if any, possess functional significance in protein-crystal interactions. Enamel proteins, including the intact full-length amelogenins and their degraded polypeptides, were isolated from the secretory enamel of male and female pigs. To identify the amelogenins among the separated pools of male- and female-matrix proteins, rabbit anti-C13 and C25 peptide sera were used, which reacted specifically with the conserved C-terminal domain. Immunoblotting showed that a few extra members of the amelogenins, sharing common epitopes at the C-terminus, were recognized in male products. The apparent yield of the male amelogenins was only marginal, on the basis of their stained intensities on the gel, but the secreted male amelogenins demonstrated selective (probably the strongest among the amelogenins) adsorption properties onto apatite crystals. Reflecting the general symmetric electrophoretic profiles of the male- and female-enamel proteins in toto, there were no sex-linked differences in the protein-crystal interaction and the resulting regulatory function of crystal precipitation.
RESUMO
The stress-responsive p38 MAPK, when activated by genotoxic stresses such as UV radiation, enhances p53 activity by phosphorylation and leads to cell cycle arrest or apoptosis. Here we report that a member of the protein phosphatase type 2C family, Wip1, has a role in down-regulating p38-p53 signaling during the recovery phase of the damaged cells. Wip1 was originally identified as a gene whose expression is induced following gamma or UV radiation in a p53-dependent manner. We found that Wip1 is also inducible by other environmental stresses, such as anisomycin, H(2)O(2) and methyl methane sulfonate. UV-induction of Wip1 requires p38 activity in addition to the wild-type p53. Wip1 selectively inactivates p38 by specific dephosphorylation of its conserved threonine residue. Furthermore, Wip1 expression attenuates UV-induced p53 phosphorylation at Ser33 and Ser46, residues previously reported to be phosphorylated by p38. Wip1 expression also suppresses both p53-mediated transcription and apoptosis in response to UV radiation. These results suggest that p53-dependent expression of Wip1 mediates a negative feedback regulation of p38-p53 signaling and contributes to suppression of the UV-induced apoptosis.
Assuntos
Regulação para Baixo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Plantas/fisiologia , Inibidores de Serina Proteinase , Proteína Supressora de Tumor p53/metabolismo , Animais , Anisomicina/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose , Northern Blotting , Células COS , Núcleo Celular/metabolismo , Dano ao DNA , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Raios gama , Glutationa Transferase/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Imidazóis/farmacologia , Luciferases/metabolismo , Metanossulfonato de Metila/farmacologia , Microscopia de Fluorescência , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Transdução de Sinais , Treonina/metabolismo , Fatores de Tempo , Transfecção , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Our recent work has shown that activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. We have extended these observations by investigating two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, which are known to be regulated by mitogen stimulation. We now show that the phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas ras/metabolismo , Sequência de Aminoácidos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Quinase 3 da Glicogênio Sintase , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfopeptídeos/isolamento & purificação , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Serina/metabolismoRESUMO
Through direct cloning of p53 binding sequences from human genomic DNA, we have isolated a novel gene, designated p53AIP1 (p53-regulated Apoptosis-Inducing Protein 1), whose expression is inducible by wild-type p53. Ectopically expressed p53AIP1, which is localized within mitochondria, leads to apoptotic cell death through dissipation of mitochondrial A(psi)m. We have found that upon severe DNA damage, Ser-46 on p53 is phosphorylated and apoptosis is induced. In addition, substitution of Ser-46 inhibits the ability of p53 to induce apoptosis and selectively blocks expression of p53AIP1. Our results suggest that p53AIP1 is likely to play an important role in mediating p53-dependent apoptosis, and phosphorylation of Ser-46 regulates the transcriptional activation of this apoptosis-inducing gene.
Assuntos
Apoptose/fisiologia , Proteínas/genética , Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adenocarcinoma , Animais , Proteínas Reguladoras de Apoptose , Neoplasias da Mama , Células COS , Carcinoma Pulmonar de Células não Pequenas , Clonagem Molecular , Neoplasias Colorretais , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Dano ao DNA/fisiologia , Fibroblastos/citologia , Regulação Neoplásica da Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Pulmonares , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutagênese/fisiologia , Osteossarcoma , Fosforilação , Ligação Proteica/fisiologia , Serina/metabolismo , Transdução de Sinais/genética , Pele/citologia , Ativação Transcricional/fisiologia , Células Tumorais CultivadasRESUMO
The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) can inhibit the growth and induce apoptosis of tumor cells. In this study we analysed the growth suppressive effect of HPR on human breast cancer cell lines in vitro and the role of the retinoblastoma protein (pRb) in this response. Treatment of MCF7, T47D and SKBR3 for 24 - 48 h with 3 microM HPR, a concentration attainable in vivo, resulted in growth inhibition and marked dephosphorylation of pRb involving Ser612, Thr821, Ser795 and Ser780, target residues for cyclin-dependent kinase 2 (Cdk2) the former two, and Cdk4 the latter two. Interestingly, this dephosphorylation of pRb occurred in S-G2-M phase cells, as revealed by experiments on cells fractionated by FACS according to the cell cycle phase, hence suggesting that the retinoid interferes with the regulation of pRb phosphorylation. The in vitro phosphorylation of a GST-pRb recombinant substrate by Cdk2 immunocomplexes from MCF7, T47D and SKBR3 was markedly suppressed after HPR treatment, whereas that by Cdk4 complexes was suppressed in T47D and SKBR3 but not in MCF7. The steady-state levels of Cdk2, Cdk4 and Cyclin A proteins were unaffected by HPR, while those of Cyclin D1 were significantly reduced in all three cell lines. Interestingly, Cyclin D1 downregulation by HPR correlated with transcriptional repression, but not with enhanced proteolysis of Cyclin D1 typically elicited by other retinoids. Collectively, our data suggest that the antiproliferative activity of HPR arises from its capacity to maintain pRb in a de-phosphorylated growth-suppressive status in S-G2/M, possibly through Cyclin D1 downregulation and inhibition of pRb-targeting Cdks. Oncogene (2000) 19, 4035 - 41.