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1.
Atherosclerosis ; 233(1): 178-85, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24529141

RESUMEN

The transcription of the Low-density lipoprotein receptor-related protein (LRP1) is upregulated by aggregated LDL (agLDL) and angiotensin II (AngII) in human vascular smooth muscle cells (hVSMC). The polymorphism c.1-25C>G creates a new GC-box in the LRP1 promoter recognized by Sp1/Sp3 transcription factors. The aims of this study were 1) to evaluate the impact of c.1-25C>G polymorphism on LRP1 transcriptional activity and expression, and 2) to examine the response of c.1-25C>G LRP1 promoter to LDL and AngII. EMSA and Luciferase assays in HeLa cells showed that -25G promoter has enhanced basal transcriptional activity and specific Sp1/Sp3 binding. hVSMC with GG genotype (GG-hVSMC) had higher LRP1 mRNA and protein levels, respectively than CC genotype (CC-hVSMC). EMSA assays showed that the polymorphism determines scarce amount of SRE-B/SREBP-2 complex formation and the failure of agLDL to further reduce these SRE-B/SREBP-2 complexes. Taken together, these results suggest that c.1-25C>G, by difficulting SREBP-2 binding, prevents SREBP-2 displacement required for LRP1 promoter response to LDL. In contrast, c.1-25C>G strongly favours Sp1/Sp3 binding and AngII-induced activity in Sp1/Sp3 dependent manner in GG-hVSMC. This increase is functionally translated into a higher capacity of GG-hVSMC to become foam cells from agLDL in presence of AngII. These results suggest that c.1-25C>G determines a lack of response to agLDL and an exacerbated response to AngII. It is thus conceivable that the presence of the polymorphism would be easily translated to vascular alterations in the presence of the pro-hypertensive autacoid, AngII.


Asunto(s)
Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Regiones Promotoras Genéticas/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Angiotensina II/fisiología , Sitios de Unión , Células HeLa , Humanos , Lipoproteínas LDL/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Polimorfismo Genético , Proteína 2 de Unión a Elementos Reguladores de Esteroles/biosíntesis , Activación Transcripcional
4.
Actas dermo-sifiliogr. (Ed. impr.) ; 102(8): 623-626, oct. 2011.
Artículo en Español | IBECS | ID: ibc-92565

RESUMEN

El diagnóstico de las queratosis actínicas pigmentadas puede resultar complicado en la práctica clínica. El diagnóstico diferencial con el lentigo maligno melanoma plantea dificultades, tanto clínica como dermatoscópicamente, ya que ambas entidades pueden presentar características en común. Presentamos 5 casos de queratosis actínicas pigmentadas en 4 pacientes. El hallazgo dermatoscópico más frecuente fue una granulación marrón-grisácea de distribución perifolicular presente en todas las lesiones, seguido de estructuras romboidales en 4 y un patrón anular-granular en 3. En ningún caso se apreciaron salidas foliculares asimétricamente pigmentadas. Destacamos algunas claves que facilitan el diagnóstico preoperatorio de las queratosis actínicas pigmentadas (AU)


The diagnosis of pigmented actinic keratosis can be complicated in clinical practice. The differential diagnosis with lentigo maligna melanoma can be difficult due to common clinical and dermoscopic characteristics. We present 5 cases of pigmented actinic keratosis in 4 patients. The most common dermoscopic finding was a grayish-brown granulation with a perifollicular distribution, present in all lesions, followed by rhomboidal structures in 4 cases, and an annular-granular pattern in 3. In no case were asymmetrical pigmented follicular openings observed. We draw attention to key findings that aid preoperative diagnosis of pigmented actinic keratosis (AU)


Asunto(s)
Humanos , Anciano , Anciano de 80 o más Años , Masculino , Femenino , Persona de Mediana Edad , Dermoscopía , Epidermis/química , Epidermis/patología , Dermatosis Facial/diagnóstico , Queratosis Actínica/diagnóstico , Nariz/patología , Pigmentación de la Piel , Diagnóstico Diferencial , Dermatosis Facial/patología , Dermatosis Facial/cirugía , Queratosis Actínica/patología , Queratosis Actínica/cirugía , Melaninas/análisis
5.
Actas Dermosifiliogr ; 102(8): 623-6, 2011 Oct.
Artículo en Español | MEDLINE | ID: mdl-21349475

RESUMEN

The diagnosis of pigmented actinic keratosis can be complicated in clinical practice. The differential diagnosis with lentigo maligna melanoma can be difficult due to common clinical and dermoscopic characteristics. We present 5 cases of pigmented actinic keratosis in 4 patients. The most common dermoscopic finding was a grayish-brown granulation with a perifollicular distribution, present in all lesions, followed by rhomboidal structures in 4 cases, and an annular-granular pattern in 3. In no case were asymmetrical pigmented follicular openings observed. We draw attention to key findings that aid preoperative diagnosis of pigmented actinic keratosis.


Asunto(s)
Dermoscopía , Dermatosis Facial/diagnóstico , Queratosis Actínica/diagnóstico , Nariz/patología , Pigmentación de la Piel , Anciano , Anciano de 80 o más Años , Diagnóstico Diferencial , Epidermis/química , Epidermis/patología , Dermatosis Facial/patología , Dermatosis Facial/cirugía , Femenino , Humanos , Peca Melanótica de Hutchinson/diagnóstico , Queratinocitos/ultraestructura , Queratosis Actínica/patología , Queratosis Actínica/cirugía , Masculino , Melaninas/análisis , Persona de Mediana Edad
8.
Acta pediatr. esp ; 66(3): 135-137, mar. 2008. ilus, tab
Artículo en Es | IBECS | ID: ibc-64853

RESUMEN

El pilomatricoma es el segundo tumor benigno superficial más frecuentemente extirpado en la infancia 1. Su aspecto clínico característico es casi diagnóstico. La presencia de múltiples pilomatricomas nos debe hacer descartar ciertas patologías asociadas a este tumor. Presentamos dos casos de pilomatricomatosis múltiple (AU)


Pilomatricoma is the second most commonly excised superficial mass in childhood 1. Its typical clinical appeareance is of diagnostic value. Multiple pilomatricomas prompt clinical evaluation for related syndromes. We present two cases of multiple pilomatricomas(AU)


Asunto(s)
Humanos , Masculino , Femenino , Adolescente , Pilomatrixoma/complicaciones , Pilomatrixoma/diagnóstico , Pilomatrixoma/terapia , Diagnóstico Diferencial , Neoplasias del Oído/diagnóstico , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/terapia , Inhibidores de la Enzima Convertidora de Angiotensina , Oído Externo/patología , Oído Externo/cirugía
9.
Mol Ecol Resour ; 8(2): 415-7, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21585807

RESUMEN

Twelve polymorphic microsatellite loci were developed for Dendrocopos medius. Polymorphism was assessed for 27 individuals from the southwesternmost population of this woodpecker species. The number of alleles per locus ranged from three to seven, with observed heterozygosity values from 0.444 to 0.852. Genotypic frequencies conformed to Hardy-Weinberg equilibrium, and no evidence for linkage disequilibrium was observed. Multilocus genotypes resulting from this set of markers will be useful to determine genetic diversity and differentiation within and among habitat patches inhabited by D. medius. Three of the loci were polymorphic for Picoides articus.

10.
Int J Gynecol Cancer ; 17(2): 484-91, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17309674

RESUMEN

Cervical carcinoma (CC) is one of the most common cancers among women worldwide and the first cause of death among the Mexican female population. Human papillomavirus (HPV) infection is the most important etiologic factor for CC. Of the oncogenic types, HPV16 and HPV18 are found in 60-70% of invasive CCs worldwide. HPV18 appears to be associated with a more aggressive form of cervical neoplasia than HPV16 infection. At present, there are no studies on differentially expressed cellular genes between transformed cells harboring HPV16 and HPV18 sequences. Based on previous complementary DNA microarray data from our group, 13 genes were found to be differentially overexpressed between HPV16- and HPV18-transformed cells. These genes were as follows: E6BP, UBE4A, C20orf14, ATF7, ABCC8, SLC6A12, WASF3, SUV39H1, SPAG8, CCNC, E2FFE, BIRC5, and DEDD. Differential expression of six selected genes was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). All real-time RT-PCRs confirmed differential expression between HPV18 and HPV(-) samples. The present work identifies genes from signaling pathways triggered by HPV transformation that could be differentially deregulated between HPV16(+) and HPV18(+) samples.


Asunto(s)
Transformación Celular Viral/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Infecciones por Papillomavirus/genética , Lesiones Precancerosas/genética , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Línea Celular Tumoral , Sondas de ADN de HPV/análisis , Femenino , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Infecciones por Papillomavirus/complicaciones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Neoplasias del Cuello Uterino/genética , Displasia del Cuello del Útero/genética
11.
Bioinorg Chem Appl ; : 98732, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18364995

RESUMEN

Four new metal complexes {M = Pd(II) or Pt(II)} containing the ligand 9-aminoacridine (9AA) were prepared. The compounds were characterized by FT-IR and (1)H, (13)C, and (195)Pt NMR spectroscopies. Crystal structure of the palladium complex of formulae [Pd(9AA)(mu-Cl)](2) . 2DMF was determined by X-ray diffraction. Two 9-acridine molecules in the imine form bind symmetrically to the metal ions in a bidentate fashion through the imine nitrogen atom and the C(1) atom of the aminoacridine closing a new five-membered ring. By reaction with phosphine or pyridine, the Cl bridges broke and compounds with general formulae [Pd(9AA)Cl(L)] (where L = PPh(3) or py) were formed. A mononuclear complex of platinum of formulae [Pt(9AA)Cl(DMSO)] was also obtained by direct reaction of 9-aminoacridine and the complex [PtCl(2)(DMSO(2)]. The capacity of the compounds to modify the secondary and tertiary structures of DNA was evaluated by means of circular dichroism and electrophoretic mobility. Both palladium and platinum compounds proved active in the modification of both the secondary and tertiary DNA structures. AFM images showed noticeable modifications of the morphology of the plasmid pBR322 DNA by the compounds probably due to the intercalation of the complexes between base pairs of the DNA molecule. Finally, the palladium complex was tested for antiproliferative activity against three different human tumor cell lines. The results suggest that the palladium complex of formula [Pd(9AA)(mu-Cl)](2) has significant antiproliferative activity, although it is less active than cisplatin.

12.
Oncology ; 67(3-4): 277-90, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15557790

RESUMEN

We analyzed the differential gene expression in the pancreatic cancer cell line NP-18 upon induction of apoptosis caused by cyclin-dependent kinase inhibition triggered by either overexpression of the tumor suppressor gene p16(INK4A)using an adenoviral construction or incubation with the chemical inhibitors, roscovitine or olomoucine. Screening was performed using cDNA arrays from Clontech that allowed the determination of the expression of 1,176 genes specifically related with cancer. The analysis was carried out using the Atlas Image 2.01 (Clontech) and GeneSpring 4.2 (Silicon Genetics) softwares. Among the differentially expressed genes, we chose for further validation histone deacetylase 1 (HDAC1), von Hippel Lindau and decorin as upregulated genes, and Sp1, hypoxia-inducible factor-1 alpha and DNA primase as downregulated genes. The changes in the expression of these genes to mRNA were validated by quantitative RT-PCR and the final translation into protein by Western blot analysis. Inhibition of HDAC activity, Sp1 binding and DNA primase expression led to an increase in the level of apoptosis, both in parental cells and in doxorubicin-resistant cells. Therefore, these proteins could constitute possible targets to develop modulators in cancer chemotherapy that would increase or restore apoptosis.


Asunto(s)
Apoptosis , Biomarcadores de Tumor/análisis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Perfilación de la Expresión Génica , Genes p16 , Neoplasias Pancreáticas/química , Inhibidores de Proteínas Quinasas/farmacología , Adenoviridae , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , ADN Primasa/análisis , Proteínas de Unión al ADN/análisis , Decorina , Regulación hacia Abajo , Proteínas de la Matriz Extracelular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes p16/efectos de los fármacos , Vectores Genéticos , Histona Desacetilasa 1 , Histona Desacetilasas/análisis , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Cinetina , Proteínas Nucleares/análisis , Neoplasias Pancreáticas/tratamiento farmacológico , Proteoglicanos/análisis , Purinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Roscovitina , Factores de Transcripción/análisis , Proteínas Supresoras de Tumor/análisis , Ubiquitina-Proteína Ligasas/análisis , Regulación hacia Arriba , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau
13.
Cell Mol Life Sci ; 61(6): 709-20, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15052413

RESUMEN

GLUT1 glucose transporters are highly expressed in proliferating and transformed cells and serum and cAMP or the transcription factor Sp1 induce GLUT1 gene transcription. Here we identified a cis element situated at -46/-37 (MG1E - muscle-specific GLUT1 element) to which muscle-specific nuclear factors bind, and the DNA-protein complexes showed electrophoretic mobility of 41 and 32 kDa. MyoD over-expression induced the generation of MG1E-protein complexes characteristic of myoblast cells. MG1E does not bind any known factors defined in databases. Mutation of the MG1E sequence impaired transcriptional activity of the GLUT1 promoter specifically in skeletal or cardiac muscle cells. The transcriptional activity of the GLUT1 promoter induced by either Sp1, cAMP or serum was markedly reduced when MG1E was inactivated. We propose that the MG1E sequence permits the binding of muscle-specific nuclear factors and a maximal transcriptional activity in muscle cells in response to Sp1, cAMP or serum.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Animales , Cloranfenicol O-Acetiltransferasa , AMP Cíclico/farmacología , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 1 , Proteínas de Transporte de Monosacáridos/metabolismo , Músculo Esquelético/citología , Mutagénesis Sitio-Dirigida , Proteína MioD/metabolismo , Miocardio/citología , Factores Reguladores Miogénicos , Proteínas Nucleares/metabolismo , Unión Proteica , Ratas , Elementos de Respuesta , Eliminación de Secuencia , Transcripción Genética/efectos de los fármacos
14.
Eur J Biochem ; 268(11): 3163-73, 2001 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11389717

RESUMEN

4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.


Asunto(s)
Metotrexato/farmacología , Factor de Transcripción Sp1/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Células CHO , Cricetinae , Resistencia a Medicamentos , Activación Enzimática/efectos de los fármacos , Plicamicina/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Proteína Quinasa C/metabolismo , Factor de Transcripción Sp1/biosíntesis , Tetrahidrofolato Deshidrogenasa/biosíntesis , Tetrahidrofolato Deshidrogenasa/genética , Transfección , Regulación hacia Arriba
15.
J Biol Chem ; 276(25): 22126-32, 2001 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-11294852

RESUMEN

The 5'-flanking region of the human Sp1 gene was cloned and characterized. Sequence analysis of this region showed the absence of both CAAT and TATA boxes and an initiator element. The proximal promoter of the Sp1 gene is a GC-rich region that contains multiple GC boxes and Ap2 binding sites. The major transcription start site is located 63 base pairs upstream of the translation start site. Transfection experiments demonstrate that all the elements necessary to achieve significant basal transcription activity are located between positions -443 and -20 relative to the translational start. Sp1 and Sp3 proteins bind to the downstream GC box located in the proximal promoter of Sp1. Furthermore, we demonstrate that the Sp1 protein activates Sp1 transcription activity; thus the Sp1 gene is autoregulated.


Asunto(s)
Factor de Transcripción Sp1/genética , Secuencia de Bases , Línea Celular Transformada , Clonación Molecular , ADN , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/química
16.
Biochem Pharmacol ; 61(3): 357-64, 2001 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-11172741

RESUMEN

We studied whether two typical effects of fibrates, induction of stearoyl-CoA desaturase (EC 1.14.99.5) and peroxisome proliferation, are related. The effect of bezafibrate on the activity and mRNA of stearoyl-CoA desaturase and acyl-CoA oxidase in the liver and epididymal white adipose tissue of male Sprague-Dawley rats was determined. The same parameters were measured in HepG2 cells, a cell line resistant to peroxisome proliferation, following incubation with ciprofibrate. Bezafibrate increased the hepatic mRNA levels (14.5-fold on day 7) and activity (9.3-fold on day 15) of acyl-CoA oxidase. Stearoyl-CoA desaturase mRNA levels were transiently increased (2.7-fold on day 7), while its activity remained increased at the end of the treatment (2.4-fold). In white adipose tissue, bezafibrate increased the mRNA (5-fold) and activity (1.9-fold) of acyl-CoA oxidase, while stearoyl-CoA desaturase was not modified. Ciprofibrate addition to HepG2 cells cultured in 7% fetal bovine serum (FBS) only increased the stearoyl-CoA desaturase mRNA (1.9-fold). When cells were cultured in 0.5% FBS, ciprofibrate increased acyl-CoA oxidase mRNA (2.2-fold), while the increase in stearoyl-CoA desaturase mRNA was identical (1.9-fold). Further, its activity was also increased (1.5-fold). Incubation of HepG2 cells in the presence of cycloheximide did not alter the capacity of ciprofibrate to induce stearoyl-CoA desaturase mRNA, whereas the presence of actinomycin abolished the induction. In addition, preincubation of HepG2 cells with ciprofibrate increased the rate of stearoyl-CoA desaturase mRNA degradation. The results presented in this study suggest that fibrates induce stearoyl-CoA desaturase activity and mRNA levels independently of peroxisome proliferation.


Asunto(s)
Tejido Adiposo/efectos de los fármacos , Bezafibrato/farmacología , Ácido Clofíbrico/farmacología , Oxidorreductasas/biosíntesis , Estearoil-CoA Desaturasa/biosíntesis , Acil-CoA Oxidasa , Tejido Adiposo/enzimología , Animales , Ácido Clofíbrico/análogos & derivados , Medios de Cultivo/análisis , Inducción Enzimática/efectos de los fármacos , Ácidos Fíbricos , Humanos , Hipolipemiantes/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Oxidorreductasas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Estearoil-CoA Desaturasa/genética , Células Tumorales Cultivadas
17.
Am J Physiol Endocrinol Metab ; 280(2): E229-37, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11158925

RESUMEN

The increased availability of saturated lipids has been correlated with development of insulin resistance, although the basis for this impairment is not defined. This work examined the interaction of saturated and unsaturated fatty acids (FA) with insulin stimulation of glucose uptake and its relation to the FA incorporation into different lipid pools in cultured human muscle. It is shown that basal or insulin-stimulated 2-deoxyglucose uptake was unaltered in cells preincubated with oleate, whereas basal glucose uptake was increased and insulin response was impaired in palmitate- and stearate-loaded cells. Analysis of the incorporation of FA into different lipid pools showed that palmitate, stearate, and oleate were similarly incorporated into phospholipids (PL) and did not modify the FA profile. In contrast, differences were observed in the total incorporation of FA into triacylglycerides (TAG): unsaturated FA were readily diverted toward TAG, whereas saturated FA could accumulate as diacylglycerol (DAG). Treatment with palmitate increased the activity of membrane-associated protein kinase C, whereas oleate had no effect. Mixture of palmitate with oleate diverted the saturated FA toward TAG and abolished its effect on glucose uptake. In conclusion, our data indicate that saturated FA-promoted changes in basal glucose uptake and insulin response were not correlated to a modification of the FA profile in PL or TAG accumulation. In contrast, these changes were related to saturated FA being accumulated as DAG and activating protein kinase C. Therefore, our results suggest that accumulation of DAG may be a molecular link between an increased availability of saturated FA and the induction of insulin resistance.


Asunto(s)
Diglicéridos/metabolismo , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Acetatos/metabolismo , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Ácidos Grasos/análisis , Ácidos Grasos/farmacología , Humanos , Lípidos/biosíntesis , Músculo Esquelético/citología , Fosfolípidos/química , Proteína Quinasa C/metabolismo , Triglicéridos/química , Triglicéridos/metabolismo
18.
Mol Pharmacol ; 58(1): 185-93, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10860941

RESUMEN

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is responsible for the hypolipidemic, peroxisome proliferation and carcinogenic effects of fibrates. Rats and mice are responsive, but guinea pigs and primates are resistant to the proliferative and carcinogenic effects of these drugs, but the hypolipidemic effect is still manifest. It is not yet clear whether humans should be considered unresponsive, and there is concern about the long-term safety of fibrates. We present molecular evidence for the reported resistance of human cells to peroxisome proliferation by describing a deficient interaction of nuclear extracts from human cells with an acyl-CoA oxidase (ACO)-peroxisome proliferator response element probe upon fibrate addition. Electrophoretic mobility shift assay analysis showed that ciprofibrate elicited a concentration-dependent increase in the binding of nuclear extracts from cells of rat (Morris) and human (HepG2) origin to an ACO-peroxisome proliferator response element probe, although in HepG2 cells the increase was of marginal statistical significance. In Morris cells, the increase was more marked than in HepG2 cells (4-fold versus 1.5-fold at 0.2 mM ciprofibrate), and maximal binding was achieved earlier in Morris (30 min) than in HepG2 cells (3 h). Morris cells responded to the addition of ciprofibrate by increasing the levels of ACO mRNA, whereas HepG2 did not. The ratio between PPARbeta/PPARalpha mRNAs was higher in HepG2 cells than in Morris cells (3.2 versus 1.9), pointing to an antagonizing effect of PPARbeta on PPARalpha activity. These results were obtained in untransfected cells expressing their own basal set of receptors. We also provide evidence of the translocation of PPARalpha from the cytosol to the nucleus upon activation by ciprofibrate.


Asunto(s)
Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacología , Hipolipemiantes/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/metabolismo , Acil-CoA Oxidasa , Animales , Bezafibrato/farmacología , Biopolímeros , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Ácidos Fíbricos , Cobayas , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Proteínas Nucleares/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores de Ácido Retinoico/efectos de los fármacos , Receptor alfa de Ácido Retinoico , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/efectos de los fármacos
19.
Biochim Biophys Acta ; 1495(3): 319-26, 2000 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-10699469

RESUMEN

RNA-based arbitrarily primed PCR (RAP-PCR) was used to identify sequences in CHO K1 cells that were differentially expressed upon methotrexate incubation during the development of resistance to this drug. Ten different RAP products were isolated, cloned and sequenced. Among these, we identified one sequence that showed 84% identity with the nucleotide sequence of rat cytochrome c oxidase subunit II, and 90% identity with the amino acid sequence of this protein. This RAP fragment was up-regulated in a dose- and time-dependent manner. The overexpression of cytochrome c oxidase subunit II mRNA as a result of methotrexate incubation was corroborated by quantitative RT-PCR and Northern blot analysis. Incubation of cells with sodium azide, a specific cytochrome c oxidase inhibitor, decreased the number of resistant colonies after methotrexate treatment. Thus, overexpression of cytochrome c oxidase is involved in the development of resistance to methotrexate. These results suggest that sodium azide may be used as a modulator in chemotherapy with methotrexate.


Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Complejo IV de Transporte de Electrones/metabolismo , Metotrexato/farmacología , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Interacciones Farmacológicas , Resistencia a Medicamentos/fisiología , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/genética , Inhibidores Enzimáticos/farmacología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis
20.
J Biol Chem ; 274(39): 27807-14, 1999 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-10488126

RESUMEN

We have constructed tetracycline-responsive dhfr minigenes and transferred them to a Chinese hamster ovary cell DHFR-deficient deletion mutant to obtained cells in which dhfr transcription can be repressed by tetracycline (tet-off). DHFR mRNA half-life measured after the repression of transcription by tetracycline in these transfectants is about 1.5 h, which is significantly shorter than previously reported. In addition, we observed that DHFR mRNA is less stable in serum-starved cells than in exponentially growing cells. Given that the dhfr gene contains multiple polyadenylation sites, we analyzed the role of polyadenylation site usage on the stability of the resultant mRNA molecules. We found that DHFR mRNA is more stable when a strong polyadenylation site is used. Finally, we have observed that the relative lengths of the poly(A) tails for the different DHFR mRNA species correlated with their relative stability in growing versus resting cells.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tetraciclina/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Transcripción Genética/efectos de los fármacos , Animales , Células CHO , División Celular , Cricetinae , Represión Enzimática , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Semivida , Cinética , Mutagénesis , Poli A/metabolismo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Eliminación de Secuencia , Tetrahidrofolato Deshidrogenasa/biosíntesis , Transfección
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