RESUMO
Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced three-dimensional (3D) liver coculture platforms offer the potential for extended hepatocyte functionality and allow for the study of more complex biologic interactions, which can improve and refine human drug safety evaluations. Here, we use a perfusion flow 3D microreactor platform for the coculture of cryopreserved primary human hepatocytes and Kupffer cells to study the regulation of cytochrome P450 3A4 isoform (CYP3A4) activity by chronic interleukin 6 (IL-6)-mediated inflammation over 2 weeks. Hepatocyte cultures remained stable over 2 weeks, with consistent albumin production and basal IL-6 levels. Direct IL-6 stimulation that mimics an inflammatory state induced a dose-dependent suppression of CYP3A4 activity, an increase in C-reactive protein (CRP) secretion, and a decrease in shed soluble interleukin-6 receptor (IL-6R) levels, indicating expected hepatic IL-6 bioactivity. Tocilizumab, an anti-IL-6R monoclonal antibody used to treat rheumatoid arthritis, has been demonstrated clinically to impact small molecule drug pharmacokinetics by modulating cytochrome P450 enzyme activities, an effect not observed in traditional hepatic cultures. We have now recapitulated the clinical observation in a 3D bioreactor system. Tocilizumab was shown to desuppress CYP3A4 activity while reducing the CRP concentration after 72 hours in the continued presence of IL-6. This change in CYP3A4 activity decreased the half-life and area under the curve up to the last measurable concentration (AUClast) of the small molecule CYP3A4 substrate simvastatin hydroxy acid, measured before and after tocilizumab treatment. We conclude that next-generation in vitro liver culture platforms are well suited for these types of long-term treatment studies and show promise for improved drug safety assessment.
Assuntos
Anticorpos Monoclonais/metabolismo , Interações Medicamentosas/fisiologia , Fígado/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Anticorpos Monoclonais Humanizados/metabolismo , Proteína C-Reativa/metabolismo , Células Cultivadas , Técnicas de Cocultura/métodos , Citocromo P-450 CYP3A/metabolismo , Meia-Vida , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-6/metabolismo , Células de Kupffer/metabolismoRESUMO
Off-target effects of drugs on nuclear hormone receptors (NHRs) may result in adverse effects in multiple organs/physiological processes. Reliable assessments of the NHR activities for drug candidates are therefore crucial for drug development. However, the highly permissive structures of NHRs for vastly different ligands make it challenging to predict interactions by examining the chemical structures of the ligands. Here, we report a detailed investigation on the agonistic and antagonistic activities of 615 known drugs or drug candidates against a panel of 6 NHRs: androgen, progesterone, estrogen α/ß, and thyroid hormone α/ß receptors. Our study revealed that 4.7 and 12.4% compounds have agonistic and antagonistic activities, respectively, against this panel of NHRs. Nonetheless, potent, unintended NHR hits are relatively rare among the known drugs, indicating that such interactions are perhaps not tolerated during drug development. However, we uncovered examples of compounds that unintentionally agonize or antagonize NHRs. In addition, a number of compounds showed multi-NHR activities, suggesting that the cross-talk between multiple NHRs co-operate to elicit in vivo effects. These data highlight the merits of counter screening drug candidate against NHRs during drug discovery/development.
Assuntos
Descoberta de Drogas/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Disruptores Endócrinos/toxicidade , Antagonistas de Hormônios/toxicidade , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Disruptores Endócrinos/química , Genes Reporter , Antagonistas de Hormônios/química , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Medição de Risco , Espectrometria de Fluorescência , TransfecçãoRESUMO
Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability.
Assuntos
Glutationa/metabolismo , Substâncias Perigosas/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Células HL-60 , Substâncias Perigosas/química , Ensaios de Triagem em Larga Escala , Humanos , Mitocôndrias/metabolismo , OxirreduçãoRESUMO
Seven novel and potent Raf small molecule kinase inhibitors (C1-7) were evaluated in seven-day oral repeat dose rat toxicity studies. All compounds tested induced hyperplasia in multiple tissues. Consistently affected was stratified squamous epithelium at a number of sites and transitional epithelium of urinary bladder and kidney. A seven-day time course study in rats showed morphologic evidence of epithelial proliferation in the nonglandular stomach within four to five hours after a single dose of C-1. Similar indications of cellular proliferation were observed in the urinary bladder by day 2 and in the heart, kidney, and liver by day 3. Transcriptional evidence of proliferation in the urinary bladder was detected within four to five hours after a single dose consistent with activation of the PI3K/AKT and ERK/MAPK pathways. In a twenty-eight-day rat toxicity study of C-1, hyperplasia was observed in the esophagus, nonglandular stomach, skin, urinary bladder, kidney, and heart. Hyperplasia of transitional epithelium of the urinary bladder was particularly severe and in one female rat was accompanied by the presence of a transitional cell carcinoma. These results suggest that these Raf inhibitors induce early transcriptional changes driving unchecked cell proliferation, resulting in marked tissue hyperplasia that can progress to carcinoma within a short time frame.
Assuntos
Inibidores de Proteínas Quinases/toxicidade , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/enzimologia , Animais , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Feminino , Histocitoquímica , Hiperplasia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Mutação , Miocárdio/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologia , Estômago/efeitos dos fármacos , Estômago/patologia , Distribuição Tecidual , Testes de Toxicidade Crônica , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
The key to the discovery of new pharmaceuticals is to develop molecules that interact with the intended target and minimize interaction with unintended molecular targets, therefore minimizing toxicity. This is aided by the use of various in vitro selectivity assays that are used to select agents most potent for the desired target. Typically, molecules from similar chemical series, with similar in vitro potencies, are expected to yield comparable in vivo pharmacological and toxicological profiles, predictive of target effects. However, in this study, we investigated the in vivo effects of two analogue compounds that similarly inhibit several receptor tyrosine kinases such as vascular endothelial growth factor receptor 1 (VEGFR/Flt1), vascular endothelial growth factor 2 (VEGFR2/kinase domain receptor/Flk-1), vascular endothelial growth factor receptor 3 (VEGFR3/Flt4), platelet-derived growth factor receptor (PDGFR), and Kit receptors, which bear similar chemical structures, have comparable potencies, but differ markedly in their rodent toxicity profiles. Global gene expression data were used to generate hypotheses regarding the existence of toxicity triggers that would reflect the perturbation of signaling in multiple organs such as the liver, adrenal glands, and the pancreas in response to compound treatment. We concluded that differences in pharmacokinetic properties of the two analogues, such as volume of distribution, half-life, and organ concentrations, resulted in marked differences in the chemical burden on target organs and may have contributed to the vast differences in toxicity profiles observed with the two otherwise similar molecules. We propose including select toxicokinetic parameters such as V(ss), T(1/2), and T(max) as additional criteria that could be used to rank order compounds from the same pharmacological series to possibly minimize organ toxicity. Assessment of toxicokinetics is not an atypical activity on toxicology studies, even in early screening studies; however, these data may not always be used in decision making for selecting or eliminating one compound over another. Finally, we illustrate that in vivo gene expression profiles can serve as a complementary assessor of this activity and simultaneously help provide an assessment of on or off-target biological activity.
Assuntos
Expressão Gênica/efeitos dos fármacos , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Genômica , Masculino , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
A vast majority of pharmacological compounds and their metabolites are excreted via the urine, and within the complex structure of the kidney,the proximal tubules are a main target site of nephrotoxic compounds. We used the model nephrotoxicants mercuric chloride, 2-bromoethylamine hydrobromide, hexachlorobutadiene, mitomycin, amphotericin, and puromycin to elucidate time- and dose-dependent global gene expression changes associated with proximal tubular toxicity. Male Sprague-Dawley rats were dosed via intraperitoneal injection once daily for mercuric chloride and amphotericin (up to 7 doses), while a single dose was given for all other compounds. Animals were exposed to 2 different doses of these compounds and kidney tissues were collected on day 1, 3, and 7 postdosing. Gene expression profiles were generated from kidney RNA using 17K rat cDNA dual dye microarray and analyzed in conjunction with histopathology. Analysis of gene expression profiles showed that the profiles clustered based on similarities in the severity and type of pathology of individual animals. Further, the expression changes were indicative of tubular toxicity showing hallmarks of tubular degeneration/regeneration and necrosis. Use of gene expression data in predicting the type of nephrotoxicity was then tested with a support vector machine (SVM)-based approach. A SVM prediction module was trained using 120 profiles of total profiles divided into four classes based on the severity of pathology and clustering. Although mitomycin C and amphotericin B treatments did not cause toxicity, their expression profiles were included in the SVM prediction module to increase the sample size. Using this classifier, the SVM predicted the type of pathology of 28 test profiles with 100% selectivity and 82% sensitivity. These data indicate that valid predictions could be made based on gene expression changes from a small set of expression profiles. A set of potential biomarkers showing a time- and dose-response with respect to the progression of proximal tubular toxicity were identified. These include several transporters (Slc21a2, Slc15, Slc34a2), Kim 1, IGFbp-1, osteopontin, alpha-fibrinogen, and Gstalpha.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Perfilação da Expressão Gênica , Marcadores Genéticos , Rim/efeitos dos fármacos , Rim/patologia , Análise em Microsséries , Animais , Antibióticos Antineoplásicos/toxicidade , Butadienos/toxicidade , Desinfetantes/toxicidade , Relação Dose-Resposta a Droga , Etilaminas/toxicidade , Fungicidas Industriais/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Masculino , Cloreto de Mercúrio/toxicidade , Valor Preditivo dos Testes , Puromicina Aminonucleosídeo/toxicidade , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , ToxicogenéticaRESUMO
BACKGROUND: Arsenic is a nonmutagenic carcinogen affecting millions of people. The cellular impact of this metalloid in Saccharomyces cerevisiae was determined by profiling global gene expression and sensitivity phenotypes. These data were then mapped to a metabolic network composed of all known biochemical reactions in yeast, as well as the yeast network of 20,985 protein-protein/protein-DNA interactions. RESULTS: While the expression data unveiled no significant nodes in the metabolic network, the regulatory network revealed several important nodes as centers of arsenic-induced activity. The highest-scoring proteins included Fhl1, Msn2, Msn4, Yap1, Cad1 (Yap2), Pre1, Hsf1 and Met31. Contrary to the gene-expression analyses, the phenotypic-profiling data mapped to the metabolic network. The two significant metabolic networks unveiled were shikimate, and serine, threonine and glutamate biosynthesis. We also carried out transcriptional profiling of specific deletion strains, confirming that the transcription factors Yap1, Arr1 (Yap8), and Rpn4 strongly mediate the cell's adaptation to arsenic-induced stress but that Cad1 has negligible impact. CONCLUSIONS: By integrating phenotypic and transcriptional profiling and mapping the data onto the metabolic and regulatory networks, we have shown that arsenic is likely to channel sulfur into glutathione for detoxification, leads to indirect oxidative stress by depleting glutathione pools, and alters protein turnover via arsenation of sulfhydryl groups on proteins. Furthermore, we show that phenotypically sensitive pathways are upstream of differentially expressed ones, indicating that transcriptional and phenotypic profiling implicate distinct, but related, pathways.
Assuntos
Arsênio/toxicidade , Poluentes Ambientais/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina Básica , Biodegradação Ambiental , Cisteína/biossíntese , Proteínas de Ligação a DNA/fisiologia , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glutationa/biossíntese , Glutationa/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Metionina/metabolismo , Estresse Oxidativo , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Selênio/metabolismo , Enxofre/metabolismo , Testes de Toxicidade/métodos , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
Identifying a minimal dose capable of eliciting a biological response is a fundamental issue in a number of scientific fields, including: drug development, signal transduction research, and environmental toxicology. Frequently, proliferation, viability, and other assays based on the cellular response to a treatment are used to assess the threshold dose for minimal activity. Here we propose a novel approach for identifying the effects of low dose treatments and pinpointing the threshold dose. Using microarrays, we examined the transcriptional response of a hormone responsive breast cancer cell line (MCF-7) stimulated with various concentrations of estrogen. Previous studies have focused on transcriptional responses to physiologically relevant concentrations of estrogen. However, relatively few studies have examined the transcriptional effects of concentrations below normal physiologic levels. These doses may not stimulate the expression of any genes or, alternatively, may regulate a different subset of genes that had not been previously characterized as estrogen responsive. We used gene expression profiling, coupled with a detailed analysis of replicates, to measure estrogen effects on many transcriptional targets and found that only physiologically relevant doses of estrogen (1 x 10(-10) M and higher) were capable of inducing a transcriptional response. This study demonstrates the utility of gene expression profiling as a means to identify concentrations that do not elicit a change in gene expression, or simply a No Observed Transcriptional Effect Level (NOTEL). The identification of a NOTEL for a given compound may be beneficial in several different scientific disciplines. For example, in the development of therapeutic drugs, a NOTEL could be used to identify doses of pharmaceutical compounds that are no longer effective at modulating the expression of biomarkers of efficacy.
Assuntos
Células Epiteliais/efeitos dos fármacos , Estrogênios/toxicidade , Regulação Neoplásica da Expressão Gênica , Transcrição Gênica/efeitos dos fármacos , Análise de Variância , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estrogênios/genética , Feminino , Humanos , Nível de Efeito Adverso não Observado , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Breast tissue from healthy women contains variant mammary epithelial cells (vHMEC) exhibiting p16INK4a promoter hypermethylation both in vivo and in vitro. When continuously cultured, vHMEC acquire telomeric dysfunction and produce the types of chromosomal abnormalities seen in premalignant lesions of cancer. We find that late passage vHMEC express elevated prostaglandin cyclo-oxygenase 2 (COX-2), which contributes to increased prostaglandin synthesis, angiogenic activity, and invasive ability. These data demonstrate the existence of human mammary epithelial cells with the potential to acquire multiple genomic alterations and phenotypes associated with malignant cells. Moreover, COX-2 overexpression coincides with focal areas of p16INK4a hypermethylation in vivo, creating ideal candidates as precursors to breast cancer. These putative precursors can be selectively eliminated upon exposure to COX-2 inhibitors in vitro.
Assuntos
Mama/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Isoenzimas/metabolismo , Lesões Pré-Cancerosas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Apoptose/fisiologia , Mama/fisiopatologia , Aberrações Cromossômicas , Ciclo-Oxigenase 2 , Dano ao DNA/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Proteínas de Membrana , Metilação , Neovascularização Patológica/metabolismo , Prostaglandinas/metabolismo , Telomerase/metabolismoRESUMO
Microarrays allow for the simultaneous measurement of changes in the levels of thousands of messenger RNAs within a single experiment. As such, the potential for the application of transcription profiling to preclinical safety assessment and mechanism-based risk assessment is profound. However, several practical and technical challenges remain. Among these are nomenclature issues, platform-specific data formats, and the lack of uniform analysis methods and tools. Experiments were designed to address biological, technical, and methodological variability, to evaluate different approaches to data analysis, and to understand the application of the technology to other profiling methodologies and to mechanism-based risk assessment. These goals were addressed using experimental information derived from analysis of the biological response to three mechanistically distinct nephrotoxins: cisplatin, gentamicin, and puromycin aminonucleoside. In spite of the technical challenges, the transcription profiling data yielded mechanistically and topographically valuable information. The analyses detailed in the articles from the Nephrotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute suggest at least equal sensitivity of microarray technology compared to traditional end points. Additionally, microarray analysis of these prototypical nephrotoxicants provided an opportunity for the development of candidate bridging biomarkers of nephrotoxicity. The potential future extension of these applications for risk assessment is also discussed.
Assuntos
Perfilação da Expressão Gênica , Rim/efeitos dos fármacos , Rim/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Antibacterianos/toxicidade , Antimetabólitos Antineoplásicos/toxicidade , Cisplatino/toxicidade , Relação Dose-Resposta a Droga , Gentamicinas/toxicidade , Masculino , Puromicina/toxicidade , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Medição de RiscoRESUMO
Within the International Life Sciences Institute Committee on Genomics, a working group was formed to focus on the application of microarray technology to preclinical assessments of drug-induced nephrotoxicity. As part of this effort, Sprague-Dawley rats were treated with the nephrotoxicant cisplatin at doses of 0.3-5 mg/kg over a 4- to 144-hr time course. RNA prepared from these animals was run on a variety of microarray formats at multiple sites. A set of 93 differentially expressed genes associated with cisplatin-induced renal injury was identified on the National Institute of Environmental Health Sciences (NIEHS) custom cDNA microarray platform using quadruplicate measurements of pooled animal RNA. The reproducibility of this profile of statistically significant gene changes on other platforms, in pooled and individual animal replicate samples, and in an independent study was investigated. A good correlation in response between platforms was found among the 48 genes in the NIEHS data set that could be matched to probes on the Affymetrix RGU34A array by UniGene identifier or sequence alignment. Similar results were obtained with genes that could be linked between the NIEHS and Incyte or PHASE-1 arrays. The degree of renal damage induced by cisplatin in individual animals was commensurate with the number of differentially expressed genes in this data set. These results suggest that gene profiles linked to specific types of tissue injury or mechanisms of toxicity and identified in well-performed replicated microarray experiments may be extrapolatable across platform technologies, laboratories, and in-life studies.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Perfilação da Expressão Gênica/métodos , Rim/efeitos dos fármacos , Rim/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants--cisplatin, gentamicin, and puromycin--to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg). Groups of rats were sacrificed at various times after administration of these compounds for standard clinical chemistry, urine analysis, and histological evaluation of the kidney. RNA was extracted from the kidney for microarray analysis. Principal component analysis and gene expression-based clustering of compound effects confirmed sample separation based on dose, time, and degree of renal toxicity. In addition, analysis of the profile components revealed some novel changes in the expression of genes that appeared to be associated with injury in specific portions of the nephron and reflected the mechanism of action of these various nephrotoxicants. For example, although puromycin is thought to specifically promote injury of the podocytes in the glomerulus, the changes in gene expression after chronic exposure of this compound suggested a pattern similar to the known proximal tubular nephrotoxicants cisplatin and gentamicin; this prediction was confirmed histologically. We conclude that renal gene expression profiling coupled with analysis of classical end points affords promising opportunities to reveal potential new mechanistic markers of renal toxicity.
Assuntos
Perfilação da Expressão Gênica , Marcadores Genéticos , Rim/efeitos dos fármacos , Rim/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Antibacterianos/toxicidade , Antimetabólitos Antineoplásicos/toxicidade , Antineoplásicos/toxicidade , Cisplatino/toxicidade , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Gentamicinas/toxicidade , Masculino , Puromicina/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
Estrogens and glucocorticoids have opposing effects on the female reproductive tract, but the molecular basis for this antagonism is poorly understood. We therefore examined the biological and transcriptional programs induced by estrogens and glucocorticoids in the uterus of immature female rats. Estradiol 17beta (E2) rapidly induced morphological changes reminiscent of an acute inflammatory response, including infiltration of eosinophils, edema in the stroma and myometrium, and a decrease in the height of luminal epithelial cells, whereas dexamethasone (Dex) only altered stromal cell morphology. When coadministered with E2, Dex completely blocked the proinflammatory effects of E2. Surprisingly, examination of E2 and Dex effects on gene expression using cDNA microarrays and real-time PCR revealed that these hormones had similar effects on the expression of many genes and that very few genes displayed antagonistic regulation. Together, these results indicate strong discord between the early biologic and genomic actions of estrogens and glucocorticoids and highlight a complex regulatory role for glucocorticoids and GR in the mammalian uterus.
Assuntos
Dexametasona/farmacologia , Antagonistas de Estrogênios/farmacologia , Glucocorticoides/farmacologia , Útero/efeitos dos fármacos , Animais , Interações Medicamentosas , Estradiol/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Imuno-Histoquímica , Ratos , Receptores de Estrogênio/análise , Receptores de Estrogênio/imunologia , Receptores de Glucocorticoides/análise , Receptores de Glucocorticoides/imunologia , Fatores de Tempo , Útero/anatomia & histologia , Útero/metabolismoRESUMO
Increasing evidence continues to emerge supporting the early hypothesis that BRCA1 might be involved in transcriptional processes. BRCA1 physically associates with more than 15 different proteins involved in transcription and is paradoxically involved in both transcriptional activation and repression. However, the underlying mechanism by which BRCA1 affects the gene expression of various genes remains speculative. In this study, we provide evidence that BRCA1 protein complexes interact with specific DNA sequences. We provide data showing that the upstream stimulatory factor 2 (USF2) physically associates with BRCA1 and is a component of this DNA-binding complex. Interestingly, these DNA-binding complexes are downregulated in breast cancer cell lines containing wild-type BRCA1, providing a critical link between modulations of BRCA1 function in sporadic breast cancers that do not involve germline BRCA1 mutations. The functional specificity of BRCA1 tumor suppression for breast and ovarian tissues is supported by our experiments, which demonstrate that BRCA1 DNA-binding complexes are modulated by serum and estrogen. Finally, functional analysis indicates that missense mutations in BRCA1 that lead to subsequent cancer susceptibility may result in improper gene activation. In summary, these findings establish a role for endogenous BRCA1 protein complexes in transcription via a defined DNA-binding sequence and indicate that one function of BRCA1 is to co-regulate the expression of genes involved in various cellular processes.
Assuntos
Proteína BRCA1/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Mama , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Ativação TranscricionalRESUMO
The physiological responses of the rodent uterus to acute estrogen (E) dosing can be divided into early and late events. Examples of early responses include increased RNA transcription, hyperemia, and water imbibition 2 and 6 h following E administration respectively, whereas later responses include cycles of DNA synthesis and mitosis of epithelial cells beginning 10 and 16 h after E. The development of estrogen receptor (ER) knockout (ERKO) mice, combined with microarray technology, has allowed us to design a genomic approach to study the acute response of the rodent reproductive tract to E. To determine whether early and late biological responses are correlated with altered regulation of a single set of genes or distinct sets of genes characteristic of early and late responses, uterine RNA was obtained from ovariectomized mice that were treated with vehicle or with estradiol for 2 h (early) or 24 h (late). Samples were also prepared from identically treated mice that lacked either ERalpha (alphaERKO) or ERbeta (betaERKO) to address the relative contributions of the ERs in the uterine responses. Microarray analysis of the relative expression of 8700 mouse cDNAs indicated distinct clusters of genes that were regulated both positively and negatively by E in the early or late phases as well as clusters of genes regulated at both times. Both early and late responses by the betaERKO samples were indistinguishable from those of WT samples, whereas the alphaERKO showed little change in gene expression in response to E, indicating the predominant role for ERalpha in the genomic response. Further studies indicated that the genomic responses in samples from intermediate time points (6 h, 12 h) fall within the early or late clusters, rather than showing unique clusters regulated in the intermediary period. The use of this genomic approach has illustrated how physiological responses are reflected in genomic patterns. Furthermore, the identification of functional gene families that are regulated by E in the uterus combined with the utilization of genetically altered experimental animal models can help to uncover and define novel mechanisms of E action.
Assuntos
Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Estrogênio/fisiologia , Útero/metabolismo , Animais , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Estradiol/análogos & derivados , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Ovariectomia , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
The mechanisms underlying the anti-tumorigenic properties of cyclooxygenase inhibitors are not well understood. One novel hypothesis is alterations in gene expression. To test this hypothesis sulindac sulfide, which is used to treat familial adenomatous polyposis, was selected to detect gene modulation in human colorectal cells at physiological concentrations with microarray analysis. At micromolar concentrations, sulindac sulfide stimulated apoptosis and inhibited the growth of colorectal cancer cells on soft agar. Sulindac sulfide (10 microm) altered the expression of 65 genes in SW-480 colorectal cancer cells, which express cyclooxygenase-1 but little cyclooxygenase-2. A more detailed study of 11 genes revealed that their expression was altered in a time- and dose-dependent manner as measured by real-time RT-PCR. Northern analysis confirmed the expression of 9 of these genes, and Western analysis supported the conclusion that sulindac sulfide altered the expression of these proteins. Cyclooxygenase-deficient HCT-116 cells were more responsive to sulindac sulfide-induced gene expression than SW-480 cells. However, this response was diminished in HCT-116 cells overexpressing cyclooxygenase-1 compared with normal HCT-116 cells suggesting the presence of cyclooxygenase attenuates this response. However, prostaglandin E2, the main product of cyclooxygenase, only suppressed the sulindac sulfide-induced expression of two genes, with little known biological function while it modulated the expression of two more. The most likely explanation for this finding is the metabolism of sulindac sulfide to inactive metabolites by the peroxidase activity of cyclooxygenase. In conclusion, this is the first report showing sulindac sulfide, independent of cyclooxygenase, altered the expression of several genes possibly linked to its anti-tumorigenic and pro-apoptotic activity.
Assuntos
Neoplasias Colorretais/enzimologia , Inibidores de Ciclo-Oxigenase/farmacologia , Sulindaco/análogos & derivados , Sulindaco/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose , Northern Blotting , Western Blotting , Divisão Celular/efeitos dos fármacos , Separação Celular , Clonagem Molecular , Neoplasias Colorretais/tratamento farmacológico , Ciclo-Oxigenase 1 , DNA/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indometacina/farmacologia , Isoenzimas/biossíntese , Proteínas de Membrana , Análise de Sequência com Séries de Oligonucleotídeos , Prostaglandina-Endoperóxido Sintases/biossíntese , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
We propose an algorithm for selecting and clustering genes according to their time-course or dose-response profiles using gene expression data. The proposed algorithm is based on the order-restricted inference methodology developed in statistics. We describe the methodology for time-course experiments although it is applicable to any ordered set of treatments. Candidate temporal profiles are defined in terms of inequalities among mean expression levels at the time points. The proposed algorithm selects genes when they meet a bootstrap-based criterion for statistical significance and assigns each selected gene to the best fitting candidate profile. We illustrate the methodology using data from a cDNA microarray experiment in which a breast cancer cell line was stimulated with estrogen for different time intervals. In this example, our method was able to identify several biologically interesting genes that previous analyses failed to reveal.
Assuntos
Algoritmos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Análise por Conglomerados , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular Tumoral , Simulação por Computador , Estradiol/farmacologia , Humanos , Modelos Genéticos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Estatística como Assunto , Fatores de Tempo , Transcrição Gênica/genéticaRESUMO
Tamoxifen is a widely used breast cancer therapeutic and preventative agent. Although functioning as an estrogen antagonist at the cellular level, transcriptional profiling revealed that at the molecular level, tamoxifen functions largely as an agonist, virtually recapitulating the gene expression profile induced in breast cancer cells by estrogen. Remarkably, tamoxifen induces transcription factors and genes involved in promoting cell cycle progression including fos, myc, myb, cdc25a, cyclins E and A2, and stk15 with kinetics that paralleled that of cells cycling in response to estrogen, even though tamoxifen-treated cells are not transiting through the cell cycle. Induction of cell cycle-associated genes was specific for tamoxifen, and did not occur with raloxifene. However, cyclin D1 was a key estrogen-induced gene not expressed in response to tamoxifen or raloxifene but constitutively expressed in tamoxifen-resistant cells.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes cdc/fisiologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Estrogênios/metabolismo , Estrogênios/farmacologia , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tamoxifeno/metabolismo , Fatores de TempoRESUMO
Dilated cardiomyopathy, a disease of unknown etiology and pathogenesis, is associated with heart failure and compensatory hypertrophy. Although cell and animal models suggest a role for altered gene expression in the transition to heart failure, there is a paucity of data derived from the study of human heart tissue. In this study, we used DNA microarray profiling to investigate changes in the expression of genes involved in apoptosis that occur in human idiopathic dilated cardiomyopathic hearts that had progressed to heart failure. We observed altered gene expression consistent with a proapoptotic shift in the TNF-alpha signaling pathway. Specifically, we found decreased expression of TNF-alpha- and NF-kappaB-induced antiapoptotic genes such as growth arrest and DNA damage-inducible (GADD)45beta, Flice inhibitory protein (FLIP), and TNF-induced protein 3 (A20). Consistent with a role for apoptosis in heart failure, we also observed a significant decrease in phosphorylation of BAD at Ser-112. This study identifies several pathways that are altered in human heart failure and provides new targets for therapy.
Assuntos
Apoptose/fisiologia , Baixo Débito Cardíaco/fisiopatologia , Cardiomiopatia Dilatada/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular , Transdução de Sinais/fisiologia , Adulto , Idoso , Antígenos de Diferenciação/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Baixo Débito Cardíaco/genética , Baixo Débito Cardíaco/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Proteínas Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/metabolismo , Proteína de Morte Celular Associada a bclRESUMO
Human exposure to arsenic, a ubiquitous and toxic environmental pollutant, is associated with an increased incidence of skin cancer. However, the mechanism(s) associated with AsIII-mediated toxicity and carcinogenesis at low levels of exposure remains elusive. Aberrations in cell proliferation, oxidative damage, and DNA-repair fidelity have been implicated in sodium arsenite (AsIII)-mediated carcinogenicity and toxicity, but these events have been examined in isolation in the majority of biological models of arsenic exposure. We hypothesized that the simultaneous interaction of these effects may be important in arsenic-mediated neoplasia in the skin. To evaluate this, normal human epidermal keratinocytes (NHEK) were exposed to nontoxic doses (0.005-5 micro M) of AsIII and monitored for several physiological endpoints at the times when cells were harvested for gene expression measurements (1-24 h). Two-fluor cDNA microarray analyses indicated that AsIII treatment decreased the expression of genes associated with DNA repair (e.g., p53 and Damage-specific DNA-binding protein 2) and increased the expression of genes indicative of the cellular response to oxidative stress (e.g., Superoxide dismutase 1, NAD(P)H quinone oxidoreductase, and Serine/threonine kinase 25). AsIII also modulated the expression of certain transcripts associated with increased cell proliferation (e.g., Cyclin G1, Protein kinase C delta), oncogenes, and genes associated with cellular transformation (e.g., Gro-1 and V-yes). These observations correlated with measurements of cell proliferation and mitotic measurements as AsIII treatment resulted in a dose-dependent increase in cellular mitoses at 24 h and an increase in cell proliferation at 48 h of exposure. Data in this manuscript demonstrates that AsIII exposure simultaneously modulates DNA repair, cell proliferation, and redox-related gene expression in nontransformed, normal NHEK. It is anticipated that data in this report will serve as a foundation for furthering our knowledge of AsIII-regulated gene expression in skin and other tissues and contribute to a better understanding of arsenic toxicity and carcinogenesis.