RESUMEN

The photosensitizer 9-capronyloxytetrakis (methoxyethyl) porphycene localizes predominantly in the endoplasmic reticulum (ER) and, to a lesser extent, in mitochondria of murine leukemia L1210 cells. Subsequent irradiation results in the loss of ER > mitochondrial Bcl-2 and an apoptotic response. Although an increase in cytosolic Ca(2+) was observed after irradiation, apoptosis was not inhibited by either the presence of the calcium chelator BAPTA or by the mitochondrial uniporter inhibitor ruthenium amino binuclear complex (Ru360). Moreover, neither reagent prevented the loss of Bcl-2. Ruthenium red (RR) devoid of Ru360 prevented Bcl-2 loss, release of Ca(2+) from the ER and the initiation of apoptosis. Since RR was significantly more sensitive than Ru360 to oxidation by singlet oxygen, we attribute the protective effect of RR to the quenching of reactive oxygen species. Although cytosolic and (to a lesser extent) mitochondrial Ca(2+) levels were elevated after photodynamic therapy, these changes were apparently insufficient to contribute to the development of apoptosis.

Asunto(s)

Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Fármacos Fotosensibilizantes/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rojo de Rutenio/farmacología , Animales , Apoptosis , Western Blotting , Relación Dosis-Respuesta a Droga , Leucemia L1210/radioterapia , Potenciales de la Membrana , Ratones , Mitocondrias/fisiología , Fotoquimioterapia , Células Tumorales Cultivadas

RESUMEN

Ursodeoxycholic acid (UDCA), a relatively nontoxic bile acid, enhanced the apoptotic response of tumor cells to both photosensitizers that cause photodamage to Bcl-2 and to the nonpeptidic Bcl-2/Bcl-x(L) antagonist HA14-1. The latter agent binds to the surface pocket formed by the BH1, BH2 and BH3 domains of Bcl-2 and Bcl-x(L). Fluorescence polarization studies indicated that affinity of HA14-1 for Bcl-2 was enhanced in the presence of UDCA. Moreover, Bcl-2 photodamage was promoted by UDCA using a photosensitizing agent with affinity for the endoplasmic reticulum, a site of Bcl-2 localization. Fluorescence resonance energy transfer (FRET) studies revealed that the proximity of Bcl-2 to a hydrophobic photosensitizing agent embedded in liposomes was enhanced by UDCA. Since photodamage will occur only if a protein is in close contact with a photosensitizing agent, we propose that these findings support the hypothesis that UDCA causes a conformational change in Bcl-2, promoting HA14-1 binding and enhancing affinity for certain membrane-bound photosensitizers.

Asunto(s)

Apoptosis/fisiología , Benzopiranos/farmacología , Retículo Endoplásmico/metabolismo , Nitrilos/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Ácido Ursodesoxicólico/farmacología , Animales , Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/farmacología , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Polarización de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Liposomas/química , Ratones , Conformación Molecular , Fármacos Fotosensibilizantes/farmacología , Proteínas Proto-Oncogénicas c-bcl-2 , Células Tumorales Cultivadas

RESUMEN

Photodynamic therapy (PDT) protocols employing lysosomal sensitizers induce apoptosis via a mechanism that causes cytochrome c release prior to loss of mitochondrial membrane potential (DeltaPsi(m)). The current study was designed to determine how lysosomal photodamage initiates mitochondrial-mediated apoptosis in murine hepatoma 1c1c7 cells. Fluorescence microscopy demonstrated that the photosensitizer N-aspartyl chlorin e6 (NPe6) localized to the lysosomes. Irradiation of cultures preloaded with NPe6 induced the rapid destruction of lysosomes, and subsequent cleavage/activation of Bid, pro-caspases-9 and -3. Pro-caspase-8 was not activated. Release of cytochrome c occurred at about the time of Bid cleavage and preceded the loss of DeltaPsi(m). Extracts of purified lysosomes catalyzed the in vitro cleavage of cytosolic Bid, but not pro-caspase-3 activation. Pharmacological inhibition of cathepsin B, L and D activities did not suppress Bid cleavage or pro-caspases-9 and -3 activation. These studies demonstrate that photodamaged lysosomes trigger the mitochondrial apoptotic pathway by releasing proteases that activate Bid.

Asunto(s)

Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Precursores Enzimáticos/metabolismo , Lisosomas/enzimología , Mitocondrias/enzimología , Neoplasias/terapia , Fotoquimioterapia , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/efectos de los fármacos , Caspasa 8 , Caspasa 9 , Caspasas/efectos de los fármacos , Catepsina D/efectos de los fármacos , Catepsina D/metabolismo , Extractos Celulares/farmacología , Grupo Citocromo c/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Neoplasias/enzimología , Neoplasias/fisiopatología , Porfirinas/farmacología , Porfirinas/efectos de la radiación , Células Tumorales Cultivadas

RESUMEN

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances or suppresses the transcriptional activation of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a cell/tissue-specific manner. The basis for these effects is not known. Exposure of the immortalized human breast epithelial cell line MCF10A-Neo to TPA at the time of, or up to 12 hr prior to, the addition of TCDD strongly suppressed the transcriptional activation of CYP1A1 and CYP1B1 (IC(50) approximately 0.5 nM). A recent study (Carcinogenesis 2000;21:1303-12) demonstrated that TPA-treated MCF10A-Neo cells rapidly activate the latent transforming growth factor beta (TGFbeta) in the serum used to supplement the culture medium. The suppressive effects of TPA on CYP1A1 induction by TCDD in MCF10A-Neo cultures could be partially suppressed by: (a) co-incubation of TCDD + TPA-treated cultures with a neutralizing TGFbeta pan antibody; (b) prior removal of latent TGFbeta from the culture medium; or (c) switching cultures to serum- and growth factor-free medium immediately before the addition of TPA and TCDD. Exposure of cultures to TPA 24-48 hr prior to subsequent TPA + TCDD treatment not only inhibited the suppressive effects of TPA, but markedly enhanced CYP1A1 mRNA accumulation. TPA caused a rapid and protracted activation of extracellular signal-regulated kinases (ERKs). Pretreatment of cultures with the mitogen-activated protein kinase kinase (MEK) inhibitor PD184352 [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropyl-methoxy-3,4-difluoro-benzamide] completely inhibited ERK activation by TPA. However, PD184352 did not prevent the suppressive effects of TPA on CYP1A1 activation by TCDD. These studies demonstrate that TPA initiates protein kinase C-dependent, ERK-independent processes that suppress CYP1A1 activation by TCDD in MCF10A-Neo cells. Furthermore, TGFbeta mediates a small portion of this suppressive activity.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP1A1/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/fisiología , Dibenzodioxinas Policloradas/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador beta/fisiología , Benzamidas/farmacología , Carcinógenos/farmacología , Células Cultivadas , Citocromo P-450 CYP1A1/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Interacciones Farmacológicas , Activación Enzimática , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Activación Transcripcional/efectos de los fármacos

RESUMEN

T cells are believed to be critical functional targets of Pb immunotoxicity. In this study, low concentrations of lead (i.e., as low as 0.1 microM approximately 2 microg/dl) were found to markedly enhance the allogeneic mixed lymphocyte reaction-an assay of CD4(+) T cell responsiveness. Cell cycle analysis of cells recovered from allogeneic mixed lymphocyte cultures revealed that Pb stimulated a substantial increase in the proportion of cycling alloreactive CD4(+) T cells. The enhanced alloproliferative response was characterized by an increased population of lymphoblasts expressing heightened cell surface expression of CD4 (i.e., CD4(high) cells). Successive rounds of cell division were monitored using the cell division dye 5- (and 6)-carboxyfluorecein diacetate succinimyl ester and it was determined that the CD4(high) subpopulation comprised the expanding alloreactive T cells, which ultimately took on the phenotype of memory/effector T cells (i.e., CD44(high), CD45RB(low), CD69(high), and CD162(high)). Enhancement of T cell proliferation by lead was selective for responsiveness to alloantigen, as lead had no effect on T cell proliferation induced by mitogens or superantigen, processes that unlike alloreactivity are not dependent on antigen presentation. Collectively, these data suggest that Pb enhances alloantigen-specific T cell proliferation through an indirect mechanism involving altered antigen processing/presentation, resulting in marked clonal expansion or repertoire expansion of alloreactive T cell clones. Consistent with this suggestion was the finding that a single exposure to Pb during alloantigen priming elicited a population of CD4(+) T cells that was hyperresponsive to further alloantigen stimulation and neither lead dependent nor lead responsive.

Asunto(s)

Linfocitos T CD4-Positivos/efectos de los fármacos , Isoantígenos/inmunología , Plomo/toxicidad , Animales , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Recuento de Células , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Citometría de Flujo , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/inmunología , Inmunofenotipificación , Plomo/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos , Mitógenos/inmunología , Mitógenos/farmacología , Superantígenos/inmunología , Superantígenos/farmacología

RESUMEN

Analyses of CYP1A1 mRNA were used to monitor the responsiveness of murine hepatoma 1c1c7 and human monocytic U937 cells in different phases of the cell cycle to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Concentrations of TCDD capable of inducing CYP1A1 were not cytostatic to either cell line. Steady-state CYP1A1 mRNA contents were reduced (45-90%) in TCDD-treated cultures arrested in G2/M as a consequence of exposure to microtubule disrupters (Colcemid, estramustine, vinblastine) or the microtubule stabilizer Taxol, relative to TCDD-treated asynchronous 1c1c7 cultures. The accumulation of mRNAs corresponding to Nmo1, another TCDD-inducible gene of the Ah battery, was also reduced in TCDD-treated G2/M cultures. Quantitative reverse transcriptase-polymerase chain reaction analyses of CYP1A1 heterogeneous nuclear RNA (hnRNA) revealed that Cyp1a1 transcription was suppressed in G2/M cells. This suppression reflected neither changes in the relative content of the proteins comprising the aryl hydrocarbon receptor (AHR) complex nor a suppression of AHR activation and translocation to the nucleus. Release of 1c1c7 cultures arrested in G2/M restored TCDD responsiveness. Centrifugal elutriation of TCDD-treated asynchronously growing U937 cells was used to prepare populations of cells in specific phases of the cell cycle. Within 3 h of TCDD exposure late G1/early S phase cells had CYP1A1 mRNA contents approximately 1.4- and 3-fold higher than the contents of asynchronous/early G1 and G2/M cultures, respectively. These studies suggest that the transcriptional activation of members of the Ah battery by TCDD is cell cycle-dependent, and markedly suppressed in G2/M cells.

Asunto(s)

Citocromo P-450 CYP1A1/biosíntesis , Dioxinas/farmacología , Western Blotting , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/efectos de los fármacos , Centrifugación , Cromosomas/efectos de los fármacos , Cromosomas/ultraestructura , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Ácido Orótico/metabolismo , Dibenzodioxinas Policloradas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Nuclear/biosíntesis , Receptores de Hidrocarburo de Aril/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

While some epidemiological risk factors for breast cancer have been identified, the environmental factors responsible for transformation of mammary epithelial cells are not clear. We have exposed the spontaneously immortalized human mammary epithelial cell line MCF-10A to benzo[a]pyrene and selected transformed clones based on a loss of contact inhibition and anchorage-dependent growth. Cytogenetic studies showed that each of the transformed sublines possess an isochromosome 8q aberration. The c-Myc proto-oncogene, which is positioned at 8q24, was analyzed for changes in expression. Both c-Myc mRNA and protein levels were increased in the transformed clones relative to the parental cells. The transformed clones were not able to grow as tumors in vivo when injected into nude or SCID mice. To determine whether the involvement of chromosome 8 in BP-induced mutagenesis was a reproducible event, transformed clones were selected from three additional independently treated sets of BP-exposed MCF-10A cultures and analyzed by spectral karyotyping (SKY). These transformed sublines also harbored the isochromosome 8q abnormality. Data from this model show that benzo[a]pyrene, a ubiquitous procarcinogen, can induce selectable morphologic changes in a human mammary epithelial cell line, and that these transformed cells possess chromosomal aberrations frequently found in human breast tumors.

Asunto(s)

Benzo(a)pireno/efectos adversos , Mama/patología , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 8/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Animales , Mama/efectos de los fármacos , Pruebas de Carcinogenicidad , Línea Celular , Línea Celular Transformada/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Aberraciones Cromosómicas/genética , Cromosomas Humanos Par 8/genética , Células Clonales , Análisis Citogenético , Análisis Mutacional de ADN , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Genes myc/genética , Humanos , Ratones , Proto-Oncogenes Mas , ARN Mensajero/metabolismo

RESUMEN

Arsenite treatment has been found to induce clinical remission in patients with acute promyelocytic leukemia. Although the potential therapeutic value of arsenite may lie in triggering apoptosis, it has not been established that cytotoxicity is the sole mechanism of action. We have used a myelomonocytic leukemia cell line (U937) to characterize the concentration-dependent effects of arsenite on cell growth, viability, apoptosis, and differentiation. Arsenite has multiple effects on U937 cells. Low concentrations of arsenite (i.e., < or = 1 microM) potentiate vitamin-D(3)-induced differentiation. Two markers of monocyte differentiation, Mac-1 expression and nitroblue tetrazolium reduction, are increased in arsenite-exposed, D(3)-costimulated cells. Concentrations of arsenite >10 microM rapidly induce the death of cells irrespective of cell cycle phase. Intermediate concentrations of arsenite (i.e., 5 to 10 microM) are cytostatic initially. Cell cycle analysis using elutriated, synchronous cell populations revealed that intermediate concentrations of arsenite delay both G(1) and G(2) transit. G(2) cells appear to be most sensitive to arsenite, in that transit through G(2)/M is more delayed than transit through G(1), and apoptosis is induced in these cells as they emerge from an aberrant G(2)/M. Arsenite-induced apoptosis was caspase-3 dependent. Arsenite-mediated cytotoxicity was reduced in the presence of the broad caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone; however, caspase inhibition did not reverse arsenite-induced cytostasis. Thus, arsenite has multiple effects on U937 cells that are dependent on concentration and cell cycle phase. Specifically, cell cycle transit and differentiation are more sensitive to arsenite than is the induction of apoptosis.

Asunto(s)

Apoptosis/efectos de los fármacos , Arsenitos/farmacología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Apoptosis/fisiología , Calcitriol/farmacología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Factores de Tiempo , Células U937

RESUMEN

Numerous flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and function as AHR antagonists and/or agonists. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] is a widely used inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), and is structurally related to members of the flavonoid family. Concentrations of LY294002 >/= 10 microM were cytostatic, but not cytotoxic, to cultures of the immortalized human breast epithelial cell line MCF10A-Neo. Treatment of MCF10A-Neo cultures with the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) stimulated the transcriptional activation of CYP1A1, as monitored by measurements of steady-state CYP1A1 mRNA. Pretreatment of cultures with >/= 10 microM LY294002 suppressed the TCDD activation of CYP1A1 (IC(50) approximately 10 microM). Electrophoretic mobility shift assays employing rat liver cytosol demonstrated that concentrations of LY294002 /= 50 microM and Wortmannin >/= 10 nM completely suppressed PI 3-kinase activity. Hence, the ability of LY294002 to suppress TCDD-dependent activation of CYP1A1 is unrelated to PI 3-kinase inhibition. Instead, this activity reflects LY294002 functioning as an AHR antagonist. Furthermore, most of the cytostatic activity of LY294002 towards MCF10A-Neo cells is unrelated to the inhibition of PI 3-kinase.

Asunto(s)

Cromonas/farmacología , Citocromo P-450 CYP1A1/biosíntesis , Inhibidores Enzimáticos/farmacología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Dibenzodioxinas Policloradas/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Androstadienos/farmacología , División Celular/efectos de los fármacos , Línea Celular Transformada , Citocromo P-450 CYP1A1/genética , ADN/efectos de los fármacos , ADN/metabolismo , Interacciones Farmacológicas , Inducción Enzimática , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Hidrocarburo de Aril/fisiología , Activación Transcripcional/efectos de los fármacos , Wortmanina

RESUMEN

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGF beta supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

Asunto(s)

Antineoplásicos/farmacología , Mama/efectos de los fármacos , Mama/metabolismo , Genes ras/fisiología , Proteínas Proto-Oncogénicas c-mos/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Antineoplásicos/antagonistas & inhibidores , Aprotinina/farmacología , Mama/citología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Transformada , Medios de Cultivo Condicionados , Interacciones Farmacológicas , Humanos , Proteína Oncogénica p21(ras)/biosíntesis , Proteína Oncogénica p21(ras)/fisiología , Inhibidor 1 de Activador Plasminogénico/farmacología , Receptores de Estrógenos/fisiología , Inhibidores de Serina Proteinasa/farmacología , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Transformación Genética , Factor de Crecimiento Transformador beta/farmacología

RESUMEN

Cultured cells are commonly exposed to trypsin-containing solutions in order to prepare cell suspensions suitable for subculture. Conditions used to release and disperse monolayers of cultured murine hepatoma 1c1c7 and human breast epithelial MCF10A cells caused the loss (40-95%) of cellular glutathione (GSH), but did not affect viability. Glutathione contents returned to pretrypsinization values within 24 h of replating. In contrast, the GSH contents of cultured rat hepatoma 5L cells were not affected by trypsinization. Exposure of 1c1c7 cultures to H(2)O(2) or etoposide 1 or 24 h after replating resulted in concentration-dependent cytostatic and cytotoxic effects. The concentration-response curves defining the cytostatic and cytotoxic effects of etoposide, and the cytostatic effects of H(2)O(2) were not influenced by the timing of toxicant addition. However, 1c1c7 cultures treated with H(2)O(2) 1 h after replating were more susceptible to the cytotoxic actions of the peroxide than cultures treated 24 h after plating. These studies show that conditions commonly used for the passaging of cultured cells can lead to a transient, but profound loss of GSH in some cell lines. Furthermore, the outcome of cytotoxicity analyses can be influenced by the time elapsed between the plating of cultures and the addition of toxicant.

Asunto(s)

Glutatión/metabolismo , Células Tumorales Cultivadas/patología , Animales , Antineoplásicos/toxicidad , Neoplasias de la Mama/patología , Carcinoma Hepatocelular/patología , Comunicación Celular , Técnicas de Cultivo de Célula/métodos , Muerte Celular , Etopósido/toxicidad , Femenino , Glutatión/farmacocinética , Humanos , Peróxido de Hidrógeno/toxicidad , Neoplasias Hepáticas/patología , Oxidantes/toxicidad , Ratas , Pruebas de Toxicidad

RESUMEN

Studies with mouse leukemia L1210 cells revealed that selective lysosomal photodamage caused by any of three photosensitizing agents was followed by a gradual loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol, increased DEVDase activity (a measure of levels of caspase-3) and a limited apoptotic response. Similar effects were observed in the murine hepatoma 1c1c7 cell line. Immunofluorescence techniques employing 1c1c7 cells demonstrated the immediate release of the lysosomal enzyme cathepsin B following lysosomal photodamage. These studies suggest that the cytotoxic effects of lysosomal photodamage are initiated by released lysosomal proteases that either directly and/or indirectly activate caspases as a consequence of the induction of mitochondrial damage.

Asunto(s)

Apoptosis/efectos de los fármacos , Lisosomas/efectos de los fármacos , Fármacos Fotosensibilizantes/toxicidad , Animales , Apoptosis/efectos de la radiación , Catepsina B/análisis , Membranas Intracelulares/efectos de los fármacos , Leucemia L1210 , Luz , Neoplasias Hepáticas Experimentales , Lisosomas/enzimología , Lisosomas/efectos de la radiación , Potenciales de la Membrana/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Células Tumorales Cultivadas

RESUMEN

Ursodeoxycholic acid (UDCA) protects cells from the apoptotic effects of hydrophobic bile acids and some other cytotoxic agents. We observed the opposite result when assessing the effects of UDCA on the apoptotic response to mitochondrial photodamage induced by photodynamic therapy (PDT). Two photosensitizers with predominantly mitochondrial specificity were used: a porphycene we have designated CPO; and the tin etiopurpurin SnET2. UDCA potentiated the loss of mitochondrial potential, release of cytochrome c into the cytosol, activation of caspase-3, and apoptotic cell death after irradiation of photosensitized murine leukemia L1210 or hepatoma 1c1c7 cells. These effects were not observed when UDCA was added after irradiation. Glyco-UDCA and tauro-UDCA, conjugated forms of UDCA that are formed in vivo, were as effective as UDCA in promoting PDT phototoxicity. Because UDCA does not act by enhancing intracellular accumulation of the photosensitizing agents used in this study, we propose that the mode of action of UDCA involves the sensitization of mitochondrial membranes to photodamage. UDCA is used currently in gastroenterology for several indications. The drug may offer a means for promoting the efficacy of PDT with minimal adverse effects.

Asunto(s)

Fotoquimioterapia , Ácido Ursodesoxicólico/análogos & derivados , Ácido Ursodesoxicólico/uso terapéutico , Animales , Apoptosis , Western Blotting , Caspasa 3 , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Colagogos y Coleréticos/uso terapéutico , Grupo Citocromo c/metabolismo , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Activación Enzimática , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/efectos de la radiación , Ratones , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Mitocondrias/efectos de la radiación , Células Tumorales Cultivadas

RESUMEN

Some flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and cause cell cycle arrest. The dependency of the cytostatic effects of five flavonoids (flavone, alpha-naphthoflavone, apigenin, 3'-methoxy-4'-nitroflavone and 2'-amino-3'-methoxyflavone) on a functional AHR was examined in AHR-containing rat hepatoma 5L cells and an AHR-deficient cell line (BP8) derived from the 5L line. The potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was cytostatic to the 5L line due to the induction of a G(1) arrest and dramatically elevated steady-state levels of CYP1A1 mRNA. TCDD affected neither the proliferation nor CYP1A1 mRNA contents of BP8 cells. With the exception of apigenin, the flavonoids under study induced G(1) arrest in both 5L and BP8 cells when used at concentrations at which they functioned as AHR agonists, but not antagonists. Apigenin-treated 5L and BP8 cultures primarily arrested in G(2)/M. The AHR-containing murine hepatoma cell line 1c1c7 arrested following exposure to AHR agonist concentrations of flavone and alpha-naphthoflavone, but not TCDD. Unlike the G(1) arrest observed in 5L cultures, the latter two flavonoids caused principally G(2)/M arrest in 1c1c7 cells. These studies demonstrate that the cytostatic activities of flavonoids do not require the AHR and the site of checkpoint arrest with a specific flavonoid can vary with cell type.

Asunto(s)

Ciclo Celular/efectos de los fármacos , Contaminantes Ambientales/farmacología , Flavonoides/farmacología , Dibenzodioxinas Policloradas/farmacología , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Animales , Benzoflavonas/química , Benzoflavonas/metabolismo , Benzoflavonas/farmacología , División Celular/efectos de los fármacos , Manzanilla , Citocromo P-450 CYP1A1/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Contaminantes Ambientales/metabolismo , Flavonas , Flavonoides/química , Flavonoides/metabolismo , Fase G1/efectos de los fármacos , Aceites Volátiles/química , Aceites Volátiles/metabolismo , Aceites Volátiles/farmacología , Plantas Medicinales , Dibenzodioxinas Policloradas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Receptores de Hidrocarburo de Aril/metabolismo , Células Tumorales Cultivadas

RESUMEN

The relationship between aryl hydrocarbon receptor (AHR) content and susceptibility to apoptosis was examined in the murine hepatoma 1c1c7 cell line and a series of variants having different levels of AHR expression. Exposure of 1c1c7 cultures to N-acetylsphingosine (C2-ceramide) caused a concentration-dependent inhibition of cell proliferation, loss of viability, and induction of apoptosis as monitored by analyses of DNA fragmentation and caspase activation. A variant cell line (Tao) having approximately 10% of the AHR content of 1c1c7 cells also arrested following exposure to C2-ceramide, but did not undergo apoptosis. Modulation of 1c1c7 and Tao AHR contents by transfection of Ahr antisense and sense constructs, respectively, confirmed the relationship between AHR content and susceptibility to C2-ceramide-induced apoptosis. C2-ceramide also induced the apoptosis of an AHR-containing cell line lacking the aryl hydrocarbon receptor nuclear translocator protein. AHR ligands (i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin and alpha-naphthoflavone) neither induced apoptosis nor modulated the development of apoptosis in C2-ceramide-treated 1c1c7 cultures. AHR content did not affect staurosporine- or doxorubicin-induced apoptosis. These results suggest the AHR modulates aspects of ceramide signaling associated with the induction of apoptosis but not cell cycle arrest, and does so by a mechanism that is independent of its interaction with aryl hydrocarbon receptor nuclear translocator and exogenous AHR ligands.

Asunto(s)

Apoptosis/efectos de los fármacos , Proteínas de Unión al ADN , Neoplasias Hepáticas Experimentales/patología , Receptores de Hidrocarburo de Aril/fisiología , Esfingosina/análogos & derivados , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Doxorrubicina/farmacología , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Esfingosina/farmacología , Estaurosporina/farmacología , Factores de Transcripción/fisiología , Células Tumorales Cultivadas

RESUMEN

The effect of oral administration of lead, as Pb-acetate, via the drinking water on the murine delayed type hypersensitivity (DTH) response was investigated. The DTH response of BALB/c mice sensitized intravenously with sheep red blood cells (SRBC) was found to be suppressed markedly in lead-intoxicated mice. Suppression of the DTH correlated with increasing blood Pb concentration. Suppression of the DTH response by Pb intoxication depended on the route of administration of the sensitizing antigen, as Pb intoxication did not impair the DTH reaction when mice were sensitized to SRBC via intraperitoneal injection. Since DTH reactions are regulated in large part by Th1 cells, these data establish an in vivo model system based on a rational route of Pb exposure in which to study further the modulation of Th1-mediated immune effector function by Pb.

Asunto(s)

Hipersensibilidad Tardía/inmunología , Plomo/toxicidad , Células TH1/efectos de los fármacos , Células TH1/inmunología , Administración Oral , Animales , Relación Dosis-Respuesta Inmunológica , Eritrocitos/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Ovinos , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología

RESUMEN

PD98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] is a flavonoid and a potent inhibitor of mitogen-activated protein kinase kinase (MEK). Concentrations of PD98059 of /=10 microM. In vivo exposure of cultures to 95%) of the dually phosphorylated forms of extracellular signal-regulated kinase (IC50 = 1 muM). Treatment of cultures with PD98059 of >/=1 muM either at the time of addition or up to 48 hr before the addition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed in a concentration-dependent manner the accumulation of induced steady state CYP1A1, CYP1B1, and NQO1 mRNAs. The addition of PD98059 to rat liver cytosol just before the addition of TCDD suppressed TCDD binding (IC50 = 4 muM) and aryl hydrocarbon receptor (AHR) transformation (IC50 = 1 muM), as measured by sucrose gradient centrifugation and electrophoretic mobility shift assays. Flavone and flavanone, two closely related structural analogs of PD98059, inhibited AHR transformation by TCDD with IC50 values similar to that obtained with PD98059. However, neither analog was as potent as PD98059 in inhibiting MEK (IC50 approximately 190 muM for both). These results suggest that PD98059 is a ligand for the AHR and functions as an AHR antagonist at concentrations commonly used to inhibit MEK and signaling processes that entail MEK activation.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Inhibidores de Proteínas Quinasas , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Animales , Unión Competitiva , Línea Celular , ADN/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Dibenzodioxinas Policloradas/metabolismo , Dibenzodioxinas Policloradas/farmacología , Ratas , Receptores de Hidrocarburo de Aril/fisiología , Activación Transcripcional/efectos de los fármacos

RESUMEN

Products of several phase I and II genes transcriptionally activated by ligands of the aryl hydrocarbon receptor (AHR) were quantitated in cutaneous samples isolated from non-tumor-bearing SENCAR or SSIN mice, and animals bearing skin tumors generated in initiation-promotion protocols. The constitutive 7-ethoxyresorufin O-deethylase (EROD) activities in papillomas and squamous cell carcinomas were less than or equal to 37% of the values measured in the adjacent normal cutaneoustissue. Dermal and epidermal EROD specific activities in microsomal samples prepared from both tumor-bearing and non-tumor-bearing mice were elevated 9- to 14- and 43- to 77-fold, respectively, above constitutive levels 16-20 h after a single topical application of 100 nmol of dibenz[a,c]anthracene (DB[a,c]A). EROD specific activities in tumors were maximally elevated two-fold after topical application of DB[a,c]A. Western blot, northern blot, and reverse transcription (RT)-polymerase chain reaction (PCR) analyses confirmed that the EROD measurements reflected cutaneous cytochrome P450 (CYP) 1A1 protein, mature mRNA, and heterogeneous nuclear RNA contents, respectively. Analyses of CYP1A1, CYP1B1, cytosolic aldehyde dehydrogenase class 3, and NAD(P)H:menadione oxidoreductase (NMO1) mRNA content by RT-PCR revealed significant increases in all four mRNAs in the normal tissue adjacent to papillomas after exposure to 4 nmol of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but no increases in the tumors. NMO1 mRNA content in acetone-treated papillomas approached the levels detected in TCDD-treated normal skin. RT-PCR analyses also demonstrated elevated constitutive aryl hydrocarbon receptor nuclear translocator mRNA content (an approximately two-fold increase) in skin tumors. In contrast, AHR mRNA content in the tumors was about 20% of that measured in adjacent normal tissue. Collectively, these studies demonstrated that ligand-induced, AHR-mediated processes are absent in murine skin tumors that develop in initiation-promotion protocols.

Asunto(s)

Aldehído Deshidrogenasa/genética , Hidrocarburo de Aril Hidroxilasas , Carcinoma de Células Escamosas/genética , Citocromo P-450 CYP1A1/genética , Sistema Enzimático del Citocromo P-450/genética , Proteínas Quinasas Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/genética , Receptores de Hidrocarburo de Aril/fisiología , Neoplasias Cutáneas/genética , Animales , Benzo(a)Antracenos/farmacología , Carcinógenos , Citocromo P-450 CYP1B1 , Elementos de Facilitación Genéticos , Inducción Enzimática , Epidermis/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Ligandos , Ratones , Papiloma/genética , Dibenzodioxinas Policloradas/farmacología , ARN Mensajero/genética , ARN Neoplásico/genética

RESUMEN

Primary murine keratinocytes were cultured in a chemically defined, serum-free medium which facilitated manipulation of their differentiation status. Exposure of basal cell and differentiating cultures to >/= 0.1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) preferentially elevated 7-ethoxyresorufin O-deethylase specific activities in differentiating cultures (28-fold versus 4-fold increases after 36 h of exposure). Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analyses demonstrated the presence of constitutive mRNA transcripts corresponding to four known TCDD-inducible genes (e.g., Cyp1a1, Cyp1b1, Ahd4, and Nmo1) in both differentiating and proliferating cultures of murine keratinocytes. All four genes were induced in differentiating cultures following exposure to TCDD. No induction occurred in comparably treated basal cell cultures. Indirect immunofluorescence analyses demonstrated the presence of aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins in both basal and differentiating keratinocytes. Both proteins appeared to be associated with the nucleus and their nuclear association was independent of prior exposure to TCDD. These studies suggest that AHR activation in murine skin is regulated as a function of the keratinocyte differentiation program.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Queratinocitos/efectos de los fármacos , Dibenzodioxinas Policloradas/farmacología , Piel/efectos de los fármacos , Aldehído Deshidrogenasa/biosíntesis , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Compartimento Celular , Diferenciación Celular , Núcleo Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/biosíntesis , Femenino , Ratones , Ratones Endogámicos SENCAR , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Receptores de Hidrocarburo de Aril/aislamiento & purificación , Receptores de Hidrocarburo de Aril/metabolismo , Piel/citología , Factores de Transcripción/aislamiento & purificación , Transcripción Genética

RESUMEN

SSIN mice are very sensitive to tumor promoters in two-stage skin carcinogenesis protocols. It was recently reported that SSIN mice have fewer CD8+ T-cells than other strains of mice and develop a weaker cytotoxic T-cell response upon challenge with an allogeneic tumor transplant. The significance of this muted immune response to processes involved in two-stage carcinogenesis depends on the immunogenicity of the tumors generated in such protocols. Although they have low CD8+ T-cell contents, SSIN rejected a variety of subcutaneously transplanted allogeneic murine tumors. Analyses of the growth of primary papillomas derived from 7,12-dimethylbenz[a]anthracene-initiated/12-O-tetradecanoylphorbol-13-ac etate-promoted SSIN mice and then subcutaneously transplanted into triple-deficient (bg-nu-xid), athymic nude and immune-competent and immunosuppressed SSIN mice revealed that few tumors took and tumor takes were not markedly influenced by the immumological status of the transplant recipient. Two tumor cell lines (RS1 and RS2) were derived from the transplantation studies and could be passaged in normal SSIN mice (H-2q haplotype). Both tumors were squamous cell carcinomas (SCCs) by the second in vivo passage and were rejected in allogeneic mice (BALB/c) but grew in FVB/N mice, a strain having the H-2q haplotype. Transplantation studies revealed that prior exposure to RS1 and RS2 did not prime SSIN mice to reject a subsequent tumor challenge. Three primary SCC tumors derived from SSIN mice in a two-stage carcinogenesis protocol also grew when subcutaneously transplanted in SSIN mice and could be serially passaged. Consequently, the epidermal SCCs that develop in two-stage carcinogenesis protocols appear to be nonimmunogenic.

Asunto(s)

Carcinoma de Células Escamosas/inmunología , Cocarcinogénesis , Papiloma/inmunología , Neoplasias Cutáneas/inmunología , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinógenos , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/patología , División Celular/fisiología , Femenino , Rechazo de Injerto/inmunología , Ratones , Ratones Endogámicos SENCAR , Ratones Desnudos , Trasplante de Neoplasias/inmunología , Papiloma/inducido químicamente , Papiloma/patología , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol , Células Tumorales Cultivadas