RESUMO
The study is focused on the hydrogeological conditions and the chemistry of groundwater of the Vendian aquifer in the western part of the Leningrad oblast (Karelian Isthmus and the area near Sosnovy Bor town) and St. Petersburg City, where groundwater features higher radioactivity, but nevertheless it is used for drinking water supply. Data on the radiological characteristics, which have been determined in the estimation of the quality of groundwater used for drinking are generalized and analyzed. These characteristics include the gross alpha and gross beta activity and the specific activity of natural radionuclides 222Rn, 226Ra, 228Ra, 210Pb, 210Po, 238U, and 234U. The data were subjected to statistical and correlation analysis to determine the hygienic criteria for the use of groundwater of this aquifer for drinking water supply and to study the sources and the processes of formation of the natural radiological background. Groundwater quality standards were shown to be exceeded in the majority of the analyzed wells. The brackish water in the southern, deeper, part of the aquifer system was shown to have higher radioactivity and relatively high concentrations of 226Ra, 228Ra, 210Pb, and 210Po, compared with fresh water in the northern part of the territory, of which higher, though nonuniform, 222Rn activity is typical. Relationships between the radiation characteristics of groundwater are considered along with the causes of formation of groundwater radionuclide composition as a result of the higher radioactivity of the host deposits and the chemistry of groundwater; changes in the radiological and hydrochemical background groundwater characteristics from the north to the south are characterized in accordance with the subsidence of the aquifer system and an increase in the stagnation of the hydrochemical regime. The analysis of the well-known relationship between the concentrations of radium isotopes in groundwater, uranium and thorium isotopes in the host rocks, and groundwater residence time in the aquifer, along with the comparison of the available field data with calculation results, suggested the conclusion that the concentration of uranium in the water-bearing rocks in the major portion of the area under consideration is higher than its regional mean values.
Assuntos
Água Potável , Água Subterrânea , Monitoramento de Radiação , Radioatividade , Urânio , Poluentes Radioativos da Água , Urânio/análise , Água Potável/análise , Monitoramento de Radiação/métodos , Chumbo/análise , Poluentes Radioativos da Água/análise , Água Subterrânea/química , Radioisótopos/análise , Federação RussaRESUMO
According to the Neurodevelopmental hypothesis, the long-lasting cognitive deficit in schizophrenia and other types of neuropathology may occur by injurious factors, such as hypoxia, traumas, infections that take place during pre- and postnatal development, at least at early stages. These pathological conditions are often associated with the high production of pro-inflammatory cytokine interleukin-1B (IL-1B) by the cells of immune and nervous systems. We investigated the expression of genes involved in the neuroplastic regulation (Fgf2 and Timp2) in medial prefrontal cortex and dorsal and ventral regions of hippocampus of adult rats that were treated with IL-1beta between P15 and P21. The learning impairment in IL-1beta-treated rats is accompanied by lower FGF-2 mRNA levels in medial prefrontal cortex and ventral (not dorsal) hippocampus, but TIMP-1 was not affected. No differences in TIMP-1 and FGF-2 mRNA expressions were observed in untrained IL-1beta-treated when compared to control rats.
Assuntos
Fator 2 de Crescimento de Fibroblastos/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/crescimento & desenvolvimento , Interleucina-1beta/farmacologia , Córtex Pré-Frontal/crescimento & desenvolvimento , RNA Mensageiro/biossíntese , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Animais , Hipocampo/patologia , Hipóxia Encefálica/metabolismo , Hipóxia Encefálica/patologia , Interleucina-1beta/metabolismo , Masculino , Córtex Pré-Frontal/patologia , Ratos , Ratos WistarRESUMO
Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFalpha protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFalpha) and aha-1 (HIFbeta), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2alpha-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2alpha, but not HIF-1alpha. These results identify TWIST1 as a direct target gene of HIF-2alpha, which may provide insight into the acquired metastatic capacity of hypoxic tumors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Hipóxia Celular , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Desferroxamina/farmacologia , Genoma , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 and the key factor in cellular response to low oxygen tension. Expression of HIF-1alpha protein is associated with poor patient survival and therapy resistance in many types of solid tumors. Insight into HIF-1alpha regulation in solid tumors is important for therapeutic strategies. In this study, we determined the pathophysiological relevance of HIF-1alpha regulation by the oncogenic phosphatidylinositol 3'-kinase (PI 3-kinase)/Akt signaling pathway. We modeled the physiology of hypoxic tumor regions by culturing carcinoma cells under low oxygen tension in the absence of serum. We observed that hypoxic induction of HIF-1alpha protein was decreased by serum deprivation. Overexpression of dominant-active Akt1 restored HIF-1alpha expression, whereas inhibition of PI 3-kinase activity reduced hypoxic HIF-1alpha protein levels to a similar extent as serum deprivation. Immunohistochemical analysis of 95 human breast cancers revealed that lack of Akt1 phosphorylation correlates with low HIF-1alpha levels. To our knowledge, this is the first reported comparison between HIF-1alpha expression and Akt phosphorylation in human carcinomas. We conclude that Akt activity is physiologically relevant for HIF-1alpha expression in breast cancer. This implies that HIF-1alpha function might be therapeutically targeted by inhibition of the PI 3-kinase/Akt pathway.
Assuntos
Neoplasias da Mama/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Humanos , FosforilaçãoRESUMO
The hypoxia-inducible factor 1 (HIF-1) plays a critical role in cellular responses to hypoxia. The aim of the present study was to evaluate which genes are induced by hypoxia, and whether this induction is mediated by HIF-1, by expression microarray analysis of wt and HIF-1alpha null mouse fibroblasts. Forty-five genes were up-regulated by hypoxia and 40 (89%) of these were regulated by HIF-1. Of the 114 genes down-regulated by hypoxia, 19 (17%) were HIF-1-dependent. All glycolytic enzymes were strongly up-regulated by hypoxia in a HIF-1-dependent manner. Genes already known to be related to hypoxia, such as glucose transporter 1, BNIP3, and hypoxia-induced gene 1, were induced. In addition, multiple new HIF-1-regulated genes were identified, including genes involved in metabolism (adenylate kinase 4, galactokinase), apoptosis (galectin-3 and gelsolin), and invasion (RhoA). Genes down-regulated by hypoxia were involved in cytoskeleton maintenance (Rho kinase), mRNA processing (heterogeneous nuclear ribonucleoprotein H1 and splicing factor), and DNA repair (REV3). Furthermore, seven cDNAs from genes with unknown function or expressed sequence tags (ESTs) were up-regulated and 27 such cDNAs were down-regulated. In conclusion, hypoxia causes down- rather than up-regulation of gene expression and HIF-1 seems to play a major role in the regulation of hypoxia-induced genes.
Assuntos
Proteínas de Ligação a DNA/genética , Fibroblastos/fisiologia , Hipóxia/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Regulação para Cima/genética , Animais , Apoptose/genética , Movimento Celular/genética , Citoesqueleto/genética , Reparo do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Regulação para Baixo/genética , Galactoquinase/genética , Galectina 3/genética , Gelsolina/genética , Perfilação da Expressão Gênica/métodos , Transportador de Glucose Tipo 1 , Glicólise/genética , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Proteínas de Membrana/genética , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Hypoxia has clinically been associated with resistance to chemotherapy. The aim of this study was to investigate whether hypoxia induces resistance to doxorubicin and mitoxantrone, two common drugs in cancer treatment, in MCF-7 breast cancer cells, and SW1573 non-small lung cancer cells. In addition, the role of drug transporters P-gp, BCRP and MRP1 was analysed. Hypoxia induced resistance in MCF-7 cells to mitoxantrone shifted the IC(50) value from 0.09 microM (+/-0.01) to 0.54 microM (+/-0.06) under hypoxia, whereas survival of MCF-7 and SW1573 cells in the presence of doxorubicin was not altered. Accumulation of mitoxantrone and daunorubicin, a doxorubicin fluorescent homologue, appeared to be 5.3 and 3.2 times lower in MCF-7 cells, respectively. Cytotoxicity assays showed no increased functionality of the drug transporters P-gp, BCRP and MRP1 under hypoxia. In addition, protein levels of these drug transporters were not changed. Medium of the MCF-7 cells became more acidic under hypoxia thereby causing a decreased uptake of mitoxantrone. Hypoxia induces mitoxantrone resistance in MCF-7 cells not mediated by the three major MDR transporters. Hypoxia-induced acidification may cause this resistance by decreased cellular uptake together with a lowered cytotoxicity due to pH-dependent topoisomerase type II activity.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Hipóxia , Mitoxantrona/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Daunorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Glicólise , Humanos , Concentração de Íons de Hidrogênio , Immunoblotting , Concentração Inibidora 50RESUMO
BACKGROUND: Intratumorous hypoxia triggers a broad cellular response mediated by the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1alpha concentrations increase during breast carcinogenesis, and are associated with poor prognosis. An earlier study noted two HIF-1alpha overexpression patterns: diffuse scattered throughout the tissue and confined to perinecrotic cells. AIMS: To investigate the prognostic impact of these different HIF-1alpha overexpression patterns in relation to its downstream effectors carbonic anhydrase (CA) IX and glucose transporter 1 (GLUT-1). METHODS: HIF-1alpha, CA IX, and GLUT-1 expression was studied by immunohistochemistry, including double staining for CA IX and HIF-1alpha. Clinical data included disease free survival, lymph node status, and tumour size. RESULTS: HIF-1alpha overexpression (44% of cases) had a perinecrotic (13.5%) or diffuse staining pattern (30.5%). CA IX expression was detectable in 12.5% of breast cancers, whereas GLUT-1 expression was seen in 29%, with both showing perinecrotic membrane staining. Perinecrotic HIF-1alpha overexpression was highly associated with CA IX and GLUT-1 overexpression, and double staining for HIF-1alpha and CA IX showed strong expression in the same cells. Diffusely overexpressed HIF-1alpha was not associated with CA IX or GLUT-1 expression. Patients with diffuse HIF-1alpha staining had a significantly better prognosis than patients with perinecrotically overexpressed HIF-1alpha. CONCLUSIONS: Different regulation pathways of HIF-1alpha overexpression exist in breast cancer: (1) hypoxia induced, perinecrotic HIF-1alpha overexpression with strong expression of hypoxia associated genes (CA IX and GLUT-1), which is associated with a poor prognosis; and (2) diffuse HIF-1alpha overexpression lacking major hypoxia associated downstream effects, resulting in a more favourable prognosis.
Assuntos
Neoplasias da Mama/química , Proteínas de Neoplasias/análise , Fatores de Transcrição/análise , Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Anidrase Carbônica IX , Anidrases Carbônicas/análise , Feminino , Transportador de Glucose Tipo 1 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imuno-Histoquímica/métodos , Proteínas de Transporte de Monossacarídeos/análise , Necrose , Invasividade Neoplásica , Prognóstico , Análise de SobrevidaRESUMO
BACKGROUND: Recent evidence suggests that functional intratumorous lymph vessels may be absent from some human cancers. This could result from either the failure of tumours to induce lymphangiogenesis, or the collapse of lymph vessels, caused by high interstitial tumour pressure. METHODS: To differentiate between these two hypotheses, paraffin wax embedded clinical specimens from normal breast (n = 13), usual ductal hyperplasia (n = 11), ductal carcinoma in situ (n = 21), and invasive breast cancer (n = 40) were compared for lymphatic and blood vessel density by immunohistochemistry with antibodies to the lymphatic endothelial hyaluronan receptor (LYVE-1) and CD31, respectively. RESULTS: Lymph vessel density was lower than blood vessel density in normal breast tissue. Within breast lobuli, lymph vessels were absent. In premalignant lesions blood microvessel density increased, whereas no increase in lymph vessels could be seen intralesionally. In invasive cancers, lymph vessels were absent in all but a few cases, where probably some pre-existing lymph vessels remained, although blood microvessel density was once again increased. CONCLUSION: Unlike angiogenesis, lymphangiogenesis is absent during breast carcinogenesis. This, and not rising interstitial pressure caused by an increase in the size of lesions, explains the absence of intratumorous lymph vessels in invasive breast cancer.
Assuntos
Neoplasias da Mama/patologia , Transformação Celular Neoplásica/patologia , Sistema Linfático/patologia , Mama/anatomia & histologia , Mama/irrigação sanguínea , Mama/patologia , Neoplasias da Mama/irrigação sanguínea , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Progressão da Doença , Feminino , Humanos , Hiperplasia/patologia , Neovascularização Patológica/patologia , Lesões Pré-Cancerosas/patologiaRESUMO
Multiple adenovirus (Ad) early proteins have been shown to inhibit transcription activation by p53 and thereby to alter its normal biological functioning. Since these Ad proteins affect the activity of p53 via different mechanisms, we examined whether this inhibition is target gene specific. In addition, we analyzed whether the same Ad early proteins have a comparable effect on transcription activation by the recently identified p53 homologue p73. Our results show that the large E1B proteins very efficiently inhibited the activity of p53 on the Bax, p21(Waf1), cyclin G, and MDM2 reporter constructs but had no effect on the activation of the same reporter constructs by p73, with the exception of some inhibition of the Bax promoter by Ad12 E1B. The repressive effect of the E1A proteins on p53 activity is less than that seen with the large E1B proteins, but the E1A proteins inhibit the activity of both p53 and p73. We could not detect significant inhibition of p53 functions by E4orf6, but a clear repression of the transcription activation by p73 by this Ad early protein was observed. In addition, we found that stable expression of the Ad5 E1A and that of the E1B protein both caused increased p73 protein expression. The large E1B and the E4orf6 proteins together do not target the p73 protein for rapid degradation after adenoviral infection, as has previously been found for the p53 protein, probably because the large E1B protein does not interact with p73. Our results suggest that the p53 and p73 proteins are both inactivated after Ad infection and transformation but via distinct mechanisms.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Genes Reporter/genética , Genes Supressores de Tumor , Humanos , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ativação Transcricional/genética , Transformação Genética , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
We recently reported the identification of a mouse cDNA encoding a new p53-associating protein that we called Mdmx because of its structural similarity to Mdm2, a well-known p53-binding protein. Here we report the isolation of a cDNA encoding the human homolog of Mdmx. The ORF of the cDNA encodes a protein of 490 amino acids, 90% similar to mouse Mdmx. The homology between Mdmx and Mdm2 is most prominent in the p53-binding domain and the putative metal-binding domains. The Mdmx protein, which, based on SDS-PAGE, has a MW of 80 kDa, can bind p53 in vitro. The human MDMX gene is transcribed in all tissues tested, with high levels in thymus. By fluorescence in situ hybridization analysis we mapped the mouse mdmx gene to chromosome 1 (region F-G) and the human MDMX gene to chromosome 1q32.
Assuntos
Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 1/genética , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Distribuição Tecidual , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Wilms' tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family. As a result of alternative RNA splicing, the gene can be expressed as four polypeptides that differ in the presence or absence of a stretch of 17 amino acids just NH2 terminal of the four zinc fingers and a stretch of three amino acids (+/-KTS) between zinc fingers 3 and 4. In this study, cDNA constructs encoding the four human Wilms' tumor 1 splice variants were transiently transfected into the p53-negative Hep3B and the p53-positive HepG2 hepatoma cell lines. Morphological assessment of the WT1-expressing cells showed that the WT1(-KTS) splice variants induced apoptosis in both cell lines, whereas the WT1(+KTS) isoforms did not. The induction of apoptosis by the WT1(-KTS) isoforms appears to be p53 independent in the hepatoma cell lines. Furthermore, it was found that the WT1(-KTS)-induced apoptosis could not be suppressed by coexpression of either the Mr 21,000 E1B, the Bcl-2, or the BAG-1 protein. Coexpression of either the epidermal growth factor receptor or the insulin receptor, however, partially rescued the cells from apoptosis.
Assuntos
Apoptose , Carcinoma Hepatocelular/genética , Proteínas de Ligação a DNA/genética , Receptores ErbB/metabolismo , Genes p53 , Neoplasias Hepáticas/genética , Receptor de Insulina/metabolismo , Fatores de Transcrição/genética , Western Blotting , Proteínas de Ligação a DNA/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteínas WT1RESUMO
E2F transcription factors are key regulators of transcription during the cell cycle. E2F activity is regulated at the level of transcription and DNA binding and by complex formation with the retinoblastoma pocket protein family. We show here that free E2F-1 and E2F-4 transcription factors are unstable and that their degradation is mediated by the ubiquitin-proteasome pathway. Both E2F-1 and E2F-4 are rendered unstable by an epitope in the carboxyl terminus of the proteins, in close proximity to their pocket protein interaction surface. We show that binding of E2F-1 to pRb or E2F-4 to p107 or p130 protects E2Fs from degradation, causing the complexes to be stable. The increased stability of E2F-4 pocket protein complexes may contribute to the maintenance of active transcriptional repression in quiescent cells. Surprisingly, adenovirus transforming proteins, which release pocket protein-E2F complexes, also inhibit breakdown of free E2F. These data reveal an additional level of regulation of E2F transcription factors by targeted proteolysis, which is inhibited by pocket protein binding and adenovirus early region 1 transforming proteins.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo , Adenoviridae , Linhagem Celular , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F4 , Vetores Genéticos , Meia-Vida , Humanos , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma , Proteína 1 de Ligação ao Retinoblastoma , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-Like , Fator de Transcrição DP1 , Transfecção , Células Tumorais Cultivadas , Proteínas do Envelope Viral/metabolismoRESUMO
Here we report the isolation of a cDNA encoding a new p53-associating protein. This new protein has been called MDMX on the basis of its structural similarity to MDM2, which is especially notable in the p53-binding domain. In addition, the putative metal binding domains in the C-terminal part of MDM2 are completely conserved in MDMX. The middle part of the MDMX and MDM2 proteins shows a low degree of conservation. We can show by co-immunoprecipitation that the MDMX protein interacts specifically with p53 in vivo. This interaction probably occurs with the N-terminal part of p53, because the activity of the transcription activation domain of p53 was inhibited by co-transfection of MDMX. Northern blotting showed that MDMX, like MDM2, is expressed in all tissues tested, and that several mRNAs for MDMX can be detected. Interestingly, the level of MDMX mRNA is unchanged after UV irradiation, in contrast to MDM2 transcription. This observation suggests that MDMX may be a differently regulated modifier of p53 activity in comparison with MDM2. Our study indicates that at least one additional member of the MDM protein family exists which can modulate p53 function.
Assuntos
Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Ativação Transcricional/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos da radiação , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Raios UltravioletaRESUMO
p53 stimulates the transcription of a number of genes, such as MDM2, Waf1, and GADD45. We and others have shown previously that this activity of p53 can be inhibited by adenovirus type 2 or 12 large E1B proteins. Here we show that the adenovirus E1A proteins also can repress the stimulation of transcription by p53, both in transient transfections and in stably transfected cell lines. The inhibition by E1A occurs without a significant effect on the DNA-binding capacity of p53. Furthermore, the activity of a fusion protein containing the N-terminal part of p53 linked to the GAL4 DNA-binding domain can be suppressed by E1A. This indicates that E1A affects the transcription activation domain of p53, although tryptic phosphopeptide mapping revealed that the level of phosphorylation of this domain does not change significantly in E1A-expressing cell lines. Gel filtration studies, however, showed p53 to be present in complexes of increased molecular weight as a result of E1A expression. Apparently, E1A can cause increased homo- or hetero-oligomerization of p53, which might result in the inactivation of the transcription activation domain of p53. Additionally, we found that transfectants stably expressing E1A have lost the ability to arrest in G1 after DNA damage, indicating that E1A can abolish the normal biological function of p53.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/genética , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Adenovírus Humanos/metabolismo , Sequência de Bases , Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/metabolismo , Humanos , Luciferases/biossíntese , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Fosforilação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/biossínteseRESUMO
Apoptin, a small protein derived from chicken anemia virus (CAV), induces apoptosis in human tumor cell lines regardless of whether these express p53 or not. We examined whether the small adenovirus 5 E1B protein of 21 kDa (E1B-21kD), also called E1B-19kD) and Bcl-2 could inhibit apoptin-induced apoptosis in human tumor cell lines and compared this with p53-induced apoptosis. E1B-21kD, but not Bcl-2 was found to inhibit apoptin-induced apoptosis in the osteosarcoma cell lines U2OS and Saos-2. However, neither expression of E1B-21kD nor of Bcl-2 resulted in inhibition of apoptin-induced apoptosis in Hep3B hepatoma cells and kidney rhabdoid tumor G401 cells. Both Bcl-2 and Ad5 E1B-21kD were able to inhibit p53-induced apoptosis in the human tumor cell lines Saos-2 and Hep3B. In Saos-2 and U2OS, but not in Hep3B and G401, expression of E1B-21kD leads to retention of apoptin in the cytoplasm, in that way preventing its nuclear function. These results indicate that proteins inhibiting the p53-induced apoptotic pathway do not block apoptin-induced apoptosis or do so only in a cell type-specific manner. The apoptin-induced apoptotic pathway is distinct from that induced by p53 and, therefore, apoptin is a potential antitumor agent.
Assuntos
Proteínas E1B de Adenovirus/metabolismo , Apoptose , Proteínas do Capsídeo , Capsídeo/farmacologia , Genes p53 , Proteínas Proto-Oncogênicas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Humanos , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2 , Células Tumorais CultivadasRESUMO
P53 can both stimulate transcription via the p53-consensus sequence as well as inhibit gene expression via CAAT-TATA-sequences. Certain viral and cellular proteins can abrogate the p53-dependent stimulation of transcription by physical association. In addition, it has been shown that the large E1B protein of adenovirus type 12 (Ad12), E1B/54 kDa, can block the transcription activation potential of p53, without binding to p53. Here we show that this E1B/54-kDa protein also can prevent the repression of transcription of transfected and endogenous p53 in transient transfections. In cells containing wild-type p53 but stably expressing high levels of E1B/54 kDa, no induction of WAF1 mRNA after X-ray irradiation could be detected. In contrast, expression of another non-p53 binding E1B protein, Ad5 E1B/21 kDa has no effect on WAF-1 expression. Results of an electromobility shift assay indicated that the abrogation of p53-mediated transcription activation by E1B/54 kDa cannot be explained by inhibition of the DNA-binding capacity of p53. A biological consequence of expression of E1B/54 kDa is the loss of G1 cell-cycle arrest after X-ray irradiation, while cells expressing the E1B/21 kDa still arrest in G1 after DNA damage.
Assuntos
Proteínas E1B de Adenovirus/fisiologia , Fase G1 , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Adenovírus Humanos/química , Animais , Sequência de Bases , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , DNA/metabolismo , Dano ao DNA , Regulação da Expressão Gênica/fisiologia , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Ratos , Ativação Transcricional/fisiologia , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human chromosome 11 was introduced into adenovirus-transformed baby rat kidney (BRK) cells by microcell-mediated chromosome transfer. The resulting microcell hybrids (MCHs) showed a reduced ability to form tumors upon s.c. injection into athymic mice. Further analysis, with the use of defined deletion chromosomes of 11p, indicated that the presence of region 11p13-p12 is necessary for the suppression of tumorigenicity. In contrast, the presence of region 11p15-14.1 appeared to increase the rate of tumor growth. Expression studies on the human Wilms' tumor I (WTI) and the insulin-like growth factor II (IGF-II) genes, which lie in regions 11p13 and 11p15, respectively, suggested the involvement of both genes in determining the degree of suppression of tumorigenicity. Finally, stable expression of a murine WTI protein in the adenovirus-transformed cells resulted in almost complete suppression of tumorigenicity, establishing the WTI protein as a tumor suppressor in this cell system.
Assuntos
Cromossomos Humanos Par 11 , Genes do Tumor de Wilms , Neoplasias Experimentais/genética , Tumor de Wilms/genética , Adenovírus Humanos/genética , Animais , Divisão Celular , Transformação Celular Viral , DNA de Neoplasias/genética , Feminino , Técnicas de Transferência de Genes , Genes ras , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Camundongos Nus , RatosRESUMO
Loss of function of the tumor-suppressor protein p53 is, in general, either caused by mutation, inducing a conformational change, or by binding to inactivating cellular (e.g. MDM2) or viral (e.g. SV40 large T) proteins. In adenovirus type 12 (Ad12)-transformed cells, p53 is stabilized without detectable binding to the Ad12 E1B/54 kDa protein and still present in a wild-type conformation but contains a mutant-like activity in cellular transformation. In this study we examined whether the changed characteristics of p53 in Ad12-transformed cells are correlated with changes in phosphorylation or complex formation of the protein. By making tryptic phosphopeptide maps we found a significant increase in the phosphorylation of the N-terminus of p53. Furthermore, expression of E1A was found to be essential for the altered phosphorylation, while expression of only Ad12 E1B/54 kDa is sufficient to increase the protein half-life. Additionally, we observed p53 to be present in increased molecular weight complexes in Ad12-transformed cells. We conclude that both the phosphorylation and oligomerization of p53 is changed as a result of Ad12 transformation.