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1.
J Biol Chem ; 288(29): 21307-21319, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23720736

RESUMO

Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.


Assuntos
Neoplasias Ósseas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , MicroRNAs/metabolismo , Osteossarcoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Raios gama , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Osteossarcoma/genética , Osteossarcoma/patologia , Estabilidade Proteica/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/deficiência
2.
J Biol Chem ; 287(7): 4503-17, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22158627

RESUMO

Runt-related transcription factors (RUNX1, RUNX2, and RUNX3) are key lineage-specific regulators of progenitor cell growth and differentiation but also function pathologically as cancer genes that contribute to tumorigenesis. RUNX2 attenuates growth and stimulates maturation of osteoblasts during bone formation but is also robustly expressed in a subset of osteosarcomas, as well as in metastatic breast and prostate tumors. To assess the biological function of RUNX2 in osteosarcoma cells, we examined human genomic promoter interactions for RUNX2 using chromatin immunoprecipitation (ChIP)-microarray analysis in SAOS-2 cells. Promoter binding of both RUNX2 and RNA polymerase II was compared with gene expression profiles of cells in which RUNX2 was depleted by RNA interference. Many RUNX2-bound loci (1550 of 2339 total) exhibit promoter occupancy by RNA polymerase II and contain the RUNX consensus motif 5'-((T/A/C)G(T/A/C)GG(T/G). Gene ontology analysis indicates that RUNX2 controls components of multiple signaling pathways (e.g. WNT, TGFß, TNFα, and interleukins), as well as genes linked to cell motility and adhesion (e.g. the focal adhesion-related genes FAK/PTK2 and TLN1). Our results reveal that siRNA depletion of RUNX2, PTK2, or TLN1 diminishes motility of U2OS osteosarcoma cells. Thus, RUNX2 binding to diverse gene loci may support the biological properties of osteosarcoma cells.


Assuntos
Neoplasias Ósseas/metabolismo , Movimento Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Genoma Humano , Proteínas de Neoplasias/metabolismo , Osteossarcoma/metabolismo , Elementos de Resposta , Neoplasias Ósseas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Loci Gênicos , Humanos , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo
3.
Proc Natl Acad Sci U S A ; 106(30): 12359-64, 2009 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-19590016

RESUMO

Competency for DNA replication is functionally coupled to the activation of histone gene expression at the onset of S phase to form chromatin. Human histone nuclear factor P (HiNF-P; gene symbol HINFP) bound to its cyclin E/cyclin-dependent kinase 2 (CDK2) responsive coactivator p220(NPAT) is a key regulator of multiple human histone H4 genes that encode a major subunit of the nucleosome. Induction of the histone H4 transcription factor (HINFP)/p220(NPAT) coactivation complex occurs in parallel with the CDK-dependent release of pRB from E2F at the restriction point. Here, we show that the downstream CDK-dependent cell cycle effector HINFP is genetically required and, in contrast to the CDK2/cyclin E complex, cannot be compensated. We constructed a mouse Hinfp-null mutation and found that heterozygous Hinfp mice survive, indicating that 1 allele suffices for embryogenesis. Homozygous loss-of-function causes embryonic lethality: No homozygous Hinfp-null mice are obtained at or beyond embryonic day (E) 6.5. In blastocyst cultures, Hinfp-null embryos exhibit a delay in hatching, abnormal growth, and loss of histone H4 gene expression. Our data indicate that the CDK2/cyclin E/p220(NPAT)/HINFP/histone gene signaling pathway at the G1/S phase transition is an essential, nonredundant cell cycle regulatory mechanism that is established early in embryogenesis.


Assuntos
Ciclo Celular/fisiologia , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Western Blotting , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Ciclina E/genética , Quinase 2 Dependente de Ciclina/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fase G1/genética , Fase G1/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fase S/genética , Fase S/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Tempo
4.
J Cell Biochem ; 109(4): 828-37, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20082326

RESUMO

Prostate cancer cells often metastasize to bone where osteolytic lesions are formed. Runx2 is an essential transcription factor for bone formation and suppresses cell growth in normal osteoblasts but may function as an oncogenic factor in solid tumors (e.g., breast, prostate). Here, we addressed whether Runx2 is linked to steroid hormone and growth factor signaling, which controls prostate cancer cell growth. Protein expression profiling of prostate cell lines (i.e., PC3, LNCaP, RWPE) treated with 5alpha-dihydrotestosterone (DHT) or tumor growth factor beta (TGFbeta) revealed modulations in selected cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors that are generally consistent with mitogenic responses. Endogenous elevation of Runx2 and diminished p57 protein levels in PC3 cells are associated with faster proliferation in vitro and development of larger tumors upon xenografting these cells in bone in vivo. To examine whether TGFbeta or DHT signaling modulates the transcriptional activity of Runx2 and vice versa, we performed luciferase reporter assays. In PC3 cells that express TGFbetaRII, TGFbeta and Runx2 synergize to increase transcription of synthetic promoters. In LNCaP cells that are DHT responsive, Runx2 stimulates the androgen receptor (AR) responsive expression of the prostate-specific marker PSA, perhaps facilitated by formation of a complex with AR. Our data suggest that Runx2 is mechanistically linked to TGFbeta and androgen responsive pathways that support prostate cancer cell growth.


Assuntos
Androgênios/farmacologia , Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Neoplasias da Próstata/patologia , Fator de Crescimento Transformador beta/farmacologia , Linhagem Celular Tumoral , Di-Hidrotestosterona/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Neoplasias/análise , Proteômica
5.
J Cell Physiol ; 219(2): 438-48, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19170105

RESUMO

Cell cycle progression into S phase requires the induction of histone gene expression to package newly synthesized DNA as chromatin. Cyclin E stimulation of CDK2 at the Restriction point late in G1 controls both histone gene expression by the p220(NPAT)/HiNF-P pathway and initiation of DNA replication through the pRB/E2F pathway. The three CDK inhibitors (CKIs) p21(CIP1/WAF1), p27(KIP1), and p57(KIP2) attenuate CDK2 activity. Here we find that gamma-irradiation induces p21(CIP1/WAF1) but not the other two CKIs, while reducing histone H4 mRNA levels but not histone H4 gene promoter activation by the p220(NPAT)/HiNF-P complex. We also show that p21(CIP1/WAF1) is less effective than p27(KIP1) and p57(KIP2) in inhibiting the CDK2 dependent phosphorylation of p220(NPAT) at subnuclear foci and transcriptional activation of histone H4 genes. The greater effectiveness of p57(KIP2) in blocking the p220(NPAT)/HiNF-P pathway is attributable in part to its ability to form a specific complex with p220(NPAT) that may suppress CDK2/cyclin E phosphorylation through direct substrate inhibition. We conclude that CKIs selectively control stimulation of the histone H4 gene promoter by the p220(NPAT)/HiNF-P complex.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Regulação da Expressão Gênica , Histonas/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Animais , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/genética , Linhagem Celular/efeitos da radiação , Ciclina E/genética , Ciclina E/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Raios gama , Genes Reporter , Histonas/metabolismo , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética
6.
Aging Cell ; 18(3): e12921, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30790427

RESUMO

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pHc ) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pHc and the central nutrient signalling cAMP-Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pHc correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pHc showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.


Assuntos
Restrição Calórica , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/metabolismo , Longevidade , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/crescimento & desenvolvimento
7.
Methods Mol Biol ; 455: 165-76, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18463819

RESUMO

Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activation and repression of distinct sets of genes support the anabolic functions of osteoblasts and the catabolic properties of osteoclasts. Furthermore, metastasis of tumors to the bone environment is controlled by transcriptional mechanisms. Insights into the transcriptional regulation of genes in bone cells may provide a conceptual basis for improved therapeutic approaches to treat bone fractures, genetic osteopathologies, and/or cancer metastases to bone. Chromatin immunoprecipitation (ChIP) is a powerful technique to establish in vivo binding of transcription factors to the promoters of genes that are either activated or repressed in bone cells. Combining ChIP with genomic microarray analysis, colloquially referred to as "ChIP-on-chip," has become a valuable method for analysis of endogenous protein/DNA interactions. This technique permits assessment of chromosomal binding sites for transcription factors or the location of histone modifications at a genomic scale. This chapter discusses protocols for performing chromatin immunoprecipitation experiments, with a focus on ChIP-on-chip analysis. The information presented is based on the authors' experience with defining interactions of Runt-related (RUNX) transcription factors with bone-related genes within the context of the native nucleosomal organization of intact osteoblastic cells.


Assuntos
Osso e Ossos , Imunoprecipitação da Cromatina , Transcrição Gênica , Animais , Osso e Ossos/citologia , Osso e Ossos/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina/instrumentação , Imunoprecipitação da Cromatina/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
8.
FEBS J ; 274(12): 3003-12, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17509081

RESUMO

Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (DeltaPsim) and the production of reactive oxygen species. Fluorescent probes were used to assess DeltaPsim (JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide), reactive oxygen species [DCF, 5- (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] and [Ca2+][Fluo-3, glycine N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-(acetyloxy)methyl ester] in human kidney cells (HK-2 cells) and in a line of human small cell carcinoma cells (GLC4 cells), because these do not express cyclosporin A-sensitive P-glycoprotein. We used transfected GLC4 cells expressing P-glycoprotein as control for GLC4 cells. NIM811 (N-methyl-4-isoleucine-cyclosporin) and PSC833 (SDZ-PSC833) were applied as selective mitochondrial permeability transition pore and P-glycoprotein blockers, respectively. To study the effect of cyclosporin A on mitochondrial function, we isolated mitochondria from fresh pig livers. Cyclosporin A and PSC833 induced a more than two-fold increase in JC-1 fluorescence in HK-2 cells, whereas NIM811 had no effect. None of the three substances induced a significant increase in JC-1 fluorescence in GLC4 cells. Despite this, cyclosporin A, NIM811 and PSC833 induced a 1.5-fold increase in DCF fluorescence (P<0.05) and a two-fold increase in Fluo-3 fluorescence (P<0.05). Studies in isolated mitochondria showed that blockage of mitochondrial permeability transition pores by cyclosporin A affected neither DeltaPsim, ATP synthesis, nor respiration rate. The mitochondrial permeability transition pore blockers cyclosporin A and NIM811, but also the non-mitochondrial permeability transition pore blocker PSC833, induced comparable degrees of reactive oxygen species production and cytosolic [Ca2+]. Neither mitochondria, effects on P-glycoprotein nor inhibition of calcineurin therefore play a role in cyclosporin A-induced oxidative stress and disturbed Ca2+ homeostasis.


Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Estresse Oxidativo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Compostos de Anilina , Animais , Benzimidazóis , Cálcio/metabolismo , Carbocianinas , Linhagem Celular , Citoplasma/metabolismo , Fluoresceínas , Corantes Fluorescentes , Humanos , Técnicas In Vitro , Mitocôndrias Hepáticas/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Membranas Mitocondriais/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Espécies Reativas de Oxigênio/metabolismo , Suínos , Xantenos
9.
Respir Res ; 8: 49, 2007 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-17617921

RESUMO

BACKGROUND: Tobacco smoke is the principal risk factor for chronic obstructive pulmonary disease (COPD), though the mechanisms of its toxicity are still unclear. The ABC transporters multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp/MDR1) extrude a wide variety of toxic substances across cellular membranes and are highly expressed in bronchial epithelium. Their impaired function may contribute to COPD development by diminished detoxification of noxious compounds in cigarette smoke. METHODS: We examined whether triple knock-out (TKO) mice lacking the genes for Mrp1 and Mdr1a/1b are more susceptible to develop COPD features than their wild-type (WT) littermates. TKO and WT mice (six per group) were exposed to 2 cigarettes twice daily by nose-only exposure or room air for 6 months. Inflammatory infiltrates were analyzed in lung sections, cytokines and chemokines in whole lung homogenates, emphysema by mean linear intercept. Multiple linear regression analysis with an interaction term was used to establish the statistical significances of differences. RESULTS: TKO mice had lower levels of interleukin (IL)-7, KC (mouse IL-8), IL-12p70, IL-17, TNF-alpha, G-CSF, GM-CSF and MIP-1-alpha than WT mice independent of smoke exposure (P < 0.05). IL-1-alpha, IL-6, IL-8, IL-13, IL-17, TNF-alpha, G-CSF, GM-CSF and MCP-1 increased after smoke exposure in both groups, but the increase in IL-8 was lower in TKO than WT mice (P < 0.05) with a same trend for G-CSF (P < 0.10). Smoke-induced increase in pulmonary inflammatory cells in WT mice was almost absent in TKO mice. The mean linear intercept was not different between groups. CONCLUSION: Mrp1/Mdr1a/1b knock-out mice have a reduced inflammatory response to cigarette smoke. In addition, the expression levels of several cytokines and chemokines were also lower in lungs of Mrp1/Mdr1a/1b knock-out mice independent of smoke exposure. Further studies are required to determine whether dysfunction of MRP1 and/or P-gp contribute to the pathogenesis of COPD.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Inflamação/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Fumaça , Fumar/patologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Enfisema/genética , Enfisema/metabolismo , Enfisema/patologia , Predisposição Genética para Doença , Inflamação/genética , Inflamação/prevenção & controle , Pulmão/imunologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Fumar/genética , Fumar/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
10.
Virchows Arch ; 449(6): 682-8, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17072643

RESUMO

Cigarette smoke is the principal risk factor for chronic obstructive pulmonary disease (COPD). Multidrug resistance proteins, such as multidrug resistance-associated protein-1 (MRP1), P-glycoprotein (P-gp), and lung resistance-related protein (LRP), may protect against oxidative stress and toxic compounds generated by cigarette smoking. Expression of MRP1, P-gp, and LRP was evaluated in bronchial epithelium of two study groups of COPD patients and their controls and was associated with disease status and smoking history. In study group 1, MRP1, but not P-gp and LRP expression, was lower (p=0.029) in normal bronchial epithelium of COPD patients (n=11) compared to healthy controls (n=8). MRP1 expression was high in squamous metaplastic epithelium. When including expression in squamous metaplastic cells, MRP1 was still lower in total bronchial epithelium in the COPD group (p=0.038). In study group 2, expression of MRP1, but not of P-gp and LRP, was lower (p=0.047) in lung tissue of (very) severe COPD (n=10) vs mild to moderate COPD (n=9) patients. In conclusion, MRP1 expression was lower in bronchial biopsies of COPD patients than of healthy controls and was also lower in patients with severe COPD than with mild/moderate COPD. Our findings indicate that diminished MRP1 expression in normal bronchial epithelium is associated with COPD. The exact role in COPD pathogenesis is to be revealed by further functional studies.


Assuntos
Brônquios/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Doença Pulmonar Obstrutiva Crônica/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade
11.
Respir Res ; 6: 59, 2005 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-15967026

RESUMO

ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) are highly expressed in bronchial epithelium. This review aims to give new insights in the possible functions of ABC molecules in the lung in view of their expression in different cell types. Furthermore, their role in protection against noxious compounds, e.g. air pollutants and cigarette smoke components, will be discussed as well as the (mal)function in normal and pathological lung. Several pulmonary drugs are substrates for ABC transporters and therefore, the delivery of these drugs to the site of action may be highly dependent on the presence and activity of many ABC transporters in several cell types. Three ABC transporters are known to play an important role in lung functioning. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause cystic fibrosis, and mutations in ABCA1 and ABCA3 are responsible for respectively Tangier disease and fatal surfactant deficiency. The role of altered function of ABC transporters in highly prevalent pulmonary diseases such as asthma or chronic obstructive pulmonary disease (COPD) have hardly been investigated so far. We especially focused on polymorphisms, knock-out mice models and in vitro results of pulmonary research. Insight in the function of ABC transporters in the lung may open new ways to facilitate treatment of lung diseases.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Pneumopatias/metabolismo , Pulmão/metabolismo , Animais , Humanos
12.
Int J Chron Obstruct Pulmon Dis ; 3(3): 469-75, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18990976

RESUMO

BACKGROUND: Smoking is the principle risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is known to protect against toxic compounds and oxidative stress, and might play a role in protection against smoke-induced disease progression. We questioned whether MRP1-mediated transport is influenced by pulmonary drugs that are commonly prescribed in COPD. METHODS: The immortalized human bronchial epithelial cell line 16HBE14o- was used to analyze direct in vitro effects of budesonide, formoterol, ipratropium bromide and N-acetylcysteine (NAC) on MRP1-mediated transport. Carboxyfluorescein (CF) was used as a model MRP1 substrate and was measured with functional flow cytometry. RESULTS: Formoterol had a minor effect, whereas budesonide concentration-dependently decreased CF transport by MRP1. Remarkably, addition of formoterol to the highest concentration of budesonide increased CF transport. Ipratropium bromide inhibited CF transport at low concentrations and tended to increase CF transport at higher levels. NAC increased CF transport by MRP1 in a concentration-dependent manner. CONCLUSIONS: Our data suggest that, besides their positive effects on respiratory symptoms, budesonide, formoterol, ipratropium bromide, and NAC modulate MRP1 activity in bronchial epithelial cells. Further studies are required to assess whether stimulation of MRP1 activity is beneficial for long-term treatment of COPD.


Assuntos
Brônquios/citologia , Broncodilatadores/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Células Epiteliais/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Acetilcisteína/metabolismo , Acetilcisteína/uso terapêutico , Transporte Biológico/efeitos dos fármacos , Broncodilatadores/uso terapêutico , Budesonida/metabolismo , Budesonida/uso terapêutico , Antagonistas Colinérgicos/metabolismo , Antagonistas Colinérgicos/uso terapêutico , Células Epiteliais/efeitos dos fármacos , Etanolaminas/metabolismo , Etanolaminas/uso terapêutico , Expectorantes/metabolismo , Expectorantes/uso terapêutico , Citometria de Fluxo , Fumarato de Formoterol , Humanos , Técnicas In Vitro , Ipratrópio/metabolismo , Ipratrópio/uso terapêutico , Estresse Oxidativo/fisiologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico
13.
Cancer Res ; 67(21): 10334-42, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17974976

RESUMO

HiNF-P and its cofactor p220(NPAT) are principal factors regulating histone gene expression at the G(1)-S phase cell cycle transition. Here, we have investigated whether HiNF-P controls other cell cycle- and cancer-related genes. We used cDNA microarrays to monitor responsiveness of gene expression to small interfering RNA-mediated depletion of HiNF-P. Candidate HiNF-P target genes were examined for the presence of HiNF-P recognition motifs, in vitro HiNF-P binding to DNA, and in vivo association by chromatin immunoprecipitations and functional reporter gene assays. Of 177 proliferation-related genes we tested, 20 are modulated in HiNF-P-depleted cells and contain putative HiNF-P binding motifs. We validated that at least three genes (i.e., ATM, PRKDC, and CKS2) are HiNF-P dependent and provide data indicating that the DNA damage response is altered in HiNF-P-depleted cells. We conclude that, in addition to histone genes, HiNF-P also regulates expression of nonhistone targets that influence competency for cell cycle progression.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclo Celular , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Transdução de Sinais/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Sítios de Ligação , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Dano ao DNA , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Genes Reguladores , Humanos , Proteínas Nucleares/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética
14.
J Biochem Mol Toxicol ; 21(5): 243-51, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17912704

RESUMO

Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o-, MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRP1 downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o(-) expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% +/- 0.22%) with MK571 further decreased cell survival 31% (p, = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p < 0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p = 0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r = 0.99, p =0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o- cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development.


Assuntos
Brônquios/citologia , Brônquios/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fumaça/efeitos adversos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos de Neoplasias/metabolismo , Brônquios/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Citometria de Fluxo , Fluoresceínas/metabolismo , Humanos , Imuno-Histoquímica , Proteínas de Neoplasias/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
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