Asunto(s)

Apoptosis/genética , Fibroblastos/fisiología , Animales , Apoptosis/fisiología , Supervivencia Celular , Células Cultivadas , Embrión de Mamíferos/citología , Femenino , Citometría de Flujo/métodos , Colorantes Fluorescentes/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Ratones , Embarazo , Retroviridae/genética , Retroviridae/metabolismo , Transfección

RESUMEN

The p53 tumor suppressor activates either cell cycle arrest or apoptosis in response to cellular stress. Mouse embryo fibroblasts (MEFs) provide a powerful primary cell system to study both p53-dependent pathways. Specifically, in response to DNA damage, MEFs undergo p53-dependent G(1) arrest, whereas MEFs expressing the adenovirus E1A oncoprotein undergo p53-dependent apoptosis. As the p53-dependent apoptosis pathway is not well understood, we sought to identify apoptosis-specific p53 target genes using a subtractive cloning strategy. Here, we describe the characterization of a gene identified in this screen, PERP, which is expressed in a p53-dependent manner and at high levels in apoptotic cells compared with G(1)-arrested cells. PERP induction is linked to p53-dependent apoptosis, including in response to E2F-1-driven hyperproliferation. Furthermore, analysis of the PERP promoter suggests that PERP is directly activated by p53. PERP shows sequence similarity to the PMP-22/gas3 tetraspan membrane protein implicated in hereditary human neuropathies such as Charcot-Marie-Tooth. Like PMP-22/gas3, PERP is a plasma membrane protein, and importantly, its expression causes cell death in fibroblasts. Taken together, these data suggest that PERP is a novel effector of p53-dependent apoptosis.

Asunto(s)

Apoptosis/genética , Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteínas de la Membrana/genética , Proteínas de la Mielina/genética , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Regulación de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteína 1 de Unión a Retinoblastoma , Homología de Secuencia de Aminoácido , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo

Asunto(s)

Daño del ADN/genética , Genes myc , Linfoma/genética , Linfoma/terapia , Animales , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética

RESUMEN

The INK4a/ARF locus encodes upstream regulators of the retinoblastoma and p53 tumor suppressor gene products. To compare the impact of these loci on tumor development and treatment response, the Emu-myc transgenic lymphoma model was used to generate genetically defined tumors with mutations in the INK4a/ARF, Rb, or p53 genes. Like p53 null lymphomas, INK4a/ARF null lymphomas formed rapidly, were highly invasive, displayed apoptotic defects, and were markedly resistant to chemotherapy in vitro and in vivo. Furthermore, INK4a/ARF(-/-) lymphomas displayed reduced p53 activity despite the presence of wild-type p53 genes. Consequently, INK4a/ARF and p53 mutations lead to aggressive tumors by disrupting overlapping tumor suppressor functions. These data have important implications for understanding the clinical behavior of human tumors.

Asunto(s)

Genes p16 , Genes p53 , Linfoma de Células B/etiología , Linfoma de Células B/genética , Mutación , Proteínas/genética , Animales , Antineoplásicos/farmacología , Apoptosis/genética , Resistencia a Medicamentos/genética , Elementos de Facilitación Genéticos , Femenino , Genes myc , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Linfoma de Células B/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína p14ARF Supresora de Tumor

RESUMEN

The adenovirus E1A oncogene activates p53 through a signaling pathway involving the retinoblastoma protein and the tumor suppressor p19(ARF). The ability of E1A to induce p53 and its transcriptional targets is severely compromised in ARF-null cells, which remain resistant to apoptosis following serum depletion or adriamycin treatment. Reintroduction of p19(ARF) restores p53 accumulation and resensitizes ARF-null cells to apoptotic signals. Therefore, p19(ARF) functions as part of a p53-dependent failsafe mechanism to counter uncontrolled proliferation. Synergistic effects between the p19(ARF) and DNA damage pathways in inducing p53 may contribute to E1A's ability to enhance radio- and chemosensitivity.

Asunto(s)

Proteínas E1A de Adenovirus/genética , Genes Supresores de Tumor , Genes Virales , Genes p53 , Proteínas/genética , Animales , Apoptosis/genética , División Celular/genética , Células Cultivadas , Daño del ADN , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Transducción de Señal , Proteína p14ARF Supresora de Tumor

RESUMEN

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.

Asunto(s)

Apoptosis/fisiología , Caspasas , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Linfocitos B/citología , Linfocitos B/fisiología , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Complejo CD3/farmacología , Caspasa 3 , Muerte Celular/fisiología , División Celular/fisiología , Cisteína Endopeptidasas/deficiencia , Cisteína Endopeptidasas/genética , Citotoxicidad Inmunológica/genética , Citotoxicidad Inmunológica/fisiología , Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Desarrollo Embrionario y Fetal , Femenino , Expresión Génica/genética , Expresión Génica/fisiología , Longevidad/genética , Longevidad/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Mutantes , Mutación/genética , Mutación/fisiología , Neutrófilos/fisiología , Presión Osmótica , Células Madre/efectos de la radiación , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/fisiología , Células Tumorales Cultivadas/efectos de la radiación , Rayos Ultravioleta , Receptor fas/farmacología

RESUMEN

FADD (also known as Mort-1) is a signal transducer downstream of cell death receptor CD95 (also called Fas). CD95, tumor necrosis factor receptor type 1 (TNFR-1), and death receptor 3 (DR3) did not induce apoptosis in FADD-deficient embryonic fibroblasts, whereas DR4, oncogenes E1A and c-myc, and chemotherapeutic agent adriamycin did. Mice with a deletion in the FADD gene did not survive beyond day 11.5 of embryogenesis; these mice showed signs of cardiac failure and abdominal hemorrhage. Chimeric embryos showing a high contribution of FADD null mutant cells to the heart reproduce the phenotype of FADD-deficient mutants. Thus, not only death receptors, but also receptors that couple to developmental programs, may use FADD for signaling.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Proteínas Portadoras/fisiología , Desarrollo Embrionario y Fetal , Corazón/embriología , Animales , Proteínas Portadoras/genética , Transformación Celular Neoplásica , Células Cultivadas , Doxorrubicina/farmacología , Endotelio Vascular/embriología , Proteína de Dominio de Muerte Asociada a Fas , Femenino , Expresión Génica , Marcación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Oncogenes , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/fisiología , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/genética , Receptor fas/fisiología

RESUMEN

Expansion of a CTG trinucleotide repeat in the 3' untranslated region (UTR) of DMPK, the gene encoding myotonic dystrophy protein kinase, induces the dominantly inherited neuromuscular disorder myotonic dystrophy (DM). Transcripts containing the expanded trinucleotide are abundant in differentiated cultured myoblasts, and they are spliced and polyadenylylated normally. However, mutant transcripts never reach the cytoplasm in these nonmitotic cells; instead, they form stable clusters that are tightly linked to the nuclear matrix, which can prevent effective biochemical purification of these transcripts. In DM patients, reduced DMPK protein levels, consequent to nuclear retention of mutant transcripts, are probably a cause of disease development. Formation of nuclear foci is a novel mechanism for preventing transcript export and effecting a loss of gene function.

Asunto(s)

Proteínas Serina-Treonina Quinasas/genética , Transcripción Genética , Repeticiones de Trinucleótidos , Línea Celular , Humanos , Mutación , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica

RESUMEN

Many genotoxic agents kill tumor cells by inducing apoptosis; hence, mutations that suppress apoptosis produce resistance to chemotherapy. Although directly activating the apoptotic machinery may bypass these mutations, how to achieve this activation in cancer cells selectively is not clear. In this study, we show that the drug-resistant 293 cell line is unable to activate components of the apoptotic machinery-the ICE-like proteases (caspases)-following treatment with an anticancer drug. Remarkably, extracts from untreated cells spontaneously activate caspases and induce apoptosis in a cell-free system, indicating that drug-resistant cells have not only the apoptotic machinery but also its activator. Comparing extracts from cells with defined genetic differences, we show that this activator is generated by the adenovirus E1A oncogene and is absent from normal cells. We provide preliminary characterization of this oncogene generated activity (OGA) and show that partially purified OGA activates caspases when added to extracts from untransformed cells. We suggest that agents that link OGA to caspases in cells would kill tumor cells otherwise resistant to conventional cancer therapy. As this killing relies on an activity generated by an oncogene, the effect of these agents should be selective for transformed cells.

Asunto(s)

Apoptosis/genética , Resistencia a Múltiples Medicamentos/genética , Oncogenes , Adenosina Trifosfato/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Línea Celular , Sistema Libre de Células , Cromatografía por Intercambio Iónico , Cisteína Endopeptidasas/metabolismo , Activación Enzimática , Genes bcl-2 , Células HeLa , Humanos , Hidrólisis

RESUMEN

Inactivation of p53-dependent apoptosis promotes oncogenic transformation, tumor development, and resistance to many cytotoxic anticancer agents. p53 can transcriptionally activate bax, a bcl-2 family member that promotes apoptosis. To determine whether bax is required for p53-dependent apoptosis, the effects of bax deficiency were examined in primary fibroblasts expressing the E1A oncogene, a setting where apoptosis is dependent on endogenous p53. We demonstrate that bax can function as an effector of p53 in chemotherapy-induced apoptosis and contributes to a p53 pathway to suppress oncogenic transformation. Furthermore, we show that additional p53 effectors participate in these processes. These p53-controlled factors act synergistically with Bax to promote a full apoptotic response, and their action is suppressed by the Bcl-2 and E1B 19K oncoproteins. These studies demonstrate that Bax is a determinant of p53-dependent chemosensitivity and illustrate how p53 can promote apoptosis by coordinating the activities of multiple effectors.

Asunto(s)

Apoptosis , Transformación Celular Neoplásica , Resistencia a Medicamentos/genética , Genes p53 , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/deficiencia , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cisplatino/farmacología , Cruzamientos Genéticos , Doxorrubicina/farmacología , Embrión de Mamíferos , Etopósido/farmacología , Fibroblastos , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/biosíntesis , Recombinación Genética , Transcripción Genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2

RESUMEN

Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.

Asunto(s)

Quinasas CDC2-CDC28 , Proteínas Portadoras/metabolismo , Oncogenes/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas ras/genética , Animales , Biomarcadores , Northern Blotting , Proteínas de Ciclo Celular/metabolismo , Senescencia Celular/genética , Quinasa 2 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Inhibidores Enzimáticos/metabolismo , Fibroblastos/citología , Fibroblastos/enzimología , Regulación Neoplásica de la Expresión Génica/fisiología , Genes Supresores de Tumor , Humanos , Immunoblotting , Ratones , Ratones Mutantes , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/análisis , Transformación Genética

RESUMEN

We have analyzed the intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls. In affected individuals, a trinucleotide expansion in the gene results in the phenotype, the severity of which is proportional to the repeat length. A fluorochrome-conjugated probe (10 repeats of CAG) hybridized specifically to this expanded repeat. Mt-PK transcripts containing CTG repeat expansions were detected in the nucleus as bright foci in DM patient fibroblasts and muscle biopsies, but not from normal individuals. These foci represented transcripts from the Mt-PK gene since they simultaneously hybridized to fluorochrome-conjugated probes to the 5'-end of the Mt-PK mRNA. A single oligonucleotide probe to the repeat and the sense strand each conjugated to different fluorochromes revealed the gene and the transcripts simultaneously, and indicated that these focal concentrations (up to 13 per nucleus) represented predominately posttranscriptional RNA since only a single focus contained both the DNA and the RNA. This concentration of nuclear transcripts was diagnostic of the affected state, and may represent aberrant processing of the RNA.

Asunto(s)

Distrofia Miotónica/enzimología , Proteínas Quinasas/análisis , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , ARN Mensajero/análisis , Adulto , Secuencia de Bases , Células Cultivadas , ADN Complementario , Femenino , Fibroblastos/enzimología , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Músculos/enzimología , Distrofia Miotónica/patología , Proteína Quinasa de Distrofia Miotónica , Pronóstico , Secuencias Repetitivas de Ácidos Nucleicos , Piel/enzimología

RESUMEN

The genomic structure and apparently complete coding sequence of the myotonic dystrophy protein kinase gene have been determined. The gene contains 15 exons distributed over about 13 kb of genomic DNA. It codes for a protein of 624 amino acids with an N-terminal domain highly homologous to cAMP-dependent serine-threonine protein kinases, an intermediate domain with a high alpha-helical content and weak similarity to various filamentous proteins, and a hydrophobic C-terminal segment. Located in close proximity is a second gene, coding for a transcript of about 3 kb, that is homologous to the gene DMR-N9 in the corresponding mouse locus, but has no homologies to other known genes or proteins. Strong expression of the latter gene in brain suggests that it may have a role in the development of mental symptoms in severe cases of the disease.

Asunto(s)

Distrofia Miotónica/enzimología , Distrofia Miotónica/genética , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/enzimología , Exones , Expresión Génica , Biblioteca Genómica , Humanos , Intrones , Masculino , Ratones , Datos de Secuencia Molecular , Músculos/enzimología , Proteína Quinasa de Distrofia Miotónica , Especificidad de Órganos , Polimorfismo de Longitud del Fragmento de Restricción , Estructura Secundaria de Proteína , Mapeo Restrictivo

Asunto(s)

Distrofia Miotónica/genética , Proteínas Quinasas/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular

RESUMEN

Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have been 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3' untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.

Asunto(s)

Distrofia Miotónica/genética , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , AMP Cíclico , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Distrofia Miotónica/diagnóstico , Linaje , Reacción en Cadena de la Polimerasa , Alineación de Secuencia