RESUMEN
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.
Asunto(s)
Anuros , Encéfalo/efectos de los fármacos , Melatonina/farmacología , Receptor de Melatonina MT1/metabolismo , Estaciones del Año , Caracteres Sexuales , Vasotocina/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , Recuento de Células , Femenino , Masculino , Conducta Sexual Animal/efectos de los fármacos , Conducta Sexual Animal/fisiologíaRESUMEN
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.
Asunto(s)
Adaptación Ocular/fisiología , Medicina Aeroespacial , Anteojos/normas , Personal Militar , Lectura , Adulto , Simulación por Computador , Ergonomía , Humanos , Persona de Mediana Edad , Tiempo de Reacción , Factores de Riesgo , Factores de Tiempo , Estados UnidosRESUMEN
The Na+/H+ exchanger (NHE) becomes activated by hyperosmolar stress, thereby contributing to cell volume regulation. The signaling pathway(s) responsible for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases (MAPK), encompassing p42/p44 Erk, p38 MAPK and SAPK, has been implicated in a variety of cellular responses to changes in osmolarity. We therefore investigated whether these kinases similarly signal the hyperosmotic activation of NHE. The time course and osmolyte concentration dependence of hypertonic activation of NHE and of the three sub-families of MAPK were compared in U937 cells. The temporal course and dependence on osmolarity of Erk and p38 MAPK activation were found to be similar to that of NHE stimulation. However, while pretreatment of U937 cells with the kinase inhibitors PD98059 and SB203580 abrogated the osmotic activation of Erk and p38 MAPK, respectively, it did not prevent the associated stimulation of NHE. Thus, Erk1/2 and/or p38 MAPK are unlikely to mediate the osmotic regulation of NHE. The kinetics of NHE activation by hyperosmolarity appeared to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MKK4, an upstream activator of SAPK. Moreover, shrinkage-induced activation of NHE still occurred in COS-7 cells that were transiently transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to inhibit other isoforms of SEK as well. Together, these results demonstrate that the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel but independent events.
Asunto(s)
MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Células COS , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Fibroblastos , Humanos , Concentración de Iones de Hidrógeno , Soluciones Hipertónicas , Immunoblotting , MAP Quinasa Quinasa 1 , Ratones , Proteína Quinasa 8 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Mutación/genética , Concentración Osmolar , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Transfección , Células U937 , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
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.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Neovascularización Patológica/genética , Fosfoproteínas/genética , Proteínas , Animales , Northern Blotting , Línea Celular , Regulación hacia Abajo , Factores de Crecimiento Endotelial/análisis , Femenino , Regulación de la Expresión Génica , Terapia Genética , Humanos , Inmunoquímica , Linfocinas/análisis , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Neovascularización Patológica/metabolismo , Neovascularización Patológica/terapia , Fosfoproteínas/análisis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , ARN/genética , ARN/metabolismo , Proteína p130 Similar a la del Retinoblastoma , Trasplante Heterólogo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
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.
Asunto(s)
Apoptosis/genética , Resistencia a Múltiples Medicamentos/genética , Regulación Leucémica de la Expresión Génica/genética , Leucemia de Células T/genética , Mutación Missense , Proteína p53 Supresora de Tumor/genética , Sustitución de Aminoácidos , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Secuencia de Bases , Supervivencia Celular/efectos de la radiación , Secuencia Conservada , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , ADN de Neoplasias/efectos de la radiación , Dactinomicina/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Exones , Genes p53/genética , Humanos , Leucemia de Células T/metabolismo , Leucemia de Células T/patología , Polimorfismo Conformacional Retorcido-Simple , Tolerancia a Radiación/genética , Serina/genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiación , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Vinblastina/farmacologíaRESUMEN
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.
Asunto(s)
Sensibilidad de Contraste/fisiología , Adaptación a la Oscuridad/fisiología , Adulto , Factores de Edad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Percepción Espacial/fisiología , Factores de Tiempo , Agudeza Visual/fisiologíaRESUMEN
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.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras , Ciclina E/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Fosfoproteínas/fisiología , Proteínas , Proteína de Retinoblastoma/biosíntesis , Proteína de Retinoblastoma/fisiología , Proteínas Supresoras de Tumor , Animales , Células COS , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Genes Supresores de Tumor , Humanos , Modelos Biológicos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína 1 de Unión a Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Terapia Genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Virus de la Leucemia Murina de Moloney , Mutación , Fosfoproteínas/genética , Proteínas , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/terapia , Sustitución de Aminoácidos , Animales , Línea Celular , Codón de Terminación , Técnicas de Transferencia de Gen , Vectores Genéticos , Heterocigoto , Homocigoto , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Mutagénesis Sitio-Dirigida , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , Proteína de Retinoblastoma/genética , Proteína p130 Similar a la del Retinoblastoma , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Linfoma de Burkitt/genética , Núcleo Celular/metabolismo , Mutación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas , Sustitución de Aminoácidos , Núcleo Celular/patología , Exones , Femenino , Mutación del Sistema de Lectura , Humanos , Inmunohistoquímica , Células Jurkat , Leucemia , Linfoma , Masculino , Mutagénesis Sitio-Dirigida , Fosfoproteínas/análisis , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Proteína p130 Similar a la del Retinoblastoma , Transfección , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
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.
Asunto(s)
Mutación del Sistema de Lectura , Genes de Retinoblastoma/genética , Neoplasias Nasofaríngeas/genética , Proteína de Retinoblastoma/genética , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Down syndrome (DS; trisomy 21) is associated with a wide range of variable clinical features, one of the most common being congenital heart defects (CHD). We used molecular genetic techniques to study the inheritance of genes on chromosome 21 in children with DS and CHD. Polymorphic markers on the long arm of chromosome 21 were analysed in 99 families who had a child with DS. Of these, 60 children had a CHD and 39 children had no CHD. Heterotrisomy describes the inheritance of an allele from each of three different grandparents. In some cases heterotrisomy will involve the inheritance of three different alleles. Heterotrisomic regions were defined as those showing retention of non-disjoining parental heterozygosity at polymorphic loci in the non-disjoined chromosomes of children with DS. Using polymorphic non-coding markers, we identified a consistent 9.6-cM minimum region (D21S167-HMG14) of heterotrisomy in children with DS and ventricular septal defect (VSD). Comparing individuals with DS and VSD to all others with DS (those either with no CHD or with any other CHD combined) shows the individuals with DS and VSD to have significantly more non-reduction or heterotrisomy in this region (P=0.006, Fisher's exact test, two-tailed). We postulate that heterotrisomy for a gene or genes in this region is a contributing factor to the pathogenesis of VSD in trisomy 21 either through the presence of three different specific alleles or through the presence of specific combinations of alleles.
Asunto(s)
Cromosomas Humanos Par 21 , Síndrome de Down/genética , Defectos del Tabique Interventricular/genética , Polimorfismo Genético , Trisomía , Adulto , Niño , Síndrome de Down/complicaciones , Femenino , Tamización de Portadores Genéticos , Marcadores Genéticos , Impresión Genómica , Cardiopatías Congénitas/complicaciones , Cardiopatías Congénitas/genética , Defectos del Tabique Interventricular/complicaciones , Humanos , Masculino , Núcleo FamiliarRESUMEN
Smooth muscle cell (SMC) proliferation that results in neointima formation is implicated in the pathogenesis of atherosclerotic plaques and accounts for the high rates of restenosis that occur after percutaneous transluminal coronary angioplasty, a widespread treatment for coronary artery disease. Endothelial lesions trigger intense proliferative signals to the SMCs of the subintima, stimulating their reentry into the cell cycle from a resting G(0) state, resulting in neointima formation and vascular occlusion. Cellular proliferation is negatively controlled by growth-regulatory or tumor-suppressor genes, or both, such as the retinoblastoma gene family members (RB/p105, p107, RB2/p130). In the present study, we show that RB2/p130 inhibited SMC proliferation in vitro and in vivo. We used the rat carotid artery model of restenosis to demonstrate that adenovirus-mediated localized arterial transduction of RB2/p130 at the time of angioplasty significantly reduced neointimal hyperplasia and prevented restenosis. Furthermore, the ability of pRb2/p130 to block proliferation correlated with its ability to bind and sequester the E2F family of transcription factors, which are important mediators of cell cycle progression. These results imply that RB2/p130 could be an important target for vascular gene therapy.
Asunto(s)
Adenoviridae/genética , Estenosis Carotídea/prevención & control , Proteínas Portadoras , Cateterismo/efectos adversos , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Terapia Genética , Vectores Genéticos/uso terapéutico , Músculo Liso Vascular/patología , Fosfoproteínas/fisiología , Proteínas , Túnica Íntima/lesiones , Angioplastia Coronaria con Balón , Animales , Traumatismos de las Arterias Carótidas/etiología , Traumatismos de las Arterias Carótidas/patología , Estenosis Carotídea/patología , Estenosis Carotídea/terapia , Ciclo Celular , División Celular , Células Cultivadas , Factores de Transcripción E2F , Genes de Retinoblastoma , Fosfoproteínas/genética , Arteria Pulmonar/citología , Ratas , Recurrencia , Proteína 1 de Unión a Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Activación Transcripcional , Túnica Íntima/metabolismo , Cicatrización de HeridasRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/patología , Proteína Quinasa CDC2/análisis , Ciclina B/análisis , Fase G2 , Mitosis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/química , Ciclina B1 , Femenino , Humanos , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Neoplasias Ováricas/patología , Fosfoproteínas/biosíntesis , Proteínas , Receptor ErbB-2/metabolismo , Animales , Femenino , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Mutagénesis , Trasplante de Neoplasias , Fosfoproteínas/genética , Proteína p130 Similar a la del Retinoblastoma , Transfección , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/virología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes de Retinoblastoma/efectos de los fármacos , Genes Supresores de Tumor/efectos de los fármacos , Fosfoproteínas/biosíntesis , Fosfoproteínas/farmacología , Proteínas , Animales , Antígenos Virales de Tumores/biosíntesis , Northern Blotting , Western Blotting , Cricetinae , ADN de Neoplasias/análisis , Modelos Animales de Enfermedad , Citometría de Flujo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Virus JC/inmunología , Ratones , Ratones Desnudos , Pruebas de Precipitina , Inhibidores de la Síntesis de la Proteína/farmacología , Proteína p130 Similar a la del Retinoblastoma , Tetraciclina/farmacología , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
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.
Asunto(s)
Arginina/metabolismo , Plaquetas/metabolismo , Disulfuro de Glutatión/farmacología , Glutatión/análogos & derivados , Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Transporte Biológico/efectos de los fármacos , Cobre/metabolismo , Glutatión/farmacología , Humanos , Activación Plaquetaria , S-NitrosoglutatiónRESUMEN
OBJECTIVE: Our purpose was to determine whether the RhD gene is expressed in trophoblast at any stage of gestation. STUDY DESIGN: Trophoblast and fetal tissue were obtained from 18 pregnancies at 8 to 40 weeks' gestation. Deoxyribonucleic acid and ribonucleic acid were extracted from trophoblast. Complementary deoxyribonucleic acid was synthesized from ribonucleic acid, and reverse transcriptase-polymerase chain reaction was performed using primers specific for the RhD gene. Deoxyribonucleic acid was extracted from fetal tissue to determine the fetal RhD status by means of polymerase chain reaction. Antigen expression was also sought by analytic cytometric analysis (flow cytometry and immunocytochemistry) using a monoclonal anti-D antibody. RESULTS: Trophoblast was studied from various combinations of RhD-positive and RhD-negative fetuses (on deoxyribonucleic acid) from mothers to find no RhD gene expression in any sample. Flow cytometry and immunocytochemistry confirmed this by demonstrating no RhD antigen sites on trophoblast cells. CONCLUSION: Contrary to a previous report, we conclude that the RhD gene is not expressed in human trophoblast in any trimester.
Asunto(s)
ADN/análisis , Expresión Génica , ARN/análisis , Sistema del Grupo Sanguíneo Rh-Hr/genética , Trofoblastos/inmunología , Femenino , Citometría de Flujo , Edad Gestacional , Humanos , Inmunohistoquímica , Reacción en Cadena de la Polimerasa , Embarazo , ADN Polimerasa Dirigida por ARN , Sistema del Grupo Sanguíneo Rh-Hr/análisisRESUMEN
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.
Asunto(s)
Neoplasias Endometriales/genética , Fosfoproteínas/genética , Proteínas , Proteína de Retinoblastoma/análisis , Anciano , ADN de Neoplasias/análisis , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/patología , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Análisis Multivariante , Ploidias , Pronóstico , Modelos de Riesgos Proporcionales , Proteína p130 Similar a la del Retinoblastoma , Tasa de SupervivenciaRESUMEN
The Cdk2 kinase has long been known to be involved in the progression of mammalian cells past the G1 phase restriction point and through DNA replication in the cell cycle. The Rb family of proteins, consisting of pRb, p107, and pRb2/p130, has also been shown to monitor progression of G1 phase, mostly through their interaction with E2F family members. p107 is able to inhibit Cdk2 kinase activity through this interaction via a p21-related domain present in the C terminus of the protein. We show here that pRb2/p130 also possesses this activity, but through a separate domain. Moreover, we correlate the increased expression of pRb2/p130 during various cellular processes with the decreased kinase activity of Cdk2. We hypothesize that pRb2/p130 may act not only to bind and modify E2F activity, but also to inhibit Cdk2 kinase activity in concert with p21 in a manner different from p107.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Proteínas de Unión al ADN , Fosfoproteínas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas , Animales , Sitios de Unión , Ciclo Celular , Diferenciación Celular , Quinasa 2 Dependiente de la Ciclina , Factores de Transcripción E2F , Ratones , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Recombinantes , Proteína de Retinoblastoma/metabolismo , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismoRESUMEN
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.