RESUMEN

Peptides and proteins have been largely neglected in the analysis of insect tarsal adhesives. After extraction of the protein fraction of the tarsal secretion of the desert locust, Schistocerca gregaria, and Madagascar hissing cockroach, Gromphadorhina portentosa, we combined Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analyses for protein mass detection. In both these insects, SDS-PAGE analysis revealed several protein bands ranging from 8-190 kDa in both the tarsal secretion and the tibia control sample. Two (S. gregaria) and one (G. portentosa) protein bands exclusively occurred in the tarsal secretion and can be considered to belong to peptides and proteins specific to this secretion. MALDI-TOF analyses revealed 83 different proteins/peptides of 1-7 kDa in S. gregaria, and 48 of 1-11 kDa in G. portentosa. 59 (S. gregaria) and 27 (G. portentosa) proteins exclusively occurred in the tarsal secretion. In G. portentosa, a characteristic series of signal peaks occurred in the range of c. 10-12 kDa, each peak being approximately 160 Da apart. Such a pattern is indicative of proteins modified by glycosylation. Our approach demonstrates that extensive sampling involving considerable time and manpower to sample the adhesive fluid directly from the tarsi opens up a perspective for extracting peptides and proteins in sufficient quantities. This makes them accessible to the field of proteomics and thus to elucidate their possible function in the adhesive process.

Asunto(s)

Cucarachas/química , Saltamontes/química , Proteínas de Insectos/análisis , Animales , Electroforesis en Gel de Poliacrilamida , Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

The anti-apoptotic Bcl-2 protein, which confers oncogenic transformation and drug resistance in most human cancers, including breast cancer, has recently been shown to effectively counteract autophagy by directly targeting Beclin1, an essential autophagy mediator and tumor suppressor. However, it remains unknown whether autophagy inhibition contributes to Bcl-2-mediated oncogenesis. Here, by using a loss-of-function mutagenesis study, we show that Bcl-2-mediated antagonism of autophagy has a critical role in enhancing the tumorigenic properties of MCF7 breast cancer cells independent of its anti-apoptosis activity. A Bcl-2 mutant defective in apoptosis inhibition but competent for autophagy suppression promotes MCF7 breast cancer cell growth in vitro and in vivo as efficiently as wild-type Bcl-2. The growth-promoting activity of this Bcl-2 mutant is strongly correlated with its suppression of Beclin1-dependent autophagy, leading to sustained p62 expression and increased DNA damage in xenograft tumors, which may directly contribute to tumorigenesis. Thus, the anti-autophagic property of Bcl-2 is a key feature of Bcl-2-mediated oncogenesis and may in some contexts, serve as an attractive target for breast and other cancer therapies.

Asunto(s)

Apoptosis , Autofagia , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Regulación hacia Abajo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Neoplasias de la Mama/ultraestructura , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Daño del ADN , Femenino , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Proteínas Mutantes/metabolismo , Células 3T3 NIH , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteína Sequestosoma-1 , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The stringent regulation of cell cycle progression helps to maintain genetic stability in cells. MicroRNAs (miRNAs) are critical regulators of gene expression in diverse cellular pathways, including developmental patterning, hematopoietic differentiation and antiviral defense. Here, we show that two c-Myc-regulated miRNAs, miR-17 and miR-20a, govern the transition through G1 in normal diploid human cells. Inhibition of these miRNAs leads to a G1 checkpoint due to an accumulation of DNA double-strand breaks, resulting from premature temporal accumulation of the E2F1 transcription factor. Surprisingly, gross changes in E2F1 levels were not required to initiate the DNA damage response and checkpoint, as these responses could occur with a less than twofold change in E2F1 protein levels. Instead, our findings indicate that the precise timing of E2F1 expression dictates S-phase entry and that accurate timing of E2F1 accumulation requires converging signals from the Rb/E2F pathway and the c-Myc-regulated miR-17 and miR-20a miRNAs to circumvent a G1 checkpoint arising from the untimely accumulation of E2F1. These data provide a mechanistic view of miRNA-based regulation of E2F1 in the context of the emerging model that miRNAs coordinate the timing of cell cycle progression.

Asunto(s)

Factor de Transcripción E2F1/metabolismo , Fase G1 , MicroARNs/metabolismo , Ciclo Celular/genética , Línea Celular , Daño del ADN , Diploidia , Factor de Transcripción E2F1/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Fase G1/genética , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética

RESUMEN

Deregulation of the Rb/E2F pathway in human fibroblasts results in an E2F1-mediated apoptosis dependent on Atm, Nbs1, Chk2 and p53. Here, we show that E2F1 expression results in MRN foci formation, which is independent of the Nbs1 interacting region and the DNA-binding domain of E2F1. E2F1-induced MRN foci are similar to irradiation-induced foci (IRIF) that result from double-strand DNA breaks because they correlate with 53BP1 and gammaH2AX foci, do not form in NBS cells, do form in AT cells and do not correlate with cell cycle entry. In fact, we find that in human fibroblasts deregulated E2F1 causes a G1 arrest, blocking serum-induced cell cycle progression, in part through an Nbs1/53BP1/p53/p21(WAF1/CIP1) checkpoint pathway. This checkpoint protects against apoptosis because depletion of 53BP1 or p21(WAF1/CIP1) increases both the rate and extent of apoptosis. Nbs1 and p53 contribute to both checkpoint and apoptosis pathways. These results suggest that E2F1-induced foci generate a cell cycle checkpoint that, with sustained E2F1 activity, eventually yields to apoptosis. Uncontrolled proliferation due to Rb/E2F deregulation as well as inactivation of both checkpoint and apoptosis programs would then be required for transformation of normal cells to tumor cells.

Asunto(s)

Ciclo Celular/fisiología , Reparación del ADN/fisiología , Factor de Transcripción E2F1/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , Ácido Anhídrido Hidrolasas , Apoptosis/fisiología , Proteínas de Ciclo Celular/fisiología , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Daño del ADN/fisiología , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F1/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteína Homóloga de MRE11 , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteína 1 de Unión al Supresor Tumoral P53

RESUMEN

Although it is unclear which cellular factor(s) is responsible for the genetic instability associated with initiating and sustaining cell transformation, it is known that many cancers have mutations that inactivate the Rb-mediated proliferation pathway. We show here that pRb inactivation and the resultant deregulation of one E2F family member, E2F1, leads to DNA double-strand break (DSB) accumulation in normal diploid human cells. These DSBs occur independent of Atm, p53, caspases, reactive oxygen species, and apoptosis. Moreover, E2F1 does not contribute to c-Myc-associated DSBs, indicating that the DSBs associated with these oncoproteins arise through distinct pathways. We also find E2F1-associated DSBs in an Rb mutated cancer cell line in the absence of an exogenous DSB stimulus. These basal, E2F1-associated DSBs are not observed in a p16(ink4a) inactivated cancer cell line that retains functional pRb, unless pRb is depleted. Thus, Rb status is key to regulating both the proliferation promoting functions associated with E2F and for preventing DNA damage accumulation if E2F1 becomes deregulated. Taken together, these data suggest that loss of Rb creates strong selective pressure, via DSB accumulation, for inactivating p53 mutations and that E2F1 contributes to the genetic instability associated with transformation and tumorigenesis.

Asunto(s)

Daño del ADN , Factor de Transcripción E2F1/fisiología , Genes de Retinoblastoma , Secuencia de Bases , Transformación Celular Neoplásica , Células Cultivadas , Humanos , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Interferente Pequeño

RESUMEN

UNLABELLED: Islet transplantation offers a potential cure for type I diabetes, although its success has been limited, due to loss of cells by apoptosis stimulated by the procurement, ischemia, and the isolation process itself. RNA interference (RNAi) as mediated by short interfering RNAs (siRNAs) has become a potent tool to manipulate gene expression in mammalian cells. We describe the first successful introduction of siRNA directly into pancreatic islet cells both during in situ perfusion and from intravenous tail vein injection (in vivo). METHODS: siRNA was targeted to the pancreatic islets of BALB/c mice by retrograde portal vein perfusion or tail vein injection. Cy3-labeled siRNA was dissolved in University of Wisconsin (UW) solution at 2 microg/mL. After delivery pancreata were placed in cold storage at 4 degrees C in UW solution for 24 hours, followed by processing for immunofluorescent staining for insulin. Fluorescent imaging was obtained using a Nikon DIAPHOT 300 Inverted Micoscope with a Zeiss AxioCam and OpenLab image capturing software. RESULTS: In situ delivery of siRNA was demonstrated by fluorescent imaging composites of (red) siRNA in and along (green) insulin stained islets from pancreas sections as compared with untreated control sections. The siRNA was detected mainly in and along venous structures throughout the pancreatic tissue. In vivo delivery of siRNA into islets was observed by fluorescent images taken of isolated islets in culture. CONCLUSIONS: We have described the successful delivery of siRNA to pancreatic islets via a novel in situ pancreas perfusion technique and in vivo delivery via tail vein injection.

Asunto(s)

Trasplante de Islotes Pancreáticos/fisiología , Islotes Pancreáticos/fisiología , ARN Interferente Pequeño/metabolismo , Adenosina , Alopurinol , Animales , Secuencia de Bases , Glutatión , Inyecciones Intravenosas , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Soluciones Preservantes de Órganos , ARN Interferente Pequeño/administración & dosificación , Rafinosa

RESUMEN

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in several disorders, including an association between HCMV reactivation and the overproliferation of arterial smooth muscle cells observed in restenosis. Although HCMV can mediate a growth-arrest phenotype in infected cells, the virus can also promote an environment conducive to proliferation. Here, we present evidence that the HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, may be responsible for inducing this proliferative environment by altering cell cycle control. We find that expression of either of these IE proteins can alter the cell cycle distribution of randomly cycling cells towards S and G(2)/M phases. Additionally, we find that expression of IE2-86, but not IE1-72, induces quiescent cells into S phase and delays cell cycle exit. In the absence of p53, IE1-72 expression can induce S phase and delay cell cycle exit. We also demonstrate that p53 protein levels increase in fibroblasts following the expression of IE1-72. The observed accumulation of p53 protein in IE1-72-expressing cells may account for the inability of IE1-72 to induce S phase and delay cell cycle exit. Our data suggest that expression of HCMV IE1-72 and IE2-86 is sufficient to alter the cell cycle to generate an environment conducive to proliferation.

Asunto(s)

Citomegalovirus/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Glicoproteínas de Membrana , Transactivadores , Proteínas del Envoltorio Viral , Proteínas Virales , Adenoviridae/genética , Animales , Western Blotting , Ciclo Celular/fisiología , División Celular/fisiología , Línea Celular , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/virología , Citometría de Flujo , Humanos , Proteínas Inmediatas-Precoces/genética , Ratas , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

In astrocytes, thyroxine modulates type II iodothyronine 5'-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC(50) levels approximately 1 nm) were >100-fold more potent than 3,5,3'-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH(2)-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.

Asunto(s)

Proteínas de Unión a Calmodulina/metabolismo , Endocitosis/efectos de los fármacos , Yoduro Peroxidasa/metabolismo , Miosina Tipo V , Proteínas del Tejido Nervioso/metabolismo , Hormonas Tiroideas/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/genética , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Inmunohistoquímica , Yoduro Peroxidasa/clasificación , Yoduro Peroxidasa/inmunología , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Unión Proteica/efectos de los fármacos , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Tiroxina/farmacología , Triyodotironina/farmacología , Triyodotironina Inversa/farmacología

RESUMEN

Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.

Asunto(s)

Yoduro Peroxidasa/química , Secuencia de Aminoácidos , Animales , Astrocitos/metabolismo , Secuencia de Bases , Northern Blotting , Encéfalo/metabolismo , Sistema Libre de Células , Células Cultivadas , Corteza Cerebral/metabolismo , Clonación Molecular , AMP Cíclico/metabolismo , ADN Complementario/metabolismo , Biblioteca de Genes , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Yoduro Peroxidasa/biosíntesis , Yoduro Peroxidasa/genética , Cinética , Proteínas Luminiscentes/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Plásmidos/metabolismo , Pruebas de Precipitina , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Distribución Tisular , Yodotironina Deyodinasa Tipo II

RESUMEN

Previous work has demonstrated a role for E2F transcription factor activity in the regulation of cell growth during the G0/G1-S phase transition. Indeed, overexpression of E2F proteins, including the E2F1 and E2F2 products, induces DNA synthesis in quiescent fibroblasts. Other experiments have shown that E2F1 expression also induces apoptosis, dependent on p53. Although this could represent a response to aberrant cell cycle progression, we show that only E2F1 induces apoptosis and that this coincides with an ability of E2F1 to induce accumulation of p53 protein. We also find that coexpression of Mdm2, which is known to regulate p53 activity, blocks the E2F1-mediated induction of apoptosis and also blocks the E2F1-mediated accumulation of p53. We propose that E2F1 acts as a specific signal for the induction of apoptosis by affecting the accumulation of p53, which under normal proliferative conditions may be controlled by Mdm2.

Asunto(s)

Apoptosis , Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteínas Nucleares , Proteínas Proto-Oncogénicas/fisiología , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/biosíntesis , Adenoviridae/genética , Animales , Línea Celular , Clonación Molecular , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Expresión Génica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Ratas , Proteína 1 de Unión a Retinoblastoma , Transducción de Señal , Factor de Transcripción DP1 , Factores de Transcripción/farmacología

RESUMEN

The adenovirus early gene product E1A is a potent stimulator of cellular proliferation, which when overexpressed can overcome the growth-inhibitory effects of the polypeptide hormone transforming growth factor beta (TGF-beta). The ability of TGF-beta to arrest cell growth in G1 correlates with the transcriptional induction of the cyclin-dependent kinase inhibitors, p15/INK4B and p21/WAF1/Cip1; an inhibition of the G1 cyclin-Cdk complexes; and a maintenance of the retinoblastoma susceptibility gene product, Rb, in a hypophosphorylated state. The ability of E1A to overcome TGF-beta-mediated growth inhibition derives, in part, from its ability to sequester Rb and Rb family members. We report here that E1A also acts upstream of Rb by blocking the TGF-beta-mediated induction of p15 and p21. Consistent with these findings, E1A expression also blocks the ability of TGF-beta to inhibit Cdk2 kinase activity, as well as its ability to hold Rb in a hypophosphorylated state. The effect of E1A on the induction of p15 and p21 is independent of E1A's Rb binding activity. The E1A-mediated decrease in p15 levels is primarily the result of a block at the level of transcriptional activation by TGF-beta. This effect is dependent on E1A's ability to bind p300, one of E1A's target proteins. Thus, the ability of E1A to affect p15 and p21 expression represents an additional possible mechanism by which E1A can circumvent the negative regulation of cell cycle progression.

Asunto(s)

Proteínas E1A de Adenovirus/genética , Proteínas Portadoras/biosíntesis , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Ciclinas/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Proteínas Supresoras de Tumor , Proteínas Portadoras/genética , Línea Celular , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/genética , Inhibidores Enzimáticos/metabolismo , Humanos , Modelos Biológicos , Fosforilación , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/metabolismo , Activación Transcripcional

RESUMEN

The Rb-related p107 protein has been implicated as an important control element in proper cell cycle progression. The p107 protein is thought to restrict cellular proliferation in part through its interaction with the E2F family of transcription factors and is, therefore, a specific target for regulation by several DNA viruses. Here, we demonstrate that p107 protein levels are induced in a biphasic manner in human fibroblasts during productive infection by the human cytomegalovirus (HCMV). Expression patterns of p107 protein levels during HCMV infection of human embryonic lung cells (HELs) demonstrate a sustained induction from early to late times of infection. We also demonstrate that the HCMV immediate-early protein IE1-72 complexes in vivo with the p107 protein and that this interaction can be reconstituted in an in vitro system by using reticulocyte-translated protein. Our data demonstrate that the interaction between p107 and the IE1-72 protein occurs at times of infection that temporally match the second tier of p107 protein induction and the phosphorylation pattern of the IE1-72 protein. Furthermore, we show here that the ability of p107 to transcriptionally repress E2F-responsive promoters can be overcome by expression of the IE1-72 protein. This effect appears to be specific, since the IE1-72 protein is not capable of relieving Rb-mediated repression of an E2F-responsive promoter. Finally, our data demonstrate that HCMV infection can induce cellular proliferation in quiescent cells and that IE1-72 expression alone can, to a degree, drive a similar progression through the cell cycle. These data suggest that IE1-72-mediated transactivation of E2F-responsive promoters through alleviation of p107 transcriptional repression may play a key role in the cell cycle progression stimulated by HCMV infection.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular , Citomegalovirus/metabolismo , Proteínas de Unión al ADN , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Proteínas Virales , Ciclo Celular , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Citomegalovirus/genética , Factores de Transcripción E2F , Genes Reporteros , Humanos , Proteínas Inmediatas-Precoces/genética , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Transcripción Genética , Células Tumorales Cultivadas

RESUMEN

During human cytomegalovirus (HCMV) infection, a series of regulated events take place following virus binding and entry into the cell, including the upregulation of cellular transcription factors, such as NF-kappa B, which play an essential role in the viral life cycle. We show here that NF-kappa B message is induced during HCMV infection and that the induction is biphasic, suggesting an initial induction at immediate-early (IE) times and a second round of induction at early times. This hypothesis is supported by experiments using cyclohexamide, which showed that the first tier of induction was drug insensitive, while the second tier was drug sensitive. We then show that virus binding alone is sufficient to stimulate NF-kappa DNA binding activity, supporting its role in the initial induction of NF-kappa B. To begin to elucidate the mechanism(s) for the second tier of NF-kappa B regulation, we examined promoter constructs from the NF-kappa B subunits (p105/p50 and p65) for responsiveness following HCMV infection. HCMV infection transactivated the p105/p50 and p65 promoters. The viral IE proteins (IE1-72, IE2-55, and IE2-86) are expressed during the time we see NF-kappa B induction, so we examined their role in NF-kappa B induction. The IE1-72, IE2-55, and IE2-86 proteins transactivated the p65 promoter, while only the IE2-55 protein transactivated the p105/p50 promoter. The p105/p50 promoter has NF-kappa B sites; therefore, upregulation could also be caused by an autoregulatory mechanism. The p65 promoter, however, has been demonstrated to contain only Sp1 sites. To investigate the potential role of SP1, we examined nuclear extracts from HCMV-infected cells. Here, we show that there is a biphasic increase in SP1 activity during viral infection and that there is apparently an absolute requirement for SP1 in the transactivation of the p65 promoter. In conclusion, we suggest a model in which the initial induction of NF-kappa B occurs through viral modulation of cellular factors and the sustained levels of NF-kappa B induction are regulated by a combination of cellular and viral factors.

Asunto(s)

Citomegalovirus/genética , Citomegalovirus/metabolismo , FN-kappa B/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/análisis , Cloranfenicol O-Acetiltransferasa/biosíntesis , Fibroblastos , Regulación Viral de la Expresión Génica , Genes Virales , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , FN-kappa B/biosíntesis , FN-kappa B/metabolismo , Sondas de Oligonucleótidos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Transfección

RESUMEN

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular , ADN Polimerasa II/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Interfase/genética , Factores de Transcripción/metabolismo , Adenoviridae/genética , Secuencia de Bases , Northern Blotting , Ciclo Celular/efectos de la radiación , ADN Polimerasa II/biosíntesis , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Fase G1/genética , Rayos gamma , Vectores Genéticos , Modelos Biológicos , Datos de Secuencia Molecular , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas Recombinantes/metabolismo , Proteína 1 de Unión a Retinoblastoma , Ribonucleótido Reductasas/biosíntesis , Ribonucleótido Reductasas/genética , Fase S/genética , Timidilato Sintasa/biosíntesis , Timidilato Sintasa/genética , Factores de Transcripción/genética

RESUMEN

Various experiments have demonstrated a role for the E2F transcription factor in the regulation of cell growth during the G0/G1/S phase transition. Indeed, overexpression of the E2F1 product, a component of the cellular E2F activity, induces DNA synthesis in quiescent fibroblasts. To provide an approach to a more detailed biochemical analysis of these events, we have made use of a recombinant adenovirus containing the E2F1 cDNA in order to efficiently express the E2F1 product in an entire population of cells. We demonstrate an induction of DNA synthesis when quiescent cells are infected with the E2F1 recombinant virus. However, we also find that the induction does not lead to a complete replication of the cellular genome, as revealed by flow cytometry. The incomplete nature of cellular DNA replication is due, at least in part, to the fact that E2F1 overexpression leads to massive cell death that is characteristic of apoptosis. This E2F1-mediated induction of apoptosis is largely dependent on endogenous wild-type p53 activity and can be subverted by introducing mutant forms of p53 into these cells or by overexpressing E2F1 in fibroblasts derived from p53-null mouse embryos. We conclude that E2F1 can induce events leading to S phase but that the process is not normal and appears to result from the activation of a cell death pathway.

Asunto(s)

Apoptosis/genética , Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , ADN/biosíntesis , Factores de Transcripción/biosíntesis , Adenoviridae/genética , Células Cultivadas , ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Fibroblastos/virología , Rayos gamma , Hidrólisis , Recombinación Genética , Proteína 1 de Unión a Retinoblastoma , Fase S , Transducción de Señal , Factor de Transcripción DP1 , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Cultured endometrial stromal cells were susceptible to productive human cytomegalovirus (HCMV) infection. Infection of endometrial stromal cells resulted in pronounced cytopathic effects including cell rounding and aggregation, fusions, and some lysis, although not in the synchronous fashion observed in infected fibroblasts. The aggregation events were reminiscent of normal endometrial stromal cell responses to cyclical estrogen/progesterone levels. Immunofluorescence analysis demonstrated expression of viral gene products suggesting a productive virus infection. One-step growth analysis showed that infectious virus was produced but the titers were two logs lower than those obtained in fibroblasts even though HCMV DNA accumulated to similar levels in both cell types. In contrast, viral DNA replication was greatly reduced in endometrial stromal cells immortalized with a temperature-sensitive SV40 large T gene at both permissive and nonpermissive temperatures. A more detailed analysis of viral gene expression by Northern blotting revealed earlier appearances and greater initial levels of viral transcripts in endometrial stromal cells. No HCMV gene expression was observed at 120 hpi in these cells even though half of the cells were still intact and cellular gene expression was functional. Since this was the time of peak virus production, it seems plausible that reduced viral gene expression at late times p.i. was a major contributor to the reduced titers observed in endometrial stromal cells. These in vitro results coupled with in vivo observations by others of endometritis associated with HCMV suggest that further investigation into the effects of HCMV on the endometrium is warranted.

Asunto(s)

Citomegalovirus/fisiología , Endometrio/microbiología , Secuencia de Bases , Células Cultivadas , Citomegalovirus/genética , Replicación del ADN , ADN Viral/biosíntesis , Endometrio/citología , Endometrio/patología , Femenino , Fibroblastos , Regulación de la Expresión Génica , Genes Virales , Humanos , Datos de Secuencia Molecular , Células del Estroma/microbiología , Replicación Viral

RESUMEN

The human cytomegalovirus immediate-early gene product 2 (IE2) is able to transactivate homologous and heterologous promoters alone or augmented by immediate-early gene product 1 (IE1). IE2 has also been shown to autoregulate the major immediate-early promoter by directly binding to a cis repression signal located between the TATA box and the cap site. However, IE2 has not been shown to act directly through a specific DNA sequence in transactivating various promoters. To understand whether IE2 can be indirectly involved in DNA sequence-specific transactivation through interactions with other transcriptional factors, we performed a study of the interactions of IE2 with cellular proteins. In order to study these interactions, IE cDNAs were subcloned into a bacterial expression vector, pGEX2T, by polymerase chain reaction amplification to produce fusion proteins which were full-length as well as proteins which contained various functional domains. We were able to demonstrate IE2's ability to interact directly or indirectly with several cellular proteins ranging from > 200 to 14 kDa through glutathione S-transferase-fusion protein precipitation and far-Western analysis. These interactions have been mapped to domains within IE2 which are known to be necessary for either transactivation or both transactivation and autoregulation. All of the IE2-associated proteins are nuclear proteins, and a subset are phosphorylated. In vitro-synthesized 35S-IE2 protein and bacterially expressed IE2 fusion proteins were used to study IE2-IE2 interaction by binding assay and far-Western analysis. IE2-IE2 interactions were mapped to a domain containing a putative helix-turn-helix motif located near the C terminus of IE2, between amino acids 456 and 539. However, IE2 was unable to directly interact with either IE1, an alternatively spliced variant of IE2 (55 kDa), or IE2 deletion mutants that did not contain the multimerization domain.

Asunto(s)

Citomegalovirus/metabolismo , Genes Virales , Proteínas Inmediatas-Precoces/metabolismo , Glicoproteínas de Membrana , Transactivadores , Activación Transcripcional , Proteínas del Envoltorio Viral , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Línea Celular , Clonación Molecular , Citomegalovirus/genética , Vectores Genéticos , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/aislamiento & purificación , Glutatión Transferasa/metabolismo , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/aislamiento & purificación , Pulmón , Metionina/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Radioisótopos de Fósforo , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Radioisótopos de Azufre , TATA Box

RESUMEN

The molecular identity of mouse sperm acrosome antigen recognized by HS-63 monoclonal antibody was analyzed by various biochemical, immunological and molecular biological methods. When its cognate antigen, MSA-63 was isolated from mouse testis by immunoaffinity chromatography, a group of protein spots with wide range of molecular sizes and isoelectric points were identified. Through previous studies, it was established that most of these protein spots were actin-like molecules co-purified with MSA-63 protein from mouse testis. To analyze the molecular size heterogeneity of the isolated MSA-63 proteins, rabbit antisera against a computer-predicted antigenic synthetic peptide (amino acid residue No. 160-171) and a recombinant glutathione S-transferase (GST) fusion protein (GST-63) were raised. These two antisera and those raised against the isolated MSA-63 protein were used as the probes in comparative Western blot assay, indirect immunofluorescent assay and enzyme-linked immunosorbent assay (ELISA). Using ELISA, antisera against GST-63 and computer-predicted antigenic synthetic peptides were shown to cross-react with affinity-isolated MSA-63 protein coated on microwells. However, little immunological cross-reactivity was observed between GST-63 fusion protein and the synthetic peptide. By using a Western blot assay, two major protein bands of 22 and 32 kDa, respectively were commonly detected on mouse testis homogenate strips by both anti-MSA-63 and anti-GST-63. In addition, anti-MSA-63 also recognized several protein bands with molecular masses greater than 35 kDa. The results of this study suggested that the molecular heterogeneity of MSA-63 protein isolated from mouse testis and sperm, is due to a series of post-translational modifications on a single gene product. These modifications may include glycosylations, proteolytic digestions and tight non-covalent associations with other testicular cytoskeletal proteins, such as actins.

Asunto(s)

Acrosoma/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos/análisis , Animales , Antígenos/inmunología , Antígenos/fisiología , Secuencia de Bases , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Hormonas Esteroides Gonadales/análisis , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Conejos

RESUMEN

Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Realizador del VIH/genética , VIH/genética , Proteínas I-kappa B , Transcripción Genética , Animales , Linfocitos B/citología , Secuencia de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/análisis , Sondas de ADN , Proteínas de Unión al ADN/farmacología , Humanos , Ratones , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , FN-kappa B/farmacología , Conformación Proteica , Proteínas Recombinantes/metabolismo , Factor de Transcripción ReIA , Transcripción Genética/efectos de los fármacos , Transfección

RESUMEN

Infection-induced activation of the human cytomegalovirus major immediate early enhancer/promoter has been shown to be regulated primarily by transcription factor NF-kappa B cis elements. However, the mechanism(s) by which human cytomegalovirus induces NF-kappa B activity is unknown. A study was therefore undertaken to determine how this virus would affect normal NF-kappa B regulation. Viral infection of fibroblasts resulted in the specific stimulation of promoters containing major histocompatibility complex NF-kappa B cis elements fused upstream of the chloramphenicol acetyltransferase reporter gene. Electrophoretic mobility shift assays of nuclear extracts derived from mock- and virus-infected cells showed dramatic and sustained increases in DNA-binding proteins specific for these NF-kappa B sequences. Experiments using MAD-3 I kappa B, a specific inhibitor of NF-kappa B, and antibodies directed against rel family members demonstrated that the induced binding activities contained p50 and p65 proteins but not c-rel. Northern analysis indicated maximal levels of p50 mRNA by 4 h postinfection, whereas p65 and MAD-3 I kappa B mRNA accumulation peaked at 48-72 h postinfection, suggesting different regulatory mechanisms for p50 and p65/I kappa B genes. Electrophoretic mobility shift assays with deoxycholate-treated cytoplasmic extracts demonstrated a 3- to 4-fold decrease in the cytosolic stores of NF-kappa B binding activity by 4 h postinfection. Western blots probed with antibodies directed against MAD-3 I kappa B or pp40 (a protein isolated from chicken with sequence and biochemical properties similar to those of MAD-3 I kappa B) indicated that a cross-reactive peptide of 39 kDa was no longer detectable after 24 h postinfection. These results demonstrate that the activation and maintenance of nuclear NF-kappa B DNA binding and enhancer activities upon human cytomegalovirus infection occurs by multiple mechanisms.

Asunto(s)

Infecciones por Citomegalovirus/metabolismo , Elementos de Facilitación Genéticos , Proteínas I-kappa B , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Secuencia de Bases , Núcleo Celular/metabolismo , Reacciones Cruzadas , Infecciones por Citomegalovirus/genética , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibroblastos , Humanos , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , FN-kappa B/genética , ARN Mensajero/metabolismo , Proteínas Virales/inmunología