RESUMEN
The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser(2). This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Flavanonas/toxicidad , Flavonas/toxicidad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Antineoplásicos/uso terapéutico , Sitios de Unión , Línea Celular Tumoral , Simulación por Computador , Quinasa 9 Dependiente de la Ciclina/metabolismo , Flavanonas/uso terapéutico , Flavonas/uso terapéutico , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , Scutellaria baicalensis/química , Transcripción GenéticaRESUMEN
We studied the nuclear topography of RNA transcription and DNA replication in mammalian cell types with super-resolution fluorescence microscopy, which offers a resolution beyond the classical Abbe/Raleigh limit. Three-dimensional structured illumination microscopy (3D-SIM) demonstrated a network of channels and wider lacunas, called the interchromatin compartment (IC). The IC starts at nuclear pores and expands throughout the nuclear space. It is demarcated from the compact interior of higher-order chromatin domains (CDs) by a 100-200-nm thick layer of decondensed chromatin, termed the perichromatin region (PR). Nascent DNA, nascent RNA, RNA polymerase II (RNA Pol II), as well as histone modifications for transcriptionally competent/active chromatin, are highly enriched in the PR, whereas splicing speckles are observed in the interior of the IC. In line with previous electron microscopic evidence, spectral precision distance/position determination microscopy (SPDM) confirmed the presence of RNA Pol II clusters indicative of transcription factories. Still, a substantial part of transcription apparently takes place outside of such factories. Previous electron microscopic evidence has suggested that the functional nuclear organization of DNA replication depends on brownian movements of chromatin between the CD interior and the PR. As an incentive for future studies, we hypothesize that such movements also take place during transcription, i.e., only the actually transcribed part of a gene may be located within the PR, whereas its major part, including previously or later transcribed sequences, is embedded in a higher-order chromatin configuration in the interior of the CD.
Asunto(s)
Compartimento Celular , Cromatina/química , Cromatina/metabolismo , Replicación del ADN/genética , Transcripción Genética , Animales , ADN/química , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Microscopía , Matriz Nuclear/metabolismo , Procesamiento Proteico-Postraduccional , ARN/química , ARN Polimerasa II/química , Empalme del ARN/genéticaAsunto(s)
Huesos/citología , Proteínas de la Matriz Extracelular/fisiología , Osteocitos/fisiología , Fosfoproteínas/fisiología , Animales , Proteínas de la Matriz Extracelular/genética , Humanos , Ratones , Ratones Noqueados , Microscopía de Fuerza Atómica , Osteocitos/ultraestructura , Fosfoproteínas/genéticaRESUMEN
The C-terminal domain (CTD) of mammalian RNA polymerase II consists of 52 repeats of the consensus hepta-peptide YSPTSPS, and links transcription to the processing of pre-mRNA. Although Pol II with a CTD shortened to five repeats (Pol II Delta5) is transcriptionally inactive on chromatin templates, it is not clear whether CTD is required for promoter recognition in vivo. Here, we demonstrate that in the context of chromatin, Pol II Delta5 can bind to the c-myc promoter with the same efficiency as wild type Pol II. However, Pol II Delta5 does not form a stable initiation complex, and does not transcribe promoter proximal sequences. Fluorescence recovery after photobleaching (FRAP) experiments with cells expressing enhanced green fluorescent protein (EGFP)-tagged Delta5 or wildtype Pol II revealed a single, highly mobile Pol II Delta5 fraction whereas wildtype Pol II yielded less mobile fractions. These data suggest that CTD is not required for promoter recognition, but rather for subsequent formation of a stable initiation complex and isomerization to an elongation competent complex.
Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa II/química , Transcripción Genética , Sitios de Unión , Línea Celular Tumoral , Núcleo Celular/enzimología , Secuencia de Consenso , Recuperación de Fluorescencia tras Fotoblanqueo , Genes myc , Proteínas Fluorescentes Verdes/análisis , Humanos , Estructura Terciaria de Proteína , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Secuencias Repetitivas de Aminoácido , Eliminación de SecuenciaRESUMEN
Upregulation of the proto-oncoprotein Myc, a basic, helix-loop-helix, leucin zipper domain transcription factor has profound consequences on cell proliferation, cell growth and apoptosis. Cell cultures of somatic c-myc-/- rat fibroblasts show extremely prolonged doubling times of 52 h. Using time-lapse microscopy, we show here that individual c-myc-/- cells proceeded within approximately 24 h through the cell cycle as fast as c-myc+/+ cells. However, c-myc-/- cells were highly sensitive to contact inhibition and readily arrested in the cell cycle already at low density. Activation of conditional MycER overcame cell cycle arrest in c-myc-/- cells and led to continuous proliferation at the expense of increased apoptosis at high cell density. Conditional expression of Mad1, a Myc antagonist, represses proliferation of different cell types including U2OS cells. In analogy to the effect of Myc, this occurs mainly by reducing the probability of cells remaining in the cycle. Our data demonstrate that the Myc/Max/Mad network does not regulate the duration of the cell cycle, but the decision of cells to enter or exit the cell cycle.
Asunto(s)
Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción , Animales , Apoptosis/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Recuento de Células , Proteínas de Ciclo Celular , División Celular , Línea Celular , Células Cultivadas , Inhibición de Contacto/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Regulación de la Expresión Génica , Microscopía por Video , Proteínas Nucleares , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Ratas , Proteínas Represoras/genética , Factores de TiempoRESUMEN
The objective of this study was to identify through quantum mechanical quantitative structure activity relationships (Q-QSARs) chemical structures in dental monomers that influence their mutagenicity. AMPAC, a semiempirical computer program that provides quantum mechanical information for chemical structures, was applied to three series of reference chemicals: a set of methacrylates, a set of aromatic and a set of aliphatic epoxy compounds. QSAR models were developed using this chemical information together with mutagenicity data (Salmonella TA 100, Ames Test). CODESSA, a QSAR program that calculates quantum chemical descriptors from information generated by AMPAC and statistically matches these descriptors with observed biological properties was used. QSARs were developed which had r2 values exceeding 0.90 for each study series. These QSARs were used to accurately predict the mutagenicity of BISGMA. a monomer commonly used in dentistry, and two epoxy monomers with developing use in dentistry, GY-281 and UVR-6105. The Q-QSAR quantum mechanical descriptors correctly predicted the level of mutagenicity for all three compounds. The descriptors in the correlation equation pointed to components of structure that may contribute to mutagenesis. The QSARs also provided 'dose windows' for testing mutagenicity, circumventing the need for extensive dose exploration in the laboratory. The Q-QSAR method promises an approach for biomaterials scientists to predict and avoid mutagenicity from the chemicals used in new biomaterial designs.
Asunto(s)
Esmalte Dental/química , Mutágenos , Relación Dosis-Respuesta a Droga , Metacrilatos/química , Modelos Químicos , Pruebas de Mutagenicidad , Teoría Cuántica , Programas Informáticos , Relación Estructura-ActividadRESUMEN
The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.
Asunto(s)
Cromatina/química , Genes myc , Regiones Promotoras Genéticas , Secuencia de Bases , Línea Celular , ADN Polimerasa II/metabolismo , Cartilla de ADN , Humanos , Mutación , Plásmidos/genética , Unión Proteica , Conformación Proteica , Proto-Oncogenes MasRESUMEN
The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.
Asunto(s)
Linfocitos B/metabolismo , Genes myc/genética , Transcripción Genética , Linfocitos B/patología , Northern Blotting , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Técnicas de Cultivo de Célula , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Marcación de Gen , Humanos , Cinética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proto-Oncogenes Mas , Activación Transcripcional/genética , Transfección , Células Tumorales CultivadasRESUMEN
The product of the proto-oncogene c-myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B-cell tumor, myc is consistently found to be transcriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B-cells is not known. As a model for myc activation in BL cells, we have established a human EBV-EBNA1 positive B-cell line, P493-6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493-6 cells arrest in G0/G1 in the presence of serum. Re-expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E-associated kinase and hyper-phosphorylation of Rb. The transcription factor E2F-1 is expressed in proliferating and arrested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493-6 cells by promoting the expression of a set of cell cycle activators but not by inactivating cell cycle inhibitors.
Asunto(s)
Linfoma de Burkitt/fisiopatología , Proteínas de Ciclo Celular/fisiología , Ciclo Celular/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Linfoma de Burkitt/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosforilación , Inhibidores de la Síntesis de la Proteína/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteína de Retinoblastoma/metabolismo , Proteína p107 Similar a la del Retinoblastoma , Tetraciclina/farmacología , Células Tumorales CultivadasRESUMEN
The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3'-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an alpha-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Delta5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of alpha-amanitin. Moreover, pol II harboring LS*Delta5 failed to transcribe up to the promoter-proximal pause sites in the hsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA.
Asunto(s)
ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Regiones no Traducidas 3'/genética , Amanitinas/farmacología , Animales , Linfoma de Burkitt , Clonación Molecular , Genes fos , Proteínas HSP70 de Choque Térmico/genética , Humanos , Sustancias Macromoleculares , Ratones , Fosforilación , Regiones Promotoras Genéticas , ARN Polimerasa II/química , Empalme del ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Células Tumorales CultivadasRESUMEN
The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, we have identified the cyclin-dependent kinase 4 (CDK4) gene as a transcriptional target of c-MYC. c-MYC induced a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay was associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviated the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation.
Asunto(s)
Quinasas Ciclina-Dependientes/genética , Regulación Enzimológica de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas , Animales , Secuencia de Bases , Células Cultivadas , Quinasa 4 Dependiente de la Ciclina , ADN Complementario , Humanos , Neoplasias Renales/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
The c-Myc protein (Myc) is a transcription factor, and deregulated expression of the c-myc gene (myc) is frequently found in tumours. In Burkitt's lymphoma (BL), myc is transcriptionally activated by chromosomal translocation. We have used a B-cell line called P493-6 that carries a conditional myc allele to elucidate the role of Myc in the proliferation of BL cells. Regulation of proliferation involves the coordination of cell growth (accumulation of cell mass) and cell division [1] [2] [3]. Here, we show that division of P493-6 cells was strictly dependent on the expression of the conditional myc allele and the presence of foetal calf serum (FCS). More importantly, cell growth was regulated by Myc without FCS: Myc alone induced an increase in cell size and positively regulated protein synthesis. An increase in protein synthesis is thought to be one of the causes of cell mass increase. Furthermore, Myc stimulated metabolic activities, as indicated by the acidification of culture medium and the activation of mitochondrial enzymes. Our results confirm the model that Myc is involved in the regulation of cell growth [4] and provide, for the first time, direct evidence that Myc induces cell growth, that is, an increase in cell size, uncoupled from cell division.
Asunto(s)
División Celular/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Línea Celular Transformada , Tamaño de la Célula/fisiología , Medios de Cultivo/metabolismo , HumanosRESUMEN
Transcriptional regulation can occur at the level of initiation and RNA elongation. We report that the rearranged, nontranscribed Ig kappa gene in the pre-B cell line 70Z/3 harbors a paused RNA polymerase II (pol II) at a position between 45 and 89 bp downstream of the transcription initiation site. LPS, an inducer of NF-kappa B, activated Ig kappa gene transcription by increasing the processivity of pol II. TGF-beta inhibited the LPS-induced transcription of the Ig kappa gene, but not initiation and pausing of pol II. A rearranged copy of the Ig kappa gene was introduced into 70Z/3 cells using an episomal vector system. The episomal Ig kappa was regulated by LPS and TGF-beta like the endogenous gene and established a paused pol II, whereas a construct with a deletion of the intron enhancer and the C region did not establish a paused pol II. Two distinct functions can therefore be assigned to the deleted DNA elements: loading of pol II to its pause site and induction of processive transcription upon LPS stimulation. It had been proposed that somatic hypermutation of Ig genes is connected to transcription. The pause site of pol II described in this work resides upstream of the previously defined 5' boundary of mutator activity at Ig kappa genes. The possible role of pausing of pol II for somatic hypermutation is discussed.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Genes de Inmunoglobulinas/genética , Cadenas kappa de Inmunoglobulina/genética , Regiones Promotoras Genéticas/inmunología , ARN Polimerasa II/genética , Transcripción Genética/inmunología , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Secuencia de Bases , Reordenamiento Génico de Cadena Ligera de Linfocito B , Vectores Genéticos/inmunología , Humanos , Linfoma de Células B , Ratones , Datos de Secuencia Molecular , FN-kappa B/fisiología , Factor de Crecimiento Transformador beta/fisiología , Células Tumorales CultivadasRESUMEN
In normal cells, the proto-oncogene c-myc is regulated by promoter-proximal pausing of RNA polymerase II (pol II). In Burkitt lymphoma cells, c-myc is chromosomally translocated to one of the three immunoglobulin (Ig) gene loci and its transcription is driven constitutively by Ig enhancers. Promoter-proximal pausing of pol II is abolished on the translocated c-myc allele. This raised the question whether induction of Ig gene transcription also involves activation of promoter-proximal paused pol II. Here we have studied the transcriptional activation of a functionally rearranged Ig kappa gene in the mouse pre B cell line 70Z/3. We show that pol II pauses approximately 50 bp downstream of the transcriptional start site of the uninduced Ig kappa gene.
Asunto(s)
Genes myc , Cadenas kappa de Inmunoglobulina/genética , ARN Polimerasa II/metabolismo , Animales , Linfocitos B/inmunología , Línea Celular , Regulación de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Transcripción GenéticaRESUMEN
Epstein-Barr virus (EBV) infects and transforms primary B lymphocytes in vitro. Viral infection initiates the cell cycle entry of the resting B lymphocytes. The maintenance of proliferation in the infected cells is strictly dependent on functional EBNA2. We have recently developed a conditional immortalization system for EBV by rendering the function of EBNA2, and thus proliferation of the immortalized cells, dependent on estrogen. This cellular system was used to identify early events preceding induction of proliferation. We show that LMP1 and c-myc are directly activated by EBNA2, indicating that all cellular factors essential for induction of these genes by EBNA2 are present in the resting cells. In contrast, induction of the cell cycle regulators cyclin D2 and cdk4 are secondary events, which require de novo protein synthesis.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas , Línea Celular Transformada , Ciclina D2 , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proto-Oncogenes Mas , Proteínas de la Matriz Viral/genéticaRESUMEN
The carboxy-terminal domain of the large subunit of mouse and human RNA polymerase II contains 52 repeats of a heptapeptide which are the targets for a variety of kinases. We have used an alpha-amanitin resistant form of the large subunit of pol II to study the role of the carboxy-terminal domain in the expression of chromosomal genes. The large subunit of RNA polymerase II and deletion mutants thereof, which contain only 31 (LSdelta31) and 5 (LSdeltaS) repeats, were expressed in 293 cells. Subsequently, the endogenous large subunit of RNA polymerase II was inhibited by alpha-amanitin and the induction of chromosomal c-fos and hsp70A genes was determined. Cells expressing the large subunit of RNA polymerase II and LSdelta31 were able to transcribe the c-fos and hsp70A genes after treatment with the phorbolester TPA and after heat-shock, respectively. In contrast, cells expressing LSdelta5 failed to induce expression of both genes.
Asunto(s)
Genes fos , Proteínas HSP70 de Choque Térmico/genética , ARN Polimerasa II/genética , Activación Transcripcional , Animales , Ácidos Carboxílicos , Línea Celular , Humanos , Ratones , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Eliminación de SecuenciaRESUMEN
Confluent 3T3-L1 preadipocytes differentiate to adipocytes in the presence of insulin, dexamethasone, and isobutylmethylxanthine (IDI). A transient increase of DNA synthesis is induced in 3T3-L1 cells 18 h after addition of IDI, followed by an arrest in the G1 phase of the cell cycle. Growth arrested cells express the proto-oncogene c-myc and the gene for the CCAAT/enhancer binding protein (C/EBPalpha) between day 2 and 5. While c-Myc is strongly implicated in cell proliferation, C/EBPalpha: is a differentiation-specific transcription factor with antiproliferative activity. Here we have characterized the cell cycle arrest in differentiating 3T3-L1 cells. Arrested cells express the Cdk inhibitors p21 and p27, but, at the same time, show hyperphosphorylation of Rb and expression of the E2F-regulated thymidine kinase gene. The addition of new serum to arrested cells resulted in cyclin A expression and Cdk2 activity, but not in DNA synthesis. Simian virus 40 large tumor antigen (LTAg) is a potent mitogen. The mutant LTAg-K1, deficient in binding of pocket proteins and unable to induce DNA synthesis in serum-starved 3T3-L1 cells, efficiently induced DNA synthesis in differentiating 3T3-L1 cells. This indicates that pocket proteins are probably not involved in the control of the cell cycle arrest during 3T3-L1 cell differentiation. Our data suggest that the differentiation-specific cell cycle block in 3T3-L1 cells is resistant to high levels of c-Myc, inactivation of pocket proteins, upregulation of cyclin A levels, and Cdk2 activation, but can be abolished by a function of LTAg that is independent of binding to pocket proteins.
Asunto(s)
Adipocitos/citología , Ciclo Celular , Diferenciación Celular , Células 3T3 , Animales , Antígenos Transformadores de Poliomavirus/inmunología , Secuencia de Bases , Cartilla de ADN , Replicación del ADN/inmunología , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína de Retinoblastoma/metabolismo , Timidina Quinasa/genéticaRESUMEN
Organization of DNA into chromatin has been shown to contribute to a repressed state of gene transcription. Disruption of nucleosomal structure is observed in response to gene induction, suggesting a model in which RNA polymerase II (pol II) is recruited to the promoter upon reorganization of nucleosomes. Here we show that induction of c-myc transcription correlates with the disruption of two nucleosomes in the upstream promoter region. This nucleosomal disruption, however, is not necessary for the binding of pol II to the promoter. Transcriptionally engaged pol II complexes can be detected when the upstream chromatin is in a more closed configuration. Thus, upstream chromatin opening is suggested to affect activation of promoter-bound pol II rather than entry of polymerases into the promoter. Interestingly, pol II complexes are detectable in both sense and antisense transcriptional directions, but only complexes in the sense direction respond to activation signals resulting in processive transcription.
Asunto(s)
Genes myc/genética , Nucleosomas , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Transcripción Genética , Linfocitos B , Línea Celular Transformada , Cromatina/química , Huella de ADN , Células HL-60 , Humanos , Nucleosomas/enzimología , ARN Polimerasa II/análisis , Activación Transcripcional , TransfecciónRESUMEN
Phospholipase C(gamma)2 (PLCgamma2) is a phospholipid-converting enzyme which, upon receptor stimulation, is activated within membrane-bound signalling complexes. In contrast to the highly ubiquitous PLCgamma1, PLCgamma2 is expressed predominantly in B-lymphocytes. Associated with antigen-coupling receptors it is activated by tyrosine phosphorylation after the triggering of B-cell surface immunoglobulin. We have cloned and sequenced the human PLCgamma2 promoter. Primer extension analysis reveals the existence of a major transcriptional start site. The TATA-less promoter contains G+C-rich stretches with a cluster of contiguous SP1 consensus sites, an NF1, and an AP2 site between bp -220 to -70. A construct containing the region from -189 to +78 confers full promoter activity, as shown by fusion to a luciferase reporter gene construct. The distal part of the promoter between bp -662 to -293 containing an SRE, EBF and CACCC box contributed negatively to promoter activity in the B-cell line Raji but not in three adherent cell lines. In Raji cells, PLCgamma2 mRNA is expressed at low levels with a half life greater than 4 h. After treatment with serum, TPA, retinoic acid, or with 5-azacytidine increased levels of PLCgamma2 mRNA were induced in B-cells.
Asunto(s)
Médula Ósea/metabolismo , Isoenzimas/genética , Regiones Promotoras Genéticas , Fosfolipasas de Tipo C/genética , Células 3T3 , Animales , Secuencia de Bases , Clonación Molecular , ADN Recombinante , Activación Enzimática , Humanos , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Fosfolipasa C gamma , Eliminación de Secuencia , Transcripción Genética , Activación Transcripcional , Fosfolipasas de Tipo C/metabolismoRESUMEN
The nucleosomal structure of active and inactive c-myc genes has been analyzed in detail in undifferentiated and differentiated cells of the promyelocytic leukemia cell line HL60. The c-myc P2 promoter was never found in nucleosomal configuration, no matter whether c-myc was expressed or not. Differences in the nucleosomal structure, however, were found in the promoter upstream region proximal to a previously described DNase I-hypersensitive site I, at the P0 promoter, and at the P1 promoter and upstream thereof. In these regions nucleosomes were detected in differentiated but not undifferentiated HL60 cells. Similar patterns of nucleosomes as found for active and inactive c-myc genes in HL60 cells were found for active and inactive episomal c-myc genes in stably transfected B cell lines. In these cell lines three activation stages could be described for episomal c-myc constructs: (i) uninducible, (ii) inducible, and (iii) induced. Significant differences in the nucleosomal structure of c-myc were observed for the uninducible and inducible stages, but not for the inducible and induced stages.