RESUMEN

Oncomirs are microRNAs (miRNA) associated with carcinogenesis and malignant transformation. They have emerged as potential molecular targets for anti-cancer therapy. We hypothesize that grape seed procyanidin extract (GSE) exerts antineoplastic effects through modulations of oncomirs and their downstream targets. We found that GSE significantly down-regulated oncomirs miR-19a and -19b in a variety of lung neoplastic cells. GSE also increased mRNA and protein levels of insulin-like growth factor II receptor (IGF-2R) and phosphatase and tensin homolog (PTEN), both predicted targets of miR-19a and -19b. Furthermore, GSE significantly increased PTEN activity and decreased AKT phosphorylation in A549 cells. Transfection of miR-19a and -19b mimics reversed the up-regulations of IGF2R and PTEN gene expression and abrogated the GSE induced anti-proliferative response. Additionally, oral administration of leucoselect phytosome, comprised of standardized grape seed oligomeric procyanidins complexed with soy phospholipids, to athymic nude mice via gavage, significantly down-regulated miR-19a, -19b and the miR-17-92 cluster host gene (MIR17HG) expressions, increased IGF-2R, PTEN, decreased phosphorylated-AKT in A549 xenograft tumors, and markedly inhibited tumor growth. To confirm the absorption of orally administered GSE, plasma procyanidin B1 levels, between 60 and 90 min after gavage of leucoselect phytosome (400 mg/kg), were measured by LC/MS at week 2 and 8 of treatment; the estimated concentration that was associated with 50% growth inhibition (IC50) (1.3 µg/mL) in vitro was much higher than the IC50 (0.032-0.13 µg/ml) observed in vivo. Our findings reveal novel antineoplastic mechanisms by GSE and support the clinical translation of leucoselect phytosome as an anti-neoplastic and chemopreventive agent for lung cancer.

Asunto(s)

Antineoplásicos Fitogénicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/prevención & control , Suplementos Dietéticos , Extracto de Semillas de Uva/uso terapéutico , Neoplasias Pulmonares/prevención & control , MicroARNs/antagonistas & inhibidores , Proantocianidinas/uso terapéutico , ARN Neoplásico/antagonistas & inhibidores , Animales , Antineoplásicos Fitogénicos/metabolismo , Antioxidantes/metabolismo , Antioxidantes/uso terapéutico , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Extracto de Semillas de Uva/metabolismo , Humanos , Absorción Intestinal , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones Desnudos , MicroARNs/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Proantocianidinas/metabolismo , ARN Neoplásico/metabolismo , Distribución Aleatoria , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Emerging preclinical data suggests that tea possess anticarcinogenic and antimutagenic properties. We therefore hypothesize that white tea extract (WTE) is capable of favorably modulating apoptosis, a mechanism associated with lung tumorigenesis. We examined the effects of physiologically relevant doses of WTE on the induction of apoptosis in non-small cell lung cancer cell lines A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cells. We further characterized the molecular mechanisms responsible for WTE-induced apoptosis, including the induction of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and the 15-lipoxygenase (15-LOX) signaling pathways. We found that WTE was effective in inducing apoptosis in both A549 and H520 cells, and inhibition of PPAR-gamma with GW9662 partially reversed WTE-induced apoptosis. We further show that WTE increased PPAR-gamma activation and mRNA expression, concomitantly increased 15(S)-hydroxy-eicosatetraenoic acid release, and upregulated 15-LOX-1 and 15-LOX-2 mRNA expression by A549 cells. Inhibition of 15-LOX with nordihydroguaiaretic acid (NGDA), as well as caffeic acid, abrogated WTE-induced PPAR-gamma activation and upregulation of PPAR-gamma mRNA expression in A549 cells. WTE also induced cyclin-dependent kinase inhibitor 1A mRNA expression and activated caspase-3. Inhibition of caspase-3 abrogated WTE-induced apoptosis. Our findings indicate that WTE is capable of inducing apoptosis in non-small cell lung cancer cell lines. The induction of apoptosis seems to be mediated, in part, through the upregulation of the PPAR-gamma and 15-LOX signaling pathways, with enhanced activation of caspase-3. Our findings support the future investigation of WTE as an antineoplastic and chemopreventive agent for lung cancer.

Asunto(s)

Apoptosis/efectos de los fármacos , Araquidonato 15-Lipooxigenasa/fisiología , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , PPAR gamma/fisiología , Extractos Vegetales/farmacología , Té , Anilidas/farmacología , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/prevención & control , Catequina/análogos & derivados , Catequina/farmacología , Catequina/uso terapéutico , Evaluación Preclínica de Medicamentos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevención & control , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Extractos Vegetales/uso terapéutico , Té/química , Células Tumorales Cultivadas

RESUMEN

Leukotrienes have been implicated to play a prominent inductive role in carcinogenesis. We previously reported that bronchoalveolar lavage (BAL) cells from smokers manifested higher levels of leukotriene B4 (LTB4) production than ex-smokers. This study aims to elucidate the underlying mechanism(s). BAL cells from current and former smokers were exposed to lipopolysaccharide (LPS) for up to 7 days. LPS induced the release of LTB4 from BAL cells and down-regulated 5-lipoxygenase (5-LOX) mRNA expression in a dose-dependent manner, followed by a decrease in 5-LOX protein production and normalization of LTB4 levels. Exogenous LTB4 inhibited LPS-induced 5-LOX activity and accentuated the down-regulation of 5-LOX mRNA, whereas suppression of 5-LOX abrogated the LPS-induced changes, suggesting a negative feedback mechanism. LPS concomitantly induced expression and activity of the LTB4 metabolizing enzyme LTB4 omega-hydroxylase (LTB4OH) in ex-smokers' BAL cells, but not in smokers' BAL cells. In vitro smoke exposure of ex-smokers' BAL cells also abrogated the LPS-induced up-regulation of LTB4OH mRNA expression. Furthermore, ex-smokers' BAL cells expressed significantly higher LTB4OH mRNA levels than smokers' BAL cells. Such differential modulation of LTB4 synthesis and degradation by LPS in the setting of tobacco smoke exposure suggests that mechanisms responsible for sustained elevation of LTB4 levels in the lung microenvironment may contribute to the pathogenesis of tobacco-related respiratory diseases such as lung cancer. By regulating the balance of LTB4 in the lung, LTB4OH may function as a suppressor of lung carcinogenesis.

Asunto(s)

Líquido del Lavado Bronquioalveolar/citología , Leucotrieno B4/metabolismo , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Fumar , Anciano , Anciano de 80 o más Años , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Líquido del Lavado Bronquioalveolar/química , Carcinoma/enzimología , Carcinoma/genética , Carcinoma/patología , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 4 del Citocromo P450 , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Leucotrieno B4/análisis , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/patología , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Persona de Mediana Edad , ARN Mensajero/metabolismo , Fumar/efectos adversos , Fumar/metabolismo

RESUMEN

We studied the effect of tumor necrosis factor (TNF)-alpha exposure on cysteinyl leukotriene (LT) synthesis by cells of monocyte/macrophage lineage. TNF-alpha conditioning of monocytic THP-1 cells and primary human monocytes resulted in a decreased capacity for LTC(4) release. TNF-alpha exposure (for 16-24 h) decreased LTC(4) synthase mRNA in THP-1 cells, primary mouse bone marrow-derived macrophages, and eosinophilic AML14.3D10 cells. TNF-alpha downregulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with downregulation observed as early as 4 h. The effect of TNF-alpha on LTC(4) synthase mRNA expression was mediated via the MEK/ERK pathway, but not via cyclooxygenase or nitric oxide synthase pathways. Conditioning of actinomycin D-treated cells with TNF-alpha did not accelerate degradation of LTC(4) synthase mRNA. TNF-alpha produced sustained activation of p50 and p65, which were previously reported by our group to decrease LTC(4) synthase promoter activity. In transiently transfected THP-1 cells, TNF-alpha decreased promoter activity via an element located within the first 620 bp of the promoter. We conclude that TNF-alpha exposure downregulates the synthetic capacity for cysteinyl LT release and LTC(4) synthase gene expression in monocytes/macrophages via a transcriptional mechanism.

Asunto(s)

Glutatión Transferasa/genética , Fagocitos/efectos de los fármacos , Fagocitos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Calcimicina/farmacología , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Humanos , Técnicas In Vitro , Ionóforos/farmacología , Leucotrieno C4/biosíntesis , Sistema de Señalización de MAP Quinasas , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología

RESUMEN

We examined induced expression of the 5-lipoxygenase-activating protein (FLAP), which is critical for leukotriene synthesis in mononuclear phagocytes. Prolonged exposure to the bacterial component, lipopolysaccharide (LPS), increased FLAP gene transcription, mRNA expression, and protein expression in the human monocyte-like THP-1 cell line. Activation and inhibition of the NF-kappaB pathway modulated LPS induction of FLAP gene expression. An NF-kappaB-mediated mechanism of action was supported by overexpression of dominant-negative IkappaBalpha and p50/p65 proteins. EMSA/supershift and DNase I footprint analyses revealed that p50 binds to an NF-kappaB site located in the proximal FLAP promoter, while chromatin immunoprecipitation assays demonstrated that LPS induced binding of p50 but not of p65. Moreover, EMSA/supershift analyses demonstrated that LPS induced time-dependent binding of THP-1 nuclear extracts (containing p50) to this promoter region. Mutation of the NF-kappaB site decreased basal promoter activity and abolished the p50- and p65-associated induction. EMSA/supershift analyses also demonstrated that LPS induced binding of THP-1 nuclear extracts [containing CCAAT/enhancer binding protein (C/EBP)-alpha, -delta, and -epsilon] to a C/EBP site located adjacent to the NF-kappaB site in the FLAP promoter. We conclude that LPS enhances FLAP gene expression via both NF-kappaB- and C/EBP-mediated transcriptional mechanisms in mononuclear phagocytes.

Asunto(s)

Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Proteínas de la Membrana/metabolismo , Monocitos/metabolismo , Regiones Promotoras Genéticas/fisiología , Proteínas Activadoras de la 5-Lipooxigenasa , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Portadoras/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genes Reporteros/genética , Genes Reporteros/fisiología , Humanos , Leucotrienos/biosíntesis , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética

RESUMEN

PURPOSE: Emerging data continue to link carcinogenesis to inflammatory events involving the eicosanoid metabolic pathways. We therefore evaluated the effects of cyclooxygenase (COX)-2 inhibition on leukotriene (LT) B(4) synthesis in the lungs of active smokers, as part of a pilot lung cancer chemoprevention study with celecoxib (Celebrex), an oral COX-2 inhibitor. EXPERIMENTAL DESIGN: Bronchoalveolar lavage was performed before celecoxib treatment and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from ex-smoker control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation. RESULTS: Treatment with oral celecoxib only modestly increased LTB(4) levels in bronchoalveolar lavage, without increasing the mRNA transcription of 5-lipoxygenase (5-LOX) or 5-LOX-activating protein in AMs, whereas the acute calcium ionophore-stimulated LTB(4) production from smokers' AMs was markedly increased by 10.6-fold. In addition, smokers' AMs were twice as responsive in producing LTB(4) when exposed to lipopolysaccharide compared with ex-smokers' AMs. Concomitant COX-2 inhibition with SC58236, however, did not significantly impact these changes, whereas the 5-LOX inhibitor Zileuton blocked the generation of LTB(4) in a dose-responsive manner. Finally, cycloheximide increased the production of LTB(4) under all conditions, suggesting a shunting phenomenon and/or the presence of pathway inhibitors. CONCLUSIONS: Our findings suggest that whereas oral celecoxib is capable of modulating LTB(4) production in the lung microenvironment, under physiologic conditions, this effect is probably not functionally significant.

Asunto(s)

Inhibidores de la Ciclooxigenasa/farmacología , Leucotrieno B4/biosíntesis , Lipopolisacáridos/farmacología , Pirazoles/farmacología , Sulfonamidas/farmacología , Administración Oral , Anciano , Lavado Broncoalveolar , Celecoxib , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Pulmón/fisiología , Macrófagos Alveolares , Masculino , Persona de Mediana Edad , ARN Mensajero/biosíntesis , Fumar

RESUMEN

PURPOSE: Preclinical data suggest that the cyclooxygenase (COX)-2/prostaglandin (PG) E2 signaling pathway plays an essential role in conferring the malignant phenotype in non-small cell lung cancer. We hypothesized that treatment with oral celecoxib, a selective COX-2 inhibitor, would favorably alter biomarkers of lung cancer risk. This study evaluated the feasibility of COX-2 inhibition as a form of chemoprevention for lung cancer. EXPERIMENTAL DESIGN: Heavy active smokers were enrolled into a pilot study and treated with celecoxib. Bronchoscopy with bronchoalveolar lavage was performed both before and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from nonsmoking control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation. RESULTS: Treatment with celecoxib significantly reduced calcium ionophore-stimulated PGE2 production from AMs recovered from smokers. AMs recovered from smokers, but not nonsmokers, were primed to produce high levels of PGE2 and interleukin (IL-10) when stimulated with lipopolysaccharide, and SC58236 significantly abrogated the production of these factors. Moreover, both plasma and bronchoalveolar lavage fluid obtained from treated subjects significantly reduced the production of PGE2 that resulted when a lung cancer cell line, A549, was stimulated with IL-1beta or A23187. CONCLUSIONS: Our findings suggest that oral celecoxib is capable of inhibiting the overproduction of PGE2, as well as modulating the production of IL-10 in the lung microenvironment in individuals at risk for lung cancer.

Asunto(s)

Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/metabolismo , Interleucina-10/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Fumar/metabolismo , Sulfonamidas/farmacología , Anciano , Lavado Broncoalveolar , Broncoscopía , Calcimicina/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudios de Casos y Controles , Celecoxib , Estudios de Factibilidad , Femenino , Humanos , Ionóforos/farmacología , Lipopolisacáridos/farmacología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Macrófagos Alveolares/metabolismo , Masculino , Persona de Mediana Edad , Proyectos Piloto , Pirazoles/farmacología , Células Tumorales Cultivadas

Asunto(s)

Asma/genética , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Glutatión Transferasa/efectos de los fármacos , Glutatión Transferasa/genética , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Asma/enzimología , Línea Celular , Humanos , Técnicas In Vitro , Monocitos/enzimología

RESUMEN

We examined expression of the 5-lipoxygenase activating protein (FLAP), which is critical for inflammatory cell leukotriene synthesis. A 3.4-kb segment of the FLAP gene 5'-untranslated region accounted for a 22-fold increase in promoter activity when transfected into the monocyte-like cell line, THP-1, and demonstrated no activity in non-inflammatory cells. Virtually all of the promoter activity was mediated by the first 134 bp upstream of the transcription start site, a region that contains CCAAT/enhancer-binding proteins (C/EBP) consensus binding sites, at -36 to -28 bp (distal) and -25 to -12 bp (proximal). DNase I footprint analyses demonstrated THP-1 nuclear extract proteins bind to the proximal site. Electrophoretic mobility shift assay analyses revealed that C/EBP alpha, delta, and epsilon bind to the proximal site and C/EBP alpha and epsilon bind to the distal site, constitutively. Transfection studies indicated that mutation of both the proximal and distal sites decreased constitutive FLAP promoter activity. Overexpression of C/EBP alpha, beta, and delta transactivated promoter activity and increased native FLAP mRNA accumulation. Mutation of both C/EBP sites essentially abolished promoter induction by C/EBP overexpression. Tumor necrosis factor (TNF) alpha induced FLAP mRNA expression, FLAP promoter activity, and C/EBP alpha, delta, and epsilon binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBP alpha, delta, and epsilon bound to this region of the 5'-untranslated region, whereas C/EBP beta does not bind even under conditions of overexpression and stimulation. We conclude that the FLAP gene is transactivated by members of the C/EBP family of transcription factors in inflammatory cells and that these factors play an important role in FLAP gene induction by TNFalpha.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regiones no Traducidas 5' , Proteínas Activadoras de la 5-Lipooxigenasa , Sitios de Unión , Northern Blotting , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Desoxirribonucleasa I/metabolismo , Genes Reporteros , Células HeLa , Humanos , Luciferasas/metabolismo , Mutación , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/metabolismo , Transcripción Genética , Activación Transcripcional , Transfección

RESUMEN

We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC(4) synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC(4) release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC(4) synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC(4) synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-alpha and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-kappaB activation resulted in inhibition of the LPS effect, while activation of NF-kappaB and p50/p65 overexpression down-regulated the LTC(4) synthase gene. LPS down-regulates cysteinyl LT release and LTC(4) synthase gene expression in mononuclear phagocytes by an NF-kappaB-mediated mechanism.

Asunto(s)

Regulación hacia Abajo/inmunología , Proteínas de Drosophila , Glutatión Transferasa/antagonistas & inhibidores , Glutatión Transferasa/genética , Lipopolisacáridos/farmacología , Monocitos/enzimología , Monocitos/inmunología , Anticuerpos Bloqueadores/farmacología , Calcimicina/farmacología , Línea Celular , Relación Dosis-Respuesta Inmunológica , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/metabolismo , Humanos , Sueros Inmunes/farmacología , Ionóforos/farmacología , Leucotrieno C4/antagonistas & inhibidores , Leucotrieno C4/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/biosíntesis , FN-kappa B/genética , FN-kappa B/metabolismo , FN-kappa B/fisiología , Subunidad p50 de NF-kappa B , Regiones Promotoras Genéticas/inmunología , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Procesamiento Postranscripcional del ARN/inmunología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/biosíntesis , Factores de Tiempo , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Transcripción ReIA , Transfección , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunología , Compuestos de Vanadio/farmacología

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Glutatión Transferasa/genética , Fagocitos/enzimología , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Genes Reporteros , Células HeLa , Humanos , Luciferasas/genética , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , ARN Mensajero/genética , Factor de Transcripción Sp1/química , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3 , Factores de Transcripción/química , Factores de Transcripción/genética , Transfección