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1.
Pharm Res ; 31(3): 635-48, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24190631

RESUMEN

PURPOSE: Study the impact of CXCL13 neutralization on germinal center (GC) response in vivo, and build quantitative relationship between target coverage and pharmacological effects at the target tissue. METHODS: An anti-CXCL13 neutralizing monoclonal antibody was dosed in vivo in a T-dependent mouse immunization (TDI) model. A quantitative site-of-action (SoA) model was developed to integrate antibody PK and total CXCL13 levels in serum and spleen towards estimating target coverage as a function of dose. To aid in the SoA model development, a radio-labeled study using [I(125)] CXCL13 was conducted in mice. Model estimated target coverage was linked to germinal center response using a sigmoidal inhibitory effect model. RESULTS: In vivo studies demonstrated that CXCL13 inhibition led to an architectural change in B-cell follicles, dislocation of GCs and a significant reduction in the GC absolute numbers per square area (GC/mm(2)). The SoA modeling analysis indicated that ~79% coverage in spleen was required to achieve 50% suppression of GC/mm(2). The 3 mg/kg dose with 52% spleen coverage resulted in no PD suppression, whereas 30 mg/kg with 93% coverage achieved close to maximum PD suppression, highlighting the steepness of PD response. CONCLUSIONS: This study showcases an application of SoA modeling towards a quantitative understanding of CXCL13 pharmacology.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Quimiocina CXCL13/inmunología , Centro Germinal/efectos de los fármacos , Linfocitos T/inmunología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Femenino , Centro Germinal/inmunología , Centro Germinal/ultraestructura , Inmunización , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/efectos de los fármacos
2.
Interdiscip Toxicol ; 7(3): 123-33, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26109889

RESUMEN

An earlier age at onset of Parkinson's disease (PD) has been reported to be associated with occupational exposures to manganese and hydrocarbon solvents suggesting that exposure to neurotoxic chemicals may hasten the progression of idiopathic PD. In this study the role of occupational exposure to metals and pesticides in the progression of idiopathic PD was assessed by looking at age at disease onset. The effects of heritable genetic risk factors, which may also influence age at onset, was minimized by including only sporadic cases of PD with no family history of the disease (n=58). Independent samples Student t-test revealed that subjects with occupational exposure to metals and/or pesticides (n=36) were significantly (p=0.013) younger than unexposed controls (n=22). These subjects were then divided into three groups [high (n=18), low (n=18), and unexposed (n=22)] to ascertain if duration of exposure further influenced age at onset of PD. One-way ANOVA revealed that subjects in the high exposure group were significantly (p=0.0121) younger (mean age: 50.33 years) than unexposed subjects (mean age: 60.45 years). Subjects were also stratified by exposure type (metals vs. pesticides). These results suggest that chronic exposure to metals and pesticides is associated with a younger age at onset of PD among patients with no family history of the disease and that duration of exposure is a factor in the magnitude of this effect.

3.
Rapid Commun Mass Spectrom ; 25(12): 1715-24, 2011 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-21598331

RESUMEN

P-Glycoprotein (P-gp/ABCB1) is expressed in membrane barriers to exclude pharmacological substrates from cells, and therefore influences the ADME/Tox properties and efficacy of therapeutics. In the present study, a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-mediated targeted proteomics was developed to quantitate P-gp protein. With the aid of in silico predictive tools, a unique 9-mer tryptic peptide of P-gp protein was synthesized (with the stable isotope labeled (SIL) peptide as internal standard) and applied for quantitative LC/MS/MS method development. For LC/MS/MS quantification, the N-glycosylation of the peptide, polymorphism and transmembrane region was intended to be excluded during the peptide selection. The lower limit of quantification was established to be 0.025 nM with the linearity of the standard curve ranging to 20 nM of P-gp signature peptides in the matrix digested surrogate bovine serum albumin. The digestion efficiency, both the accuracy (relative error) and the precision (coefficient of variation) of the method, was verified by using the synthetic quantification peptide and the synthetic surrogate substrate peptide that mimics the sequence of tryptic peptide and associated flanking tryptic cleavage sites at the N- and C-terminals. By applying the method developed, the absolute amounts of human, dog and mouse P-gp (Mdr1a) were quantified in various biological samples. LC/MS/MS-mediated P-gp quantification was achieved as a highly sensitive, selective and reproducible assay and could be directly applicable to many current research needs related to P-gp.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Cromatografía Liquida/métodos , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Subfamilia B de Transportador de Casetes de Unión a ATP/análisis , Animales , Extractos Celulares/química , Línea Celular , Simulación por Computador , Perros , Humanos , Modelos Lineales , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Alineación de Secuencia , Albúmina Sérica Bovina/análisis
4.
Biomark Med ; 4(3): 475-83, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20550481

RESUMEN

Certain compounds that induce liver injury clinically are not readily identified from earlier preclinical studies. Novel biomarkers are being sought to be applied across the pharmaceutical pipeline to fill this knowledge gap and to add increased specificity for detecting drug-induced liver injury in combination with aminotransferases (alanine and aspartate aminotransferase)--the current reference-standard biomarkers used in the clinic. The gaps in the qualification process for novel biomarkers of regulatory decision-making are assessed and compared with aminotransferase activities to guide the determination of safe compound margins for drug delivery to humans where monitoring for potential liver injury is a cause for concern. Histopathologic observations from preclinical studies are considered the principal reference standard to benchmark and assess subtle aminotransferase elevations. This approach correlates quite well for many developmental compounds, yet cases of discordance create dilemmas regarding which standard(s) indicates true injury. Concordance amongst a broader set of biomarker injury signals in a qualification paradigm will increase confidence, leading to accepted and integrated translational biomarker signals during safety assessment processes across the pharmaceutical industry, with academia, in government and in contractor laboratories.


Asunto(s)
Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Alanina Transaminasa/sangre , Aspartato Aminotransferasas/sangre , Biomarcadores/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Diagnóstico Precoz , Glutamato Deshidrogenasa/metabolismo , Humanos , L-Iditol 2-Deshidrogenasa/sangre , Valor Predictivo de las Pruebas
5.
Nat Biotechnol ; 28(5): 446-54, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20458314

RESUMEN

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.


Asunto(s)
Biomarcadores , Descubrimiento de Drogas , Preparaciones Farmacéuticas , Animales , Descubrimiento de Drogas/legislación & jurisprudencia , Descubrimiento de Drogas/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Humanos , Preparaciones Farmacéuticas/normas
6.
Nat Biotechnol ; 28(5): 455-62, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20458315

RESUMEN

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.


Asunto(s)
Biomarcadores Farmacológicos , Aprobación de Drogas/legislación & jurisprudencia , Riñón , Animales , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Europa (Continente) , Humanos , Riñón/efectos de los fármacos , Riñón/lesiones , Preparaciones Farmacéuticas/normas , Estados Unidos , United States Food and Drug Administration
7.
Nat Biotechnol ; 28(5): 470-7, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20458317

RESUMEN

The capacities of urinary trefoil factor 3 (TFF3) and urinary albumin to detect acute renal tubular injury have never been evaluated with sufficient statistical rigor to permit their use in regulated drug development instead of the current preclinical biomarkers serum creatinine (SCr) and blood urea nitrogen (BUN). Working with rats, we found that urinary TFF3 protein levels were markedly reduced, and urinary albumin were markedly increased in response to renal tubular injury. Urinary TFF3 levels did not respond to nonrenal toxicants, and urinary albumin faithfully reflected alterations in renal function. In situ hybridization localized TFF3 expression in tubules of the outer stripe of the outer medulla. Albumin outperformed either SCr or BUN for detecting kidney tubule injury and TFF3 augmented the potential of BUN and SCr to detect kidney damage. Use of urinary TFF3 and albumin will enable more sensitive and robust diagnosis of acute renal tubular injury than traditional biomarkers.


Asunto(s)
Albuminuria/orina , Biomarcadores Farmacológicos/orina , Enfermedades Renales , Túbulos Renales/efectos de los fármacos , Neuropéptidos/orina , Animales , Carbapenémicos/toxicidad , Cisplatino/toxicidad , Gentamicinas/toxicidad , Histocitoquímica , Glicósidos Iridoides , Iridoides/toxicidad , Enfermedades Renales/inducido químicamente , Enfermedades Renales/diagnóstico , Túbulos Renales/patología , Modelos Logísticos , Curva ROC , Ratas , Factor Trefoil-3
8.
Nat Biotechnol ; 28(5): 478-85, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20458318

RESUMEN

Kidney toxicity accounts both for the failure of many drug candidates as well as considerable patient morbidity. Whereas histopathology remains the gold standard for nephrotoxicity in animal systems, serum creatinine (SCr) and blood urea nitrogen (BUN) are the primary options for monitoring kidney dysfunction in humans. The transmembrane tubular protein kidney injury molecule-1 (Kim-1) was previously reported to be markedly induced in response to renal injury. Owing to the poor sensitivity and specificity of SCr and BUN, we used rat toxicology studies to compare the diagnostic performance of urinary Kim-1 to BUN, SCr and urinary N-acetyl-beta-D-glucosaminidase (NAG) as predictors of kidney tubular damage scored by histopathology. Kim-1 outperforms SCr, BUN and urinary NAG in multiple rat models of kidney injury. Urinary Kim-1 measurements may facilitate sensitive, specific and accurate prediction of human nephrotoxicity in preclinical drug screens. This should enable early identification and elimination of compounds that are potentially nephrotoxic.


Asunto(s)
Biomarcadores Farmacológicos/orina , Moléculas de Adhesión Celular/orina , Pruebas de Función Renal/métodos , Riñón , Acetilglucosaminidasa/orina , Animales , Biomarcadores Farmacológicos/metabolismo , Nitrógeno de la Urea Sanguínea , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cisplatino/toxicidad , Creatinina/sangre , Ciclosporina/toxicidad , Evaluación Preclínica de Medicamentos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Gentamicinas/toxicidad , Histocitoquímica , Riñón/efectos de los fármacos , Riñón/lesiones , Pruebas de Función Renal/normas , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Curva ROC , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Daño por Reperfusión , Tioacetamida/toxicidad
9.
Nat Biotechnol ; 28(5): 486-94, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20458319

RESUMEN

The Predictive Safety Testing Consortium's first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.


Asunto(s)
Biomarcadores Farmacológicos , Cistatina C/sangre , Enfermedades Renales/diagnóstico , Pruebas de Función Renal/métodos , Animales , Biomarcadores Farmacológicos/sangre , Biomarcadores Farmacológicos/metabolismo , Biomarcadores Farmacológicos/orina , Nitrógeno de la Urea Sanguínea , Carbapenémicos/toxicidad , Creatinina/sangre , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Femenino , Gentamicinas/toxicidad , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Curva ROC , Ratas , Ratas Sprague-Dawley , Ratas Wistar
10.
Drug Discov Today ; 15(3-4): 142-7, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20026239

RESUMEN

Guidance for the use of biomarkers in pharmaceutical development and clinical trial optimization will reduce developmental cycle time. A 'fit-for-purpose' guidance for biomarker use is considered herein when the same biomarker is applied in very different contexts in drug development and after regulatory approval. Recent approved use of renal safety biomarkers in Good Laboratory Practice studies lacks sufficient guidance for the use of these markers across the drug development pipeline. In lead optimization, renal injury biomarkers are possible anchors for promising new prodromal metabolic biomarkers, which are applied before lead candidate selection. Renal injury biomarkers can now be evaluated as potential efficacy and pharmacodynamic biomarkers in clinical trial proof-of-concept studies for diabetic nephropathy.


Asunto(s)
Biomarcadores Farmacológicos/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Riñón/metabolismo , Investigación Biomédica Traslacional/métodos , Albúminas/metabolismo , Animales , Enfermedades Autoinmunes/metabolismo , Ácidos y Sales Biliares/metabolismo , Catepsina B/orina , Ciclosporina/efectos adversos , Cistatina C/sangre , Cisteína/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/orina , Aprobación de Drogas , Humanos , Inmunosupresores/efectos adversos , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Leucotrienos/metabolismo , Modelos Animales
11.
Regul Toxicol Pharmacol ; 56(3): 237-46, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19903504

RESUMEN

Drug-induced liver injury (DILI) is the most frequent cause of discontinuation of new chemical entities during development. DILI can either be intrinsic/predictable or an idiosyncratic type. These two forms of DILI are contrasted in their manifestation and diagnosis. Even with regulatory guidance (FDA, 2009), there is still a gap in our ability to identify predictable DILI, both specifically and sensitively. Alanine aminotransferase (ALT) is the principal reference standard biomarker to diagnose DILI, yet its current application in preclinical to clinical translation for decision-making purposes has imperfections: (1) analytical ALT assay uniformity across industry would be aided by common analytical processes; (2) assessment of ALT toxicological performance in a large preclinical analysis would help to establish a true threshold of elevation for predictable DILI and improve translational use across various stages of pharmaceutical development and finally, (3) clinical evaluation of ALT elevations prospectively and retrospectively is recommended to define and manage variations in clinical study subjects including rising body mass index (BMI) range and ALT upper limit of normal (ULN) in the broader population over time. The emergence of new hepatotoxicity biomarkers necessitates a parallel and equivalent assessment to the aminotransferases in a regulatory qualification model.


Asunto(s)
Alanina Transaminasa/normas , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Alanina Transaminasa/metabolismo , Biomarcadores/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Humanos , Estándares de Referencia
13.
J Chromatogr B Analyt Technol Biomed Life Sci ; 877(22): 2052-60, 2009 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-19535304

RESUMEN

A sensitive and selective liquid chromatography tandem mass spectrometry (LC\MS\MS) method has been developed for the simultaneous quantification in human plasma of the endocannabinoid anandamide (AEA) and three other related ethanolamides, linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The analytical methodology requires 50 microL of human plasma which is processed via protein precipitation using a 96-well protein precipitation plate. Chromatographic separation of plasma extract was achieved with a Phenomenex Gemini C6-Phenyl HPLC column (2.1 mm x 50 mm, 5 microm) at a flow rate of 0.30 mL/min using gradient elution and a mobile phase consisting of acetonitrile and 5 mM ammonium formate. All four fatty acid ethanolamides were quantified by positive ion electrospray ionization tandem mass spectrometry, with the detection of ion current signal generated from the selected reaction monitoring (SRM) transition of [M+H](+)-->m/z 62. Deuterated anandamide (AEA-d8) was used as an internal standard for all four ethanolamides. The lower limit of quantitation was 0.05 ng/mL for AEA and LEA, 0.5 ng/mL for OEA and 1.0 ng/mL for PEA. Inter-assay precision and accuracy were typically within 12% for the four endogenous analytes and overall extraction recoveries ranged between 40% and 100%.


Asunto(s)
Ácidos Araquidónicos/sangre , Moduladores de Receptores de Cannabinoides/sangre , Cromatografía Líquida de Alta Presión/métodos , Ácidos Linoleicos/sangre , Alcamidas Poliinsaturadas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Endocannabinoides , Humanos
14.
Toxicology ; 245(3): 194-205, 2008 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-18291570

RESUMEN

The level of serum alanine aminotransferase (ALT) activity reflects damage to hepatocytes and is considered to be a highly sensitive and fairly specific preclinical and clinical biomarker of hepatotoxicity. However, an increase in serum ALT activity level has also been associated with other organ toxicities, thus, indicating that the enzyme has specificity beyond liver in the absence of correlative histomorphologic alteration in liver. Thus, unidentified non-hepatic sources of serum ALT activity may inadvertently influence the decision of whether to continue development of a novel pharmaceutical compound. To assess the risk of false positives due to extraneous sources of serum ALT activity, additional biomarkers are sought with improved specificity for liver function compared to serum ALT activity alone. Current published biomarker candidates are reviewed herein and compared with ALT performance in preclinical and on occasion, clinical studies. An examination of the current state of hepatotoxic biomarkers indicates that serum F protein, arginase I, and glutathione-S-transferase alpha (GSTalpha) levels, all measured by ELISA, may show utility, however, antibody availability and high cost per run may present limitations to widespread applicability in preclinical safety studies. In contrast, the enzymatic markers sorbitol dehydrogenase, glutamate dehydrogenase, paraxonase, malate dehydrogenase, and purine nucleoside phosphorylase are all readily measured by photometric methods and use reagents that work across preclinical species and humans and are commercially available. The published literature suggests that these markers, once examined collectively in a large qualification study, could provide additional information relative to serum ALT and aspartate aminotransferase (AST) values. Since these biomarkers are found in the serum/plasma of treated humans and rats, they have potential to be utilized as bridging markers to monitor acute drug-induced liver injury in early clinical trials.


Asunto(s)
Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Alanina Transaminasa/sangre , Animales , Humanos , Isoenzimas/sangre , Isoenzimas/aislamiento & purificación , Pruebas de Función Hepática
15.
Stem Cells Dev ; 15(2): 175-90, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16646664

RESUMEN

The levels of General Transcription Factor (TF) IIA were examined during mammalian brain development and in rat embryo fibroblasts and transformed cell lines. The large TFIIA subunit paralogues alphabeta and tau are largely produced in unsynchronized cell lines, yet only TFIIA alphabeta is observed in a number of differentiated tissue extracts. Steady-state protein levels of the TFIIA tau, alphabeta, and gamma subunits were significantly reduced when human embryonal (ec) and hepatic carcinoma cell lines were stimulated to differentiate with either all-trans-retinoic acid (ATRA) or sodium butyrate. ATRA-treated NT2-ec cells required replating to induce a neuronal phenotype and loss of detectable TFIIA tau and gamma proteins. High levels of TFIIA tau, alphabeta, and gamma and Sp factors were identified in extracts from human fetal and rat embryonic day-18 brains, but not in human and rat adult brain extracts. A high histone H3 Lys9/Lys4 methylation ratio was observed in the TFIIA tau promoter of primary hippocampal neurons from day-18 rat embryos, suggesting that repressive epigenetic marks of chromatin prevent TFIIA tau from being transcribed in neurons. We conclude that TFIIA tau is associated with undifferentiated cells during development, yet is down-regulated at the chromatin level upon cellular differentiation.


Asunto(s)
Diferenciación Celular/fisiología , Cromatina/metabolismo , Neuronas/metabolismo , Factor de Transcripción TFIIA/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Butiratos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Línea Celular Tumoral , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Células HT29 , Células HeLa , Histonas/metabolismo , Humanos , Células Jurkat , Masculino , Datos de Secuencia Molecular , Neuronas/citología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Factores de Transcripción Sp/metabolismo , Testículo/metabolismo , Factor de Transcripción TFIIA/genética , Tretinoina/farmacología
16.
Gene ; 323: 31-42, 2003 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-14659877

RESUMEN

The factors that bind to the hepatic-specific human apolipoprotein AI (apoAI) 48-bp downstream enhancer (DSE) were identified and characterized by electrophoretic mobility shift assays. A significant homology was shown between the histone 4 (H4) promoters and the hepatic-specific human apoAI DSE at Sp1 and H4TF2 binding sites. Human HepG2 nuclear extracts were used to form four specific complexes with the DSE (referred to as apoAI DSE-1, -2, -3, and -4). The apoAI DSE-1 and -2 complexes showed similar binding specificity to the Sp/H4TF1 consensus site within the apoAI DSE. The apoAI DSE-1 complex was predominantly recognized by anti-Sp1 and Sp3 sera in gel shift assays, indicating that the DSE was recognized by multiple Sp family members. Nuclear extracts that were prepared from retinoic acid treated HepG2 cells showed increased levels of Sp factors in gel shift and Western blot assays. The apoAI DSE-2 complex was identified as H4TF1 and formed in the absence of magnesium chloride. The apoAI DSE-3 complex bound to a consensus GATA element within the DSE that was recognized by recombinant human GATA-6 as well. The apoAI DSE-3 complex was completely disrupted by a GATA-4 antibody in EMSA. GATA-4 and -6 were detected in nuclear extracts prepared from retinoic acid treated HepG2 cells using Western blot assays. The highest apoAI DSE-3 levels were observed with retinoic acid treated HepG2 cell nuclear extracts in EMSA. ApoAI DSE-4 is a multi-factor complex that includes an Sp/H4TF1 factor and either H4TF2 or apoAI DSE-3. Because apoAI DSE mutations revealed transcription defects in transient transfection assays, we conclude that the entire DSE sequence is required for full apoAI transcriptional activity in HepG2 cells.


Asunto(s)
Apolipoproteína A-I/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Factor de Transcripción GATA4 , Factor de Transcripción GATA6 , Regulación Neoplásica de la Expresión Génica , Humanos , Hígado/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica , Factor de Transcripción Sp1/metabolismo , Transfección
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