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1.
Int J Toxicol ; 28(3): 162-76, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19546255

RESUMO

Hepatic toxicity remains a major concern for drug failure; therefore, a thorough examination of chemically induced liver toxicity is essential for a robust safety evaluation. Current hypotheses suggest that the metabolic activation of a drug to a reactive intermediate is an important process. In this article, we describe a new high-throughput GADD45beta reporter assay developed for assessing potential liver toxicity. Most importantly, this assay utilizes a human cell line and incorporates metabolic activation and thus provides significant advantage over other comparable assays used to determine hepatotoxicity. Our assay has low compound requirement and relies upon 2 reporter genes cotransfected into the HepG(2) cells. The gene encoding Renilla luciferase is fused to the CMV promoter and provides a control for cell numbers. The firefly luciferase gene is fused to the GADD45beta promoter and used to report an increase in DNA damage. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. Results are expressed as the ratio of the 2 luciferase activities; increases over the control are interpreted as evidence of stress responses. This mammalian dual luciferase reporter has been characterized with, and without, metabolic activation using positive and negative control agents. Our data demonstrate that this assay provides for an assessment of potential toxic metabolites, is adaptable to a high-throughput platform, and yields data that accurately and reproducibly detect hepatotoxicants.


Assuntos
Antígenos de Diferenciação/genética , Biotransformação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Hepatócitos/efeitos dos fármacos , Luciferases/genética , Testes de Toxicidade , Xenobióticos/toxicidade , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Hepatócitos/metabolismo , Humanos , Luciferases/metabolismo , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Testes de Mutagenicidade , Frações Subcelulares/enzimologia , Transfecção , Xenobióticos/metabolismo
2.
Cancer Res ; 67(21): 10528-37, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17974997

RESUMO

Despite the potency of dendritic cells (DC) as antigen-presenting cells for priming adaptive immunity, DC-based cancer vaccines have been largely insufficient to effectively reduce tumor burden or prevent tumor progression in most patients. To enhance DC-based vaccines, we used the combination of a synthetic ligand-inducible CD40 receptor (iCD40) along with Toll-like receptor-4 (TLR-4) ligation in human monocyte-derived DCs. The iCD40 receptor permits targeted, reversible activation of CD40 in vivo, potentially bypassing the essential role of CD4(+) T cells for activation of DCs. As a rigorous preclinical study of this approach, we evaluated key parameters of DC activation and function. Whereas neither iCD40 nor TLR-4 signaling alone led to high levels of interleukin (IL)-12p70 and IL-6, using iCD40 in combination with lipopolysaccharide (LPS) or monophosphoryl lipid A led to strongly synergistic production of both. Furthermore, this approach led to high expression of DC maturation markers, epitope-specific CTL and T helper 1 responses, as well as DC migration in vitro and in vivo. Moreover, use of iCD40-modified and LPS-stimulated DCs led to targeted expansion of autologous T cells against tumor-associated antigens, including prostate-specific membrane antigen, and elimination of preestablished tumors, supporting this technology as a potent strategy for DC-based cancer immunotherapy.


Assuntos
Antígenos CD40/fisiologia , Células Dendríticas/imunologia , Receptor 4 Toll-Like/fisiologia , Animais , Antígenos de Superfície/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Polaridade Celular , Glutamato Carboxipeptidase II/imunologia , Humanos , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR7/análise , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia , Células Th1/fisiologia
3.
Nat Med ; 11(2): 130-7, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15665830

RESUMO

Modest clinical outcomes of dendritic-cell (DC) vaccine trials call for the refinement of DC vaccine design. Although many potential antigens have been identified, development of methods to enhance antigen presentation by DCs has lagged. We have engineered a potent, drug-inducible CD40 (iCD40) receptor that permits temporally controlled, lymphoid-localized, DC-specific activation. iCD40 is comprised of a membrane-localized cytoplasmic domain of CD40 fused to drug-binding domains. This allows it to respond to a lipid-permeable, high-affinity dimerizer drug while circumventing ectodomain-dependent negative-feedback mechanisms. These modifications permit prolonged activation of iCD40-expressing DCs in vivo, resulting in more potent CD8(+) T-cell effector responses, including the eradication of previously established solid tumors, relative to activation of DCs ex vivo (P < 0.01), typical of most clinical DC protocols. In addition, iCD40-mediated DC activation exceeded that achieved by stimulating the full-length, endogenous CD40 receptor both in vitro and in vivo. Because iCD40 is insulated from the extracellular environment and can be activated within the context of an immunological synapse, iCD40-expressing DCs have a prolonged lifespan and should lead to more potent vaccines, perhaps even in immune-compromised patients.


Assuntos
Antígenos CD40/metabolismo , Vacinas Anticâncer/metabolismo , Células Dendríticas/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Animais , Antígenos CD40/genética , Células Cultivadas , Células Dendríticas/citologia , Humanos , Células Jurkat , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/fisiologia
4.
Mol Carcinog ; 40(4): 189-200, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15264211

RESUMO

Recent work from our laboratory has shown that elevated src kinase activity enhances tumor promotion, malignant progression, and metastasis during multistage skin carcinogenesis. In this study, we have generated "gene-switch" src(530) transgenic mice to further analyze the role of this nonreceptor tyrosine kinase in multistage carcinogenesis. Target transgenic mice that have an activated form of the human c-src (src(530)) gene fused with GAL4 binding sites upstream of the thymidine kinase (TK) promoter were generated. Two lines of epidermis-specific transactivator mice were used that targeted the expression of GLVPc or GLp65 transactivators, fusion molecules containing a truncated progesterone receptor with a GAl4-DNA binding domain, with either a mouse loricrin (ML) or human keratin 14 (HK14) promoter, respectively. The transactivator mice (ML.GLVPc or HK14.GLp65) and the target mice (TK.src(530)) were mated to generate bitransgenic mice, and src(530) transgene expression was induced by topical application of RU486 (mifepristone, a progesterone receptor antagonist). In both ML.GLVPc/TK.src(530) and HK14.GLp65/TK.src(530) bitransgenic mice, histological analysis revealed that only the bitransgenic mice had marked epidermal hyperplasia and hyperkeratosis after treatment with RU486. Neither the nontransgenic mice nor the mice hemizygous for either the transactivator transgene or the target transgene alone showed any response to treatment with RU486. In addition, no differences were observed in the skin of the bitransgenic mice versus nontransgenic littermates without treatment of RU486. Interestingly, in HK14.GLp65/TK.src(530) bitransgenic mice, squamous cell carcinomas (SCCs) arose along the periphery of the area of the punch biopsies in 25% of the bitransgenic mice several weeks after taking the biopsy and subsequent to RU486 treatment. Collectively, the data support a role of c-src activation in epidermal hyperproliferation. Furthermore, the data support the conclusion that src activation can substitute for an initiating event in the presence of a tumor promoting stimulus (i.e., wounding). Finally, inducible src(530) transgenic mice provide a new tool for dissecting the role of src activation in multistage carcinogenesis by allowing temporal control of the expression of this oncogene.


Assuntos
Epiderme/metabolismo , Proteínas Tirosina Quinases/genética , Animais , Proteína Tirosina Quinase CSK , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Epiderme/efeitos dos fármacos , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/fisiologia , Antagonistas de Hormônios/farmacologia , Camundongos , Camundongos Transgênicos , Mifepristona/farmacologia , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src
5.
Cancer Res ; 63(16): 4819-28, 2003 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-12941801

RESUMO

In this study, we generated transgenic mice that overexpressed either a constitutively active human c-src mutant (src(530)) or a wild-type human c-src (src(wt)) in epidermal basal cells driven by human keratin 14 (HK14) or bovine keratin 5 (BK5) promoters, respectively. HK14.src(530) transgenic mice developed severe epidermal hyperplasia and hyperkeratosis, and did not survive beyond 3 weeks of age. Four transgenic founders were obtained after injection of a BK5.src(wt) construct with variable phenotypes, and three lines (lines A-C) were established. BK5.src(wt) founder D exhibited a severe skin phenotype similar to HK14.src(530) transgenic mice and died 5 days after birth. Line C transgenic mice also exhibited significant epidermal hyperplasia and hyperkeratosis, and developed spontaneous squamous cell carcinomas (SCCs) of the skin beginning at approximately 3 months of age (70% incidence at 1 year). Mice from lines A and B did not show a marked phenotype; however, elevated human src(wt) protein in the epidermis of line B mice was clearly evident. Additional analyses of line B transgenic mice showed an enhanced responsiveness to 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia and cell proliferation. Analysis of the susceptibility of line B mice to two-stage skin carcinogenesis revealed that papillomas and SCCs arose earlier and in greater numbers compared with nontransgenic littermates. In addition, malignant conversion occurred more rapidly, and the SCCs that developed in line B transgenic mice had a greater propensity to metastasize to peripheral lymph nodes and other organs. These observations support the hypothesis that c-src plays an important role in skin tumor promotion. In addition, the data show that elevated c-src activity enhances malignant progression and metastasis in this model system.


Assuntos
Carcinoma de Células Escamosas/etiologia , Proteínas Tirosina Quinases/fisiologia , Neoplasias Cutâneas/etiologia , 9,10-Dimetil-1,2-benzantraceno , Sequência de Aminoácidos , Animais , Proteína Tirosina Quinase CSK , Carcinoma de Células Escamosas/patologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Metástase Neoplásica , Proteínas Tirosina Quinases/análise , Pele/patologia , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/farmacologia , Quinases da Família src
6.
Mol Carcinog ; 33(3): 146-55, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11870880

RESUMO

Transgenic mice were developed to study the role of c-src in epithelial tumorigenesis through targeted expression of a constitutively active form of murine c-src (src(529)). Src(529) was targeted to the interfollicular epidermis with the human keratin 1 (HK1) promoter. The skin phenotype of these mice was characterized by exaggerated epidermal hyperplasia and hyperkeratosis within the first week after birth. The severity of this phenotype correlated with overall src kinase activity, both of which subsided with age. Treatment of adult HK1.src(529) transgenic mice with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate resulted in an increase in epidermal hyperplasia and labeling index significantly greater than that seen in nontransgenic littermates. In addition, HK1.src(529) transgenic mice developed papillomas earlier and in significantly greater numbers compared with nontransgenic littermates in a standard initiation-promotion experiment. The data support the hypothesis that activation of c-src kinase plays a role in skin tumor promotion.


Assuntos
Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Neoplasias Cutâneas/etiologia , Animais , Carcinógenos , Epiderme/enzimologia , Epiderme/patologia , Hiperplasia , Queratinas/genética , Cinética , Camundongos , Camundongos Transgênicos , Mutação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol
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