RESUMEN
The interesting discovery reported here that soluble adenovirus serotype 5 (Ad5) fiber proteins enter cells without the virus was a serendipitous result during our development of Ad5 capsid proteins as nonviral gene transfer vectors. The Ad5 capsid fiber and penton proteins mediate infection. The fiber docks to a noninternalizing cell surface protein called the coxsackievirus-Ad receptor (CAR), followed by penton binding to integrins, triggering integrin-mediated endocytosis of the virus. In our previous work, we assembled the nonviral complex, 3PO, which utilized the penton to mediate gene transfer through integrin binding and endocytosis. Here, we tested whether incorporating the fiber targets 3PO to CAR, thus recapitulating the Ad5 infection pathway. As CAR is not an endocytic receptor, we were surprised to find that the fiber alone, without the penton, enabled gene transfer by binding CAR, but internalizing through an unknown mechanism. We show here that the fiber distributes to the nucleus and cytoplasm after temperature-independent uptake, whereas the penton accumulates around the nucleus after temperature-dependent uptake. Fiber uptake by HeLa cells is also actin-dependent, requires the fiber tail/shaft region, and is largely inhibited by heparin. This study raises the possibility that alternative pathways may enable both viral and nonviral cell entry.
Asunto(s)
Proteínas de la Cápside/genética , Terapia Genética/métodos , Integrinas/metabolismo , Receptores Virales/metabolismo , Transducción Genética/métodos , Proteínas de la Cápside/metabolismo , Línea Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Endocitosis , Células HeLa , Humanos , Microscopía Confocal , Unión ProteicaRESUMEN
We describe a functional and biochemical link between the myogenic activator MyoD, the deacetylase HDAC1, and the tumor suppressor pRb. Interaction of MyoD with HDAC1 in undifferentiated myoblasts mediates repression of muscle-specific gene expression. Prodifferentiation cues, mimicked by serum removal, induce both downregulation of HDAC1 protein and pRb hypophosphorylation. Dephosphorylation of pRb promotes the formation of pRb-HDAC1 complex in differentiated myotubes. pRb-HDAC1 association coincides with disassembling of MyoD-HDAC1 complex, transcriptional activation of muscle-restricted genes, and cellular differentiation of skeletal myoblasts. A single point mutation introduced in the HDAC1 binding domain of pRb compromises its ability to disrupt MyoD-HDAC1 interaction and to promote muscle gene expression. These results suggest that reduced expression of HDAC1 accompanied by its redistribution in alternative nuclear protein complexes is critical for terminal differentiation of skeletal muscle cells.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasas/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/crecimiento & desarrollo , Proteína MioD/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Medio de Cultivo Libre de Suero , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Histona Desacetilasa 1 , Histona Desacetilasas/genética , Factores de Transcripción MEF2 , Microscopía Fluorescente , Modelos Biológicos , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Proteína MioD/genética , Factores Reguladores Miogénicos , Fosforilación , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Retinoblastoma/genética , Factores de Transcripción/metabolismoRESUMEN
Notch signaling dictates cell fate and critically influences cell proliferation, differentiation, and apoptosis in metazoans. Ligand binding initiates the signal through regulated intramembrane proteolysis of a transmembrane Notch receptor which releases the signal-transducing Notch intracellular domain (NICD). The HES/E(spl) gene family is a primary target of Notch and thus far the only known Notch effector. A newly isolated HERP family, a HES-related basic helix-loop-helix protein family, has been proposed as a potential target of Notch, based on its induction following NICD overexpression. However, NICD is physiologically maintained at an extremely low level that typically escapes detection, and therefore, nonregulated overexpression of NICD-as in transient transfection-has the potential of generating cellular responses of little physiological relevance. Indeed, a constitutively active NICD indiscriminately up-regulates expression of both HERP1 and HERP2 mRNAs. However, physiological Notch stimulation through ligand binding results in the selective induction of HERP2 but not HERP1 mRNA and causes only marginal up-regulation of HES1 mRNA. Importantly, HERP2 is an immediate target gene of Notch signaling since HERP2 mRNA expression is induced even in the absence of de novo protein synthesis. HERP2 mRNA induction is accompanied by specific expression of HERP2 protein in the nucleus. Furthermore, using RBP-Jk-deficient cells, we show that an RBP-Jk protein, a transcription factor that directly activates HES/E(spl) transcription, also is essential for HERP2 mRNA expression and that expression of exogenous RBP-Jk is sufficient to rescue HERP2 mRNA expression. These data establish that HERP2 is a novel primary target gene of Notch that, together with HES, may effect diverse biological activities of Notch.
Asunto(s)
Regulación de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Proteínas de la Membrana/metabolismo , Proteínas Nucleares , Proteínas Represoras/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Núcleo Celular/metabolismo , ADN Complementario , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Ligandos , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Unión Proteica , ARN Mensajero , Ratas , Receptores Notch , Transducción de SeñalRESUMEN
HERP1 and -2 are members of a new basic helix-loop-helix (bHLH) protein family closely related to HES/E(spl), the only previously known Notch effector. Like that of HES, HERP mRNA expression is directly up-regulated by Notch ligand binding without de novo protein synthesis. HES and HERP are individually expressed in certain cells, but they are also coexpressed within single cells after Notch stimulation. Here, we show that HERP has intrinsic transcriptional repression activity. Transcriptional repression by HES/E(spl) entails the recruitment of the corepressor TLE/Groucho via a conserved WRPW motif, whereas unexpectedly the corresponding-but modified-tetrapeptide motif in HERP confers marginal repression. Rather, HERP uses its bHLH domain to recruit the mSin3 complex containing histone deacetylase HDAC1 and an additional corepressor, N-CoR, to mediate repression. HES and HERP homodimers bind similar DNA sequences, but with distinct sequence preferences, and they repress transcription from specific DNA binding sites. Importantly, HES and HERP associate with each other in solution and form a stable HES-HERP heterodimer upon DNA binding. HES-HERP heterodimers have both a greater DNA binding activity and a stronger repression activity than do the respective homodimers. Thus, Notch signaling relies on cooperation between HES and HERP, two transcriptional repressors with distinctive repression mechanisms which, either as homo- or as heterodimers, regulate target gene expression.
Asunto(s)
Secuencias Hélice-Asa-Hélice , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas , Proteínas Represoras/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión , Células COS , Chlorocebus aethiops , Proteínas Co-Represoras , ADN/metabolismo , Dimerización , Regulación de la Expresión Génica , Células HeLa , Histona Desacetilasas/metabolismo , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear , Receptores Notch , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Complejo Correpresor Histona Desacetilasa y Sin3 , Soluciones , Factor de Transcripción HES-1 , Transcripción GenéticaRESUMEN
Cell transplantation has been proposed as a future therapy for various myocardial diseases. It is unknown, however, whether the encouraging results obtained in animal models of ischemia and reperfusion, cryoinjury or cardiomyopathy can be reproduced in the setting of permanent coronary artery occlusion and extensive myocardial infarction (MI). Embryonic cardiac cells were isolated and cultured for 3 days to confirm viability, morphology and to label cells with BrdU or the reporter gene LacZ. Seven days after extensive MI, rats were randomized to cell (1.5x10(6)) transplantation (n=11) or culture medium injection (n=16) into the myocardial scar. Echocardiography study was performed before and 53+/-3 days after implantation to assess left ventricular (LV) remodeling and function. During follow-up, there was no mortality among cell-treated rats v 4 of 16 control rats (P=0.12). X-gal staining, BrdU and alpha -SMA immunohistochemistry identified the engrafted cells 1 week, 4 weeks and 8 weeks after transplantation, respectively. Antibodies against alpha -SMA, connexin-43, fast and slow myosin heavy chain revealed grafts in various stages of differentiation in 10 of 11 cell-treated hearts. Many of them, however, kept their embryonic phenotype and were isolated from the host myocardium by scar tissue. Serial echocardiography studies revealed that cell transplantation prevented scar thinning, LV dilatation and dysfunction while control animals developed scar thinning, significant LV dilatation accompanied by progressive deterioration in LV contractility. Transplantation of embryonic cardiomyocytes after extensive MI in a rat model attenuate LV dilatation, infarct thinning, and myocardial dysfunction. Still, many grafts remain isolated and do not differentiate into an adult phenotype, even when studied 2 months after grafting.
Asunto(s)
Trasplante de Células/fisiología , Corazón Fetal/patología , Infarto del Miocardio/terapia , Animales , Diferenciación Celular , Supervivencia Celular , Trasplante de Células/métodos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ecocardiografía/instrumentación , Femenino , Infarto del Miocardio/complicaciones , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Neovascularización Patológica , Ratas , Ratas Sprague-Dawley , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/etiologíaRESUMEN
This study describes the development of 3PO, a nonviral, protein-based gene delivery vector which utilizes the highly evolved cell-binding, cell-entry and intracellular transport functions of the adenovirus serotype 5 (Ad5) capsid penton protein. A penton fusion protein containing a polylysine sequence was produced by recombinant methods and tested for gene delivery capability. As the protein itself is known to bind integrins through a conserved consensus motif, the penton inherently possesses the ability to bind and enter cells through receptor-mediated internalization. The ability to lyse the cellular endosome encapsulating internalized receptors is also attributed to the penton. The recombinant protein gains the additional function of DNA binding and transport with the appendage of a polylysine motif. This protein retains the ability to form pentamers and mediates delivery of a reporter gene to cultured cells. Interference by oligopeptides bearing the integrin binding motif suggests that delivery is mediated specifically through integrin receptor binding and internalization. The addition of protamine to penton-DNA complexes allows gene delivery in the presence of serum.
Asunto(s)
Adenoviridae , Proteínas de la Cápside , Cápside , Ingeniería Genética , Vectores Genéticos , Línea Celular , ADN/metabolismo , Células HeLa , Humanos , Integrinas/metabolismo , Protaminas , Unión Proteica , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de FusiónRESUMEN
The capsid proteins of adenovirus serotype 5 (Ad5) are key to the virus' highly efficient cell binding and entry mechanism. In particular, the penton base plays a significant role in both viral internalization and endosome penetration. We have produced an adenovirus penton fusion protein (HerPBK10) containing moieties for DNA transport and targeted delivery to breast cancer cells. HerPBK10 binds DNA through a polylysine appendage, while the EGF-like domain of the heregulin-alpha(1) isoform is used as the targeting ligand. This ligand binds with high affinity to HER2/3 or HER2/4 heterodimers, which are overexpressed on certain aggressive breast cancers. In addition, this ligand is rapidly internalized after binding, thus adding to the utility of heregulin for targeting. HerPBK10 binds MDA-MB-453 breast cancer cells in a receptor-specific manner, and mediates the entry of a reporter plasmid in MDA-MB-453 cells in culture. Delivery can be competed by excess heregulin peptide, thus confirming receptor specificity. Importantly, the penton segment appears to contribute significantly to enhanced delivery. Complexes containing HerPBK10 and DNA have been optimized to provide targeted gene delivery to breast cancer cells in vitro. We demonstrate that delivery can be accomplished in the presence of serum, thus suggesting a potential use for in vivo delivery.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de la Cápside , Cápside/genética , Marcación de Gen/métodos , Técnicas de Transferencia de Gen , Adenoviridae/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cápside/biosíntesis , ADN/metabolismo , Endopeptidasas/sangre , Femenino , Vectores Genéticos , Humanos , Neurregulina-1/metabolismo , Protaminas/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , Células Tumorales CultivadasRESUMEN
Cardiomyocytes are terminally differentiated and are unable to proliferate in response to injury. Genetic modulation, cell transplantation and tissue engineering promise a revolutionary approach for myocardial regeneration and tissue repair after myocardial injury. Current data derived from animal models suggest that it may be possible to treat heart failure by inserting genetic materials or myogenic cells into injured myocardium. Success with animal models has raised the hope for new treatment after heart attacks and could prove an alternative to transplantation, particularly in elderly patients for whom there is often a lack of donor hearts. This exciting research, however, still faces significant difficulties before it can develop into a clinical therapeutic tool and many challenges need to be overcome before cell transplantation, gene therapy and tissue engineering can be considered efficient, therapeutic strategies for myocardial regeneration.
Asunto(s)
Cardiopatías/terapia , Miocitos Cardíacos/fisiología , Regeneración , Animales , Trasplante de Células/métodos , Cardiopatías/patología , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/terapia , Infarto del Miocardio/patología , Infarto del Miocardio/terapia , Miocitos Cardíacos/trasplante , Ingeniería de Tejidos/métodosRESUMEN
In a previous study, we observed that one injection of 500 microg of DNA for the plasmid encoding for vascular endothelial growth factor (ph VEGF(165)) into one site in a rat myocardial infarction model resulted in neovascularization confined to angiomatous structures that did not contribute to regional myocardial blood flow. The purpose of the present study was to determine whether a lower dose (125 microg DNA), which is the same as that being used in some clinical trials, injected into four separate sites could enhance collateral flow and vascularity to the ischemic bed without inducing angiomas. Rats received injections of 125 microg DNA of the plasmid encoding phVEGF(165) or control DNA at four separate sites within the anterior free wall of the left ventricle (LV) supplied by the left coronary artery. The left coronary artery was ligated and hearts analyzed at 4 weeks. In vitro studies confirmed that the phVEGF(165) used was capable of producing VEGF polypeptide in mammalian cells. The infarct size (percentage of endocardial circumference that infarcted) was similar in controls (42+/-6%) and treated hearts (39+/-7%); the LV cavity area did not differ between groups. The number of vascular structures per high-power field within the infarct scar was 10.50+/-0.68 in controls and 10.00+/-0.85 in phVEGF(165)-treated rats. Relative regional myocardial blood flow determined by radioactive microspheres and expressed as a ratio of radioactive counts within the scar divided by radioactive counts in the noninfarcted ventricular septum was similar in control (0.74+/-0.25) and treated hearts (0.88+/-0.30) (p=not significant). No angiomatous structures were observed. Injections of 125 microg of DNA of phVEGF(165) into myocardium to become ischemic had no effect on infarct size or LV cavity size. Unlike higher doses of 500 microg of DNA, it did not cause gross angiomatous structures; however, it failed to improve neovascularization or regional myocardial blood flow in this rodent model of acute myocardial infarction.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Infarto del Miocardio/terapia , Vacunas de ADN/administración & dosificación , Animales , Línea Celular , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/uso terapéutico , Ventrículos Cardíacos , Hemangioma/inducido químicamente , Inyecciones Intraarteriales , Linfocinas/biosíntesis , Linfocinas/uso terapéutico , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Neovascularización Fisiológica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacos , Transfección , Vacunas de ADN/efectos adversos , Vacunas de ADN/síntesis química , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Remodelación Ventricular/efectos de los fármacosRESUMEN
The transcription of tissue-specific genes is controlled by regulatory factors and cofactors and is suppressed in cardiac cells by the antineoplastic agent doxorubicin. Here we show that exposure of cultured cardiomyocytes to doxorubicin resulted in the rapid depletion of transcripts for MEF2C, dHAND, and NKX2.5, three pivotal regulators of cardiac gene expression. Delivery of exogenous p300, a coactivator of MEF2C and NKX2.5 in cardiomyocytes, restored cardiac transcription despite the presence of doxorubicin. Furthermore, p300 also restored the accumulation of transcripts for MEF2C itself. Importantly, cardiocytes exposed to doxorubicin displayed reduced levels of p300 proteins. This was not due to alterations in the level of p300 transcripts; rather, and surprisingly, doxorubicin promoted selective degradation of p300 mediated by the 26S-proteasome machinery. Doxorubicin had no effect on the general level of ubiquitinated proteins or on the levels of beta-catenin, a protein known to be degraded by proteasome-mediated degradation. These results provide evidence for a new mechanism of transcriptional repression caused by doxorubicin in which the selective degradation of p300 results in reduced p300-dependent transcription, including production of MEF2C mRNA.
Asunto(s)
Regulación de la Expresión Génica , Miocardio/metabolismo , Proteínas Nucleares/metabolismo , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Transactivadores/metabolismo , Transcripción Genética , Proteínas de Xenopus , Animales , Animales Recién Nacidos , Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Doxorrubicina/farmacología , Proteína p300 Asociada a E1A , Corazón/efectos de los fármacos , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/biosíntesis , Factores de Transcripción MEF2 , Modelos Genéticos , Proteínas Musculares/biosíntesis , Miocardio/citología , Factores Reguladores Miogénicos/biosíntesis , Proteínas Nucleares/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transactivadores/genética , Factores de Transcripción/biosíntesis , Proteínas de Pez CebraRESUMEN
We have developed a simple scheme for characterizing ligand-receptor binding and post-binding activity on living cells. Our approach makes use of green fluorescent protein (GFP) as an auto-fluorescent tag to label protein ligands. We have constructed GFP-tagged ligands that can be expressed in bacteria as soluble fusion proteins. A cell-binding assay using fluorescence-activated cell sorting (FACS) demonstrates that GFP-tagged proteins retain their wild-type receptor-binding specificity. Using this assay, we measure ligand binding on unfixed cells and demonstrate receptor specificity using specific competitors. To determine the ability of receptor targets to internalize, we developed a second FACS-based assay to detect the rate and percentage of internalized ligand in living cells. Noninternalizing control ligands and fluorescence microscopy of treated cells confirm that our assay is reliable for determining receptor internalization activity.
Asunto(s)
Endocitosis , Proteínas Luminiscentes/genética , Neurregulina-1/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión , Sitios de Unión , Unión Competitiva , Neoplasias de la Mama , Dimerización , Citometría de Flujo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Neurregulina-1/metabolismo , Péptidos/metabolismo , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias , Células Tumorales CultivadasRESUMEN
OBJECTIVES: The effects of direct intramyocardial injection of the plasmid encoding vascular endothelial growth factor (phVEGF165) in the border zone of myocardial infarct tissue in rat hearts were investigated. BACKGROUND: Controversy exists concerning the ability of VEGF to induce angiogenesis and enhance coronary flow in the myocardium. METHODS: Sprague-Dawley rats received a ligation of the left coronary artery to induce myocardial infarction (MI). At 33.1 +/- 6.5 days, the rats were injected with phVEGF165 at one location and control plasmid at a second location (500 microg DNA, n = 24) or saline (n = 16). After 33.1 +/- 5.7 days, the hearts were excised for macroscopic and histologic analysis. Regional blood flow ratios were measured in 18 rats by radioactive microspheres. RESULTS: phVEGF165-treated sites showed macroscopic angioma-like structures at the injection site while control DNA and saline injection sites did not. By histology, 21/24 phVEGF165-treated hearts showed increased focal epicardial blood vessel density and angioma-like formation. Quantitative morphometric evaluation in 20 phVEGF165-treated hearts revealed 44.4 +/- 10.5 vascular structures per field in phVEGF165-treated hearts versus 21.4 +/- 4.7 in control DNA injection sites (p < 0.05). Regional myocardial blood flow ratios between the injection site and noninfarcted area did not demonstrate any difference between phVEGF,165-treated hearts (0.9 +/- 0.2) and saline-treated hearts (0.7 +/- 0.1). CONCLUSIONS: Injection of DNA for VEGF in the border zone of MI in rat hearts induced angiogenesis. Angioma formation at the injection sites did not appear to contribute to regional myocardial blood flow, which may be a limitation of gene therapy for this application.
Asunto(s)
Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/uso terapéutico , Terapia Genética/métodos , Neoplasias Cardíacas/inducido químicamente , Hemangioma/inducido químicamente , Linfocinas/genética , Linfocinas/uso terapéutico , Infarto del Miocardio/terapia , Neovascularización Fisiológica/efectos de los fármacos , Plásmidos/genética , Plásmidos/uso terapéutico , Animales , Circulación Coronaria/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Factores de Crecimiento Endotelial/efectos adversos , Terapia Genética/efectos adversos , Neoplasias Cardíacas/patología , Hemangioma/patología , Inyecciones , Linfocinas/efectos adversos , Infarto del Miocardio/patología , Plásmidos/efectos adversos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Human immunodeficiency virus (HIV, lentivirus) type-1 based vectors have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and long term transgene expression. We used a three-plasmid expression system to generate pseudotyped lentivirus-based vectors by transient transfection of human embryonic kidney 293T cells in the presence of sodium butyrate, which is known to activate the long terminal repeat-directed expression of HIV. Using this system we successfully generated versatile high titer lentivirus at titers of up to 2 x 10(8) transducing units/ml (TU/ml), and improved transduction efficiency in various cell types from seven to over twenty fold. We demonstrate its applicability of these vectors for the efficient transduction of non-dividing cells, including post mitotic beating rat cardiac myocytes and well-differentiated rat L6 myofibers. While both lentivirus-based and murine retrovirus-based vectors effectively transduced dividing cardiac fibroblasts and L6 muscle myoblasts in culture, lentivirus-based vectors also efficiently transduced cardiac myocytes and yielded titers of (6.3 +/- 1.2) x 10(5) TU/ml; however murine retrovirus-based vectors showed low transduction efficiency with titers reaching only (8.9 +/- 2.1) x 10(2) TU/ml. Furthermore, even 12 days after induction of differentiation of L6 myofibers, lentivirus-mediated transduction of beta-galactosidase (beta-Gal) at approximately 30-40% of the maximum expression levels achieved in replicating myoblasts. In contrast, the expression of beta-Gal following transduction of the myofibers by murine retrovirus-based vectors fell to less than 1% of an already reduced level of transduction in undifferentiated confluent myoblasts. These results demonstrate that lentivirus-based vectors can efficiently transduce both well-differentiated cardiac myocytes and differentiated myofibers. This appears to be an efficient method and provides a new tool for research and therapy for cardiovascular diseases.
Asunto(s)
Vectores Genéticos , VIH-1/fisiología , Corazón/fisiología , Miocardio/citología , Transfección/métodos , Animales , Animales Recién Nacidos , Butiratos/farmacología , Diferenciación Celular , Línea Celular , Células Cultivadas , Duplicado del Terminal Largo de VIH , VIH-1/efectos de los fármacos , Corazón/virología , Humanos , Riñón , Músculo Esquelético/virología , Ratas , Retroviridae/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
A full-length rabbit cDNA of cardiac adriamycin responsive protein (CARP) has been cloned. It shows high levels of identity at the amino acid sequence level (>86%) with the rat, mouse and human homologues. CARP mRNA levels are highly regulated in adriamycin-cardiomyopathy in rabbits.
Asunto(s)
Cardiomiopatías/genética , ADN Complementario/genética , Regulación de la Expresión Génica , Proteínas Musculares/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cardiomiopatías/inducido químicamente , Clonación Molecular , Doxorrubicina , Humanos , Ratones , Datos de Secuencia Molecular , Conejos , Ratas , Proteínas Represoras , Alineación de SecuenciaRESUMEN
Oncogene activation increases susceptibility to apoptosis. Thus, tumorigenesis must depend, in part, on compensating mutations that protect from programmed cell death. A functional screen for cDNAs that could counteract the proapoptotic effects of the myc oncogene identified two related bHLH family members, Twist and Dermo1. Both of these proteins inhibited oncogene- and p53-dependent cell death. Twist expression bypassed p53-induced growth arrest. These effects correlated with an ability of Twist to interfere with activation of a p53-dependent reporter and to impair induction of p53 target genes in response to DNA damage. An underlying explanation for this observation may be provided by the ability of Twist to reduce expression of the ARF tumor suppressor. Thus, Twist may affect p53 indirectly through modulation of the ARF/MDM2/p53 pathway. Consistent with a role as a potential oncoprotein, Twist expression promoted colony formation of E1A/ras-transformed mouse embryo fibroblasts (MEFs) in soft agar. Furthermore, Twist was inappropriately expressed in 50% of rhabdomyosarcomas, a tumor that arises from skeletal muscle precursors that fail to differentiate. Twist is known to block myogenic differentiation. Thus, Twist may play multiple roles in the formation of rhabdomyosarcomas, halting terminal differentiation, inhibiting apoptosis, and interfering with the p53 tumor-suppressor pathway.
Asunto(s)
Apoptosis , Proteínas Nucleares , Oncogenes , Proteínas Represoras , Factores de Transcripción/metabolismo , Animales , División Celular , Línea Celular , Transformación Celular Neoplásica , Fibroblastos , Eliminación de Gen , Genes myc , Genes p53 , Genes ras , Secuencias Hélice-Asa-Hélice , Humanos , Ratones , Ratas , Rabdomiosarcoma/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 Relacionada con TwistRESUMEN
Few studies have addressed the molecular differences that exist between muscles of the body and those of the craniofacial apparatus. In this study, we characterize the molecular events associated with determination and differentiation of the tongue musculature. We assess the expression of myogenic regulatory factors as well as the developmentally regulated myosin heavy chain, (MHC), genes which serve as markers of differentiation. These results suggest that tongue and limb muscle form by distinct molecular pathways. The myoblasts that contribute to the formation of the tongue preferentially express Myf-5 during myoblast determination rather than MyoD. Subsequently, isolated regions of myogenin expression mark the differentiation of first, the small primary myofibers and later, the larger secondary myofibers. Analysis of differentiation markers demonstrates that the tongue muscle also assumes a unique profile of MHC expression as compared to that of the muscles of the body. Unlike the myoblasts of the developing limb, which express embryonic and neonatal forms of MHC and later express MHC-slow, the tongue myoblasts co-express MHC-embryonic, MHC-slow and MHC-fast isoforms from gestational age E12. Proteins for MHC embryonic and MHC fast isoforms are detected almost simultaneously. Interestingly, MHC-slow transcripts do not appear to be translated into a detectable MHC slow protein at any developmental stage assayed. These results provide further evidence to suggest that skeletal tongue muscle represents a myoblast lineage that develops differently than the limb.
Asunto(s)
Músculos/embriología , Factores Reguladores Miogénicos/fisiología , Lengua/embriología , Factores de Edad , Animales , Western Blotting , Expresión Génica , Inmunohistoquímica , Operón Lac , Ratones , Ratones Transgénicos , Músculos/anatomía & histología , Músculos/metabolismo , Cadenas Pesadas de Miosina/genética , Factores de Tiempo , Lengua/anatomía & histología , Lengua/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Activation of the human cardiac alpha-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.
Asunto(s)
Actinas/genética , Secuencias Hélice-Asa-Hélice , Músculo Esquelético/metabolismo , Factores Reguladores Miogénicos/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción , Proteínas de Unión al ADN/metabolismo , Dimerización , Humanos , Proteína MioD/metabolismo , Miogenina/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Factor de Respuesta Sérica , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
Histone acetyltransferases (HAT) play a critical role in transcriptional control by relieving repressive effects of chromatin, and yet how HATs themselves are regulated remains largely unknown. Here, it is shown that Twist directly binds two independent HAT domains of acetyltransferases, p300 and p300/CBP-associated factor (PCAF), and directly regulates their HAT activities. The N terminus of Twist is a primary domain interacting with both acetyltransferases, and the same domain is required for inhibition of p300-dependent transcription by Twist. Adenovirus E1A protein mimics the effects of Twist by inhibiting the HAT activities of p300 and PCAF. These findings establish a cogent argument for considering the HAT domains as a direct target for acetyltransferase regulation by both a cellular transcription factor and a viral oncoprotein.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas E1A de Adenovirus/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Factores de Transcripción/fisiología , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/fisiología , Proteínas E1A de Adenovirus/metabolismo , Animales , Células COS , Células Cultivadas , Proteína p300 Asociada a E1A , Activación Enzimática , Histona Acetiltransferasas , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/fisiología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína 1 Relacionada con TwistRESUMEN
p300/CBP and PCAF coactivators have acetyltransferase activities and regulate transcription, cell cycle progression, and differentiation. They are both required for MyoD activity and muscle differentiation. Nevertheless, their roles must be different since the acetyltransferase activity of PCAF but not of p300 is involved in controlling myogenic transcription and differentiation. Here, we provide a molecular explanation of this phenomenon and report that MyoD is directly acetylated by PCAF at evolutionarily conserved lysines. Acetylated MyoD displays an increased affinity for its DNA target. Importantly, conservative substitutions of acetylated lysines with nonacetylatable arginines impair the ability of MyoD to stimulate transcription and to induce muscle conversion indicating that acetylation of MyoD is functionally critical.
Asunto(s)
Acetiltransferasas/metabolismo , Músculos/citología , Músculos/metabolismo , Proteína MioD/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Activación Transcripcional/genética , Acetilación , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular , Núcleo Celular/metabolismo , Secuencia Conservada , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Dimerización , Fibroblastos/citología , Fibroblastos/metabolismo , Histona Acetiltransferasas , Lisina/genética , Lisina/metabolismo , Ratones , Desarrollo de Músculos , Músculos/enzimología , Proteína MioD/química , Proteína MioD/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
We have characterized the specific DNA regulatory elements responsible for the function of the human cardiac troponin C gene (cTnC) muscle-specific enhancer in myogenic cells. We used functional transient transfection assays with deletional and site-specific mutagenesis to evaluate the role of the conserved sequence elements. Gel electrophoresis mobility shift assays (EMSA) demonstrated the ability of the functional sites to interact with nuclear proteins. We demonstrate that three distinct transcription activator binding sites commonly found in muscle-specific enhancers (a MEF-2 site, a MEF-3 site, and at least four redundant E-box sites) all contribute to full enhancer activity but a CArG box does not. Mutation of either the MEF-2 or MEF-3 sites or deletion of the E-boxes reduces expression by 70% or more. Furthermore, the MEF-2 site and the E-boxes specifically bind, respectively, to MEF-2 and myogenic determination factors derived from nuclear extracts. EMSA assays using a MEF-3 containing oligonucleotide revealed indistinguishable separation patterns with extracts from myogenic cells and nonmyogenic cells. These data suggest that expression of the cTnC gene in slow-twitch skeletal muscle is sustained through complex interactions at the 3'Ile enhancer between muscle-specific and nontissue-specific transcription factors: either a myogenic bHLH complex or MEF-2 can activate transcription but only in the presence of a third transcriptional activator that appears not to be muscle specific. We conclude from these observations that the cTnC 3'Ile element is a composite enhancer that functions through the combined interactions of at least five regulatory elements and their cognate binding factors: three or four E-boxes, a MEF-2 site, and a MEF-3 site. The data support the notion that all of these sites contribute to enhancer function in cell systems in an additive way but that none are absolutely required for enhancer activity. The data imply that the levels of transcription of cTnC in myogenic tissues in which the activities of one of the transcriptional factors is lacking would be partially but not wholly suppressed. Our data support the critical role of E-box sites in conjunction with the adjacent elements. Hence, we assign CTnC gene regulation to the "ordinary" rather than to the "novel" category of transcriptional regulation during skeletal myogenesis.