RESUMEN
Proper functioning of trophoblastic cells is essential for maintenance of the placenta and development of the embryo/fetus. Exposure of trophoblasts to toxic exogenous factors, such as cadmium (Cd), perturbs placental function and affects fetal outcome. Cellular responses to Cd exposure include induction of the metal-binding protein, metallothionein (MT), and initiation of apoptosis. To analyze the functional relationship between cellular MT levels and apoptosis in trophoblasts, we have examined the effects of DNA transfection-mediated alterations in MT levels on trophoblastic function and apoptosis, with and without Cd exposure, using the trophoblast-like JEG-3 human choriocarcinoma cell line. JEG-3 cells stably transfected with human MT-IIa cDNA expression constructs, in either sense or antisense orientation, were unchanged in human chorionic gonadotropin (hCG) production or expression of the apoptotic markers, bcl-2 and CPP-32. However, MT overexpression significantly prolonged the recovery time of intracellular Ca flux, whereas reduced basal MT increased the incidence of apoptosis as determined by morphology and terminal deoxynucleotidyl end labeling (TUNEL) staining. Upon Cd exposure, a dose-dependent decrease in hCG secretion was seen in all JEG-3 cultures, without any correlation to basal MT expression. Basal MT levels, however, significantly affected the extent of apoptosis, the incidence being inversely related to basal MT level. These results suggest that while MT does not ameliorate heavy-metal induced perturbation of some trophoblastic functions, its expression is critical for protection of these cells from Cd-induced apoptosis and could act to maintain placental integrity in cases of maternal Cd exposure.
Asunto(s)
Apoptosis/efectos de los fármacos , Cadmio/toxicidad , Metalotioneína/biosíntesis , Trofoblastos/metabolismo , Caspasa 3 , Caspasas/análisis , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Gonadotropina Coriónica/análisis , Medios de Cultivo Condicionados/química , Daño del ADN , Relación Dosis-Respuesta a Droga , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Etiquetado Corte-Fin in Situ , Metalotioneína/genética , Proteínas Proto-Oncogénicas c-bcl-2/análisis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Trofoblastos/efectos de los fármacos , Trofoblastos/patología , Células Tumorales CultivadasRESUMEN
Oxidative stress induces cellular apoptosis. Many agents producing intracellular oxidative stress, including H(2)O(2) and steroid hormones, have also been found to induce metallothionein (MT) expression. Recently, MT has been recognized as potentially having antioxidant activity. This action may be essential for survival of terminally differentiated cells subject to oxidative stress, such as syncytiotrophoblasts, placental cells producing pregnancy hormones and forming the maternal-fetal barrier. We previously demonstrated an inverse relationship between basal MT expression and apoptotic incidence in the trophoblastic cell line, JEG-3. Using JEG-3 cells transfected with MT in sense or antisense orientation, we have examined here the effect of altered basal MT levels on trophoblastic function and apoptosis following treatment with H(2)O(2) or diethylstilbestrol (DES). Induction of MT mRNA was observed in control and transfected JEG-3 cells following exposure to severe oxidative stress. Changes in the localization of MT protein, however, were apparent after a low oxidative stress challenge. Exposure to H(2)O(2) resulted in a dose-dependent decrease in human chorionic gonadotropin secretion in all JEG-3 cultures regardless of basal MT expression, whereas no change was detected following DES treatment. With respect to apoptosis, a significant protective effect was observed proportional to the basal MT level. These results suggest that although MT does not ameliorate oxidative stress-induced perturbation of some trophoblastic functions, its expression is critical for protection of these cells from severe oxidative stress-induced apoptosis. MT thus appears to act as an anti-apoptotic antioxidant in trophoblastic cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Dietilestilbestrol/toxicidad , Peróxido de Hidrógeno/toxicidad , Metalotioneína/biosíntesis , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/metabolismo , Línea Celular , Humanos , Técnicas In Vitro , Metalotioneína/genética , Estrés Oxidativo/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Transfección , TrofoblastosRESUMEN
A murine monoclonal antibody (7E3) directed against the platelet glycoprotein IIb/IIIa was engineered to reduce immunogenicity by substituting human for murine constant regions. The chimeric antibody is functionally identical to the murine antibody in vitro. Results from clinical trials with 7E3 Fab antibody fragments, however, show that the 7E3 variable region, which elicits the vast majority of the immune response to murine 7E3 Fab, is rendered dramatically less immunogenic (incidence reduced from 17% to 1%) when the identical variable region is linked to human rather than murine constant regions. Neither murine nor human constant regions were highly immunogenic themselves. We conclude that the constant regions of the Fab fragments are critical in modulating the immune response elicited by the linked 7E3 variable region. Because naturally occurring anti-human Fab fragment antibodies are prevalent both in the normal human population and in the patient population studied here, murine 7E3 Fab and chimeric 7E3 Fab may be fundamentally different in their interactions with the human immune system. This difference may be related to the dramatic difference in immunogenicity observed between murine 7E3 Fab and chimeric 7E3 Fab.