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1.
Toxicol Appl Pharmacol ; 281(3): 303-9, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25448049

RESUMO

NSC-743380 (1-[(3-chlorophenyl)-methyl]-1H-indole-3-carbinol) is in early stages of development as an anticancer agent. Two metabolites reflect sequential conversion of the carbinol functionality to a carboxaldehyde and the major metabolite, 1-[(3-chlorophenyl)-methyl]-1H-indole-3-carboxylic acid. In an exploratory toxicity study in rats, NSC-743380 induced elevations in liver-associated serum enzymes and biliary hyperplasia. Biliary hyperplasia was observed 2 days after dosing orally for 2 consecutive days at 100mg/kg/day. Notably, hepatotoxicity and biliary hyperplasia were observed after oral administration of the parent compound, but not when major metabolites were administered. The toxicities of a structurally similar but pharmacologically inactive molecule and a structurally diverse molecule with a similar efficacy profile in killing cancer cells in vitro were compared to NSC-743380 to explore scaffold versus target-mediated toxicity. Following two oral doses of 100mg/kg/day given once daily on two consecutive days, the structurally unrelated active compound produced hepatic toxicity similar to NSC-743380. The structurally similar inactive compound did not, but, lower exposures were achieved. The weight of evidence implies that the hepatotoxicity associated with NSC-743380 is related to the anticancer activity of the parent molecule. Furthermore, because biliary hyperplasia represents an unmanageable and non-monitorable adverse effect in clinical settings, this model may provide an opportunity for investigators to use a short-duration study design to explore biomarkers of biliary hyperplasia.


Assuntos
Doença Aguda , Doenças Biliares/induzido quimicamente , Sistema Biliar/efeitos dos fármacos , Indóis/efeitos adversos , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Sistema Biliar/metabolismo , Sistema Biliar/patologia , Doenças Biliares/sangue , Doenças Biliares/metabolismo , Doenças Biliares/patologia , Biomarcadores/sangue , Biotransformação , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Drogas em Investigação/administração & dosagem , Drogas em Investigação/efeitos adversos , Drogas em Investigação/metabolismo , Drogas em Investigação/farmacocinética , Hiperplasia , Indóis/administração & dosagem , Indóis/sangue , Indóis/metabolismo , Indóis/farmacocinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Masculino , Distribuição Aleatória , Ratos Endogâmicos F344 , Relação Estrutura-Atividade
2.
Toxicol Pathol ; 42(8): 1188-96, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24670817

RESUMO

Endoxifen (4-hydroxy-N-desmethyl-tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for 3 days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was 3-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen.


Assuntos
Ovariectomia , Tamoxifeno/análogos & derivados , Tamoxifeno/toxicidade , Útero/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Endométrio/química , Endométrio/efeitos dos fármacos , Feminino , Histocitoquímica , Hipertrofia/induzido quimicamente , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Útero/química
3.
Clin Cancer Res ; 17(21): 6641-5, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22046025

RESUMO

There has been an explosion of technology-enabled scientific insight into the basic biology of the causes of adverse events. This has been driven, in part, by the development of the various "omics" tools (e.g., genomics, proteomics, and metabolomics) and associated bioinformatics platforms. Meanwhile, for decades, changes in preclinical testing protocols and guidelines have been limited. Preclinical safety testing currently relies heavily on the use of outdated animal models. Application of systems biology methods to evaluation of toxicities in oncology treatments can accelerate the introduction of safe, effective drugs. Systems biology adds insights regarding the causes and mechanisms of adverse effects, provides important and actionable information to help understand the risks and benefits to humans, focuses testing on methods that add value to the safety testing process, and leads to modifications of chemical entities to reduce liabilities during development. Leveraging emerging technologies, such as genomics and proteomics, may make preclinical safety testing more efficient and accurate and lead to better safety decisions. The development of a U.S. Food and Drug Administration guidance document on the use of systems biology in clinical testing would greatly benefit the development of drugs for oncology by communicating the potential application of specific methodologies, providing a framework for qualification and application of systems biology outcomes, and providing insight into the challenges and limitations of systems biology in the regulatory decision-making process.


Assuntos
Antineoplásicos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Biologia de Sistemas/métodos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Pesquisa Translacional Biomédica
4.
Nat Biotechnol ; 28(5): 455-62, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20458315

RESUMO

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


Assuntos
Biomarcadores Farmacológicos , Aprovação de Drogas/legislação & jurisprudência , Rim , Animais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Europa (Continente) , Humanos , Rim/efeitos dos fármacos , Rim/lesões , Preparações Farmacêuticas/normas , Estados Unidos , United States Food and Drug Administration
5.
Toxicol Pathol ; 37(5): 617-28, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19549929

RESUMO

Cardiac troponin I is a useful biomarker of myocardial injury, but its use in mice and application to early drug discovery are not well described. The authors investigated the relationship between cTnI concentration in serum and histologic lesions in heart tissue from mice treated with isoproterenol (ISO). Cardiac TnI concentrations in serum increased in a dose-dependant manner and remained increased twenty-four to forty-eight hours after a single administration of isoproterenol. Increased cTnI concentration was of greater magnitude and longer duration than increased fatty acid binding protein 3 concentration, aspartate aminotransferase activity, and creatine kinase activity in serum. Isoproterenol-induced increases in cTnI concentrations were both greater and more sustained in BALB/c than in CD1 mice and correlated with incidence and severity of lesions observed in heart sections from both strains. In drug development studies in BALB/c mice with novel kinase inhibitors, cTnI concentration was a reliable stand-alone biomarker of cardiac injury and was used in combination with measurements of in vivo target inhibition to demonstrate an off-target contribution to cardiotoxicity. Additional attributes, including low cost and rapid turnaround time, made cTnI concentration in serum invaluable for detecting cardiotoxicity, exploring structure-activity relationships, and prioritizing development of compounds with improved safety profiles early in drug discovery.


Assuntos
Descoberta de Drogas/métodos , Cardiopatias/sangue , Cardiopatias/induzido quimicamente , Isoproterenol/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Troponina I/sangue , Animais , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Cardiotônicos/toxicidade , Creatina Quinase/antagonistas & inibidores , Creatina Quinase/sangue , Relação Dose-Resposta a Droga , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/sangue , Feminino , Ventrículos do Coração/efeitos dos fármacos , Histocitoquímica , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/metabolismo , Miocárdio/patologia , Necrose
7.
Toxicol Pathol ; 36(2): 190-203, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18349427

RESUMO

Homozygous p53 deficient knockout mice were used to assess the role of p53 in tumor promotion by the protein phosphatase inhibitor and hepatic tumor promoter microcystin-LR (MCLR). More than 50% of human cancers bear mutations in the p53 gene, and in particular, p53 tumor suppressor gene mutations have been shown to play a major role in hepatocarcinogenesis. Trp53 homozygous (inactivated p53) and age-matched wild-type control mice were assigned to vehicle or MCLR-treated groups. MCLR or saline was administered daily for up to 28 days. RNA from the 28-day study was hybridized onto Mouse Genome GeneChip arrays. Selected RNA from 28 days and earlier time points was also processed for quantitative polymerase chain reaction (PCR). Livers from the 28-day, Trp53-deficient, MCLR group displayed greater hyperplastic and dysplastic changes morphologically and increases in Ki-67 and phosphohistone H3 (mitotic marker) immunoreactivity. Gene-expression analysis revealed significant increases in expression of cell-cycle regulation and cellular proliferation genes in the MCLR-treated, p53-deficient mutant mice compared to controls. These data suggest that regulation of the cell cycle by p53 is important in preventing the proliferative response associated with chronic, sublethal microcystin exposure, and therefore, conclude that p53 plays an important role in MCLR-induced tumor promotion.


Assuntos
Toxinas Bacterianas/toxicidade , Ciclo Celular/genética , Expressão Gênica/efeitos dos fármacos , Genes p53/genética , Hepatócitos/efeitos dos fármacos , Microcistinas/toxicidade , Mitose/genética , Animais , Carcinógenos/toxicidade , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Inativação Gênica , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Toxinas Marinhas , Camundongos , Camundongos Knockout , Índice Mitótico , Fosfoproteínas Fosfatases/antagonistas & inibidores , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
8.
Toxicol Pathol ; 35(4): 594-605, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17654400

RESUMO

Microcystin-LR (MCLR) is an acute hepatotoxicant and suspected carcinogen. Previous chronic studies have individually described hepatic morphologic changes, or alterations in the cytoskeleton, cell signaling or redox pathways. The objective of this study was to characterize chronic effects of MCLR in wild-type mice utilizing gene array analysis, morphology, and plasma chemistries. MCLR was given daily for up to 28 days. RNA from the 28-day study was hybridized onto mouse genechip arrays. RNA from 4 hours, 24 hours, 4 days, 1 day, and 28 days for selected genes was processed for quantitative-PCR. Increases in plasma hepatic enzyme activities and decreases in total protein, albumin and glucose concentrations were identified in MCLR-treated groups at 14 and 28 days. Histologically, marked hepatokaryomegaly was identified in the 14-day MCLR group with the addition of giant cells at 28 days. Major gene transcript changes were identified in the actin organization, cell cycle, apoptotic, cellular redox, cell signaling, albumin metabolism, and glucose homeostasis pathways, and the organic anion transport polypeptide system. Using toxicogenomics, we have identified key molecular pathways involved in chronic sublethal MCLR exposure in wild-type mice, genes participating in those critical pathways and related them to cellular and morphologic alterations seen in this and other studies.


Assuntos
Carcinógenos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Microcistinas/toxicidade , Animais , Análise Química do Sangue , Peso Corporal/efeitos dos fármacos , DNA Complementar/biossíntese , DNA Complementar/genética , Genes p53/genética , Genes p53/fisiologia , Fígado/patologia , Toxinas Marinhas , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão/efeitos dos fármacos , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxicogenética
9.
J Pharmacol Exp Ther ; 318(2): 589-95, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16687476

RESUMO

Therapeutic strategies focused on kinase inhibition rely heavily on surrogate measures of kinase inhibition obtained from in vitro assay systems. There is a need to develop methodology that will facilitate measurement of kinase inhibitor activity or specificity in tissue samples from whole animals treated with these compounds. Many of the current methods are limited by the use of antibodies, many of which do not cross-react between several species. The proteomics approach described herein has the potential to reveal novel tissue substrates, potential new pathway interconnections, and inhibitor specificity by monitoring differences in protein phosphorylation. We used the protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide) as a tool to determine whether differential profiling of tissue phosphoproteins can be used to detect treatment-related effects of a protein kinase A (PKA) inhibitor in vivo. With a combination of phosphoprotein column enrichment, high-throughput two-dimensional gel electrophoresis, differential gel staining with Pro-Q Diamond/SYPRO Ruby, statistical analysis, and matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis, we were able to show clear differences between the phosphoprotein profiles of rat liver protein extract from control and treated animals. Moreover, several proteins that show a potential change in phosphorylation were previously identified as PKA substrates or have putative PKA phosphorylation sites. The data presented support the use of differential proteomic methods to measure effects of kinase inhibitor treatment on protein phosphorylation in vivo.


Assuntos
Inibidores Enzimáticos/farmacologia , Isoquinolinas/farmacologia , Fígado/metabolismo , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Sulfonamidas/farmacologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Bases de Dados Genéticas , Eletroforese em Gel Bidimensional , Inibidores Enzimáticos/farmacocinética , Processamento de Imagem Assistida por Computador , Isoquinolinas/farmacocinética , Fígado/efeitos dos fármacos , Masculino , Fosforilação , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sulfonamidas/farmacocinética
10.
Toxicol Sci ; 88(2): 331-9, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16150885

RESUMO

Although the precise role for the tuberous sclerosis complex-2 tumor suppressor gene (Tsc2) in tumor suppression is not clear, many studies have implicated Tsc2 in the regulation of cell differentiation, cell cycle control, GTPase activity, transcription, polycystin-1 localization, and translation initiation. We propose that Tsc2 also increases susceptibility to apoptosis, and that this functional role may contribute to the tumor suppressor activity of Tsc2. We previously characterized the apoptotic response of a Tsc2-null renal tumor cell line (ERC-18) to the tumor promoter okadaic acid (OKA). In the present study, we expressed Tsc2 in ERC-18 cells and compared the effect of Tsc2 expression on apoptotic induction. Tsc2 expression increased the susceptibility of ERC-18 cells to apoptosis induced by OKA and the phosphatidylinositol-3' kinase inhibitor, LY294002. In addition, Tsc2 expression abrogated OKA-induced cell detachment of ERC-18 cells. These results indicate that the OKA-induced, caspase-independent detachment previously observed in ERC-18 cells is Tsc2-dependent, and may support an additional role for the Tsc2 in regulating cell adhesion.


Assuntos
Apoptose/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Caspase 3 , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromonas/toxicidade , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/toxicidade , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Mesotelina , Morfolinas/toxicidade , Ácido Okadáico/toxicidade , Ratos , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética
11.
Toxicol Sci ; 81(1): 233-42, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15178807

RESUMO

Loss of tumor suppressor function dramatically alters the cellular response to chemicals. The phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulates cell proliferation through rapid activation of protein kinase C (PKC), followed by gradual degradation of the kinase. TPA also activates the GTPase Rap1 in some cell types. The tumor suppressor protein Tsc2 has a proposed GTPase activating protein (GAP) function for Rap1, providing a common mechanistic target for Tsc2 and TPA. We compared the cellular response of Tsc2-null (ERC-18) and Tsc2-competent (NRK-52E) renal epithelial cells to TPA treatment. Treatment of ERC-18 cells with 100 ng/ml TPA for 24 h resulted in loss of cell-cell contact, retraction of the cell periphery and rounding. These changes were reversed 1 h after treatment in NRK-52E cells and were apparent 24 h after treatment of ERC-18 cells. Expression of Tsc2 in ERC-18 cells abrogated the prolonged morphologic response. TPA treatment rapidly increased phosphorylation of ERK, a reported downstream effector of both PKC and Rap1, in ERC-18 cells, but induced weak Rap1 activation. TPA-induced ERK phosphorylation was prolonged in ERC-18 cells compared to NRK-52E cells and expression of Tsc2 in ERC-18 cells did not inhibit prolonged ERK activation. The selective PKC inhibitor, bisindolylmaleimide VIII, however, inhibited TPA-induced changes in morphology and ERK activation. These results imply that TPA-induced changes in morphology and ERK activation are mediated primarily through PKC and not Rap1 in renal epithelial cells. These data also imply that Tsc2 expression modulates TPA-induced changes in renal epithelial cell morphology via an ERK-independent mechanism.


Assuntos
Carcinógenos/toxicidade , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Repressoras/genética , Acetato de Tetradecanoilforbol/toxicidade , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Immunoblotting , Imuno-Histoquímica , Mesotelina , Fosforilação , Plasmídeos/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Ratos , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor
12.
Toxicol Pathol ; 32 Suppl 1: 131-5, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15209413

RESUMO

Cell signaling is a term used to describe a complex interactive system of signals that act to regulate or mediate a cellular response. Therapies that target cell signaling pathways have the potential to effectively reverse molecular deregulation underlying disease. The inherent complexity of cell signaling presents a major challenge to designing such therapies however, because perturbation of pathways has the potential to produce dramatic adverse effects. Pathologists are in the primary position of detecting adverse responses in drug development and are essential members of teams whose goal is to determine the mechanisms underlying tissue responses. The pathologist therefore will be expected to integrate morphologic interpretation with data obtained from several laboratory-based methods and data derived from novel technologies. Approaches being used include several in silico tools that provide access to public databases and signal pathway visualization that can serve to focus on key mechanistic hypotheses. The main objective of this article is to discuss a basic mechanistic approach and methods that can be used to associate modulation of cell signaling pathways with pathologic endpoints. The approach suggested begins with diagnostic pathology and uses global gene expression analysis in conjunction with transcription factor profiling and confirmatory protein technologies, to elucidate pathways relevant to the biological mechanism. Another important objective is to highlight the use of in silico technologies to prioritize laboratory efforts and focus these efforts on key hypotheses.


Assuntos
Células/metabolismo , Patologia , Transdução de Sinais , Animais , Western Blotting , Regulação da Expressão Gênica , Humanos , Lasers , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfotransferases/metabolismo , Regiões Promotoras Genéticas
13.
Cancer Res ; 62(19): 5463-9, 2002 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-12359754

RESUMO

The vascular endothelial growth factor receptor (VEGFR) is an important angiogenic target for cancer gene therapy. In this study, we designed an mRNA-cleaving oligodeoxynucleotide that targets the VEGF receptor 2 (VEGFR2) transcript (VEGFR2 DNAzyme). This DNAzyme was found to digest efficiently mRNA substrates of VEGFR2 in a concentration- and time-dependent manner. We also showed that the DNAzyme induces apoptosis and markedly inhibits endothelial cell growth compared with a disabled DNAzyme and untreated controls. In contrast, the DNAzyme did not inhibit the growth of MDA-MB-435 cells in vitro. The DNAzyme in complex with a nonviral carrier also significantly inhibited tumor growth in vivo. After the fourth injection, there was nearly a 75% reduction of tumor size in the DNAzyme-treated group compared with the saline-injected control group (P = 0.024). Marked cell death in the peripheral regions of the tumor accompanied by a reduction in blood vessel density is consistent with the antiangiogenic mechanism of the DNAzyme. This study indicates that DNAzymes, targeting angiogenic growth factors of tumors, show promise as antitumor agents.


Assuntos
Inibidores da Angiogênese/farmacologia , DNA Catalítico/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Bovinos , Divisão Celular/efeitos dos fármacos , DNA Catalítico/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Antígeno Ki-67/biossíntese , Camundongos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Conformação de Ácido Nucleico , Oligonucleotídeos/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
14.
Toxicol Pathol ; 30(2): 235-46, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-11950167

RESUMO

Several tumor suppressor genes have been shown to regulate cellular susceptibility to proliferation or apoptotic cell death. An essential first step in studies with the long-range goal of determining the effect of a tumor suppressor gene on cellular susceptibility to apoptosis is careful characterization of the cell's response to an apoptotic stimulus. The goals of this study were to characterize the apoptotic response of a tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene-null cell line, to establish valid biochemical events that can be used as apoptosis markers, and to determine how these events correlate with apoptosis-specific morphologic changes. For characterization of apoptosis, we treated Tsc2-null renal epithelial tumor cells (ERC-18) with okadaic acid (OKA, 0.1-0.25 microM), and measured the biochemical and morphologic events during the apoptotic response. Electron microscopic and immunocytochemical evaluation showed an early loss of microvilli and a loss of vinculin and talin staining from focal adhesions within 1 hour. During the first 2 hours of treatment with 0.25 microM OKA, ERC-18 cells rounded and approximately 50% detached from the culture vessel with minimal membrane bleb formation. Phosphatidylserine externalization, chromatin margination and fragmentation, cytochrome C release, and caspase-3 and -7 cleavage were evident at 6 hours. Maximal membrane bleb formation occurred between 6 and 10 hours. Cells progressed to secondary oncotic necrosis between 10 and 24 hours of OKA treatment. Almost all cells had an oncotic phenotype after 24 hours, and 17.5% lost cell membrane integrity. A small subpopulation (< or = 5%) of OKA-treated cells underwent primary oncotic necrosis within 6 hours. Interestingly, the caspase-3 and -7 inhibitor Z-DEVD-FMK did not inhibit or delay OKA-induced apoptosis in these cells. Our results suggest a complex apoptotic model involving 2 or more potentially parallel death pathways. Although caspase-3 and -7 cleavage occurs during apoptosis in this model, this cleavage may not independently regulate cell death in ERC-18 cells. Therefore, measurement of apoptosis in this model requires analysis of both biochemical and morphologic events.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Renais/patologia , Ácido Okadáico/toxicidade , Proteínas Repressoras/fisiologia , Animais , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Mesotelina , Oligopeptídeos/farmacologia , Fosfatidilserinas/metabolismo , Fosforilação , Ratos , Talina/análise , Proteína 2 do Complexo Esclerose Tuberosa , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Vinculina/análise
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