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1.
Blood Cells Mol Dis ; 40(3): 370-80, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18093851

RESUMEN

This study was designed to evaluate effects of specific p38 MAP kinase inhibition on gene and protein expression of essential hematopoietic cytokines in primary human bone marrow stromal cells (HBMSC) and to identify downstream transcription factors (TF) regulated by the p38 MAP kinase signalling pathway. In vitro effects of p38 inhibitors (p38i) on cytokine regulation were compared to inhibitors of other major signalling pathways including PI3 kinase, JNK, MEK-1, NF-kappaB or protein kinase C (PKC). HBMSC were pre-treated with p38i (SB-203580) for 1 h and then stimulated with 200 ng/ml lipopolysaccharide (LPS). Supernatants and RNA were collected 6 h post LPS treatment for quantitative protein and mRNA analyses by ELISA and real-time RT-PCR, respectively, for interleukin-6 (IL-6), interleukin-11 (IL-11), granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and Activin A. Effects of the inhibitors of PI3 kinase (LY294002), JNK (synthetic inhibitory peptide), MEK-1 (PD90859), NF-kappaB (pyrrolidinedithiocarbamate (PDTC)) and protein kinase C (calphostin C) on HBMSC expression hematopoietic cytokines were evaluated and compared. SB-203580 caused dose-dependent decreases in cytokine protein expression and decreased IL-6 and IL-11 mRNA expression. Of the pathway inhibitors examined, only NF-kappaB elicited similar effects on cytokine protein and mRNA expression. p38-regulated transcription factor activity was assessed using a DNA/Protein array. Several TFs linked to cytokine regulation were modulated by SB-203580, with 10 of 21 p38-regulated TFs identified have not been previously linked to downstream p38 signalling. These observations in cultured HBMSC have illustrated the involvement of cytokine proteins, mRNA and TF activities and may improve the current understanding of the in vivo p38i suppression of erythropoiesis. In addition, these results suggest that IL-6, IL-11, GM-CSF, G-CSF and Activin A are similarly regulated by p38 and NF-kappaB and that the MEK1, JNK and PKC pathways appear to play a more limited role in modulating cytokine expression in HBMSC.


Asunto(s)
Células de la Médula Ósea/metabolismo , Citocinas/metabolismo , Hematopoyesis/fisiología , Células del Estroma/metabolismo , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adulto , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Células Cultivadas , Citocinas/genética , Inhibidores Enzimáticos/farmacología , Hematopoyesis/efectos de los fármacos , Humanos , Imidazoles/farmacología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Masculino , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis por Matrices de Proteínas , Piridinas/farmacología , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores
2.
Toxicol Pathol ; 36(3): 496-519, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18467687

RESUMEN

Transcriptional profiling of specific elements of vasculature from animal models of vascular toxicity is an approach to gain insight into molecular mechanisms of vascular injury. Feasibility of using laser capture microdissection (LCM) to evaluate differential gene expression in selected elements of mesenteric arteries (MA) from untreated rats and rats given a single vasotoxic dose of 100 mg/kg Fenoldopam and euthanized 1 or 4 hours postdose was assessed. Regions of MA (endothelial cells [EC] and vascular smooth muscle cells [VSMC]) were selectively microdissected from optimal-cutting-temperature (O.C.T.)-embedded-frozen tissue sections. RNA was isolated, linearly amplified (LA), and hybridized to Affymetrix GeneChips. Enrichment for specific vascular elements was evident by unique gene-expression profiles. Statistical analysis indicated that Fenoldopam treatment resulted in differential expression of 333 versus 458 genes in EC and 371 versus 618 genes in VSMC at the 1-hour or 4-hour time point, respectively. Analysis of regulated EC and VSMC genes common to both time points identified several gene functions or pathways affected by treatment. Several genes were identified in EC and/or VSMC that have not been previously linked to vascular structure or function. These data indicate that tissue-element-enrichment by LCM in conjunction with LA and GeneChip analysis offers a refined approach for assessment of injury-mediated transcriptome changes in distinct elements of the vasculature.


Asunto(s)
Arterias/efectos de los fármacos , Agonistas de Dopamina/toxicidad , Fenoldopam/toxicidad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Arterias/metabolismo , Arterias/patología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inyecciones Subcutáneas , Rayos Láser , Masculino , Mesenterio/irrigación sanguínea , Microdisección/métodos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley
3.
Toxicol Pathol ; 36(7): 958-71, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19126791

RESUMEN

In rodents, p38 MAP kinase inhibitors (p38is) induce bone marrow hypocellularity and reduce reticulocyte and erythrocyte counts. To identify target cell populations affected, a differentiating primary liquid erythroid culture system using sca-1(+)cells from mouse bone marrow was developed and challenged with p38is SB-203580, SB-226882, and SB-267030. Drug-related alterations in genes involved at different stages of erythropoiesis, cell-surface antigen expression (CSAE), burst-forming unit erythroid (BFU-E) colony formation, and cellular morphology (CM), growth (CG), and viability were evaluated. CSAE, CM, and decreases in BFU-E formation indicated delayed maturation, while CG and viability were unaffected. Terminal differentiation was delayed until day 14 versus day 7 in controls. CSAE demonstrated higher percentages of sca-1(+)cells after day 2 and reduced percentages of ter119(+) cells after day 7 in all treated cultures. Real-time reverse transcriptase polymerase chain reaction revealed a transient delay in expression of genes involved at early, intermediate, and late stages of erythropoiesis, followed by rebound expression at later time points. Results demonstrate p38is do not irreversibly inhibit erythrogenesis but induce a potency-dependent, transient delay in erythropoietic activity. The delay in activity is suggestive of effects on sca-1(+)bone marrow cells caused by alterations in expression of genes related to erythroid commitment and differentiation resulting in delayed maturation.


Asunto(s)
Eritropoyesis/efectos de los fármacos , Eritropoyesis/genética , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Antígenos Ly/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Precursoras Eritroides/efectos de los fármacos , Factor de Transcripción GATA2/metabolismo , Inmunofenotipificación , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 de la Leucemia Linfocítica T Aguda
4.
Toxicol In Vitro ; 19(4): 471-9, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15826805

RESUMEN

SH-SY5Y human neuroblastoma cells were incubated with 6-hydroxydopamine (6-OHDA) for 4 and 24 h to examine the mechanism of cell death and to determine the time-dependent effects of 6-OHDA on cellular glutathione status. After 4 h, 6-OHDA significantly depleted cellular ATP and GSH concentrations with only slight increases in cell death. GSH:GSSG ratios and mitochondrial membrane potential (Deltapsim) were significantly decreased during 4 h incubations with 6-OHDA. High concentrations of 6-OHDA (100 microM) induced oxidative stress and mitochondrial dysfunction in SH-SY5Y cells within 4 h leading to cell death. In 24 h incubations, 25 and 50 microM 6-OHDA significantly decreased ATP concentrations; however, significant increases in cell death were only observed with 50 microM 6-OHDA. 6-OHDA induced a concentration-dependent increase in GSH and total glutathione concentrations after 24 h. After exposure to 50 microM 6-OHDA, GSH concentrations were increased up to 12-fold after 24 h with no change in the GSH:GSSG ratio. Gene analysis suggests that the increase in GSH concentration was due to increased expression of the GSH synthesis genes glutamate cysteine ligase modifier and catalytic subunits. Our results suggest that 6-OHDA induces oxidative stress in SH-SY5Y cells resulting in an adaptive increase in cellular GSH concentrations.


Asunto(s)
Glutatión/metabolismo , Mitocondrias/metabolismo , Neuroblastoma/metabolismo , Oxidopamina/farmacología , Simpaticolíticos/farmacología , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN Complementario/biosíntesis , ADN Complementario/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo
5.
Exp Hematol ; 31(9): 760-9, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12962721

RESUMEN

OBJECTIVE: Erythropoiesis involves proliferation and differentiation of committed erythroid progenitors to mature red blood cells. The objective of this study was to characterize growth characteristics of human CD36+ erythroid progenitors and to profile temporal expression of lineage-specific transcription factors, structural proteins, and growth factor receptors involved in erythropoiesis. MATERIALS AND METHODS: Erythropoietin-induced differentiation of human cord blood CD36+ erythroid progenitors was profiled for GATA-1, GATA-2, NFE2, EKLF, SCL, PU.1, Id1, Evi-1, c-myb, Hox2.2, c-kit, EpoR, glycophorin A (GPA), CD71, beta- and gamma-globin, and protein 4.2 gene and/or protein expression and DNA content analysis on days 4, 7, and 15 of culture. RESULTS: Real-time RT-PCR analysis revealed upregulation of GATA-1, Id1, glycophorin A, and protein 4.2 mRNA expression on day 7 when compared to day 4 and decreased expression on day 15. EKLF, GATA-2, Hox2.2, c-myb, Evi-1, c-kit, and PU.1 mRNA expression decreased on days 7 and 15. NFE2, CD71, SCL, and EPO-R mRNA expression remained similar on days 4 and 7 but decreased on day 15. Expression of globin genes beta- and gamma-globin increased on both day 7 and day 15 compared to day 4. Values from flow cytometric quantitation of glycophorin A, transferrin receptor (CD71), and hemoglobin A proteins correlated with gene expression results. DNA analysis demonstrated that most cells lacked DNA content by day 15, a finding consistent with enucleation and terminal erythroid differentiation. CONCLUSION: These data indicate that in vitro liquid cultures of committed CD36+ erythroid progenitor cells retain, in part, many features of erythropoiesis at the cellular and molecular level and may provide a useful model for assessment of disease-related or drug-induced erythropoietic abnormalities.


Asunto(s)
Células Precursoras Eritroides/citología , Células Precursoras Eritroides/fisiología , Eritropoyesis , Regulación del Desarrollo de la Expresión Génica , Antígenos CD36 , Diferenciación Celular , División Celular , Linaje de la Célula/genética , Células Cultivadas , Eritropoyesis/genética , Citometría de Flujo , Humanos , Biosíntesis de Proteínas , Proteínas/genética
6.
Toxicol Sci ; 69(1): 131-8, 2002 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12215667

RESUMEN

Troglitazone (TRO), a member of the thiazolidinedione class of drugs, has been associated with hepatotoxicity in patients. The following in vitro study was conducted to investigate the effects of TRO on mitochondrial function and viability in a human hepatoma cell line, HepG2. TRO induced a concentration- and time-dependent increase in cell death, as measured by lactate dehydrogenase release. Exposure to 50 or 100 micro M TRO produced total loss of cell viability within 5 h. Preincubation of HepG2 cells with P450 inhibitors did not significantly protect against TRO-induced cell death suggesting that P450 metabolism was not required to induce cell death. Preincubation with the mitochondrial permeability transition inhibitor, cyclosporin A, provided complete protection against TRO-induced cell death. Our results also indicated that TRO produced concentration-dependent decreases in cellular ATP levels and mitochondrial membrane potential (MMP). Ultrastructural analysis demonstrated that TRO induced mitochondrial changes at concentrations of > or =10 micro M after 2 h. Decreased MMP and altered mitochondrial morphology occurred at time points that preceded cell death and at sublethal concentrations of TRO. These observations in HepG2 cells suggest that TRO disrupts mitochondrial function, leading to mitochondrial permeability transition and cell death.


Asunto(s)
Cromanos/farmacología , Hipoglucemiantes/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Tiazoles/farmacología , Tiazolidinedionas , Adenosina Trifosfato/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450 , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Microscopía Confocal , Microscopía Electrónica , Mitocondrias Hepáticas/ultraestructura , Permeabilidad , Factores de Tiempo , Troglitazona , Células Tumorales Cultivadas
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