RESUMEN
Shigella flexneri possesses multiple iron acquisition systems, including proteins involved in the synthesis and uptake of siderophores and the Feo system for ferrous iron utilization. We identified an additional S. flexneri putative iron transport gene, sitA, in a screen for S. flexneri genes that are induced in the eukaryotic intracellular environment. sitA was present in all Shigella species and in most enteroinvasive Escherichia coli strains but not in any other E. coli isolates tested. The sit locus consists of four genes encoding a potential ABC transport system. The deduced amino acid sequence of the S. flexneri sit locus was homologous to the Salmonella enterica serovar Typhimurium Sit and Yersinia pestis Yfe systems, which mediate both manganese and iron transport. The S. flexneri sit promoter was repressed by either iron or manganese, and the iron repression was partially dependent upon Fur. A sitA::cam mutation was constructed in S. flexneri. The sitA mutant showed reduced growth, relative to the wild type, in Luria broth containing an iron chelator but formed wild-type plaques on Henle cell monolayers, indicating that the sitA mutant was able to acquire iron and/or manganese in the host cell. However, mutants defective in two of these iron acquisition systems (sitA iucD, sitA feoB, and feoB iucD) formed slightly smaller plaques on Henle cell monolayers. A strain carrying mutations in sitA, feoB, and iucD did not form plaques on Henle cell monolayers.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Hierro/metabolismo , Shigella flexneri/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas de Transporte de Catión/genética , Células Cultivadas , Regulación Bacteriana de la Expresión Génica , Humanos , Asa de la Nefrona/citología , Asa de la Nefrona/microbiología , Manganeso/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Shigella flexneri/genética , Shigella flexneri/crecimiento & desarrolloRESUMEN
We describe a mouse model in which p27(Kip1) transgene expression is spatially restricted to the central nervous system neuroepithelium and temporally controlled with doxycycline. Transgene-specific transcripts are detectable within 6 h of doxycycline administration, and maximum nonlethal expression is approached within 12 h. After 18-26 h of transgene expression, the G(1) phase of the cell cycle is estimated to increase from 9 to 13 h in the neocortical neuroepithelium, the maximum G(1) phase length attainable in this proliferative population in normal mice. Thus our data establish a direct link between p27(Kip1) and control of G(1) phase length in the mammalian central nervous system and unveil intrinsic mechanisms that constrain the G(1) phase length to a putative physiological maximum despite ongoing p27(Kip1) transgene expression.
Asunto(s)
Proteínas de Ciclo Celular , Sistema Nervioso Central/fisiología , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Supresoras de Tumor , Animales , Antibacterianos/farmacología , Apoptosis , Ciclo Celular , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Corteza Cerebral/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Epitelio , Fase G1 , Expresión Génica , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Mitosis , Modelos Biológicos , Neocórtex/citología , Neocórtex/metabolismo , Neocórtex/fisiología , Células Madre , TransgenesRESUMEN
Cell culture studies have established SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) as an important factor in growth factor and cytokine-activated signaling pathways. However, the significance of SHP2 in the mammalian central nervous system (CNS) is not known since early embryonic lethality occurs in shp2 null mice. To bypass this embryonic lethality, transgenic animals containing a catalytically inactive mutant of SHP2 (SHP2-CS) under the control of a nestin intron II/thymidine kinase minimal promoter were generated. In the developing CNS of these animals, although high-level transgene expression was detected in the neuroepithelium, there was no obvious abnormality in progenitor cell proliferation or migration. In the adult brain, high-level transgene expression was detected in the subventricular zone, rostral migratory stream, dentate gyrus of hippocampus, and cerebellum. Because SHP2 function is likely important in cell survival pathways, we used a focal cerebral ischemia model to examined whether SHP2 is important during CNS injury. Ischemia-induced damage and neuronal death was found to be significantly greater in nestin-SHP2-CS mice than in wild-type littermates. These findings indicate that SHP2 is a required factor in signaling pathway(s) important for neuronal survival.
Asunto(s)
Isquemia Encefálica/fisiopatología , Mutación , Proteínas Tirosina Fosfatasas/genética , Animales , Encéfalo/embriología , Epitelio , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Transgénicos , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas con Dominio SH2 , Distribución Tisular , Dominios Homologos srcRESUMEN
Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine transplantation model by retrovirus-mediated transfer of allogeneic MHC class II genes into bone marrow cells (BMCs) of recipient animals. Graft survival correlated with transient expression of the somatic transgene (Tg) in the induction phase of tolerance. With the aim of investigating the effects of timing and threshold levels of Tg expression on induction of hyporesponsiveness to the grafted tissues, two recombinant retrovirus constructs containing the tetracycline binary regulatory system were used to achieve conditional expression of either the green fluorescent protein (tetGFP) as a control, or the porcine MHC class II DRbeta chain (tetDRB). Effective downregulation of GFP gene transcription was demonstrated in transduced murine fibroblasts after doxycycline treatment, leading to a > 90% reduction of GFP fluorescence. Similar diminution of the DRB gene transcription was achieved in transduced pig endothelial cells (ECs). Drug-dependent downregulation of DRBc gene expression in SLAd pig ECs coincided with complete inhibition of allogeneic activation of anti-class IIc-primed SLAd T cells. These in vitro results suggest that the binary tetracycline retrovirus system may also be adequate to regulate MHC class II Tg expression in vivo.
Asunto(s)
Trasplante de Médula Ósea/métodos , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Genes MHC Clase II/genética , Regiones Promotoras Genéticas , Retroviridae/genética , Trasplante Homólogo/métodos , Animales , Antibacterianos/farmacología , Northern Blotting , Southern Blotting , Línea Celular , Células Cultivadas , Regulación hacia Abajo , Doxiciclina/farmacología , Endotelio/metabolismo , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Linfocitos/metabolismo , Ratones , Modelos Genéticos , Porcinos , Tetraciclina/farmacología , Factores de Tiempo , Transcripción Genética , Transducción Genética , TransfecciónRESUMEN
To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de la Membrana/fisiología , Shigella dysenteriae/crecimiento & desarrollo , Transporte Biológico , Células Cultivadas , Humanos , Hierro/metabolismo , Regiones Promotoras Genéticas , Shigella dysenteriae/genética , Shigella dysenteriae/patogenicidad , VirulenciaRESUMEN
BACKGROUND: This study investigates elements of herpes simplex virus type 1 (HSV-1) which influence transgene expression in tetracycline-regulated expression systems. METHODS: Different HSV-1 mutants were used to infect Vero cells that had been transfected with plasmids containing the luciferase gene under the control of tet-off or tet-on tetracycline-regulation systems. RESULTS: The baseline level of luciferase expression was elevated after infection with HSV-1 mutants lacking one or more immediate early genes encoding transactivating factors: ICP27, ICP4 and ICP0. With the tet-off system, not only was baseline expression elevated, but there was a complete loss of induction upon removal of tet when this regulatory system was brought into the cell by infection with helper virus-free amplicon vectors. Elevation of luciferase expression was also observed upon infection with the same HSV-1 mutants following transfection with a plasmid containing only a CMV minimal promoter driving luciferase (pUHC13-3). Only one HSV mutant (14Hdelta3), which bears a disruption in the transactivation domain of VP16 and is deleted for both ICP4 genes, did not increase baseline luciferase expression after transfection of pUHC13-3. The disregulating effects were dependent on virus dose and were not influenced by treatment with interferon (IFN)-alpha, which suppresses viral gene expression. Additional assays involving cotransfection of pUHC13-3 with a plasmid encoding of the HSV-1 transactivating factor ICP4 revealed that ICP4 was the most potent inducer of gene expression from the tetO/CMV minimal promoter. CONCLUSION: These results indicate that proteins encoded in the HSV-1 genome, especially the transactivating immediate early gene products (ICP4, ICP27 and ICP0) and the VP16 tegument protein can activate the tetO/ minimal CMV promoter and thereby interfere with the integrity of tetracycline-regulated transgene expression.
Asunto(s)
Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Herpesvirus Humano 1/genética , Tetraciclina/farmacología , Animales , Chlorocebus aethiops , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces/genética , Luciferasas/genética , Mutación , Plásmidos/genética , Regiones Promotoras Genéticas , Transcripción Genética , Transfección , Células Vero , Proteínas Virales/metabolismoRESUMEN
Neuropoletic cytokines such as ciliary neurotrophic factor (CNTF) can activate multiple signaling pathways in parallel, including those involving Janus kinase (JAK)-signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamydn (mTOR)-p70 S6 kinase . Crosstalk occurs between these pathways, because studies have shown that STAT3 requires phosphorylation on tyrosine and serine residues by independent protein kinase activities for maximal activation of target gene transcription. Members of the JAK/Tyk family of tyrosine kinases mediate phosphorylation of STAT3 at Tyr705 during CNTF signaling; however, the kinase responsible for phosphorylation at STAT3 Tyr727 appears to depend on both the extracellular stimulus and the cellular context. Here we investigate the kinase activity responsible for phosphorylation of STAT3 on Ser727 in CNTF-stimulated neuroblastoma cells. We found that CNTF-induced phosphorylation of Ser727 was inhibited by the mTOR inhibitor rapamycin, but not by inhibitors of MAPK and protein kinase C (PKC) activation. A STAT3 peptide was efficiently phosphorylated on Ser727 in a CNTF-dependent manner by mTOR, but not by a kinase-inactive mTOR mutant or by p70 S6 kinase. In agreement with these biochemical studies, rapamycin treatment of cells transfected with a STAT-responsive promoter reporter decreased activation of the reporter to the same degree as a STAT3 Ser727Ala mutant The ability of mTOR to contribute to activation of STAT3 extends the function of mTOR in mammalian cells to include transcriptional regulation.
Asunto(s)
Factor Neurotrófico Ciliar/farmacología , Proteínas de Unión al ADN/metabolismo , Fosfoserina/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Proteínas Quinasas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Transactivadores/metabolismo , Línea Celular , Humanos , Sistema de Señalización de MAP Quinasas , Fragmentos de Péptidos/farmacología , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Factor de Transcripción STAT3 , Serina-Treonina Quinasas TORRESUMEN
Genes encoding the synthesis and transport of aerobactin, a hydroxamate siderophore associated with increased virulence of enteric bacteria, were mapped within a pathogenicity island in Shigella flexneri. The island, designated SHI-2 for Shigella pathogenicity island 2, was located downstream of selC, the site of insertion of pathogenicity islands in several other enteric pathogens. DNA sequence analysis revealed the presence of multiple insertion sequences upstream and downstream of the aerobactin genes and an integrase gene that was nearly identical to an int gene found in Escherichia coli O157:H7. SHI-2 sequences adjacent to selC were similar to sequences at the junction between selC and pathogenicity islands found in E. coli O157:H7 and in enteropathogenic E. coli, but the junctions between the island and downstream yic genes were variable. SHI-2 also encoded immunity to the normally plasmid-encoded colicins I and V, suggesting a common origin for the aerobactin genes in both S. flexneri and E. coli pColV. Polymerase chain reaction and Southern hybridization data indicate that SHI-2 is present in the same location in Shigella sonnei, but the aerobactin genes are not located within SHI-2 in Shigella boydii or enteroinvasive E. coli. Shigella dysenteriae type 1 strains do not produce aerobactin but do contain sequences downstream of selC that are homologous to SHI-2. The presence of the aerobactin genes on plasmids in E. coli pColV and Salmonella, on a pathogenicity island in S. flexneri and S. sonnei and in a different chromosomal location in S. boydii and some E. coli suggests that these virulence-enhancing genes are mobile, and they may constitute an island within an island in S. flexneri.
Asunto(s)
Proteínas Bacterianas/genética , Transporte Biológico/genética , Cromosomas Bacterianos/genética , Colicinas , Genes Bacterianos , Ácidos Hidroxámicos/metabolismo , Hierro/metabolismo , Shigella flexneri/genética , Mapeo Cromosómico , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Shigella boydii/genética , Shigella dysenteriae/genética , Shigella flexneri/patogenicidad , Shigella sonnei/genética , Especificidad de la Especie , Virulencia/genéticaRESUMEN
DNA damaging agents such as ultraviolet (UV) induce cell cycle arrest followed by apoptosis in cells where irreparable damage has occurred. Here we show that during early phase G1 arrest which occurs in UV-irradiated human U343 glioblastoma cells, there are (1) decreases in cyclin D1 and cdk4 levels which parallel a loss of S-phase promoting cyclin D1/cdk4 complexes, and (2) increases in p53 and p21 protein levels. We also show that the late phase UV-induced apoptosis of U343 cells occurs after cell cycle re-entry and parallels the reappearance of cyclin D1 and cdk4 and cyclin D1/cdk4 complexes. These findings suggest that cyclin D1 can abrogate UV-induced G1 arrest and that the p53-mediated apoptosis that occurs in these cells is dependent on cyclin D1 levels. We examined these possibilities using U343 cells that ectopically express cyclin D1 and found that indeed cyclin D1 can overcome the cell cycle arrest caused by UV. Moreover, the appearance of p53 protein and the induction of apoptosis in UV-irradiated cells was found to be dependent on the level of ectopically expressed cyclin D1. These findings, therefore, indicate that expression of cyclin D1 following DNA damage is essential for cell cycle re-entry and p53-mediated apoptosis.
Asunto(s)
Apoptosis/efectos de la radiación , Ciclina D1/fisiología , Proteínas Proto-Oncogénicas , Proteína p53 Supresora de Tumor/metabolismo , Ciclo Celular , Ciclina D1/biosíntesis , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Humanos , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
The control of cell cycle progression is orchestrated by an extraordinary diverse and dynamic in function group of proteins. Critical in the progression are the actions of the E2F family of transcription factors which regulate the expression of genes necessary for the G1/S transition and the WAF/CIP/KIP family of cdk inhibitors which can inhibit cell cycle progression. In this report, we have identified E2F binding sites in both the human and mouse p21 promoters that bind E2F protein complexes from nuclear extracts in a cell cycle-dependent manner. In ectopic expression experiments we determined that E2F1, but not E2F4, can strongly transactivate the human p21 gene through these E2F binding sites which are located in the -215/+1 region of the p21 gene. The transactivation of the p21 gene through regulatory elements within the -215/+1 region of the promoter was correlated with increased levels of endogenous E2F1 and p21 proteins at the G1/S boundary. The significance of transactivation of the p21 gene by E2F is that p21 function is important in cell cycle progression as well as for cell cycle arrest. Indeed, E2F-induced levels of p21 protein during the G1/ S transition is consistent with the recent findings demonstrating that p21 acts as an assembly factor for kinase active cyclin/cdk/p21 complexes.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclo Celular/genética , Ciclinas/genética , Proteínas de Unión al ADN , Inhibidores Enzimáticos/metabolismo , Factores de Transcripción/fisiología , Animales , Sitios de Unión/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F4 , Fase G1/genética , Regulación Neoplásica de la Expresión Génica , Glioma , Humanos , Ratones , Regiones Promotoras Genéticas/genética , Proteína 1 de Unión a Retinoblastoma , Fase S/genética , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
The src homology 2 (SH2) domain-containing protein-tyrosine phosphatase SHP-2 has been implicated as an important positive regulator of several mitogenic signaling pathways. SHP-2 has more recently been shown to be tyrosine phosphorylated and recruited to the gp130 component of the ciliary neurotrophic factor (CNTF) receptor complex upon stimulation with CNTF. CNTF does not, however, have a proliferative effect on responsive cells, but rather enhances the survival and differentiation of sympathetic, motor, and sensory neurons. In this study, expression of an interfering mutant of SHP-2 in the neuroblastoma cell line NBFL increased CNTF induction of a vasoactive intestinal peptide (VIP) reporter gene, and in cultures of sympathetic neurons, it resulted in an up-regulation of endogenous VIP and substance P (SP) gene expression. Members of the CNTF family of cytokines transmit their signal by activating signaling pathways involving both STAT and Fos-Jun transcription factors. In CNTF-stimulated NBFL cells that constitutively express the SHP-2 interfering mutant, there was increased and prolonged formation of STAT/DNA complexes, but decreased AP-1 binding activity, that mirrored a down-regulation of c-fos expression both at the mRNA and protein level. Taken together, these data indicate that SHP-2 has dual and opposing roles in a signaling cascade triggered by the same ligand, as illustrated by its ability to differentially regulate the levels of activity of both STAT and AP-1 transcription factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Factor Neurotrófico Ciliar , Ganglios Simpáticos/citología , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Neuronas/citología , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/genética , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Fosfatasas con Dominio SH2 , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transducción de Señal , Sustancia P/biosíntesis , Transactivadores/metabolismo , Factor de Transcripción AP-1/metabolismo , Péptido Intestinal Vasoactivo/biosíntesisRESUMEN
In this study we examined the distribution and developmental profile of the src homology 2 (SH2) domain-containing protein tyrosine phosphatase SHP-2 in the mouse brain. We found that SHP-2 is present in both mitotically active and postmitotic cells in the forebrains of embryonic day 12 (E12) mice. In a developmental study extending from embryonic day 12 to adulthood, Western blotting analysis demonstrated equivalent levels of SHP-2 protein at all of the ages examined. Expression of SHP-2 paralleled the level of enzymatic activity at the different developmental periods. In the adult brain SHP-2 was restricted to diverse classes of neurons, while the majority of glial cells did not express detectable levels of protein. However, reactive astrocytes in response to an ischemic brain injury showed SHP-2 immunolabelling. Our data suggest that SHP-2 may play a role in pathways of neuronal and glial progenitor cells, in a broad spectrum of neuronal responses in the adult brain and in the gliotic response to the injury.
Asunto(s)
Astrocitos/enzimología , Neuroglía/enzimología , Neuronas/enzimología , Proteínas Tirosina Fosfatasas/biosíntesis , Animales , Antimetabolitos/farmacología , Western Blotting , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Isquemia Encefálica/enzimología , Bromodesoxiuridina/farmacología , Femenino , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Mitosis , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas con Dominio SH2 , Células Madre/enzimologíaRESUMEN
Ciliary neurotrophic factor, along with other neuropoietic cytokines, signals through the shared receptor subunit gp130 [1-3], leading to the tyrosine phosphorylation of a number of substrates [4,5], including the transcription factors STAT1 and STAT3 and the protein tyrosine phosphatase SHP-2 [6,7] [8]. SHP-2 (also known as PTP1D, SHPTP2, Syp and PTP2C) is a positive regulatory molecule required for the activation of the mitogen-activated protein kinase pathway and the stimulation of gene expression in response to epidermal growth factor, insulin and platelet-derived growth factor stimulation [9-11]. We have previously shown that cytokines that signal via the gp130 receptor subunit activate transcription of the vasoactive intestinal peptide (VIP) gene through a 180 bp cytokine response element (CyRE) [12,13]. To characterize the role of SHP-2 in the regulation of gp130-stimulated gene expression, we examined the regulation of the VIP CyRE in two systems that prevented ligand-dependent SHP-2 phosphorylation. Inhibition of SHP-2, either by mutating the tyrosine residue in gp130 that mediates the SHP-2 interaction, or by expression of dominant-negative SHP-2, resulted in dramatic increases in gp130-dependent gene expression, through the VIP CyRE and more specifically through multimerized STAT-binding sites. These data suggest that SHP-2 has a negative role in gp130 signaling by modulating STAT-mediated transcriptional activation.
Asunto(s)
Regulación hacia Abajo , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Antígenos CD/metabolismo , Línea Celular , Factor Neurotrófico Ciliar , Receptor gp130 de Citocinas , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transducción de Señal , Transactivadores/metabolismo , Activación Transcripcional , Péptido Intestinal Vasoactivo/genéticaRESUMEN
Because accurate regulation of toxin gene expression is critical for safe and effective gene therapy applications, the authors have examined the regulation of diphtheria toxin A (DTA) fragment expression in human glioma cell lines using two transcriptional control systems derived from Escherichia coli: the tetracycline (Tet) system and the lactose (Lac) system. The Tet system includes a tetracycline-controlled transactivator (tTA), a tTA-responsive minimum human cytomegalovirus (hCMV) promoter controlling the expression of the DTA gene, and tetracycline as an allosteric inhibitor. The Lac system includes the lac repressor (lacR), a lacR-regulated Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter controlling the expression of the DTA gene, and isopropyl-thio-beta-D-galactoside (IPTG) as an allosteric inducer. Expression plasmids encoding either tTA or lacR were transfected into U-87MG and U-343MG glioma cells along with the responsive DTA plasmid. Cell killing was monitored by the ability of the toxin to abolish protein synthesis and was quantitated using a luciferase reporter gene. In the Tet system, tumor cell killing could be regulated by tetracycline up to 120-fold. In contrast, only a twofold IPTG-dependent regulation was obtained using the Lac system because of an incomplete repression of DTA expression in the uninduced state. Replacement of the RSV-LTR promoter with the heavy metal-inducible mouse metallothionein-1 promoter in the lacR-responsive unit, as well as the generation of a clonal glioma cell line expressing lacR, did not significantly enhance regulation of DTA in the Lac system. In conclusion, this study demonstrates that the Tet system is of potential use in gene therapy applications in which regulated expression of a therapeutic gene is an important issue.
Asunto(s)
Neoplasias Encefálicas/genética , Toxina Diftérica/genética , Regulación de la Expresión Génica , Glioma/genética , Células Procariotas/fisiología , Transcripción Genética , Animales , Línea Celular , Expresión Génica , Técnicas Genéticas , Humanos , Operón Lac , Ratones , Operón , Resistencia a la Tetraciclina/genéticaRESUMEN
Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent fibroblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identifiable and anchorage independent growth was observed in NIH3T3 cells. In a first step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can specifically transactivate the p21 promoter. In support of these findings and a direct effect of E2F on induction of p21 gene expression a putative E2F binding site was identified within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.
Asunto(s)
Proteínas Portadoras , Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas , Células 3T3 , Animales , Secuencia de Bases , Western Blotting , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Ciclina D1 , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Inhibidores Enzimáticos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Glioma/metabolismo , Glioma/patología , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Proteína 1 de Unión a Retinoblastoma , Tetraciclina/farmacología , Factores de Tiempo , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Diffuse invasion of brain tissue by single tumor cells is a characteristic feature of gliomas and a major reason why these tumors cannot be completely resected. The molecular basis of brain invasion is poorly understood. We regulated the expression of beta 1 integrins, the major group of extracellular matrix receptors, in astrocytic tumor cells by using a tetracycline-dependent transcription control system. Rat C6 glioma cells were stably transfected with (a) the tetracycline-controlled transactivator (tTA) gene, (b) antisense beta 1 cDNA under the control of a tTA/tetracycline-responsive promoter, and (c) the beta-galactosidase (lacZ) gene for histochemical identification. In one clone, C6TL beta, beta 1 protein levels were unaffected in the presence of tetracycline, but they were reduced by 60% in the absence of tetracycline because of production of antisense mRNA. C6TL beta cells were transplanted into the striatum of nude mice. After 14 days in the presence of tetracycline in the drinking water, tumors showed diffuse brain invasion, mainly along vascular basement membranes. In the absence of tetracycline, however, tumor cells were compact and generally well delineated from the surrounding brain tissue. These data, ie, blocking of brain invasion by antisense beta 1 mRNA, either because of disturbed interaction of beta 1 with brain extracellular matrix components or interference with beta 1-dependent signaling pathways, strongly suggest that beta 1 integrins are required for diffuse brain invasion of gliomas.
Asunto(s)
Neoplasias Encefálicas/patología , Encéfalo/patología , Glioma/patología , Animales , Línea Celular , Femenino , Integrina beta1/fisiología , Ratones , Ratones Endogámicos ICR , Ratones Desnudos , Invasividad Neoplásica , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes/biosíntesis , Tetraciclina/farmacología , Transactivadores/biosíntesis , Transactivadores/genética , Transcripción Genética/efectos de los fármacos , Transfección , Trasplante Heterólogo , beta-Galactosidasa/biosíntesisRESUMEN
Interleukin 1beta-converting enzyme (ICE) is a member of a growing family of cysteine proteases shown to be a crucial component in the activation of a genetic program that leads to autonomous cell death in mammalian cells. In this study, a murine ICE-lacZ fusion gene was introduced into a novel retroviral vector designed to achieve regulated ectopic expression of a foreign gene in mammalian cells. By delivering the ICE-lacZ gene within a retroviral vector and under the control of a tetracycline-regulated promoter, we were able to utilize the intrinsic cell death program of ICE as a means for tumoricidal therapy in a rat brain tumor model. Both in culture and in vivo suppression of ICE-lacZ expression was extremely tight in the presence of tetracycline, as determined by the lack of X-galactosidase-positive tumor cells and by cell viability. When tetracycline was withdrawn, ICE-lacZ gene expression was rapidly turned on and apoptosis-mediated cell death occurred in essentially all tumor cells.
Asunto(s)
Apoptosis/fisiología , Neoplasias Encefálicas/terapia , Cisteína Endopeptidasas/fisiología , Terapia Genética , Vectores Genéticos/genética , Gliosarcoma/terapia , Virus de la Leucemia Murina de Moloney/genética , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Caspasa 1 , Cuerpo Estriado , Cisteína Endopeptidasas/biosíntesis , Cisteína Endopeptidasas/genética , Inducción Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Gliosarcoma/patología , Masculino , Trasplante de Neoplasias , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Tetraciclina/farmacología , Tetraciclina/uso terapéutico , TransfecciónRESUMEN
The protein tyrosine phosphatase SH-PTP2 has been implicated in a variety of cell signaling cascades, including those mediating neuronal survival. We therefore investigated the expression of SH-PTP2 in the adult human nervous system using Western blotting, immunohistochemistry and immunoelectron microscopy. SH-PTP2 immunoreactivity was noted only in neurons, but was not restricted to a specific neuronal type or location. Immunohistochemistry showed perikaryal staining, whereas Western blotting and ultrastructural analysis suggested that SH-PTP2 is present in axons as well. While immunohistochemistry showed a Nissl-like pattern in large motor neurons, immunoelectron microscopy demonstrated a diffuse pattern of cytoplasmic staining, without apparent preferential localization. The presence of the SH2 domain-containing tyrosine-specific phosphatase SH-PTP2 in diverse neurons in the adult nervous system suggests that SH-PTP2 may play a role in a broad spectrum of neuronal responses.
Asunto(s)
Encéfalo/enzimología , Proteínas Tirosina Fosfatasas/análisis , Médula Espinal/enzimología , Adulto , Western Blotting , Encéfalo/citología , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Microscopía Electrónica , Neuronas/enzimología , Neuronas/ultraestructura , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas con Dominio SH2 , Médula Espinal/citologíaRESUMEN
Pleomorphic xanthoastrocytoma (PXA) is a low-grade glioma that may recur as a malignant diffuse astrocytoma such as glioblastoma (GBM). While the molecular genetic basis of diffuse astrocytomas has been studied extensively, PXAs have not been analyzed in detail. We, therefore analyzed DNA from archival primary and recurrent PXAs from eight patients (three grade II PXAs without recurrence, one grade II PXA with recurrence as grade II PXA, two grade II PXAs with progression to GBM, and two grade III anaplastic PXAs with recurrence as grade III anaplastic PXA or GBM) for genetic changes associated with diffuse astrocytomas. Single-strand conformation polymorphism analysis of p53 exons 5-8 revealed migration shifts in two cases, one primary PXA without recurrence and one recurrent grade II PXA in which the primary tumor did not show a shift. DNA sequencing showed two missense mutations in codons 220 (exon 6) and 292 (exon 8), respectively, mutations which have not been previously noted in astrocytomas. Differential polymerase chain reaction analysis demonstrated epidermal growth factor receptor gene amplification in only one tumor, a GBM without allelic loss of chromosome 10 that was the second GBM recurrence of an initial grade II PXA. Loss of heterozygosity studies on tumors from five patients, using three microsatellite polymorphisms on chromosome 10q and three on chromosome 19q, did not disclose allelic loss in any recurrent tumor. These findings suggest that the genetic events that underlie PXA formation and progression may differ significantly from those involved in diffuse astrocytoma tumorigenesis.
Asunto(s)
Astrocitoma/genética , Astrocitoma/patología , Eliminación de Gen , Adolescente , Adulto , Niño , Preescolar , ADN de Neoplasias/análisis , Receptores ErbB/genética , Femenino , Heterocigoto , Humanos , Masculino , Recurrencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Retroviral vectors that contain the tetracycline-inducible (Tet) system were developed. The two components of the Tet system were organized within the vectors in a manner that stringently maintains tetracycline-dependent regulation. Regulated expression of an indicator gene inserted into the retroviral vectors was examined in several different cell types. In infected NIH 3T3 cells, levels of induction in the absence of tetracycline were observed to be as much as 336-fold higher than levels in the presence of tetracycline, which were extremely low. Tetracycline-dependent regulation was observed in all other transduced cell types and ranged from 24- to 127-fold. The generation of retroviral vectors containing regulatory elements that allow for the regulated expression of heterologous genes and that have the ability to infect virtually all dividing target cells should greatly facilitate the biochemical and genetic examination of a broad range of genes. Moreover, these inducible retroviral vectors should prove useful in gene therapy applications.