RESUMEN
Polyhexamethylene guanidine phosphate (PHMG-P), a cationic biocide, is widely used in household products due to its strong bactericidal activity and low toxicity. However, it causes fatal lung damage when inhaled. In this study, we investigated why PHMG-P causes fatal lung injury when inhaled, and demonstrated that the disruption of membrane integrity through ionic interaction-a molecular initiating event of PHMG-P-determines toxicity. Mice were injected intravenously with 0.9 or 7.2 mg/kg PHMG-P (IV group), or instilled intratracheally with 0.9 mg/kg PHMG-P (ITI group); they were euthanatized at 4 h and on days 1 and 7 after treatment. Increased total BAL cell count and proinflammatory cytokine production, along with fibrotic changes in the lungs, were detected in the ITI group only. Levels of hepatic enzymes and hepatic serum amyloid A mRNA expression were markedly upregulated in the 7.2 mg/kg IV and ITI groups at 4 h or day 1 after treatment, but returned to baseline. No pathological findings were detected in the heart, liver, or kidneys. To simulate the IV injection, A549, THP-1, and HepG2 cells were treated with PHMG-P in cell culture media supplemented with different serum concentrations. Increased serum concentration was associated with an increase in cell viability. These results support the idea that direct contact between PHMG-P and cell membranes is necessary for PHMG-induced toxicity.
Asunto(s)
Desinfectantes , Lesión Pulmonar , Animales , Desinfectantes/toxicidad , Guanidinas/toxicidad , Pulmón/patología , Lesión Pulmonar/patología , RatonesRESUMEN
Polyhexamethyleneguanidine phosphate (PHMG-P) is one of the causative agents of humidifier disinfectant-induced lung injury. Direct exposure of the lungs to PHMG-P causes interstitial pneumonia with fibrosis. Epidemiological studies showed that patients with humidifier disinfectant-associated lung injuries have suffered from restrictive lung function five years after the onset of the lung injuries. We investigated whether lung damage was sustained after repeated exposure to PHMG-P followed by a long-term recovery and evaluated the adverse effects of PHMG-P on mice lungs. Mice were intranasally instilled with 0.3 mg/kg PHMG-P six times at two weeks intervals, followed by a recovery period of 292 days. Histopathological examination of the lungs showed the infiltration of inflammatory cells, the accumulation of extracellular matrix in the lung parenchyma, proteinaceous substances in the alveoli and bronchiolar-alveolar hyperplasia. From RNA-seq, the gene expression levels associated with the inflammatory response, leukocyte chemotaxis and fibrosis were significantly upregulated, whereas genes associated with epithelial/endothelial cells development, angiogenesis and smooth muscle contraction were markedly decreased. These results imply that persistent inflammation and fibrotic changes caused by repeated exposure to PHMG-P led to the downregulation of muscle and vascular development and lung dysfunction. Most importantly, this pathological structural remodeling induced by PHMG-P was not reversed even after long-term recovery.
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Psiguadial B (8), and its fluoro- (8a), chloro- (8b), and bromo- (8c) derivatives were synthesized using a sodium acetate-catalyzed single step coupling of three components: ß-caryophyllene (5), diformylphloroglucinol (11), and benzaldehyde (12). These compounds efficiently and dose-dependently decreased H2O2-induced cell death, a quantitative marker of cell death, in primary cultures of mouse cortical neurons. Psiguadial B also decreased neuronal death and accumulation of ROS induced by FeCl2 in cortical cultures. The in vitro effects of these compounds in lipopolysaccharide (LPS)-induced expression of nitric oxide (NO), and TNF-α and IL-6 by suppressing the NF-κB pathway in immune cells demonstrated their antioxidative and anti-inflammatory activity. The present findings warrant further research on the development of psiguadial B-based neuroprotective agents for the treatment of neurodegenerative diseases, acute brain injuries and immunological disorders.
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Antiinflamatorios/química , Antioxidantes/química , Fármacos Neuroprotectores/química , Terpenos/química , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Compuestos Ferrosos/farmacología , Halogenación , Peróxido de Hidrógeno/farmacología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/metabolismo , Psidium/química , Psidium/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
HL235 is a new cathepsin K inhibitor designed and synthesized to treat osteoporosis. Since HL235 has poor aqueous solubility, a self-microemulsifying drug delivery system (SMEDDS) was formulated to enhance its oral bioavailability. A solubility study of HL235 was performed to select a suitable oil, surfactant and cosurfactant. Pseudoternary phase diagrams were plotted to identify the microemulsion region and to determine the range of components in the isotropic mixture. D-optimal mixture design and a desirability function were introduced to optimize the SMEDDS formulation for the desired physicochemical characteristics, i.e., high drug concentration at 15â¯min after dilution with simulated gastric fluid (SGF) and high solubilization capacity. The optimized HL235-loaded SMEDDS formulation consisted of 5.0% Capmul MCM EP (oil), 75.0% Tween 20 (surfactant) and 20.0% Carbitol (cosurfactant). The droplet size of the microemulsion formed by the optimized formulation was 10.7⯱â¯1.6â¯nm, and the droplets were spherical in shape. Pharmacokinetic studies in rats showed that the relative oral bioavailability of the SMEDDS formulation increased up to 3.22-fold compared to its solution in DMSO:PEG400 (8:92, v/v). Thus, the formulation of SMEDDS optimized by D-optimal mixture design could be a promising approach to improve the oral bioavailability of HL235.
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Catepsina K/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos , Pirimidinas/administración & dosificación , Administración Oral , Animales , Disponibilidad Biológica , Química Farmacéutica , Diglicéridos/química , Emulsiones , Glicoles de Etileno/química , Excipientes/química , Masculino , Monoglicéridos/química , Tamaño de la Partícula , Polisorbatos/química , Pirimidinas/química , Pirimidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , SolubilidadRESUMEN
As a potential treatment of type 2 diabetes, a novel PPARγ non-TZD full agonist, compound 18 (BR102375) was identified from the original lead BR101549 by the SAR efforts of the labile metabolite control through bioisosteres approach. In vitro assessments of BR102375 demonstrated its activating potential of PPARγ comparable to Pioglitazone as well as the induction of related gene expressions. Further in vivo evaluation of BR102375 in diabetic rodent models successfully proved its glucose lowering effect as a potential antidiabetic agent, but the anticipated suppression of weight gain was not evident. The X-ray co-crystal analysis of BR102375-PPARγ LBD unexpectedly revealed binding modes totally different from those of BR101549, which was found, instead, closely resembled to those of TZD full agonists.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Descubrimiento de Drogas , Hipoglucemiantes/farmacología , Oxadiazoles/farmacología , PPAR gamma/agonistas , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Modelos Moleculares , Estructura Molecular , Oxadiazoles/química , PPAR gamma/metabolismo , Relación Estructura-ActividadRESUMEN
The new class of PPARgamma non-TZD agonist originally derived from the backbone of anti-hypertensive Fimasartan, BR101549, was identified as a potential lead for anti-diabetic drug development. The X-ray crystallography of BR101549 with PPARgamma ligand binding domain (LBD) revealed unique binding characteristics versus traditional TZD full agonists. The lead candidate, BR101549, has been found activating PPARgamma to the level of Pioglitazone in vitro and indeed has demonstrated its effects on blood glucose control in mouse proof-of-concept evaluation. The attempts to improve its metabolic stability profile through follow-up SAR including deuterium incorporation have been also described.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Oxadiazoles/uso terapéutico , PPAR gamma/agonistas , Pirimidinas/uso terapéutico , Pirimidinonas/uso terapéutico , Células 3T3-L1 , Animales , Humanos , Ratones , Prueba de Estudio Conceptual , Pirimidinonas/farmacología , Relación Estructura-ActividadRESUMEN
Inspired by the well-known PPARγ partial agonism of angiotensin II type 1 receptor (AT1R) antagonists exemplified by an antihypertensive drug, Telmisartan, efforts to identify compounds with the dual activities have been pursued in order to control the two major metabolic disorders, hypertension and hyperglycemia simultaneously. Lead compound 18 derived from the AT1R antagonist, Fimasartan, has successfully presented the possibility to control the medical conditions by a single molecule.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Compuestos de Bifenilo/farmacología , PPAR gamma/agonistas , Pirimidinas/farmacología , Tetrazoles/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacocinética , Animales , Antihipertensivos/química , Antihipertensivos/farmacocinética , Antihipertensivos/farmacología , Área Bajo la Curva , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacocinética , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Agonismo Parcial de Drogas , Semivida , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Prueba de Estudio Conceptual , Pirimidinas/química , Pirimidinas/farmacocinética , Relación Estructura-Actividad , Tetrazoles/química , Tetrazoles/farmacocinéticaRESUMEN
White-spotted flower chafer (Protaetia brevitarsis) is an edible insect and its larva was used as a traditional Asian medicine. It's a promising material as a novel food source because of its nutritional components. In this study, as part of the preclinical toxicity program, we evaluated the toxicity of freeze-dried P. brevitarsis larva powder to develop a novel food material. In a single-dose oral toxicity study in rats, there were no changes in mortality, clinical observations, and body weight in rats administered 5000â¯mg/kg P. brevitarsis larva powder. In a 13-week oral repeated dose toxicity study in rats, there were no adverse effects or changes in mortality, clinical observations, body weight, food consumption, ophthalmology, clinical pathology, necropsy, organ weight, and histopathology at doses of 300, 1000, and 3000â¯mg/kg/day. In identification of allergic reactions, P. brevitarsis larva powder induced no increases of serum immunoglobulin E and histamine concentrations over 13 weeks of oral administration in rats. In a genotoxicity assessment, P. brevitarsis larva powder didn't provoke bacterial reverse mutations, chromosomal aberrations, and micronucleated reticulocytes. Therefore, freeze-dried P. brevitarsis larva powder shows no evidence of toxic and mutagenic changes under the experimental conditions of the present in vitro and in vivo studies.
RESUMEN
The convergent and enantioselective synthesis of a highly potent human peroxisome proliferator-activated receptor delta agonist is presented. More specifically, the thiazoline structure, which constitutes the biosynthetically distinctive core structure of pulicatin (a secondary metabolite of symbiotic bacteria), was synthesized from a commercially available and inexpensive chiral pool of l-threonine.
RESUMEN
Abstract The aggregation of water molecules inside heat-gelatinized rice grain due to retrogradation of the grain was investigated by textural change and scanning electron microscopy (SEM) analysis of cooked grains after storage at 11 °C in a sealed package. Relaxation tests using a disc-type tip showed an increase in hardness (strength) of the cooked grain as the degree of retrogradation increased with increasing storage time, measured by the α-amylase-iodine method. SEM analysis of the vacuum-dried cooked rice grain showed a gradual increase in crevices, which further developed into holes at the center of the granule with increasing storage time. The results suggest that the disruption of hydrogen bonds between water and starch molecules is the first step for the retrogradation of gelatinized rice grain stored in a hermetic environment to avoid drying, resulting in its increased hardness, followed by the aggregation of starch molecules with subsequent water extrusion.
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Worldwide demand for novel food source has grown and edible insects are a promising food sources for humans. Tenebrio molitor, as known as yellow mealworm, has advantages of being rich in protein, and easy to raise as a novel food source. The objective of this study was to evaluate subchronic toxicity, including potential hypersensitivity, of freeze-dried powdered T. molitor larvae (fdTML) in male and female Sprague-Dawley rats. The fdTML was administered orally once daily at dose levels of 0, 300, 1000 and 3000 mg/kg/day for 90 days. A toxicological assessment was performed, which included mortality, clinical signs, body and organ weights, food consumption, ophthalmology, urinalysis, hematology, serum chemistry, gross findings, histopathologic examination and allergic reaction. There were no fdTML- related findings in clinical signs, urinalysis, hematology and serum chemistry, gross examination, histopathologic examination or allergic reaction. In conclusion, the No Observed Adverse Effect Level (NOAEL) for fdTML was determined to be in excess of 3000 mg/kg/day in both sexes of rats under the experimental conditions of this study.
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Alimentación Animal/toxicidad , Proteínas en la Dieta/toxicidad , Proteínas de Insectos/toxicidad , Larva/crecimiento & desarrollo , Valor Nutritivo , Tenebrio/crecimiento & desarrollo , Pruebas de Toxicidad/métodos , Administración Oral , Animales , Biomarcadores/sangre , Proteínas en la Dieta/administración & dosificación , Proteínas en la Dieta/inmunología , Femenino , Hipersensibilidad a los Alimentos/etiología , Hipersensibilidad a los Alimentos/inmunología , Liofilización , Proteínas de Insectos/administración & dosificación , Proteínas de Insectos/inmunología , Masculino , Nivel sin Efectos Adversos Observados , Tamaño de los Órganos/efectos de los fármacos , Polvos , Ratas Sprague-Dawley , Medición de Riesgo , Factores de TiempoRESUMEN
A series of 4-arylamido 3-methyl isoxazoles were synthesized and evaluated for their antiproliferative activities against the A375P melanoma and U937 hematopoietic cell lines. Most compounds showed selective antiproliferative activity toward the U937 cell line and the activities were better than that of sorafenib, the reference standard. Derivatives were made as amide 5a-b, 6a-o and urea 7a-n, 8a-g with hydrophobic moieties, and one of the most potent inhibitor 6a, 5-methyl-N-(2-methyl-5-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)benzamido)phenyl)isoxazole-4-carboxamide was found to be very potent inhibitor of FMS kinase (GI50 = 0.016 µM, IC50 = 9.95 nM) with excellent selectivity profiles and is a promising candidate for further development in therapeutics for cancer.
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Descubrimiento de Drogas , Isoxazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Isoxazoles/síntesis química , Isoxazoles/química , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Células U937RESUMEN
The objective of this study was to investigate the toxicological information of freeze-dried powder from Allomyrina dichotoma (A. dichotoma) larvae as a food ingredient. The powder, suspended in distilled water, was administered once daily by oral gavage to four groups of Sprague-Dawley (SD) rats at dose levels of 0 (vehicle control), 250, 850, and 2500 mg/kg/day. After 13 wks of repeated administration, the standard toxicological parameters such as mortality, clinical signs, body weight, food consumption, ophthalmologic examination, clinical pathology, organ weights and macro/microscopic examination were applied for assessment of general toxicity. In addition, serum IgE and histamine levels were determined to evaluate allergenicity. The freeze-dried powder from A. dichotoma larvae did not produce treatmentrelated changes or findings in any toxicological parameters in either sex of any dosed groups except for slight increases in serum histamine levels at 2500 mg/kg/day. The changes were considered not to be adverse since the magnitude was minimal. In conclusion, the NOAEL (No Observed Adverse Effect Level) of the freeze-dried powder from A. dichotoma larvae was determined to be 2500 mg/kg/day or more in both sexes of SD rats and it is considered a candidate to be edible material.
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The synthesis of a novel series of (4-aminobenzyl/benzoyl)-1H-imidazol-1-yl pyrimidin-2-yl derivatives 9, 10, 18, 19 and their in vitro antiproliferative activities against the A375P human melanoma cell line and the U937 human leukemic monocyte lymphoma cell line are described. Potent antiproliferative effects were found from 9l, 9s and 10c; 10c was found to be a highly potent and selective BRAF V600E and CRAF inhibitor (IC50=38.3 nM and 8.79 nM).
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Antineoplásicos/farmacología , Diseño de Fármacos , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Pirimidinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Estructura Molecular , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Células U937RESUMEN
1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal kinases, JNK3. Their activities were evaluated through measurement of Kd using SPR, JNK3 kinase assay, and cell-viability of human neuroblastoma cells. Most tested compounds showed high affinity (10 µM-46 nM) to JNK3. Among them, compound 16f exhibited potent activities (Kd=46 nM). Especially, 16f was also found to present a potent cell protective effect (IC50=1.09 µM) against toxicity induced by anisomycin, showing a possibility as protective therapeutics in neuronal cell apoptosis.
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Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Proteína Quinasa 10 Activada por Mitógenos/antagonistas & inhibidores , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Humanos , Modelos Moleculares , Neuroblastoma , Fosforilación , Relación Estructura-ActividadRESUMEN
Chlorpheniramine is an anti-histamine agent on IgE-mediated inflammation. In order to investigate the immunomodulatory effects of chlorpheniramine, we assessed the changes of peripheral mononuclear cell populations and other general clinical parameters, including hematology and clinical chemistry, following chlorpheniramine administration in rats. Since prednisolone is commonly co-prescribed with anti-histamine in many hypersensitive reactions, we also examined the changes to compare the results after the prednisolone administration. Chlorpheniramine (50, 100 and 200 µg/kg) and prednisolone (1, 2 and 4 mg/kg) were intramuscularly administered to female Sprague-Dawley (SD) rats 3 times, at intervals of 1 week. Except the clinical signs, such as stiffness and abnormal gait due to the local toxicity at injection sites, no other significant changes in body weights, urinalysis, and macroscopic examination were noted in the animals given chlorpheniramine. On the other hand, white blood cells, especially B cells and monocytes, showed a dose-dependent increase in the chlorpheniramine-treated animals; whereas, the numbers of both B and T cells (helper T and cytotoxic T, NKT cells) were decreased in the prednisolone-treated animals. Taken together, these results suggest that chloropheniramine administration enhances white blood cells in the peripheral blood, mostly due to increases of the B cells and monocytes, but no T cells and NK cells.
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Subgrupos de Linfocitos B/efectos de los fármacos , Clorfeniramina/farmacología , Monocitos/efectos de los fármacos , Animales , Subgrupos de Linfocitos B/metabolismo , Clorfeniramina/sangre , Relación Dosis-Respuesta a Droga , Femenino , Monocitos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), an aggressive and highly chemoresistant malignancy. Rho family GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization, transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho family GTPases is essential for cell membrane localization and activation of these proteins. It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that GGTI-298 decreased cell viability and induced G(2)/M phase accumulation of HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho family GTPases. These studies suggest that protein geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.
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Transferasas Alquil y Aril/antagonistas & inhibidores , Benzamidas/farmacología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Prenilación de Proteína/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transferasas Alquil y Aril/metabolismo , Ciclo Celular , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Transformación Celular Viral , Productos del Gen tax/efectos de los fármacos , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Humanos , Proteínas I-kappa B/metabolismo , Leucemia-Linfoma de Células T del Adulto/enzimología , Leucemia-Linfoma de Células T del Adulto/virología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Fosforilación , Transporte de Proteínas/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
A series of steroid-polyamine conjugates were synthesized and evaluated for their antimicrobial activity. This study was focused on the effect of stereochemistry at the C-3 and C-5 of steroids and types of polyamine at C-3 on activity against various human pathogens. All the conjugates exhibited strong antimicrobial activities against Gram-positive strains. Compound 18 was found to be the most potent in these series with a MIC value as low as 1 µg/mL against the bacterium Staphylococcus aureus ATCC6538P.
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Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Colanos/síntesis química , Colanos/farmacología , Bacterias Grampositivas/efectos de los fármacos , Antiinfecciosos/química , Colanos/química , Humanos , Pruebas de Sensibilidad Microbiana , Poliaminas/síntesis química , Poliaminas/química , Poliaminas/farmacología , Relación Estructura-ActividadRESUMEN
Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.
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Quinasa 9 Dependiente de la Ciclina/metabolismo , VIH/fisiología , Proteínas Nucleares/metabolismo , ARN Viral/biosíntesis , Treonina/metabolismo , Factores de Transcripción/metabolismo , Replicación Viral , Proteínas de Ciclo Celular , Inmunoprecipitación de Cromatina , Células HeLa , Humanos , Fosforilación , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica , Transcripción GenéticaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the HTLV-1-encoded Tax protein inhibits the function of tumor suppressor p53 through a Tax-induced NF-kappaB pathway. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kappaB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is "druggable" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kappaB suggests a promising strategy for the treatment of HTLV-1-transformed cells.