RESUMEN
Intrinsic apoptosis, reliant on BAX and BAK, has been postulated to be fundamental for morphogenesis, but its precise contribution to this process has not been fully explored in mammals. Our structural analysis of BOK suggests close resemblance to BAX and BAK structures. Notably, Bok-/-Bax-/-Bak-/- animals exhibited more severe defects and died earlier than Bax-/-Bak-/- mice, implying that BOK has overlapping roles with BAX and BAK during developmental cell death. By analyzing Bok-/-Bax-/-Bak-/- triple-knockout mice whose cells are incapable of undergoing intrinsic apoptosis, we identified tissues that formed well without this process. We provide evidence that necroptosis, pyroptosis, or autophagy does not substantially substitute for the loss of apoptosis. Albeit very rare, unexpected attainment of adult Bok-/-Bax-/-Bak-/- mice suggests that morphogenesis can proceed entirely without apoptosis mediated by these proteins and possibly without cell death in general.
Asunto(s)
Apoptosis , Embrión de Mamíferos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genética , Anomalías Múltiples/patología , Anomalías Múltiples/veterinaria , Animales , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/patología , Desarrollo Embrionario/genética , Feto/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in gamma-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from gamma-irradiation-induced death, because their glucocorticoid-mediated decimation in Puma-deficient mice activated cycling of stem/progenitor cells and restored thymic lymphomagenesis. Our demonstration that cycles of cell attrition and repopulation by stem/progenitor cells can drive tumorigenesis has parallels in human cancers, such as therapy-induced malignancies.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de la radiación , Rayos gamma , Linfoma/fisiopatología , Neoplasias del Timo/fisiopatología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Células Cultivadas , Dexametasona/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Leucocitos/efectos de los fármacos , Leucocitos/patología , Leucocitos/efectos de la radiación , Linfoma/genética , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Análisis de Supervivencia , Neoplasias del Timo/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Understanding the critical factors that govern recovery of the hematopoietic system from stress, such as during anticancer therapy and bone marrow transplantation, is of clinical significance. We investigated the importance of the prosurvival proteins myeloid cell leukemia-1 (MCL-1) and B-cell lymphoma-extra large (BCL-XL) in stem/progenitor cell survival and fitness during hematopoietic recovery from stress. Loss of a single Mcl-1 allele, which reduced MCL-1 protein levels, severely compromised hematopoietic recovery from myeloablative challenge and following bone marrow transplantation, whereas BCL-XL was dispensable in both contexts. We identified inhibition of proapoptotic p53 upregulated modulator of apoptosis (PUMA) as the key role of MCL-1 in both settings, with Mcl-1(+/-);Puma(-/-) mice completely protected from the deleterious effects of loss of 1 Mcl-1 allele. These results reveal the molecular mechanisms that govern cell survival during hematopoietic recovery from stress.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Células Madre Hematopoyéticas/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Estrés Fisiológico , Proteínas Supresoras de Tumor/metabolismo , Animales , Separación Celular , Supervivencia Celular/fisiología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/deficiencia , Eliminación de Gen , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Proteínas Proto-Oncogénicas/deficiencia , Trombocitopenia/genética , Proteína X Asociada a bcl-2/deficiencia , Animales , Proteínas Reguladoras de la Apoptosis/genética , Plaquetas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Genotipo , Masculino , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Trombocitopenia/sangre , Proteína X Asociada a bcl-2/genéticaRESUMEN
Apoptosis plays a role in normal lymphopoiesis and lymphoid malignancies. Pro-survival MCL-1 is essential for survival of T-cell progenitors, BCL-XL for immature thymocytes, and BCL-2 for mature T cells. Conversely, little is known about the regulators that are required for the survival of T-cell lymphomas. We used constitutive and conditionally gene-targeted mice to investigate which pro-survival BCL-2 family member is required for the sustained survival of thymic lymphomas initiated by loss of p53. Constitutive loss of a single Mcl-1 allele delayed tumor onset. In contrast, lymphomas emerging in p53(-/-) mice in which Mcl-1 could be conditionally deleted had been selected for retention of MCL-1 expression. In contrast, complete loss of BCL-XL had no impact on lymphoma development in p53(-/-) mice. These results demonstrate that thymic lymphomas elicited by loss of p53 must arise from cancer-initiating cells that require MCL-1 for their survival. Acute deletion of both Mcl-1 alleles abrogated the expansion of p53(-/-) lymphomas in mice, whereas inducible loss of BCL-XL had little impact. This reveals that MCL-1 is essential for the sustained survival of these malignant cells and suggests that targeting MCL-1 may be an attractive strategy for the treatment of T-cell lymphoma.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes p53 , Linfoma de Células T/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/fisiología , Neoplasias del Timo/metabolismo , Proteína bcl-X/fisiología , Alelos , Animales , Apoptosis , Supervivencia Celular , Trasplante de Células , Eliminación de Gen , Genotipo , Ratones , Ratones Endogámicos C57BL , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Células Madre/citología , Proteína bcl-X/genéticaRESUMEN
Impaired apoptosis is a cancer hallmark, and some types of lymphomas and other cancers harbor mutations that directly affect key cell death regulators, such as Bcl-2 family members. However, because the majority of tumors seem to lack such mutations, we are examining the hypothesis that tumorigenesis can be sustained at least initially by the normal expression of specific endogenous pro-survival Bcl-2 family members. We previously demonstrated that the lymphomagenesis in Εµ-myc transgenic mice, which constitutively overexpress the c-Myc oncoprotein in B-lymphoid cells and develop pre-B and B-cell lymphomas, does not require endogenous Bcl-2. In striking contrast, we report here that loss in these mice of its close relative Bcl-x(L) attenuated the pre-neoplastic expansion of pro-B and pre-B cells otherwise driven by c-Myc overexpression, sensitized these cells to apoptosis and ablated lymphoma formation. Remarkably, even loss of a single bcl-x allele delayed the lymphomagenesis. These findings identify Bcl-x(L) as a prerequisite for the emergence of c-Myc-driven pre-B/B lymphoma and suggest that BH3 mimetic drugs may provide a prophylactic strategy for c-Myc-driven tumors.
Asunto(s)
Linfocitos B/metabolismo , Transformación Celular Neoplásica/metabolismo , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Regulación hacia Arriba , Proteína bcl-X/metabolismo , Animales , Apoptosis , Linfocitos B/citología , Células Cultivadas , Cruzamientos Genéticos , Femenino , Heterocigoto , Linfoma de Células B/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Análisis de Supervivencia , Proteína bcl-X/genéticaRESUMEN
Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.
RESUMEN
Neural tube defects (NTDs) are common birth defects in humans and show an unexplained female bias. Female mice lacking the tumor suppressor p53 display NTDs with incomplete penetrance. We found that the combined loss of pro-apoptotic BIM and p53 caused 100% penetrant, female-exclusive NTDs, which allowed us to investigate the female-specific functions of p53. We report that female p53-/- embryonic neural tube samples show fewer cells with inactive X chromosome markers Xist and H3K27me3 and a concomitant increase in biallelic expression of the X-linked genes, Huwe1 and Usp9x. Decreased Xist and increased X-linked gene expression was confirmed by RNA sequencing. Moreover, we found that p53 directly bound response elements in the X chromosome inactivation center (XIC). Together, these findings suggest p53 directly activates XIC genes, without which there is stochastic failure in X chromosome inactivation, and that X chromosome inactivation failure may underlie the female bias in neural tube closure defects.
Asunto(s)
Defectos del Tubo Neural/genética , Proteína p53 Supresora de Tumor/deficiencia , Animales , Células Madre Embrionarias/patología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Defectos del Tubo Neural/patología , Embarazo , Procesos Estocásticos , Proteína p53 Supresora de Tumor/genética , Inactivación del Cromosoma XRESUMEN
The 'hallmarks of cancer' are generally accepted as a set of genetic and epigenetic alterations that a normal cell must accrue to transform into a fully malignant cancer. It follows that therapies designed to counter these alterations might be effective as anti-cancer strategies. Over the past 30 years, research on the BCL-2-regulated apoptotic pathway has led to the development of small-molecule compounds, known as 'BH3-mimetics', that bind to pro-survival BCL-2 proteins to directly activate apoptosis of malignant cells. This Timeline article focuses on the discovery and study of BCL-2, the wider BCL-2 protein family and, specifically, its roles in cancer development and therapy.
Asunto(s)
Terapia Molecular Dirigida/métodos , Neoplasias/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/genética , Compuestos de Bifenilo/farmacología , Muerte Celular , Regulación Neoplásica de la Expresión Génica , Genes bcl-2 , Humanos , Ratones , Proteínas de Transporte de Membrana Mitocondrial , Familia de Multigenes , Neoplasias/tratamiento farmacológico , Nitrofenoles/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
MCL-1 is critical for progenitor cell survival during emergency hematopoiesis, but its role in sustaining cells undergoing transformation and in lymphomagenesis is only poorly understood. We investigated the importance of MCL-1 in the survival of B lymphoid progenitors undergoing MYC-driven transformation and its functional interactions with pro-apoptotic BIM and PUMA and the tumor suppressor p53 in lymphoma development. Loss of one Mcl-1 allele almost abrogated MYC-driven-lymphoma development owing to a reduction in lymphoma initiating pre-B cells. Although loss of the p53 target PUMA had minor impact, loss of one p53 allele substantially accelerated lymphoma development when MCL-1 was limiting, most likely because p53 loss also causes defects in non-apoptotic tumor suppressive processes. Remarkably, loss of BIM restored the survival of lymphoma initiating cells and rate of tumor development. Thus, MCL-1 has a major role in lymphoma initiating pro-B cells to oppose BIM, which is upregulated in response to oncogenic stress.
Asunto(s)
Apoptosis , Linfoma/fisiopatología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Alelos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Médula Ósea/metabolismo , Médula Ósea/patología , Citometría de Flujo , Genotipo , Estimación de Kaplan-Meier , Linfoma/mortalidad , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Células Precursoras de Linfocitos B/citología , Proteínas Proto-Oncogénicas c-myc/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Neoplastic transformation is driven by oncogenic lesions that facilitate unrestrained cell expansion and resistance to antiproliferative signals. These oncogenic DNA lesions, acquired through errors in DNA replication, gene recombination, or extrinsically imposed damage, are thought to activate multiple tumor suppressive pathways, particularly apoptotic cell death. DNA damage induces apoptosis through well-described p53-mediated induction of PUMA and NOXA. However, loss of both these mediators (even together with defects in p53-mediated induction of cell cycle arrest and cell senescence) does not recapitulate the tumor susceptibility observed in p53(-/-) mice. Thus, potentially oncogenic DNA lesions are likely to also trigger apoptosis through additional, p53-independent processes. We found that loss of the BH3-only protein BIM accelerated lymphoma development in p53-deficient mice. This process was negated by concomitant loss of RAG1/2-mediated antigen receptor gene rearrangement. This demonstrates that BIM is critical for the induction of apoptosis caused by potentially oncogenic DNA lesions elicited by RAG1/2-induced gene rearrangement. Furthermore, this highlights the role of a BIM-mediated tumor suppressor pathway that acts in parallel to the p53 pathway and remains active even in the absence of wild-type p53 function, suggesting this may be exploited in the treatment of p53-deficient cancers.
Asunto(s)
Apoptosis , Proteína 11 Similar a Bcl2/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinogénesis/patología , Heterocigoto , Linfoma/patología , Ratones Endogámicos C57BL , Recombinasas/metabolismo , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Multicellular organisms have evolved processes to prevent abnormal proliferation or inappropriate tissue infiltration of cells, and these tumor suppressive mechanisms serve to prevent tissue hyperplasia, tumor development, and metastatic spread of tumors. These include potentially reversible processes such as cell cycle arrest and cellular senescence, as well as apoptotic cell death, which in contrast eliminates dangerous cells that may initiate tumor development. Tumor suppressive processes are organized as complex, extensive signaling networks, controlled by central "nodes." These "nodes" are prominent tumor suppressors, such as P53 or PTEN, whose loss is responsible for the development of the majority of human cancers. In this review we discuss the processes by which some of these prominent tumor suppressors trigger apoptotic cell death and how this process protects us from cancer development.