RESUMO
The oncoprotein of simian virus-40, SV40 large T-antigen (Tag), is reported to target and to inactivate growth suppressive proteins such as the retinoblastoma family and p53 (ref. 4, 5), leading to transformation of human cell lines in vitro, tumor production in rodents, and detection of Tag in several human cancers including mesotheliomas. The retinoblastoma family contains three members, pRb, p107 and pRb2/p130 (ref. 9), that are phosphorylated in a cell cycle-dependent manner, have cell growth suppressive properties and bind to specific members of the E2F family and various cyclins. Even though mesotheliomas are among the most aggressive human cancers, alterations of important cell-cycle "controllers," such as the Rb family genes, have never been reported in these tumors. We found the presence of SV40-like sequences in 86% of 35 archival specimens of mesothelioma. We also demonstrated that SV40 Tag, isolated from frozen biopsies of human mesothelioma, binds each of the retinoblastoma family proteins, pRb, p107 and pRb2/p130, in four of four specimens. We propose that the tumorigenic potential of SV40 Tag in some human mesotheliomas may arise from its ability to interact with and thereby inactivate several tumor and/or growth suppressive proteins.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Mesotelioma/genética , Mesotelioma/imunologia , Proteína do Retinoblastoma/metabolismo , Vírus 40 dos Símios/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células COS , Células HL-60 , Humanos , Imuno-Histoquímica , Mesotelioma/patologia , Pessoa de Meia-Idade , Família Multigênica , Ligação Proteica , Proteína do Retinoblastoma/genética , Células Tumorais CultivadasRESUMO
In order to study the splanchnic metabolism of blood-borne estrogens, a constant infusion of estrone-6,7-(3)H was made in a series of dogs, and arteriovenous (A-V) differences at equilibrium were determined for estrone-6,7-(3)H and for its products estradiol-17beta, estrone sulfate, estrone glucosiduronate, and estradiol-17beta glucosiduronate across the splanchnic bed (artery-hepatic vein), the small intestine (artery-superior mesenteric vein), and the spleen (artery-splenic vein). Per cent extractions (100 - [V/A] 100) were calculated. The plasma metabolic clearance rate (MCR) for estrone was measured. Principal findings were as follows: mean MCR was 731 liters/day per m(2), SEM 50. By comparison with estimated hepatic plasma flow and using the observed splanchnic extraction of estrone, 45-71% of estrone metabolism was calculated to be extrasplanchnic. The significant mean per cent extractions were as follows (SEM in parentheses): splanchnic bedestrone 85.9 (1.92), estradiol-17beta 88.11 (3.36), estrone sulfate 27.9 (5.22), estrone glucosiduronate -48.5 (9.33), estradiol-17beta glucosiduronate -33.3 (80.3); small intestine-estrone 45.3 (2.60), estradiol-17beta 46.1 (12.9), estrone glucosiduronate - 30.8 (7.9); spleen-estrone 35 (3.8), estrone glucosiduronate 12 (3.7). These results lead to the following conclusions. Both estrone and estradiol-17beta are nearly completely extracted in one passage through the splanchnic bed. There is net uptake of estrone sulfate and net production of estrone glucosiduronate and of estradiol-17beta glucosiduronate by the splanchnic bed. There is net uptake of estrone and of estradiol-17beta by the intestine, associated with substantial net production of estrone glucosiduronate. There is net uptake of estrone by the spleen and a small but significant net uptake of estrone glucosiduronate.
Assuntos
Estradiol/metabolismo , Estrona/metabolismo , Intestino Delgado/metabolismo , Baço/metabolismo , Animais , Cateterismo , Cães , Estradiol/sangue , Estrona/administração & dosagem , Estrona/sangue , Fluoroscopia , Injeções Intravenosas , Circulação Hepática , Masculino , Artérias Mesentéricas/metabolismo , Veias Mesentéricas/metabolismo , Taxa de Depuração Metabólica , Artéria Esplênica/metabolismo , Veia Esplênica/metabolismo , TrítioRESUMO
BACKGROUND: The RB/p105 and p107 genes of the retinoblastoma family are tumor suppressor genes whose proteins are inactivated by interaction with T-antigen proteins encoded by polyomaviruses (e.g., simian virus 40 and human JC virus), which have been found to be highly tumorigenic in animals. A variety of indirect evidence suggests that another member of the retinoblastoma gene family, RB2/p130, is also a tumor suppressor gene. To investigate the putative tumor suppressor activity of RB2/p130 more directly, we utilized a tetracycline-regulated gene expression system to control expression of the encoded protein pRb2/p130 in JC virus-induced hamster brain tumor cells and to study the effects of pRb2/p130 on the growth of such tumor cells in nude mice. The ability of pRb2/p130 to interact with JC virus T antigen was also studied. METHODS: Northern blot hybridization analyses were performed on samples of total cellular RNA to measure RB2/p130 and beta-actin messenger RNA levels. Immunoprecipitation and western blot analyses were used to determine T-antigen and pRb2/p130 protein levels and to assess the phosphorylation status of these proteins. Tumor cells were injected subcutaneously into nude mice, and tumor growth, with or without induced expression of pRb2/p130, was monitored. RESULTS: Induction of pRb2/p130 expression brought about a 3.2-fold, or 69% (95% confidence interval = 64%-73%), reduction in final tumor mass in nude mice. We also demonstrated that JC virus T antigen binds hypophosphorylated pRb2/p130 and that stimulation of pRb2/p130 expression overcomes cellular transformation mediated by this antigen. CONCLUSION: Our findings support the hypothesis that RB2/p130 is a tumor suppressor gene.
Assuntos
Antígenos Virais de Tumores/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/virologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes do Retinoblastoma/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Fosfoproteínas/biossíntese , Fosfoproteínas/farmacologia , Proteínas , Animais , Antígenos Virais de Tumores/biossíntese , Northern Blotting , Western Blotting , Cricetinae , DNA de Neoplasias/análise , Modelos Animais de Doenças , Citometria de Fluxo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Vírus JC/imunologia , Camundongos , Camundongos Nus , Testes de Precipitina , Inibidores da Síntese de Proteínas/farmacologia , Proteína p130 Retinoblastoma-Like , Tetraciclina/farmacologia , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
The retinoblastoma tumor suppressor gene product, as well as its related protein p107, has been shown clearly to exert its growth suppressive effects in a cell cycle dependent manner. In this study we demonstrate that the introduction of our recently cloned Rb family member p130/pRb2 causes growth arrest in three tumor cell lines. In addition, in the nasopharyngeal carcinoma derived cell line HONE-1, we identified a low level of expression of p130/pRb2, possibly due to gene rearrangement, and a drastic reduction in proliferation upon introduction of a constitutive active p130/pRb2 complementary DNA clone. Furthermore, we were able to dissect distinct properties of the Rb family by demonstrating that p130/pRb2 inhibits proliferation of the glioblastoma cell line T98G, which is resistant to the growth suppressive effects of both pRb and p107. Our studies demonstrate that the Rb family proteins identified to date may complement each other but they are not fully functionally redundant.
Assuntos
Neoplasias Nasofaríngeas/metabolismo , Proteínas Nucleares , Fosfoproteínas , Proteínas/fisiologia , Proteína do Retinoblastoma/fisiologia , Divisão Celular , Humanos , Neoplasias Nasofaríngeas/patologia , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-Like , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Nasopharyngeal carcinoma (NPC) is an endemic cancer in southern China and northern Africa, and its pathogenesis is not yet well defined at the molecular level. Although the involvement of p53 and of the retinoblastoma gene (RB/p105) in NPC has been well studied, there is paucity of mutational data regarding the retinoblastoma-related gene RB2/p130 in primary tumors and particularly in NPC. We have shown previously that RB2/p130 could be rearranged in a nasopharyngeal cell line. In the present study, we screened by single-strand conformation polymorphism and sequence analysis the retinoblastoma-related gene RB2/p130 for mutations within exons 19-22. Mutations in the RB2/p130 gene were detected in 3 of 10 primary human NPCs from Northern Africa (30%). These findings, along with previous data showing that genetic replacement of RB2/p130 restores a normal growth pathway in the nasopharyngeal cell line Hone-1, strengthen the hypothesis that genetic changes of RB2/p130 may be involved in the development and/or progression of nasopharyngeal cancer and suggest that RB2/p130 could be considered a tumor suppressor gene and may be a candidate for novel gene therapeutic approaches for NPC.
Assuntos
Mutação da Fase de Leitura , Genes do Retinoblastoma/genética , Neoplasias Nasofaríngeas/genética , Proteína do Retinoblastoma/genética , Análise Mutacional de DNA , DNA de Neoplasias/genética , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.
Assuntos
Linfoma de Burkitt/genética , Núcleo Celular/metabolismo , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas , Substituição de Aminoácidos , Núcleo Celular/patologia , Éxons , Feminino , Mutação da Fase de Leitura , Humanos , Imuno-Histoquímica , Células Jurkat , Leucemia , Linfoma , Masculino , Mutagênese Sítio-Dirigida , Fosfoproteínas/análise , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteína p130 Retinoblastoma-Like , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
The retinoblastoma family of proteins, pRb/p105, p107, and pRb2/ p130, cooperate to regulate cell cycle progression through the G1 phase of the cell cycle. Each of the family members realize their common goal of G1-S checkpoint regulation through overlapping and unique growth regulatory pathways. We took advantage of a tetracycline-regulated gene expression system to control the expression of RB2/p130 in JC virus-induced hamster brain tumor cells to study in vivo the molecular mechanisms used by pRb2/p130 to elicit its growth-suppressive function. We have previously used this system to demonstrate that induction of pRb/ p130 expression suppresses tumor growth in vivo by overcoming neoplastic transformation mediated by the large T-antigen oncoprotein of JCV (JCV TAg). Here we found that induction of pRb2/p130 in vivo specifically inhibits cyclin A- and cyclin E-associated kinase activity and by doing so induces p27Kip1 levels presumably by inhibiting p27Kip1-targeted proteolysis by cyclin E-Cdk2 phosphorylation of p27Kip1. RB2/p130 induction also decreased cyclin A and the transcription factor E2F-1 while increasing cyclin E at both the transcriptional and protein levels of expression. The growth inhibitory activity of pRb2/p130 also correlated with its E2F-binding capacity. Furthermore, p27Kip1 and pRb2/p130 were found to be targets of the JCV TAg oncoprotein and to interact in vivo with each other independently from the presence of TAg. Interestingly, pRb2/p130 expression negatively modulated the binding of p27Kip1 to JCV TAg. These data suggest that pRb2/p130 and p27Kip1 may cooperate in regulating cellular proliferation, and both may be involved in a negative feedback regulatory loop with cyclin E.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Ciclina E/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Fosfoproteínas/fisiologia , Proteínas , Proteína do Retinoblastoma/biossíntese , Proteína do Retinoblastoma/fisiologia , Proteínas Supressoras de Tumor , Animais , Células COS , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Genes Supressores de Tumor , Humanos , Modelos Biológicos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Proteína p130 Retinoblastoma-Like , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The retinoblastoma (Rb) family consists of the tumor suppressor pRb and related proteins p107 and pRb2/p130. Ectopic expression of pRb and p107 results in a growth arrest of sensitive cells in the G1 phase of the cell cycle. We demonstrated here that the growth-suppressive properties of pRb2/p130 were also specific for the G1 phase. The A-, E-, and D-type cyclins as well as transcription factor E2F1 and the E1A viral oncoprotein were able to rescue the pRb2/p130-mediated G1 growth arrest in SAOS-2 cells. The rescue with cyclins A and E correlated with their physical interaction with pRb2/p130, which surprisingly has been found to occur over all phases of the cell cycle. The phosphorylation status as well as the kinase activity associated with pRb2/p130 dramatically increased near the G1-S-phase transition. This suggests that, like the other Rb family members, pRb and p107, the phosphorylation of pRb2/p130 is controlled by the cell cycle machinery and that pRb2/p130 may indeed be another key G1-S-phase regulator.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclo Celular/genética , Ciclinas/genética , Fosfoproteínas/genética , Proteínas , Proteína do Retinoblastoma/genética , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Citometria de Fluxo , Fase G1/genética , Humanos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína p130 Retinoblastoma-Like , Fase S/genética , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Angiogenesis is an essential step in the progression of tumor formation and development. The switch to an angiogenetic phenotype can occur as a distinct step before progression to a neoplastic phenotype and is linked to genetic changes such as mutations in key cell cycle regulatory genes. The pathogenesis of the angiogenetic phenotype may involve the inactivation of tumor suppressor genes such as the "guardian of the genome," p53, and the cyclin-dependent kinase inhibitor p16. Retinoblastoma family member RB2/p130 encodes a cell cycle regulatory protein and has been found mutated in different tumor types. Overexpression of RB2/p130 not only suppresses tumor formation in nude mice but also causes regression of established tumor grafts, suggesting that RB2/p130 may modulate the angiogenetic balance. We found that induction of RB2/p130 expression using a tetracycline-regulated gene expression system as well as retroviral and adenoviral-mediated gene delivery inhibited angiogenesis in vivo. This correlated with pRb2/p130-mediated down-regulation of vascular endothelial growth factor protein expression both in vitro and in vivo.
Assuntos
Fatores de Crescimento Endotelial/genética , Linfocinas/genética , Neovascularização Patológica/genética , Fosfoproteínas/genética , Proteínas , Animais , Northern Blotting , Linhagem Celular , Regulação para Baixo , Fatores de Crescimento Endotelial/análise , Feminino , Regulação da Expressão Gênica , Terapia Genética , Humanos , Imunoquímica , Linfocinas/análise , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/genética , Neoplasias Experimentais/terapia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/terapia , Fosfoproteínas/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , RNA/genética , RNA/metabolismo , Proteína p130 Retinoblastoma-Like , Transplante Heterólogo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The retinoblastoma (Rb) family consists of the tumor suppressor pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in 235 specimens of lung cancer show the tightest inverse association between the histological grading in the most aggressive tumor types and pRb2/p130. This led us to study a panel of human lung cancers for mutations in the RB2/p130 gene. Mutations in the Rb-related gene RB2/p130 were detected in 11 of 14 (78.5%) primary lung tumors by single-strand conformation polymorphism and sequence analysis. A Moloney leukemia virus-based retroviral system was set up, and a comparable viral concentration of 1 x 10(7) infectious units/ml was obtained. Retrovirus-mediated delivery of wild-type RB2/p130 to the lung tumor cell line H23 potently inhibited tumorigenesis in vitro and in vivo, as shown by the dramatic growth arrest observed in a colony assay and the suppression of anchorage-independent growth potential and tumor formation in nude mice. The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and a 12-fold reduction in tumor growth after RB2/p130 transduction compared with the Pac-transduced tumors (92% reduction, P = 0.003) and lacZ-transduced tumors (93% reduction, P < 0.001) was found to be statistically significant. These findings provide the missing confirmation that RB2/p130 is a "bona fide" tumor suppressor gene and strengthen the hypothesis that it may be a candidate for cancer gene therapy for lung cancer.
Assuntos
Terapia Genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Vírus da Leucemia Murina de Moloney , Mutação , Fosfoproteínas/genética , Proteínas , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Substituição de Aminoácidos , Animais , Linhagem Celular , Códon de Terminação , Técnicas de Transferência de Genes , Vetores Genéticos , Heterozigoto , Homozigoto , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Mutagênese Sítio-Dirigida , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Proteína do Retinoblastoma/genética , Proteína p130 Retinoblastoma-Like , Transfecção , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
We investigated the in vitro and in vivo effects of the ectopic expression of the pRb2/p130 cell cycle regulator on c-erbB-2-associated tumorigenicity. SKOV3 ovarian cancer cells, which display c-erbB-2 gene amplification and oncoprotein (p185HER2) overexpression, were stably transfected with a plasmid containing the coding sequence for human wild-type pRb2/p130 (wtRb2), or with pcDNA3 empty vector. Three wtRb2-transfected clones (cl. 24, ci. 49, cl. 100) and one empty vector-transfected clone (cl. mock) were randomly picked and further analysed. Western blot analysis revealed high levels of pRb2/p130 in the three clones compared to mock cells. Levels of p185HER2 and the extent of its tyrosine phosphorylation were similar in all transfectant clones, as were levels of pRb1 and p107. In anchorage-independent growth assays, the number of colonies from wtRb2 clone-transfectants was about 90% less than that arising from mock cells (P<0.001). Tumor take rates of the three wtRb2-transfected clones xenografted in nu/nu mice were much lower than those of mock cells, and tumor volume was decreased by 80% (P<0.001). A mutant version of pRb2/p130 deleted of the pocket region (mut-Rb2) was also transfected into SKOV3 cells and studied in parallel with the wtRb2-transfected and pcDNA empty vector-transfected bulk populations. mut-Rb2 transfected cells showed no inhibition of in vitro colony formation and were fully tumorigenic. Together, these findings indicate that Rb2 acts as a tumor suppressor gene in vivo and in vitro in SKOV3 cells and that the intact pocket region is required for the suppressor activity.
Assuntos
Neoplasias Ovarianas/patologia , Fosfoproteínas/biossíntese , Proteínas , Receptor ErbB-2/metabolismo , Animais , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Mutagênese , Transplante de Neoplasias , Fosfoproteínas/genética , Proteína p130 Retinoblastoma-Like , Transfecção , Células Tumorais CultivadasRESUMO
The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.
Assuntos
Apoptose/genética , Resistência a Múltiplos Medicamentos/genética , Regulação Leucêmica da Expressão Gênica/genética , Leucemia de Células T/genética , Mutação de Sentido Incorreto , Proteína Supressora de Tumor p53/genética , Substituição de Aminoácidos , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Sequência de Bases , Sobrevivência Celular/efeitos da radiação , Sequência Conservada , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , DNA de Neoplasias/efeitos da radiação , Dactinomicina/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Éxons , Genes p53/genética , Humanos , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Polimorfismo Conformacional de Fita Simples , Tolerância a Radiação/genética , Serina/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Vimblastina/farmacologiaRESUMO
PURPOSE: The retinoblastoma gene is the prototype of tumor-suppressor genes and has been shown to be involved in the pathogenesis and progression of several human malignancies. In this study, we determined the relation between the expression of a newly discovered retinoblastoma-related gene Rb2/p130 and outcome in patients with endometrial carcinoma. PATIENTS AND METHODS: pRb2/p130 expression was determined immunohistochemically in specimens of endometrial carcinoma (stages I to IV) from 100 patients who underwent surgery as the first treatment. The pRb2/p130 status was analyzed in relation to the length of disease-free survival and disease-specific survival. RESULTS: Decreased levels of pRb2/p130 in endometrial cancer cells was significantly associated with a decreased probability of remaining disease-free after treatment (P = .003) and with decreased probability of survival (P < .0001). In a multivariate analysis, pRb2/p130 status (P = .004), tumor stage (P = .009), and ploidy status (P = .02) were independent predictors of clinical outcome. The risk of dying of disease was increased substantially (risk ratio, 4.91; 95% confidence interval, 1.66 to 14.54) among patients with decreased levels of pRb2/p130 in tumor cells. CONCLUSION: In patients with endometrial carcinoma who did not receive radiotherapy or chemotherapy before surgery, the presence of decreased levels of pRb2/p130 in tumor cells is associated with a significantly increased risk of recurrence and death of disease, independent of tumor stage and ploidy status.
Assuntos
Neoplasias do Endométrio/genética , Fosfoproteínas/genética , Proteínas , Proteína do Retinoblastoma/análise , Idoso , DNA de Neoplasias/análise , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/mortalidade , Neoplasias do Endométrio/patologia , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Análise Multivariada , Ploidias , Prognóstico , Modelos de Riscos Proporcionais , Proteína p130 Retinoblastoma-Like , Taxa de SobrevidaRESUMO
The Rb (retinoblastoma) gene family is composed of three members: the RB gene (one of the best-studied tumor suppressor genes) and two related genes, p107 and pRb2/p130. These three proteins share many structural and functional features and play a fundamental role in growth control. Using immunocytochemical techniques, we evaluated a variety of lung tumor specimens for the expression of this family of proteins and compared protein expression with the histological grading of the tumors and with the expression of the proliferating cell nuclear antigen. These Rb family members displayed distinctive patterns when compared and contrasted using different parameters. The highest percentage of undetectable levels in all of the specimens examined and the tightest inverse correlation (P) with the histological grading and with proliferating cell nuclear antigen expression in the most aggressive tumor types were found for pRb2/p130, which may suggest an important role for this protein in the pathogenesis and progression of lung cancer.
Assuntos
Neoplasias Pulmonares/química , Proteínas Nucleares/análise , Fosfoproteínas/análise , Proteínas , Proteína do Retinoblastoma/análise , Animais , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Antígeno Nuclear de Célula em Proliferação/análise , Coelhos , Proteína do Retinoblastoma/imunologia , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-LikeRESUMO
The neuroendocrine mechanisms by which animals synchronise their physiological state with environmental cues are vital to timing life-history events appropriately. One important endocrine transducer of environmental cues in vertebrates is the pineal hormone melatonin, the secretion of which is directly sensitive to photoperiod and temperature. Melatonin modulates arginine vasotocin (AVT)-immunoreactive (-IR) cell number in the brain of green treefrogs (Hyla cinerea) during the summer breeding season, and this modulation is sexually dimorphic. In the present study, we investigated whether the influence of melatonin on vasotocin varies seasonally. We show that treatment of nonreproductive male green treefrogs with melatonin-filled silastic implants for 4 weeks during the winter does not alter vasotocin-IR cell number in any brain region (i.e. nucleus accumbens, amygdala, preoptic area, suprachiasmatic nucleus or ventral hypothalamus). Taken together, these results suggest that the influence of melatonin on AVT is associated with sex and seasonal variation in melatonin receptor expression. We tested this hypothesis by using immunohistochemistry to characterise the distribution of melatonin receptor type 1 (MT1, also known as Mel1a) in the brain of reproductive and nonreproductive male and female frogs. We quantified MT1-IR cell number in regions known to contain AVT cell populations. Reproductive males had significantly more MT1-IR cells than nonreproductive males in all brain regions, including the combined nucleus accumbens, diagonal band of Broca and septum, striatum, amygdala, combined preoptic area and suprachiasmatic nucleus, as well as the ventral hypothalamus. In the accumbens region, where the effect of melatonin on AVT is known to be sexually dimorphic, males had significantly more MT1-IR cells than females during the summer breeding season. Based on these findings, we suggest that MT1 plays a role in mediating the interactions between melatonin and vasotocin that regulate seasonal and sexually dimorphic changes in sociosexual behaviour.
Assuntos
Anuros , Encéfalo/efeitos dos fármacos , Melatonina/farmacologia , Receptor MT1 de Melatonina/metabolismo , Estações do Ano , Caracteres Sexuais , Vasotocina/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Contagem de Células , Feminino , Masculino , Comportamento Sexual Animal/efeitos dos fármacos , Comportamento Sexual Animal/fisiologiaRESUMO
In this study, we have examined the effects of authentic nitric oxide (NO), NO+ (NOBF4), glutathione (GSH), glutathione disulphide (GSSG), and S-nitrosoglutathione (GSNO) in the presence and absence of Cu2+, which thermally releases NO from S-nitrosothiols on the transport of L-arginine into the human platelet. The K(M,apparent) was unaffected by NO, NO+, GSH, and GSNO. However, Cu2+ lowered K(M,apparent) by approximately 2.85-fold. Cu2+-dependent lowering of K(M,apparent) was also observed, albeit to a smaller extent when this ion was mixed with GSH (approximately 1.9-fold lower) and GSNO (approximately 2.0-fold). GSSG also lowered K(M,apparent) by approximately 1.5-fold. The Vmax,apparent of L-arginine uptake was unaffected by NO, NO+, GSH, and Cu2+. Vmax,apparent was stimulated by to the largest extent by GSNO (approximately 2.28-fold) and GSNO plus Cu2+ (approximately 2.7-fold). GSSG and GSH plus Cu2+ also increased Vmax,apparent by approximately 1.9-fold. When these parameters are expressed in terms of transport efficiency (Vmax/K(M)) the largest effect of nearly 4.7-fold (over controls) was obtained by a combination of GSNO plus Cu2+. These results suggest that platelet L-Arg transport is not affected either by NO or NO+ but by a thiol-disulphide exchange reactions on the platelet L-Arg transporter, brought about by GSNO and GSSG. Based on these results, a GSNO/GSSG/Cu2+ dependent regulatory mechanism for the uptake of L-arginine in human platelets has been proposed.
Assuntos
Arginina/metabolismo , Plaquetas/metabolismo , Dissulfeto de Glutationa/farmacologia , Glutationa/análogos & derivados , Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Transporte Biológico/efeitos dos fármacos , Cobre/metabolismo , Glutationa/farmacologia , Humanos , Ativação Plaquetária , S-NitrosoglutationaRESUMO
The time course of recovery of spatial resolution following adaptation to a uniform field was measured for test probes presented at lower illuminance than the adapting field. Six observers were tested in a Maxwellian-view system using 20 degrees adapting fields of 1.6-2.6 log photopic trolands. Test stimuli were 7 degrees, 250 ms Gabor patches (1 and 6 cpd) of mean retinal illuminance 2-3 log units lower than the adapting field. During the 9 s after adapting field offset, contrast thresholds for orientation discrimination followed an exponential-decay function and showed longer recovery times for larger illuminance decrements and higher spatial frequency.
Assuntos
Sensibilidades de Contraste/fisiologia , Adaptação à Escuridão/fisiologia , Adulto , Fatores Etários , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Percepção Espacial/fisiologia , Fatores de Tempo , Acuidade Visual/fisiologiaRESUMO
In this study, the expression of cyclin B1 and p34cdc2 in neoplastic and non-neoplastic breast lesions was evaluated by immunohistochemistry and quantitative analysis in relation to cellular kinetic parameters such as Mitotic Index (MI), Anatelophase Index (ATI), and Apoptotic Index (AI). The percentage of cyclin B1 and p34cdc2-positive cells was significantly higher in neoplastic glands than in their normal counterparts. This finding was paralleled by significantly higher values of MI, ATI, and AI in breast cancer than in normal glands. Furthermore, two groups with different cytokinetic characteristics were identified among infiltrating ductal carcinomas by an unsupervised learning technique of cluster analysis using the percentages of cyclin B1 and p34cdc2 positive cells and the cellular kinetic parameters (MI, ATI and AI) as variables. The final clusters, groups I and II, consisted of 42 and 13 cases respectively. The first cluster (group I) was characterized by a significantly linear correlation between the percentages of cyclin B1 and p34cdc2-positive cells. On the contrary, the second cluster (group II) revealed no correlation between these two proteins and was characterized by values of p34cdc2 largely exceeding those of cyclin B1. A positive correlation between the expression of these two proteins and the cellular kinetic parameters (MI, ATI and AI) was also found in group I but not in group II. These observations suggest that a disturbed nuclear translocation of Mitosis Promoting Factor (MPF) components is present in group II cases, resulting in a defective cellular division cycle. In fact, group I cases showed lymph node metastasis more frequently than group II cases. Our results suggest that the analysis of the cell cycle "machinery" components, such as the cyclins and their dependent kinases, can identify tumors with different levels of aggressiveness.
Assuntos
Neoplasias da Mama/patologia , Proteína Quinase CDC2/análise , Ciclina B/análise , Fase G2 , Mitose , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/química , Ciclina B1 , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Light adaptation to the intensified image provided by a night vision device may handicap pilots who have set cockpit instrument luminance too low. METHODS: Under conditions simulating night flying, subjects adapted to an NVG image at 3 or 10 footlamberts (fL), then used a joystick to indicate the position of the horizon in an ADI illuminated by NVIS-compatible light at luminances 2 to 3.5 log units lower than the NVG image. RESULTS: Response times increased no more than a few tenths of a second when the decrease in luminance was only 2 log units. Greater decreases produced correspondingly longer delays in response, reaching as much as 5.5 s for subjects in their twenties and 8-15 s for older subjects. CONCLUSIONS: While a decrease of more than 2 log units is not likely to occur under most operational conditions, it is certainly possible, and pilots should be aware that significant risk can be incurred by setting cockpit instruments to luminance levels below 0.03 fL.