RESUMEN
The retinoblastoma protein (RB) is essential for normal cell cycle control. RB function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors (E2Fs). To study the role of RB in the adult testis, a Sertoli cell (SC)-specific Rb knockout mouse line (SC-RbKO) was generated using the Cre/loxP recombination system. SC-RbKO mice exhibited an age-dependent testicular atrophy, impaired fertility, severe SC dysfunction, and spermatogenic defects. Removal of Rb in SC induced aberrant SC cycling, dedifferentiation, and apoptosis. Here we show that E2F3 is the only E2F expressed in mouse SCs and that RB interacts with E2F3 during mouse testicular development. In the absence of RB, the other retinoblastoma family members p107 and p130 began interacting with E2F3 in the adult testes. In vivo silencing of E2F3 partially restored the SC maturation and survival as well as spermatogenesis in the SC-RbKO mice. These results point to RB as a key regulator of SC function in adult mice and that the RB/E2F3 pathway directs SC maturation, cell cycle quiescence, and RB protects SC from apoptosis.
Asunto(s)
Diferenciación Celular/fisiología , Factor de Transcripción E2F3/metabolismo , Proteína de Retinoblastoma/metabolismo , Células de Sertoli/metabolismo , Animales , Apoptosis/genética , Factor de Transcripción E2F3/genética , Silenciador del Gen , Masculino , Ratones , Ratones Noqueados , Proteína de Retinoblastoma/genética , Células de Sertoli/citologíaRESUMEN
We have produced over the years several genetically modified mouse models (transgenic [TG], knockout [KO] and knockin [KI]) for the study of normal and aberrant functions of gonadotrophins and their receptors. We summarise in the present review some of our recent findings on these animal models. One is the cascade of extragonadal phenotypes triggered by ovarian hyperstimulation in TG mice overexpressing the human choriongonadotrophin (hCG) beta-subunit and presenting with elevated levels of serum luteinising hormone (LH)/hCG bioactivity. Massively elevated levels of serum progesterone, rather than oestrogens, are responsible for the induction of pituitary prolactinomas and the subsequently elevated prolactin (PRL) levels. Along with normal oestradiol and elevated progesterone levels, the increased concentration of PRL induces lobuloalveolar development of the mammary gland, with ultimate formation of oestrogen and progesterone receptor-negative malignant tumours. Another TG mouse model expressing a constitutively activating mutant form of the follicle-stimulating hormone receptor (FSHR) presents with a strong ovarian phenotype inducing advanced follicular development and depletion, haemorrhagic follicles, teratomas and infertility. A third TG mouse model, coexpressing binding- and signalling-deficient mutants of LHCGR in the KO background for the same receptor (R) gene provided convincing evidence that functional complementation through homo-di/oligomerisation is a physiologically relevant mode of activation of class A G protein-coupled receptors (GPCR). Taken together, genetically modified mouse models provide powerful tools for the elucidation of normal and pathological functions of gonadotrophins and their R.
Asunto(s)
Gonadotropinas/genética , Receptores de Gonadotropina/genética , Animales , Estradiol/fisiología , Femenino , Hormona Folículo Estimulante/genética , Hormona Liberadora de Gonadotropina/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Enfermedades del Ovario/genética , Receptores de HFE/genética , Receptores de HL/deficiencia , Receptores de HL/genéticaRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selectively pro-apoptotic in cancer cells, with minimal toxicity to normal tissues. Although this feature makes TRAIL a promising anticancer agent, not all cancer cell types are sensitive to TRAIL-induced apoptosis despite abundant expression of TRAIL receptors. Thus, combinatorial treatments to sensitize tumor cells to TRAIL-induced apoptosis have been in the focus of extensive research. Dietary lignans have shown cancer preventive and antitumorigenic activity, but the mechanisms behind these effects are poorly known. Here we observed that of the three tested lignan molecules, matairesinol (MAT) was the most effective as a death receptor-sensitizing agent. MAT sensitized the androgen-dependent LNCaP cells to TRAIL-induced apoptosis both in the presence and absence of androgens. Treatment with MAT markedly decreased Akt activity, which has been implicated as a key signaling mechanism in the TRAIL resistance of LNCaP prostate cancer cells. The involvement of the pathway in the MAT-mediated sensitization was shown in rescue experiments using ectopic expression of constitutively active Akt. Owing to the high activity of phosphatidylinositol 3-kinase/Akt signaling in cancer, targeting this survival pathway with MAT could markedly benefit TRAIL-based tumor therapies, including those aimed at prostate cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Furanos/farmacología , Lignanos/farmacología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Andrógenos/metabolismo , Animales , Antineoplásicos/farmacología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Línea Celular Tumoral , Polaridad Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
Improvement of cancer treatment is a major challenge of medical research. Despite the immense efforts made in the improvement of diagnosis and treatment, cancer remains a major concern and cause of morbidity and mortality. Most of the modern anti-neoplastic therapies have severe side effects, and tumor cells often develop drug resistance. There is promise in the new generation of treatments (gene therapy, immunotherapy, vaccines, etc.) that are under development, but the efficacies and side effects of such therapies have so far been disappointing. Receptor-based therapies are not new, but many normal cells also present the same receptors reducing the specificity of such approaches. Several lytic peptides have been investigated because of they appear to kill cancer cells due to changes of their membrane potential. Thus, linking receptor-specific ligands to lytic peptides is expected to augment the specificity of targeting and decrease the toxicity of lytic peptides on normal cells. One such polypeptide is hecate (an analogue to the bee venom main component, melittin) that preferentially kills cancer cells at low doses. When this peptide is fused with the 81-95 amino acid fragment of chorionic gonadotropin-beta (CGbeta) subunit (hecate-CGbeta), it targets cells expressing luteinizing hormone receptor (LHR), even at very low doses, or when LHR is expressed at low level. Our recent data showed that this peptide conjugate is efficient in destroying LHR-positive cells in xenografts and more importantly in transgenic mouse models developing LHR-positive somatic cell tumors in gonads. The mechanism of action of hecate-CGbeta after binding to LHR is destruction of cell membranes resulting in rapid cell death by necrosis with minimal side effects. This review summarizes our findings on the action of this novel peptide and considers the future potential of this family of targeting peptides in the treatment of neoplasias.
Asunto(s)
Meliteno/análogos & derivados , Neoplasias Ováricas/tratamiento farmacológico , Receptores de HL/metabolismo , Neoplasias Testiculares/tratamiento farmacológico , Animales , Gonadotropina Coriónica Humana de Subunidad beta/uso terapéutico , Sistemas de Liberación de Medicamentos , Femenino , Terapia Genética , Humanos , Masculino , Meliteno/uso terapéutico , Modelos Biológicos , Neoplasias Ováricas/metabolismo , Receptores de HL/antagonistas & inhibidores , Receptores de HL/genética , Neoplasias Testiculares/metabolismoRESUMEN
The in vivo toxicity of the novel copper-based anticancer agent, casiopeina II (Cu(4,7-dimethyl-1,10-phenanthroline)(glycine)NO3) (CII), was investigated. Casiopeinas are a family of copper-coordinated complexes that have shown promising anticancer activity. The major toxic effect attributed to a single i.v. administration of CII (5 mg/kg dose) in the rat was an hemolytic anemia (reduced hemoglobin concentration (HB), red blood cell (RBC) count and packed cell volume (PCV) accompanied by a marked neutrophilic leukocytosis) 12 h and 5 days after administration, attributed to a direct erythrocyte damage. Increased reticulocyte levels and presence of normoblasts in peripheral blood 5 days post-administration indicated an effective erythropoietic response with recovery at 15 days. Increase in spleen weight and the morphological evidence of congestion of the red pulp (RP) with erythrocytes (E) resulting in a higher ratio of red to white pulp (WP) was consistent with increased uptake of damaged erythrocytes by the reticuloendothelial system observed by histopathology and electron microscopy. Extramedullary hemopoiesis was markedly increased at 5 days giving further evidence of a regenerative erythropoietic response that had an effective recovery by 15 days. Morphological changes in spleen cellularity were consistent with hematotoxicity, mainly a reduction of the red pulp/white pulp ratio, increase in erythrocyte content at 12 h, and an infiltration of nucleated cells in the red pulp at 5 days, with a tendency towards recovery 15 days after administration. The erythrocyte damage is attributed to generation of free radicals and oxidative damage on the membrane and within cells resulting from the reduction of Cu(II) and the probable dissociation of the CII complex.
Asunto(s)
Antineoplásicos/toxicidad , Cobre/química , Eritrocitos/efectos de los fármacos , Leucocitos/efectos de los fármacos , Compuestos Organometálicos/toxicidad , Bazo/efectos de los fármacos , Animales , Recuento de Células Sanguíneas , Relación Dosis-Respuesta a Droga , Eritrocitos/citología , Eritrocitos/ultraestructura , Inyecciones Intravenosas , Leucocitos/citología , Leucocitos/ultraestructura , Masculino , Microscopía Electrónica , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , Bazo/ultraestructura , Factores de Tiempo , Pruebas de ToxicidadRESUMEN
The heat shock response and death receptor-mediated apoptosis are both key physiological determinants of cell survival. We found that exposure to a mild heat stress rapidly sensitized Jurkat and HeLa cells to Fas-mediated apoptosis. We further demonstrate that Hsp70 and the mitogen-activated protein kinases, critical molecules involved in both stress-associated and apoptotic responses, are not responsible for the sensitization. Instead, heat stress on its own induced downregulation of FLIP and promoted caspase-8 cleavage without triggering cell death, which might be the cause of the observed sensitization. Since caspase-9 and -3 were not cleaved after heat shock, caspase-8 seemed to be the initial caspase activated in the process. These findings could help understanding the regulation of death receptor signaling during stress, fever, or inflammation.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/fisiología , Respuesta al Choque Térmico/fisiología , Péptidos y Proteínas de Señalización Intracelular , MAP Quinasa Quinasa 4 , Receptor fas/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/análisis , Caspasa 8 , Inhibidores de Caspasas , Caspasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Proteína Ligando Fas , Citometría de Flujo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Factores de Transcripción del Choque Térmico , Calor , Humanos , Inmunoglobulina M/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Células Jurkat , Proteínas Luminiscentes/genética , Sistema de Señalización de MAP Quinasas/fisiología , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Potenciales de la Membrana/fisiología , Microscopía de Polarización , Mitocondrias/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oligopéptidos/farmacología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/fisiología , Factores de Transcripción , Receptor fas/inmunologíaRESUMEN
The activity of casiopeina II [Cu(1,4-dimethyl-1, 10-phenanthroline)(glycine)NO(3)], a novel anticancer agent, was tested in two cell lines, L1210 murine leukaemia, CH1 human ovarian carcinoma, cisplatin-resistant and sensitive. Exposure of the cells to a range of concentrations of casiopeina II indicates that this copper complex kills cells by apoptosis and necrosis. Condensed chromatin and nuclear fragmentation were observed after exposure to casiopeina II. The caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK) almost completely inhibited apoptosis induced by cisplatin; however, casiopeina II-induced apoptosis was inhibited only by 50-70%. These data are consistent with caspase activation (measured by Z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; Z-DEVD-AFC) by casiopeina II and cisplatin and confirm that caspases are activated in the apoptotic cell death induced by casiopeina II. DNA fragmentation was observed in L1210 cells, but not in CH1 cells. No difference in susceptibility to induction of apoptosis by casiopeina II was found between sensitive and cisplatin resistant cells. In this work we show that the novel copper-based antineoplastic agent casiopeina II is highly active against murine and human cancer cell lines, including cell lines resistant to cisplatin.