RESUMEN
The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Transcripción SOXB1/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular Tumoral , Células Cultivadas , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Células HEK293 , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/química , Histona Demetilasas/genética , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Metilación , Ratones Endogámicos C57BL , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Estabilidad Proteica , Proteolisis , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción SOXB1/química , Factores de Transcripción SOXB1/genética , Teratocarcinoma/enzimología , Teratocarcinoma/metabolismo , Teratocarcinoma/patologíaRESUMEN
Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.
Asunto(s)
Elementos Alu , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular/genética , Regulación Neoplásica de la Expresión Génica , Receptores de Hidrocarburo de Aril/fisiología , Teratocarcinoma/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Ratones , MicroARNs/metabolismo , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Teratocarcinoma/enzimología , Teratocarcinoma/metabolismo , Teratocarcinoma/patología , Teratoma/genética , Teratoma/metabolismo , Transcripción Genética , Tretinoina/farmacologíaRESUMEN
Rex1 (Zfp42), first identified as a gene that is transcriptionally repressed by retinoic acid (RA), encodes a zinc finger transcription factor expressed at high levels in F9 teratocarcinoma stem cells, embryonic stem cells, and other stem cells. Loss of both alleles of Rex1 by homologous recombination alters the RA-induced differentiation of F9 cells, a model of pluripotent embryonic stem cells. We identified Suppressor of Cytokine Signaling-3 (SOCS-3) as a gene that exhibits greatly increased transcriptional activation in RA, cAMP, and theophylline (RACT)-treated F9 Rex1(-/-) cells (approximately 25-fold) as compared to wild-type (WT) cells ( approximately 2.5-fold). By promoter deletion, mutation, and transient transfection analyses, we have shown that this transcriptional increase is mediated by the STAT3 DNA-binding elements located between -99 to -60 in the SOCS-3 promoter. Overexpression of STAT3 dominant-negative mutants greatly diminishes this SOCS-3 transcriptional increase in F9 Rex1(-/-) cells. This increase in SOCS-3 transcription is associated with a four- to fivefold higher level of tyrosine-phosphorylated STAT3 in the RACT-treated F9 Rex1(-/-) cells as compared to WT. Dominant-negative Src tyrosine kinase, Jak2, and protein kinase A partially reduce the transcriptional activation of the SOCS 3 gene in RACT-treated F9 Rex1 null cells. In contrast, parathyroid hormone peptide enhances the effect of RA in F9 Rex1(-/-) cells, but not in F9 WT. Thus, Rex1, which is highly expressed in stem cells, inhibits signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, thereby modulating the differentiation of F9 cells.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/metabolismo , Factor de Transcripción STAT3/genética , Proteínas Supresoras de la Señalización de Citocinas/genética , Teratocarcinoma/genética , Factores de Transcripción/deficiencia , Activación Transcripcional/genética , Animales , Sitios de Unión , Bucladesina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Madre de Carcinoma Embrionario , Genes Reporteros , Janus Quinasa 2/metabolismo , Mercaptoetanol/farmacología , Ratones , Análisis por Micromatrices , Células Madre Neoplásicas/efectos de los fármacos , Hormona Paratiroidea/farmacología , Péptidos/farmacología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Ácido Retinoico/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Teratocarcinoma/enzimología , Teofilina/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
Knocking out the regulatory beta subunit of protein kinase CK2 in mice leads to early embryonic lethality. Heterozygous CK2beta (CK2beta+/-) knockout mice do not show an obvious phenotype. However, the number of heterozygous offsprings from CK2B+/- inter-crossings is lower than expected, meaning that some heterozygous embryos do not survive. Interestingly, CK2beta+/- ES (Embryonic Stem) cells express a considerably lower level of CK2beta than wild-type ES cells, whereas the level of CK2beta in organs from heterozygous adult mice does not significantly differ from those of wild-type mice. The data suggest a compensatory mechanism that adjusts CK2beta levels during development in the majority of, but not in all, cases (Mol Cell Biol 23: 908-915, 2003). In order to find an explanation for the gene dosage effect observed for heterozygous offsprings, we analysed embryos at mid-gestation (E10.5) as well as wild-type and CK2beta+/- ES cells for differences in growth rate and response to different stress agents. Analysis of E10.5 embryos generated from heterozygous matings revealed about 20% of smaller retarded CK2beta+/- embryos. No correlation between CK2beta levels in normal looking and retarded CK2beta+/- embryos were found. However, a different post-translational form of CK2beta protein has been detected in these retarded embryos. Cellular parameters such as growth rate and G1-, G2-checkpoints in ES cells were identical in both wild-type and CK2beta+/- cells. When ES cells were injected to induce differentiated teratocarcinoma in syngenic mice, the size of the tumours correlated with the level of CK2beta.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Embrión de Mamíferos/anomalías , Dosificación de Gen , Subunidades de Proteína/metabolismo , Células Madre/fisiología , Animales , Quinasa de la Caseína II/genética , Ciclo Celular , Embrión de Mamíferos/enzimología , Heterocigoto , Ratones , Ratones Noqueados , Fenotipo , Subunidades de Proteína/genética , Células Madre/enzimología , Teratocarcinoma/enzimología , Teratocarcinoma/patologíaRESUMEN
Nitrofen is a diphenyl ether herbicide that produces a spectrum of fetal abnormalities in rodents. To characterize the molecular mechanisms of nitrofen-mediated birth defects at the cellular level, we explored its effects on undifferentiated P19 teratocarcinoma cells. Nitrofen induces a time-dependent cell death of P19 cells that is associated with increases in TUNEL-positivity and caspase-3 cleavage suggesting that nitrofen induces P19 cell apoptosis. In addition, the increase in TUNEL-positive cells was inhibited with zVAD-fmk, suggesting that nitrofen induces a caspase-dependent apoptosis. Nitrofen treatment was associated with increased p38 MAP kinase activity, though pretreatment of cells with multiple p38 inhibitors did not affect nitrofen-mediated caspase-3 cleavage, suggesting caspase-3 cleavage is p38-independent. Nitrofen induced a dose-dependent increase in reactive oxygen species (ROS), which was accompanied by a decrease in the ratio of reduced/oxidized glutathione, indicating that nitrofen alters the cellular redox state of these cells. Furthermore, pretreatment of cells with N-acetyl cysteine gave a dose- and time-dependent reduction of caspase-3 cleavage, supporting the observations that caspase-3 cleavage is cell-redox-dependent. Therefore, nitrofen induces P19 cell apoptosis that is cell-redox-dependent and is associated with increases in p38 activity and ROS and may play a role in nitrofen-mediated birth defects.
Asunto(s)
Apoptosis/efectos de los fármacos , Herbicidas/toxicidad , Éteres Fenílicos/toxicidad , Teratocarcinoma/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Línea Celular Tumoral/patología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Etiquetado Corte-Fin in Situ , Ratones , Oxidación-Reducción , Teratocarcinoma/tratamiento farmacológico , Teratocarcinoma/enzimologíaRESUMEN
Telomerase is active in about 90% of cancers and contributes to the immortality of cancer cells by maintaining the lengths of the ends of chromosomes. Undifferentiated embryonic human teratocarcinoma (HT) cells were found to express high levels of hTERT, the catalytic subunit of telomerase, and the hTERT promoter was unmethylated in these cells. Retinoic acid (RA)-induced differentiation led to hTERT gene silencing and increased methylation of the hTERT promoter. Treatment with trichostatin A, a histone deacetylase inhibitor, resulted in hTERT reactivation only in very early differentiating HT cells. After methylation patterns had been established within the hTERT promoter region in late differentiating cells, 5-azacytidine, a common demethylating agent, activated the hTERT gene but trichostatin A had no effect on hTERT transcription. These studies suggest that histone deacetylation is involved in early hTERT gene down-regulation and that DNA methylation may maintain silencing of the hTERT gene in these cells.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Telomerasa/metabolismo , Teratocarcinoma/enzimología , Antineoplásicos/metabolismo , Azacitidina/metabolismo , Dominio Catalítico , Diferenciación Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN , Inhibidores Enzimáticos/metabolismo , Humanos , Ácidos Hidroxámicos/metabolismo , Metilación , Regiones Promotoras Genéticas , Telomerasa/genética , Teratocarcinoma/genética , Teratocarcinoma/patología , Tretinoina/metabolismo , Células Tumorales CultivadasRESUMEN
Redistribution, post-translational modifications and coclustering with viral antigens contribute to the immunogenicity of apoptotic cell-derived autoantigens. Almost all known targets of the humoral autoimmune response in systemic lupus erythematosus (SLE) are cleaved by caspases or granzyme B during apoptosis. Antibodies against retroviral proteins can frequently be detected in the sera of SLE patients without overt retroviral infections. These antibodies may represent cross-reactive antibodies or may have been induced by proteins encoded by endogenous retroviral sequences. We used Tera-1 cells that abundantly express a group-specific antigen of human endogenous retroviruses, HERV-K10gag polyprotein, to investigate its processing during apoptosis. Tera-1 cells induced to undergo apoptosis showed an altered HERV-K10gag processing compared with viable cells. In addition, granzyme B was able to cleave HERV-K10gag isolated from viable Tera-1 cells. Similar to nuclear autoantigens, endogenous retroviral proteins are cleaved during the execution phase of apoptosis. These post-translational modifications may result in the generation of T-cell neoepitopes or a changed epitope hierarchy of retroviral proteins. Therefore, immunogenicity of retroviral antigens in SLE patients may result from a similar mechanism as described for nuclear autoantigens.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Retrovirus Endógenos , Productos del Gen gag/metabolismo , Lupus Eritematoso Sistémico/inmunología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Anticuerpos Antivirales/sangre , Inhibidores de Caspasas , Extractos Celulares/análisis , Inhibidores de Cisteína Proteinasa/farmacología , Productos del Gen gag/química , Granzimas , Humanos , Immunoblotting , Teratocarcinoma/enzimología , Teratocarcinoma/metabolismo , Teratocarcinoma/patología , Células Tumorales Cultivadas , Proteínas ViralesRESUMEN
Patients with Down's syndrome (DS) show elevated levels of copper, zinc-containing superoxide dismutase (SOD1) and appear to have increased lipid peroxidation and oxidative damage to DNA as well as elevated glutathione peroxidase activity. Increasing SOD1 levels by gene transfection in NT-2 and SK-N-MC cell lines also led to a rise in glutathione peroxidase activity, but this was nevertheless accompanied by decreased proliferation rates, increased lipid peroxidation and protein carbonyls, and a trend to a rise in 8-hydroxyguanine and protein-bound 3-nitrotyrosine. Transfection of these cell lines with DNA encoding two mutant SOD1 enzymes (G37R and G85R) associated with familial amyotrophic lateral sclerosis (FALS), produced similar, but more severe changes, i.e. even lower growth rates, higher lipid peroxidation, 3-nitrotyrosine and protein carbonyl levels, decreased GSH levels, raised GSSG levels and higher glutathione peroxidase activities. Since G85R has little SOD activity, these changes cannot be related to increased O(2)(-) scavenging. In no case was SOD2 (mitochondrial Mn-SOD) level altered. Our cellular systems reproduce many of the biochemical changes observed in patients with DS or ALS, and in transgenic mice overexpressing mutant SOD1. They also show the potentially deleterious effects of SOD1 overexpression on cellular proliferation, which may be relevant to abnormal development in DS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Antioxidantes/metabolismo , Síndrome de Down/genética , Guanina/análogos & derivados , Estrés Oxidativo/genética , Superóxido Dismutasa/biosíntesis , Tirosina/análogos & derivados , Aldehídos/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , División Celular/genética , Línea Celular , Supervivencia Celular/genética , Síndrome de Down/metabolismo , Expresión Génica , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Guanina/metabolismo , Humanos , Cetonas/metabolismo , Peroxidación de Lípido/genética , Mutación , Neuroblastoma/enzimología , Neuroblastoma/genética , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Teratocarcinoma/enzimología , Teratocarcinoma/genética , Transfección , Tirosina/metabolismoRESUMEN
Cell cycle regulators govern cellular proliferation, modulate differentiation and, when defective, contribute to oncogenesis. Here, we examined expression of cyclins A, B1 and E, and cyclin-dependent kinase (CDK) inhibitors p18INK4C (p18), p21WAF1/Cip1 (p21) and p27KiP1 (p27), in normal human adult testis (n = 5), and 53 testicular tumours, including 23 carcinomas in situ (CIS) and 30 germ cell tumours (GCTs). Immunohistochemical analysis revealed a correlation between proliferation and abundance of the cyclin proteins, and abundant p18 and the lack of p21 and p27 in normal spermatogenesis. Expression of p21 and/or p27 was induced in some differentiated structures seen in teratomas, and was recapitulated in cell culture, using human NTera2/D1 teratocarcinoma cells induced to differentiate into neurons. CIS lesions showed abundant p18, low cyclin E, and moderate p27, in contrast with most invasive seminomas and embryonal carcinomas with very low-to-negative p18, often elevated cyclin E, and, unexpectedly, sustained or increased p27. Our results suggest increased abundance of cyclin E, and particularly downmodulation or loss of p18INK4C as the features that correlate with progression from CIS to invasive germ cell tumours of the human testis.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Germinoma/enzimología , Germinoma/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Teratocarcinoma/enzimología , Teratocarcinoma/patología , Neoplasias Testiculares/enzimología , Neoplasias Testiculares/patología , Proteínas Supresoras de Tumor , Adulto , Carcinoma in Situ/enzimología , Carcinoma in Situ/patología , Inhibidor p18 de las Quinasas Dependientes de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Progresión de la Enfermedad , Electroforesis en Gel de Poliacrilamida , Germinoma/fisiopatología , Humanos , Immunoblotting , Mediciones Luminiscentes , Masculino , Invasividad Neoplásica , Teratocarcinoma/fisiopatología , Neoplasias Testiculares/fisiopatología , Células Tumorales CultivadasRESUMEN
Phospholipase D has been recognized as playing an important role in signal transduction in many types of cells. We investigated the expression of phospholipase D during the differentiation of F9 embryonal teratocarcinoma cells. The ADP ribosylation factor-dependent phospholipase D activity, as measured by an in vitro assay, and H2O2-induced phospholipase D activity and phospholipase D protein content in whole cells were decreased during the differentiation of F9 cells induced by a combination of dibutyryl cyclic AMP and all-trans retinoic acid. In contrast, these changes were not observed when cells were induced by retinoic acid. These results suggest that down-regulation of phospholipase D protein is associated with differentiation of F9 cells to a parietal endoderm lineage.
Asunto(s)
Fosfolipasa D/biosíntesis , Teratocarcinoma/enzimología , Teratocarcinoma/patología , Animales , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , AMP Cíclico/farmacología , Regulación hacia Abajo/efectos de los fármacos , Ratones , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
Extracellular glutathione peroxidase (EGPx) is a secreted selenium-dependent enzyme that reduces hydroperoxides and organic hydroperoxides. Selenium deficiency in females is associated with infertility and spontaneous abortion, suggesting a role for selenium-requiring proteins during embryonic development. To gain insight into functions of EGPx in vivo, we determined sites of murine EGPx synthesis by in situ hybridization during embryogenesis and in adult tissues. At E7.5 of development, high EGPx expression was found in the maternally derived deciduum, with lower levels of accumulation in the embryonic visceral endoderm. At E9.5, the major sites of expression were the yolk sac endoderm and heart musculature. By E16.5, EGPx mRNA expression persisted in yolk sac endoderm but also accumulated significantly in atrially derived myocytes, ossification centers, adipose tissue, intestinal epithelium, and in a ventral-to-dorsal gradient in developing skin. Glutathione peroxidase activity due to EGPx protein was identified in the fluids surrounding the developing mouse embryo at midgestation. The expression of EGPx in tissues at the maternal-fetal interface--deciduum, visceral yolk sac, and skin--suggests that EGPx may serve to protect the embryo from oxidant damage. In adult mice, we identified the S1 segment of the kidney proximal tubules as the primary site of EGPx mRNA accumulation, with lower EGPx levels in atrial cardiac muscle, intestine, skin, and adipose tissue. These findings suggest that EGPx may serve a wider antioxidant role than previously recognized in the interstitium of multiple localized tissues, particularly those associated with the active transport of lipids.
Asunto(s)
Antioxidantes/metabolismo , Decidua/enzimología , Espacio Extracelular/enzimología , Glutatión Peroxidasa/biosíntesis , Piel/enzimología , Saco Vitelino/enzimología , Líquido Amniótico/enzimología , Animales , Diferenciación Celular , Decidua/embriología , Femenino , Riñón/enzimología , Riñón/crecimiento & desarrollo , Ratones , Miocardio/enzimología , Especificidad de Órganos/genética , ARN Mensajero/biosíntesis , Piel/embriología , Teratocarcinoma/enzimología , Teratocarcinoma/patología , Células Tumorales Cultivadas , Saco Vitelino/embriologíaRESUMEN
Treatment with all-trans-retinoic acid (ATRA) induces complete remission in many acute promyelocytic leukemia patients. However, plasma drug levels progressively decrease following prolonged treatment with oral ATRA. This decrease is due, at least in part, to the induced cytochrome P-450-dependent metabolism of ATRA. To investigate if incorporation of ATRA in liposomes could alter its metabolism, we compared the cellular metabolism of liposomal-ATRA (L-ATRA) with free drug. Microsomes isolated from the rat liver metabolized L-ATRA to a significantly lower extent than they did free-ATRA. Similarly, in F9 cells, L-ATRA was metabolized at a slower rate than the free drug. These results suggest that L-ATRA may have important clinical implications in terms of slowing down the rate of ATRA metabolism and producing long-term remission in APL patients.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Portadores de Fármacos , Liposomas , Microsomas Hepáticos/enzimología , Teratocarcinoma/enzimología , Tretinoina/metabolismo , Animales , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Masculino , Ratas , Neoplasias Testiculares/enzimología , Células Tumorales CultivadasRESUMEN
When ornithine decarboxylase, the initial and highly regulated enzyme in polyamine biosynthesis, is irreversibly inactivated by alpha-difluoromethylornithine, F9 teratocarcinoma stem cells are depleted of putrescine and spermidine and as a result differentiate into a cell type which phenotypically resembles the parietal endoderm cells of the early mouse embryo. Simultaneously the level of decarboxylated S-adenosylmethionine (dcAdoMet), the aminopropyl group donor in spermidine and spermine synthesis, increases dramatically, as the aminopropyl group acceptor molecules (putrescine and spermidine) become limiting. When this excessive accumulation of dcAdoMet is prevented by specific inhibition of the AdoMet decarboxylase activity, the differentiative effect is counteracted, despite the fact that the extent of polyamine depletion remains almost identical. Therefore, it may be concluded that dcAdoMet plays an important role in the induction of differentiation. Moreover, this key metabolite acts as a competitive inhibitor of DNA methyltransferase and is therefore capable of interfering with the maintenance methylation of newly replicated DNA. During the course of F9 cell differentiation, the highly methylated genome is gradually demethylated, and its pattern of gene expression is changed. Our present findings, that the DNA remains highly methylated and that the differentiative process is counteracted when the build-up of dcAdoMet is prevented, provide strong evidence for a causative relation between the level of dcAdoMet and the state of DNA methylation as well as cell differentiation.
Asunto(s)
Diferenciación Celular , Metilación de ADN , Metilasas de Modificación del ADN/antagonistas & inhibidores , Poliaminas/metabolismo , Teratocarcinoma/metabolismo , Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Animales , Citosina/metabolismo , Metilasas de Modificación del ADN/genética , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Ratones , Inhibidores de la Ornitina Descarboxilasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/farmacología , Teratocarcinoma/enzimología , Teratocarcinoma/genética , Células Tumorales CultivadasRESUMEN
gamma-Glutamyl transpeptidase (gamma GT) is a crucial enzyme for the metabolism of xenobiotics and endogenous mediators of biological functions (leukotrienes, prostaglandins, and hepoxillins). Yet little is known about its potential role during development. It is a single copy gene expressed from at least seven promoters. Using histochemistry and immunohistochemistry we demonstrate that gamma GT first appears in the midgestational yolk sacs of mouse embryos. Established cell lines with phenotypic features of yolk sac endoderm (JC-44) or embryonic stem cells were also assayed for the expression of gamma GT. Significant levels were detected in JC-44 cells and higher levels were found in JC-44-derived embryoid bodies. Because this cell line appears to be a good in vitro counterpart of yolk sac differentiation, we characterized the gamma GT mRNA types expressed in JC-44 cells. By ribonuclease protection analysis, gamma GT RNA types IV and VI represent about 80% of the total gamma GT RNA in JC-44 embryoid bodies. Reverse transcription-mediated polymerase chain reaction detected low amounts of gamma GT RNA types I, III, and V. Expression of gamma GT in yolk sac follows a pattern seen in many tissues in which one or two gamma GT RNA types dominate the expression profile; however, the reason for this tissue specificity is unknown.
Asunto(s)
Tumor del Seno Endodérmico/enzimología , Neoplasias Ováricas/enzimología , Teratocarcinoma/enzimología , Saco Vitelino/enzimología , gamma-Glutamiltransferasa/biosíntesis , Animales , Línea Celular Transformada , Desarrollo Embrionario y Fetal , Femenino , Inmunoquímica , Ratones , EmbarazoRESUMEN
The effect of cis-diaminedichloroplatinum(II) (cisplatin) on the induction of p53 and protein kinase CK2 activity was studied in the mouse teratocarcinoma cell line F9. Treatment of the cells with the chemotherapeutic agent cisplatin led to the detection of p53 3 h after addition of the drug. F9 cell extracts treated with and without cisplatin were analyzed by ion exchange chromatography for protein kinase CK2 alpha/beta subunits and p53 distribution. The following results were obtained: (a) in crude extracts of cisplatin-treated cells, CK2 activity was sometimes reduced by as much as 50%; (b) after separation by anionic exchange chromatography (MA7Q, BioRad) of the crude cellular extracts from cisplatin-treated cells, lower CK2 activity was found in the peak fractions confirming the results obtained with crude cellular extracts; (c) besides the detection of CK2 alpha subunit by immunostaining, we have detected, at a concentration of approximately 200 mM NaCl, a protein of approximately 46 kDa which reacted with the CK2 alpha-specific antibody. This fraction was devoid of CK2 activity; and (d) cisplatin-treated cells exhibited p53 protein, which was mostly eluting ahead but also partly together with CK2 holoenzyme.
Asunto(s)
Cisplatino/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Teratocarcinoma/enzimología , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II , Cromatografía por Intercambio Iónico , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Fosforilación/efectos de los fármacos , Conformación Proteica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Teratocarcinoma/patología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Secretion of proteolytic enzymes by cells has been implicated in tissue remodeling during embryonic development as well as in invasive neoplastic diseases. We studied the regulation of type-IV-collagenase activity in Tera 2 human embryonal carcinoma cells, which in the undifferentiated state proliferate rapidly and are tumorigenic. The undifferentiated cells produced relatively low levels of matrix-metalloproteinase-2 (MMP-2) activity. This activity was not markedly affected by exogenous basic fibroblast growth factor (bFGF) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though the plasminogen activator activity of the cells was increased by these agents. Tera 2 cells can be induced by retinoic acid to differentiate into quiescent cells, of which many express neuronal characteristics. The type-IV-collagenase activity of the cells increased markedly during the differentiation. This increase was mainly due to increased expression of MMP-2. Expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was not markedly affected by the differentiation of Tera 2 cells. The results show that in the Tera 2 cell system, increased expression of MMP-2 is characteristic of the differentiated derivatives. This is in contrast with many other model systems, where increased type-IV-collagenase activity is associated with the malignant phenotype. This pattern of regulation may reflect the facts that Tera 2 cells resemble early embryonic cells and that their differentiation mimics related cell-differentiation processes in the developing embryo.