RESUMEN
Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin-3 gallate (EGCG), which possess anti-oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen-induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12-dimethylbenz(a)anthracene (DMBA) Sprague-Dawley (S-D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor-bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA-MB-231 estrogen receptor-negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA-transformed D3-1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27(Kip1) cyclin-dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27(Kip1) protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen-induced mammary tumorigenesis in female S-D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27(Kip1) CKI.
Asunto(s)
Antineoplásicos/farmacología , Carcinógenos/farmacología , Flavonoides , Neoplasias Mamarias Animales/inducido químicamente , Neoplasias Mamarias Animales/patología , Extractos Vegetales/farmacología , Té , Proteínas Supresoras de Tumor , 9,10-Dimetil-1,2-benzantraceno/antagonistas & inhibidores , 9,10-Dimetil-1,2-benzantraceno/farmacología , Animales , Antineoplásicos/uso terapéutico , Carcinógenos/antagonistas & inhibidores , Catequina/análogos & derivados , Catequina/farmacología , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Femenino , Citometría de Flujo , Neoplasias Mamarias Animales/enzimología , Fenoles/metabolismo , Extractos Vegetales/uso terapéutico , Polímeros/metabolismo , Probabilidad , Ratas , Células Tumorales CultivadasRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter-reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/análogos & derivados , Contaminación Ambiental/efectos adversos , Insuficiencia Ovárica Primaria/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Receptores de Hidrocarburo de Aril/metabolismo , Adulto , Animales , Apoptosis , Femenino , Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Microinyecciones , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/trasplante , Insuficiencia Ovárica Primaria/inducido químicamente , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/deficiencia , Receptores de Hidrocarburo de Aril/deficiencia , Receptores de Hidrocarburo de Aril/genética , Elementos de Respuesta , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , Proteína X Asociada a bcl-2RESUMEN
Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental chemicals that suppress the immune system at multiple levels, including at the level of B cell development in the bone marrow microenvironment. Specifically, PAH induce preB cell apoptosis in primary bone marrow cultures and in cocultures of an early preB cell line (BU-11) and a bone marrow stromal cell line (BMS2). Previous studies focused on the molecular mechanisms through which PAH induce stromal cells to deliver an apoptosis signal to adjacent preB cells. Apoptosis signaling within the preB cell itself was not investigated. Here, the role of NF-kappaB, a lymphocyte survival factor, in PAH-induced preB cell apoptosis was assessed. Analysis of DNA-binding proteins extracted from the nuclei of untreated BU-11 cells indicated DNA-binding complexes comprising NF-kappaB subunits p50, c-Rel, and/or Rel A. NF-kappaB down-regulation with previously described inhibitors induced BU-11 cell apoptosis, demonstrating that the default apoptosis pathway blocked by NF-kappaB is functional at this early stage in B cell development. Similarly, exposure of BU-11/BMS2 cocultures to 7,12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH, down-regulated nuclear Rel A and c-Rel before overt apoptosis. Finally, ectopic expression of Rel A or c-Rel rescued BU-11 cells from DMBA-induced apoptosis. These results extend previous observations by demonstrating that 1) NF-kappaB is a survival factor at an earlier stage of B cell development than previously appreciated and 2) NF-kappaB down-regulation is likely to be part of the molecular mechanism resulting in PAH-induced preB cell apoptosis. These results suggest nonclonally restricted, PAH-mediated suppression of B lymphopoiesis.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/farmacología , Apoptosis , Linfocitos B/citología , FN-kappa B/fisiología , Animales , Carcinógenos/farmacología , Supervivencia Celular , Regulación hacia Abajo , Contaminantes Ambientales/farmacología , Ratones , FN-kappa B/biosíntesis , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/biosíntesis , Proteínas Proto-Oncogénicas c-rel/fisiología , Factor de Transcripción ReIA , Células Tumorales CultivadasRESUMEN
NF-kappaB/Rel transcription factors regulate many genes involved in control of cellular proliferation, neoplastic transformation, and apoptosis, including the c-myc oncogene. Recently, we have observed that levels of NF-kappaB and aryl hydrocarbon receptor (AhR), which mediates malignant transformation by environmental carcinogens, are highly elevated and appear constitutively active in breast cancer cells. Rel factors have been found to functionally interact with other transcription factors. Here we demonstrate a physical and functional association between the RelA subunit of NF-kappaB and AhR resulting in the activation of c-myc gene transcription in breast cancer cells. RelA and AhR proteins were co-immunoprecipitated from cytoplasmic and nuclear extracts of non-malignant MCF-10F breast epithelial and malignant Hs578T breast cancer cells. In transient co-transfection, RelA and AhR gene products demonstrated cooperation in transactivation of the c-myc promoter, which was dependent on the NF-kappaB elements, and in induction of endogenous c-Myc protein levels. A novel AhR/RelA-containing NF-kappaB element binding complex was identified by electrophoretic mobility shift analysis of nuclear extracts from RelA and AhR co-transfected Hs578T cells. Thus, the RelA and AhR proteins functionally cooperate to bind to NF-kappaB elements and induce c-myc gene expression. These findings suggest a novel signaling mechanism whereby the Ah receptor can stimulate proliferation and tumorigenesis of mammary cells.
Asunto(s)
Mama/fisiología , Genes myc/genética , FN-kappa B/fisiología , Receptores de Hidrocarburo de Aril/fisiología , Activación Transcripcional/fisiología , Sitios de Unión , Mama/citología , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , División Celular/genética , Línea Celular , Transformación Celular Neoplásica/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Pruebas de Precipitina , Regiones Promotoras Genéticas/genética , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Factor de Transcripción ReIA , Transfección , Células Tumorales CultivadasRESUMEN
Exposure to ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons (PAH), may contribute to human breast cancer. In animals, PAH induce tumors in part by activating the aryl hydrocarbon receptor (AhR)/transcription factor. Historically, investigations into AhR-regulated carcinogenesis have focused on AhR-dependent transcriptional regulation of cytochrome P450 (CYP) enzymes which oxidize PAH to mutagenic intermediates. However, recent studies suggest that the AhR directly regulates cell growth. Given the postulated role of the AhR in carcinogenesis, we predicted that: (1) tissue predisposed to PAH tumorigenesis would express the AhR and (2) aberrant AhR and/or AhR-regulated gene expression would accompany malignant transformation. To test these hypotheses, AhR and CYP1 protein and/or mRNA levels were evaluated in rat mammary tumors induced with 7, 12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH and AhR ligand. Results indicate modest AhR expression in normal mammary myoepithelial and ductal epithelial cells. In contrast, high AhR levels were detected in DMBA-induced tumors. Nuclear AhR localization in tumors suggested constitutive AhR activation. In situ hybridization and quantitative RT-PCR assays indicated high AhR mRNA levels in neoplastic epithelial cells. While both AhR-regulated CYP1A1 and CYP1B1 mRNAs were induced in breast tissue within 6 h of DMBA gavage, only CYP1B1 mRNA remained elevated in tumors. These results: (1) help explain targeting of breast tissue by carcinogenic PAH, (2) imply that AhR and CYP1B1 hyper-expression represent molecular biomarkers for, at least, PAH-induced mammary cell transformation, and (3) suggest mechanisms through which the AhR may contribute to carcinogenesis well after exogenous AhR ligands have been eliminated.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP1A1/genética , Sistema Enzimático del Citocromo P-450/genética , Neoplasias Mamarias Experimentales/metabolismo , ARN Mensajero/análisis , Receptores de Hidrocarburo de Aril/análisis , 9,10-Dimetil-1,2-benzantraceno , Animales , Citocromo P-450 CYP1B1 , Femenino , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/inducido químicamente , Ratas , Ratas Sprague-DawleyRESUMEN
Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1-10 microg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1-100 ng/ml significantly enhanced cytotoxicity against the FcR(+) LB27.4, M59, and LF(+) targets, but not the FcR(-) Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR(+) A20 B lymphoma but not with the FcR(-) A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR(-) Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Sistema Libre de Células/inmunología , Glicoproteínas de Membrana/biosíntesis , Receptores Fc/fisiología , Receptor fas/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Línea Celular , Sistema Libre de Células/metabolismo , Pruebas Inmunológicas de Citotoxicidad , Proteína Ligando Fas , Humanos , Inmunoglobulina G/genética , Isotipos de Inmunoglobulinas/fisiología , Inmunosupresores/farmacología , Leucemia L5178 , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Receptores Fc/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Células Tumorales CultivadasRESUMEN
Bioflavonoids are plant compounds touted for their potential to treat or prevent several diseases including cancers induced by common environmental chemicals. Much of the biologic activity of one such class of pollutants, polycyclic aromatic hydrocarbons (PAH), is mediated by the aryl hydrocarbon receptor/transcription factor (AhR). For example, the AhR regulates PAH immunotoxicity that manifests as pre-B cell apoptosis in models of B cell development. Because bioflavonoids block PAH-induced cell transformation and are structurally similar to AhR ligands, it was postulated that some of them would suppress PAH-induced, AhR-dependent immunotoxicity, possibly through a direct AhR blockade. This hypothesis was tested using a model of B cell development in which pre-B cells are cultured with and are dependent on bone marrow stromal or hepatic parenchymal cell monolayers. Of seven bioflavonoids screened, galangin (3,5,7-trihydroxyflavone) blocked PAH-induced but not C(2)-ceramide- or H(2)O(2)-induced pre-B cell apoptosis. Because galangin blocked AhR-dependent reporter gene expression, AhR complex-DNA binding, and AhR nuclear translocation, inhibition of a relatively early step in AhR signaling was implicated. This hypothesis was supported by the ability of galangin to bind the AhR and stabilize AhR-90-kDa heat shock protein complexes in the presence of AhR agonists. These studies demonstrate the utility of pre-B cell culture systems in identifying compounds capable of blocking PAH immunotoxicity, define at least one mechanism of galangin activity (i.e., repression of AhR activation), and motivate the use of this and similar dietary bioflavonoids as relatively nontoxic inhibitors of AhR agonist activity and as pharmacologic agents with which to dissect AhR signaling pathways.
Asunto(s)
Apoptosis , Linfocitos B/efectos de los fármacos , Proteínas de Unión al ADN , Flavonoides/farmacología , Hidrocarburos Policíclicos Aromáticos/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , 9,10-Dimetil-1,2-benzantraceno/farmacología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Linfocitos B/citología , Sitios de Unión , Transporte Biológico , Carcinógenos/farmacología , Línea Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , ADN/efectos de los fármacos , ADN/metabolismo , Dimerización , Expresión Génica/efectos de los fármacos , Genes Reporteros , Proteínas HSP90 de Choque Térmico/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutágenos/farmacología , Dibenzodioxinas Policloradas/farmacología , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , TritioRESUMEN
The aryl hydrocarbon receptor (AhR), so-designated based on the ability of the protein to bind with and be activated by polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons, is part of an emerging family of ligand-activated transcriptional regulators that are distinct from the steroid-thyroid hormone receptor superfamily. Once bound by ligand, the AhR interacts with the AhR nuclear translocator (ARNT) protein to form the aryl hydrocarbon receptor complex (AHRC). Both subunits of the AHRC contain sequences corresponding to basic helix-loop-helix domains, a motif that is shared by a number of other dimeric transcription factors. Although the natural ligand(s) for the AhR remains to be elucidated, to date over fifteen genes, including enzymes, growth factors and other transcription factors, have been identified as potential targets for transcriptional regulation by the chemically-activated AHRC. In the ovary, PAH exposure is known to cause destruction of oocytes within immature follicles, implying that one function of the AhR is to mediate cell death signaling in the female germ line. To assess this possibility, we explored AhR expression patterns in the murine ovary, and then determined the impact of AhR-deficiency (gene knockout) on female germ cell dynamics. Immunohistochemical analysis of ovaries of wild-type female mice indicated that AhR protein was abundantly and exclusively expressed in oocytes and granulosa cells of follicles at all stages of development. Histomorphometric analysis of serial ovarian sections revealed a two-fold higher number of primordial follicles in Ahr-null versus wild-type females at day 4 postpartum. This phenotype likely results from a cell-intrinsic death defect in the developing germ line since AhR-deficiency attenuated the magnitude of oocyte apoptosis in fetal ovaries cultured without hormonal support for 72 h. We propose that the AhR, activated by an as yet unknown endogenous ligand(s), serves to regulate the size of the oocyte reserve endowed at birth by affecting germ cell death during female gametogenesis.
Asunto(s)
Secuencias Hélice-Asa-Hélice/genética , Ovario/fisiología , Óvulo/fisiología , Receptores de Hidrocarburo de Aril/genética , Factores de Transcripción/genética , Animales , Recuento de Células , Células Cultivadas , Femenino , Células de la Granulosa/fisiología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ovario/citologíaRESUMEN
Previous studies indicated that two prototypic PAH, benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA), suppress the developing immune system by inducing apoptosis in bone marrow pre-B lymphocytes. In bone marrow cultures consisting of pre-B cells growing on bone marrow stromal cell monolayers, pre-B cell apoptosis was shown to be dependent on the aryl hydrocarbon receptor/transcription factor (AhR) expressed in stromal cells. However, it was not determined if AhR activation alone is sufficient or if DMBA metabolism is required for induction of a stromal cell-derived apoptosis signal. To address these issues we assessed: 1) the ability of poorly metabolized AhR ligands to induce pre-B cell apoptosis and 2) the capacity for and the mechanism through which an early DMBA metabolite induces pre-B cell apoptosis. Three poorly metabolized AhR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3,3',4,4',5-pentachlorobiphenyl, and 3,3',4,4'-tetrachlorobiphenyl failed to induce pre-B cell apoptosis in bone marrow cultures, indicating that AhR activation alone is not sufficient to induce apoptosis and suggesting a role for PAH metabolism in induction of an apoptosis signal. Consistent with this hypothesis, DMBA-3, 4-dihydrodiol, an early DMBA metabolite, induced significant pre-B cell apoptosis. The ability of DMBA-3,4-dihydrodiol to activate the AhR, inhibition of DMBA-3,4-dihydrodiol-induced apoptosis by alpha-naphthoflavone, and the significantly lower levels of DMBA-3, 4-dihydrodiol-induced apoposis in pre-B cell populations maintained on AhR(-) stromal cells strongly support a role for the AhR in DMBA-3,4-dihydrodiol-induced apoptosis. Of two DMBA-metabolizing enzymes evaluated, CYP1A1 and CYP1B1, the latter appeared to be the more likely to play a role in DMBA-induced apoptosis. These data confirm a role for the AhR in PAH-induced pre-B cell apoptosis, indicate a role for DMBA metabolism, and suggest a feedback loop in which at least one product of DMBA metabolism augments AhR signaling, leading to induction of an apoptosis stimulus.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/farmacología , Apoptosis/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas , Linfocitos B/citología , Células Madre Hematopoyéticas/citología , Hidrocarburos Policíclicos Aromáticos/metabolismo , 9,10-Dimetil-1,2-benzantraceno/análogos & derivados , 9,10-Dimetil-1,2-benzantraceno/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Linfocitos B/metabolismo , Línea Celular , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Ligandos , Ratones , Modelos Biológicos , Dibenzodioxinas Policloradas/farmacología , Hidrocarburos Policíclicos Aromáticos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Células del Estroma/enzimología , Células del Estroma/metabolismo , Células del Estroma/fisiología , Triazoles/farmacologíaRESUMEN
Polycyclic aromatic hydrocarbons (PAH) are environmental chemicals that mediate immunosuppression. In long-term bone marrow B-cell lymphopoiesis models, PAH induce apoptosis in immature (preB) lymphocytes. Since the biologic function of PAH is often mediated by the aryl hydrocarbon receptor/transcription factor (AhR), the role of the AhR or AhR-regulated genes was assessed in preB cell apoptosis. Specifically, a bone marrow-derived preB cell line (BU-11) was cultured on monolayers of the AhR + bone marrow-derived stromal cell line BMS2, hepatoma sublines that express various levels of AhR activity (Hepa-1c1c7 and variants), AhR+ thymic epithelial cells, and primary bone marrow stromal cells from wildtype or AhR-/- mice. Cultures were treated with one of two prototypic PAH, 7,12-dimethylbenz[a] anthracene (DMBA) or benz[a]pyrene (B[a]P), and the percentage of cells undergoing apoptosis measured. The data demonstrated that: 1) bone marrow- and hepatic-derived stromal/adherent cells support preB cell growth and regulate apoptosis induced by DMBA or B[a]P; 2) B[a]P is more effective than DMBA when preB cells are maintained on Hepa-1c1c7 monolayers than when maintained on BMS2 monolayers; 3) DMBA is more effective than B[a]P when preB cells are cultured on BMS2 monolayers; 4) alpha-naphthoflavone, an AhR antagonist and cytochrome P-450 inhibitor, blocks preB cell apoptosis in both BU-11/Hepa-1c1c7 and BU-11/BMS2 cultures; 5) although preB cells grow well in Hepa-1c1c7 or BMS2 supernatants, addition of PAH in the absence of hepatic- or bone marrow-derived adherent cells does not result in preB cell apoptosis; 6) preB cell apoptosis is dependent on AhR activity in adherent hepatic- or bone marrow-derived stromal cells; and 7) apoptosis is induced by DMBA when preB cells are maintained on primary bone marrow stromal cell monolayers from wildtype but not from AhR-/- mice. Collectively, the data indicated that AhR-regulated activities in the hematopoietic microenvironment influence the susceptibility of immature lymphocytes to low-dose PAH exposure.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos B/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/genética , Células del Estroma/metabolismo , Factores de Transcripción/genética , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Benzo(a)pireno/toxicidad , Benzoflavonas/farmacología , Células de la Médula Ósea , Línea Celular , Fragmentación del ADN/genética , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Hematopoyesis , Hígado/efectos de los fármacos , Ratones , Ratones Noqueados , ARN Mensajero/genéticaRESUMEN
In the absence of known endogenous ligands, investigators have exploited ubiquitous environmental pollutants, including polycyclic aromatic hydrocarbons, to gain insight into the physiologic functions of the aryl hydrocarbon (dioxin) receptor/transcription factor (AhR). AhR ligands induce cell transformation and steroid-like immunosuppression, suggesting a role for the AhR in regulation of cell growth and/or function. However, mechanisms through which the AhR influences cells in general and lymphocytes in particular remain unresolved. A murine model of B cell development was created to: 1) examine a role for the AhR in immunosuppression; 2) define mechanisms of AhR ligand immunosuppression; 3) characterize AhR expression in preB cells, in bone marrow stromal cells that support preB cells, or in primary bone marrow B cells; and 4) determine if AhR ligands suppress lymphopoiesis by acting directly on preB cells or indirectly via the microenvironment, as represented by bone marrow stromal cells. Results indicate that: 1) low doses (> or = 10(-8) M) of the prototypic AhR ligand, 7,12-dimethylbenz[a]anthracene (DMBA), induce preB cell apoptosis in 12 to 24 h; 2) alpha-naphthoflavone, an AhR and cytochrome P-450 inhibitor, blocks DMBA-induced apoptosis; 3) AhR mRNA and functional AhR protein are expressed at high levels in bone marrow stromal cells (little or no AhR is present in preB cell lines), and 4) preB cells maintained in rIL-7 do not undergo DMBA-induced apoptosis unless cultured with stromal cells. Results underscore the regulatory role played by bone marrow stromal cells in lymphopoiesis and support the hypothesis that the AhR effects immunosuppression by inducing stromal cells to deliver a death signal to lymphocytes.
Asunto(s)
Apoptosis , Linfocitos B/metabolismo , Médula Ósea/metabolismo , Células Madre Hematopoyéticas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Factores de Transcripción/metabolismo , 9,10-Dimetil-1,2-benzantraceno/farmacología , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Benzoflavonas/farmacología , Unión Competitiva , Células de la Médula Ósea , Línea Celular , Inhibidores Enzimáticos del Citocromo P-450 , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/biosíntesis , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/biosíntesis , Receptores de Hidrocarburo de Aril/genética , Células del Estroma/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Numerous studies demonstrate that polycyclic aromatic hydrocarbons (PAH) suppress immunity by modifying the function of both B and T cells. Relatively few studies have assessed the effects of these common environmental chemicals on immature lymphocytes. In the present study, long-term primary bone marrow cultures were employed to investigate the effects of a prototypic PAH and aryl hydrocarbon receptor (AhR) agonist, 7,12-dimethylbenz[a]anthracene (DMBA), on immature B lymphocytes. In this system, immature preB cells are maintained in a supportive microenvironment provided by bone marrow stromal cells. Results presented here demonstrate that (1) exposure of primary bone marrow cultures to DMBA results in preB cell death by apoptosis; (2) notably low doses of DMBA (> or = 10(-8) M) induce preB cell apoptosis; (3) in long-term cultures, bone marrow stromal cells, but not preB cells, express AhR mRNA and protein as determined by in situ hybridization, RT-PCR, and immunoblotting; (4) freshly isolated unfractionated bone marrow cells, but not purified bone marrow B cells, express AhR protein as assessed by immunohistochemistry; (5) alpha-naphthoflavone, a competitive AhR inhibitor and cytochrome P450 antagonist, completely blocks DMBA-induced preB cell apoptosis in primary bone marrow cultures; and (6) DMBA or benzo[a]pyrene injection in vivo results in bone marrow cell apoptosis consistent with the death of hematopoietic cells clustered around stromal elements. The results implicate programmed cell death as a mechanism underlying DMBA-mediated immunosuppression and suggest that preB cell death is influenced by local interactions with AhR+ bone marrow stromal cells.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/farmacología , Apoptosis , Linfocitos B/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , 9,10-Dimetil-1,2-benzantraceno/antagonistas & inhibidores , Animales , Linfocitos B/fisiología , Células Cultivadas , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Fas (CD95) has been shown in multiple systems to play a critical role in deletion of autoreactive lymphocytes by transducing cell death signals. The role of Fas in clonal deletion may best be exemplified in autoimmune lpr mice, in which a defect in the lpr gene leads to persistence of autoreactive clones in the periphery. Since negative selection in the lpr thymus appears not to be ablated, it has been suggested that Fas is not essential to thymic negative selection. A recent study has shown that lpr thymocytes express low levels of Fas protein. However, it is not determined whether this low level of Fas could transduce the death signal. This is a critical issue for the hypothesis that lpr thymocyte negative selection does not involve a Fas-death pathway. Here, we demonstrate that thymocytes, but not peripheral lymphocytes, from 2- to 4-week-old C3H.MRL-lpr mice are killed by Fas-dependent cytotoxicity at levels commensurate with the low levels of Fas expression. The level of lpr thymocyte killing is approximately 20% of that observed in wild-type controls. Both Fas staining and Th1 cytotoxicity are specifically blocked by a recombinant Fas-hIgG fusion protein. Thymocyte subset analyses indicate that Fas is expressed primarily on CD4+/CD8+ lpr thymocytes and that CD4+/CD8+ lpr thymocytes are the primary targets for Th1 effector cytotoxicity. The data suggest that the lpr mutation is functionally "leaky" and that the demonstration of normal negative selection in lpr thymocytes should not be taken as evidence that Fas is not involved in clonal deletion in the thymus.
Asunto(s)
Autoinmunidad/inmunología , Citotoxicidad Inmunológica , Linfocitos T/inmunología , Receptor fas/inmunología , Animales , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Supresión Clonal , Activación de Linfocitos , Ratones , Ratones Endogámicos C3H , Ratones Mutantes , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Receptor fas/análisisRESUMEN
The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates biological responses to planar aromatic hydrocarbons such as benzo[alpha]pyrene. However, no endogenous physiological ligand for the AhR has been identified. Since the AhR regulates bioactivity to common environmental pollutants, and since it is predicted to play an important physiological function, we have investigated the expression of the AhR during the cell cycle of murine 3T3 fibroblasts. We show here that stimulation of growth-arrested 3T3 cells with serum results in increased expression of AhR protein. Serum-induced expression of AhR in synchronized, serum-stimulated cells occurs at the onset of DNA synthesis (S phase) and is maximal at time points corresponding to late S phase. Transient transfections with an AhR-promoter-luciferase construct demonstrate that reporter gene transcription from the AhR promoter is regulated in a serum-dependent manner. Serum-dependent induction of AhR expression is prevented by an inhibitor of tyrosine kinase activity. Ligand-activated growth factor receptors (platelet-derived growth factor receptor basic fibroblast growth factor receptor) as well as an ectopically expressed tyrosine kinase (the v-Src oncoprotein) induce AhR expression in the absence of serum. Therefore, tyrosine kinase signaling is both necessary and sufficient for induction of AhR expression. Studies with the G1 blocker sodium butyrate show that the signal transduction pathways mediating serum-stimulated progression through the cell cycle are distinct from those that induce AhR expression. These data suggest that transcriptional regulation of the AhR is important in determining cellular sensitivity to the actions of AhR ligand(s) and that the AhR may play a role during the cellular proliferative response.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptores de Hidrocarburo de Aril/biosíntesis , Animales , Sangre , Butiratos/farmacología , Ácido Butírico , Ciclo Celular , Línea Celular , Electroforesis en Gel de Poliacrilamida , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Citometría de Flujo , Ratones , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti-IgM) is preceded by dramatic changes in Nuclear Factor-kappaB (NF-kappaB)/ Rel binding activities. An early transient increase in NF-kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel-related factors in B cell apoptosis. Treatment of WEH1 231 cells with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF-kappaB (IkappaB)-alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF-kappaB/Rel factor binding and induced apoptosis. Bcl-XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB-alpha-GST protein or a c-Rel affinity-purified antibody induced apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TPCK or anti-IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF-kappaB/Rel binding following anti-IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF-kappaB/Rel family in control of apoptosis of normal and transformed B cells.
Asunto(s)
Apoptosis , Linfocitos B/patología , FN-kappa B/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2 , Factores de Transcripción , Animales , Inmunoglobulina M/efectos de los fármacos , Linfoma de Células B/patología , Ratones , Microinyecciones , FN-kappa B/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Bazo/patología , Clorometilcetona de Tosilfenilalanila/farmacología , Factor de Transcripción ReIB , Células Tumorales Cultivadas , Proteína bcl-XRESUMEN
Treatment of WEHI 231 immature B-lymphoma cells with an antibody against their surface immunoglobulin (anti-Ig) induces apoptosis and has been studied extensively as a model of B-cell tolerance. Anti-Ig treatment of exponentially growing WEHI 231 cells results in an early transient increase in c-myc expression that is followed by a decline to below basal levels; this decrease in c-myc expression immediately precedes the induction of cell death. Here we have modulated NF-kappaB/Rel factor activity, which regulates the rate of c-myc gene transcription, to determine whether the increase or decrease in c-Myc-levels mediates apoptosis in WEHI 231 cells. Addition of the serine/threonine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), which blocks the normally rapid turnover of the specific inhibitor of NF-kappaB/Rel IkappaBalpha in these cells, caused a drop in Rel-related factor binding. TPCK treatment resulted in decreased c-myc expression, preventing the usual increase seen following anti-Ig treatment. Whereas inhibition of the induction of c-myc expression mediated by anti-Ig failed to block apoptosis, reduction of c-myc expression in exponentially growing WEHI 231 cells induced apoptosis even in the absence of anti-Ig treatment. In WEHI 231 clones ectopically expressing c-Myc, apoptosis induced by treatment with TPCK or anti-Ig was significantly diminished and cells continued to proliferate. Furthermore, apoptosis of WEHI 231 cells ensued following enhanced expression of Mad1, which has been found to reduce functional c-Myc levels. These results indicate that the decline in c-myc expression resulting from the drop in NF-kappaB/Rel binding leads to activation of apoptosis of WEHI 231 B cells.
Asunto(s)
Apoptosis/genética , Linfocitos B/metabolismo , Proteínas Portadoras , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Represoras , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Proteínas de Ciclo Celular , Citomegalovirus/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células B/patología , Ratones , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-rel , Proteínas Recombinantes de Fusión/metabolismo , Clorometilcetona de Tosilfenilalanila/farmacología , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Recent studies suggest that environmental chemicals such as polycyclic aromatic hydrocarbons (PAH) compromise the immune system in part through the induction of programmed cell death (apoptosis). Nevertheless, mechanisms through which PAH induce apoptosis remain elusive. In particular, the role of the 8S AhR remains controversial and the nature of intracellular signal transduction in PAH-induced apoptosis remains largely undefined. To extend previous studies to the T cell compartment and to develop a clonal system in which intracellular signals leading to PAH-induced apoptosis can be dissected, the ability of fluoranthene, a ubiquitous, but less well-studied PAH, to induce apoptosis in murine T cell hybridomas was evaluated. Particular emphasis was placed on the role of the 8S AhR. The data indicate that (1) three of four hybridomas studied undergo apoptosis within 8 hr of fluoranthene exposure; (2) fluoranthene induces growth arrest concurrent with apoptosis; (3) at doses sufficient to induce lymphocyte apoptosis, fluoranthene does not induce AhR nuclear translocation in cells expressing high AhR levels; (4) fluoranthene-responsive hybridomas do not express AhR mRNA or protein; (5) the Ca2+ chelating agent EGTA partially inhibits fluoranthene-induced apoptosis. These results (1) indicate the immunosuppressive potential of fluoranthene; (2) support a role for apoptosis in PAH immunotoxicity; (3) demonstrate that fluoranthene-mediated T cell death and growth arrest are AhR independent; and (4) illustrate similarities between PAH- and antigen-specific receptor-mediated apoptosis. These findings encourage consideration of AhR- independent events in PAH risk assessment.
Asunto(s)
Apoptosis/efectos de los fármacos , Fluorenos/toxicidad , Hibridomas/citología , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Animales , Secuencia de Bases , Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quelantes/farmacología , ADN/química , ADN/metabolismo , Cartilla de ADN/química , Ácido Egtácico/farmacología , Hibridomas/efectos de los fármacos , Immunoblotting , Inmunosupresores/toxicidad , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/fisiología , Espectrometría de Fluorescencia , Linfocitos T/citología , Linfocitos T/patologíaRESUMEN
Activation-induced cell death (AICD) requires coexpression of Fas and FasL. Hybridoma T-cells express detectable FasL mRNA 3 to 5 hr after culture in anti-CD3-coated wells. High and steady expression of FasL mRNA was observed after 8-10 hr of activation. Expression of FasL cytotoxicity and AICD is consistent with the time-course of FasL mRNA induction. Fas-Ig was effective in inhibiting AICD when added no later than 5 hr after activation, but was ineffective when added after 8-10 hr of activation. These observations suggest that FasL gene activation is a critical step for AICD. By contrast, Fas was constitutively expressed and the time-course study did not support the idea that up-regulation of Fas is critical for AICD. The critical role of FasL gene activation for AICD was confirmed by studies using inhibitors of AICD. Dexamethasone (Dex) inhibited FasL induction and Fas up-regulation, but not basal Fas expression of hybridoma T-cells. All-trans retinoic acid (RA) inhibited FasL induction, but had little effect on Fas up-regulation. Both agents inhibited FasL cytotoxicity. The Fas-mediated death pathway distal to Fas/FasL interactions remained intact in the protected hybridoma T-cells. These results demonstrate that FasL gene activation, but not Fas up-regulation, is critical for AICD and that Dex and all-trans RA selectively inhibits FasL but not Fas function. The system may prove useful for the identification of critical factors regulating T-cell death genes. It may also serve as a useful system to study gene regulation in AICD-dependent phenomena.
Asunto(s)
Muerte Celular/efectos de los fármacos , Dexametasona/farmacología , Glicoproteínas de Membrana/fisiología , Linfocitos T/citología , Tretinoina/farmacología , Receptor fas/fisiología , Animales , Secuencia de Bases , Cartilla de ADN/química , Proteína Ligando Fas , Expresión Génica/efectos de los fármacos , Hibridomas , Activación de Linfocitos , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN Mensajero/genéticaRESUMEN
T cell hybridomas that are deathless upon TCR crosslinking were generated from lpr mice. The deathless hybridomas (1.4 and 5D5) expressed extremely low Fas even after anti-CD3 activation, whereas activation-induced cell death (AICD) was observed for Fas-expressing hybridomas. The deathless hybridomas were activated to produce FasL and IL-2, indicating that the intrinsic defect in Fas expression or up-regulation resulted in AICD blockade. The deathless hybridoma cells expressed longer and stronger FasL cytotoxicity than AICD-sensitive hybridomas. Although deathless, activated 5D5 cells were arrested at the G1/S border. Growth arrest lasted for at least 5 days, but some cells eventually recovered and proliferated. The deathless 5D5 cells were used to demonstrate that AICD includes a fratricidal mechanism that kills AICD-sensitive bystanders. The deathless T cell hybridomas are useful tools for studying T cell activation-dependent functions sensitive to AICD.
Asunto(s)
Muerte Celular , Hibridomas/patología , Activación de Linfocitos , Linfocitos T Citotóxicos/citología , Linfocitos T/citología , Receptor fas/fisiología , Animales , Secuencia de Bases , División Celular , Cartilla de ADN/química , Proteína Ligando Fas , Expresión Génica , Glicoproteínas de Membrana/genética , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T/fisiologíaRESUMEN
Peritoneal (PE) B cells, a large fraction of which belong to the B-1 subset (i.e., the CD5+ B cell lineage), exhibit unusual growth and/or survival characteristics. To evaluate if these characteristics reflect intrinsic properties of PE B cells or the support provided by a potentially unique peritoneal microenvironment, B cells from various organs were cultured on PE stromal cell monolayers. It was determined that sIgM+ PE B cell populations survive for notably long periods of time (at least 4 weeks) when cultured with PE stromal cells. Contact of PE B cells with stromal cell monolayers optimized B cell survival. Although expressing a similar spectrum of adhesion molecules as peritoneal stromal cells, splenic and bone marrow stromal cells were significantly inferior at supporting PE B cells. Increased survival of PE B cells was characterized by a significant but transient increase in proliferation and by an increase in the percentage of B220/CD45high/CD5+ PE B cells. PE stromal cell support of B cell populations did not extend to all B or even to all B-1 cell populations since survival of splenic B cells, only a minority of which belong to the B-1 lineage, or thymic B cells, a majority of which belong to the B-1 lineage, was not enhanced by culture on PE stromal cells. Results demonstrate: (a) a system for relatively long-term maintenance of mature B cells, (b) that the growth/survival of PE B cells is preferentially supported by PE stromal cells, and (c) that growth on PE stromal cells is a characteristic of peritoneal B cells but not necessarily of splenic B cells or B-1 cells from other organs. The suggestion that the local microenvironment contributes to unusual growth, survival, or anatomic distribution of B-1 cells is discussed.