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1.
Cell Death Discov ; 9(1): 66, 2023 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-36797268

RESUMEN

Approximately 20 TP53 retrogenes exist in the African and Asian elephant genomes (Loxodonta Africana, Elephas Maximus) in addition to a conserved TP53 gene that encodes a full-length protein. Elephant TP53-RETROGENE 9 (TP53-R9) encodes a p53 protein (p53-R9) that is truncated in the middle of the canonical DNA binding domain. This C-terminally truncated p53 retrogene protein lacks the nuclear localization signals and oligomerization domain of its full-length counterpart. When expressed in human osteosarcoma cells (U2OS), p53-R9 binds to Tid1, the chaperone protein responsible for mitochondrial translocation of human p53 in response to cellular stress. Tid1 expression is required for p53-R9-induced apoptosis. At the mitochondria, p53-R9 binds to the pro-apoptotic BCL-2 family member Bax, which leads to caspase activation, cytochrome c release, and cell death. Our data show, for the first time, that expression of this truncated elephant p53 retrogene protein induces apoptosis in human cancer cells. Understanding the molecular mechanism by which the additional elephant TP53 retrogenes function may provide evolutionary insight that can be utilized for the development of therapeutics to treat human cancers.

2.
PLoS One ; 16(2): e0244334, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33544724

RESUMEN

Wild elephant populations are declining rapidly due to rampant killing for ivory and body parts, range fragmentation, and human-elephant conflict. Wild and captive elephants are further impacted by viruses, including highly pathogenic elephant endotheliotropic herpesviruses. Moreover, while the rich genetic diversity of the ancient elephant lineage is disappearing, elephants, with their low incidence of cancer, have emerged as a surprising resource in human cancer research for understanding the intrinsic cellular response to DNA damage. However, studies on cellular resistance to transformation and herpesvirus reproduction have been severely limited, in part due to the lack of established elephant cell lines to enable in vitro experiments. This report describes creation of a recombinant plasmid, pAelPyV-1-Tag, derived from a wild isolate of African Elephant Polyomavirus (AelPyV-1), that can be used to create immortalized lines of elephant cells. This isolate was extracted from a trunk nodule biopsy isolated from a wild African elephant, Loxodonta africana, in Botswana. The AelPyV-1 genome contains open-reading frames encoding the canonical large (LTag) and small (STag) tumor antigens. We cloned the entire early region spanning the LTag and overlapping STag genes from this isolate into a high-copy vector to construct a recombinant plasmid, pAelPyV-1-Tag, which effectively transformed primary elephant endothelial cells. We expect that the potential of this reagent to transform elephant primary cells will, at a minimum, facilitate study of elephant-specific herpesviruses.


Asunto(s)
Antígenos Virales de Tumores/genética , Genoma Viral , Infecciones por Polyomavirus/veterinaria , Poliomavirus/aislamiento & purificación , Infecciones Tumorales por Virus/veterinaria , Animales , Animales Salvajes , Elefantes , Células Endoteliales/virología , Infecciones por Polyomavirus/diagnóstico , Infecciones Tumorales por Virus/diagnóstico
3.
Dis Model Mech ; 11(2)2018 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-29419415

RESUMEN

RNA splicing factors are essential for the viability of all eukaryotic cells; however, in metazoans some cell types are exquisitely sensitive to disruption of splicing factors. Neuronal cells represent one such cell type, and defects in RNA splicing factors can lead to neurodegenerative diseases. The basis for this tissue selectivity is not well understood owing to difficulties in analyzing the consequences of splicing factor defects in whole-animal systems. Here, we use zebrafish mutants to show that loss of spliceosomal components, including splicing factor 3b, subunit 1 (sf3b1), causes increased DNA double-strand breaks and apoptosis in embryonic neurons. Moreover, these mutants show a concomitant accumulation of R-loops, which are non-canonical nucleic acid structures that promote genomic instability. Dampening R-loop formation by conditional induction of ribonuclease H1 in sf3b1 mutants reduced neuronal DNA damage and apoptosis. These findings show that splicing factor dysfunction leads to R-loop accumulation and DNA damage that sensitizes embryonic neurons to apoptosis. Our results suggest that diseases associated with splicing factor mutations could be susceptible to treatments that modulate R-loop levels.


Asunto(s)
Apoptosis , Citoprotección , Daño del ADN , Neuronas/citología , Neuronas/metabolismo , Conformación de Ácido Nucleico , Empalmosomas/metabolismo , Pez Cebra/genética , Animales , Apoptosis/efectos de la radiación , Citoprotección/efectos de la radiación , Roturas del ADN de Doble Cadena , Genes Esenciales , Mutación/genética , Neuronas/efectos de la radiación , Empalme del ARN/genética , Empalme del ARN/efectos de la radiación , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de la radiación , Radiación Ionizante , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismo
4.
PLoS One ; 9(2): e88151, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24516599

RESUMEN

Ionizing radiation (IR)-induced DNA double-strand breaks trigger an extensive cellular signaling response that involves the coordination of hundreds of proteins to regulate DNA repair, cell cycle arrest and apoptotic pathways. The cellular outcome often depends on the level of DNA damage as well as the particular cell type. Proliferating zebrafish embryonic neurons are highly sensitive to IR-induced apoptosis, and both p53 and its transcriptional target puma are essential mediators of the response. The BH3-only protein Puma has previously been reported to activate mitochondrial apoptosis through direct interaction with the pro-apoptotic Bcl-2 family proteins Bax and Bak, thus constituting the role of an "activator" BH3-only protein. This distinguishes it from BH3-only proteins like Bad that are thought to indirectly promote apoptosis through binding to anti-apoptotic Bcl-2 family members, thereby preventing the sequestration of activator BH3-only proteins and allowing them to directly interact with and activate Bax and Bak. We have shown previously that overexpression of the BH3-only protein Bad in zebrafish embryos supports normal embryonic development but greatly sensitizes developing neurons to IR-induced apoptosis. While Bad has previously been shown to play only a minor role in promoting IR-induced apoptosis of T cells in mice, we demonstrate that Bad is essential for robust IR-induced apoptosis in zebrafish embryonic neural tissue. Moreover, we found that both p53 and Puma are required for Bad-mediated radiosensitization in vivo. Our findings show the existence of a hierarchical interdependence between Bad and Puma whereby Bad functions as an essential sensitizer and Puma as an essential activator of IR-induced mitochondrial apoptosis specifically in embryonic neural tissue.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de la radiación , Proteínas Proto-Oncogénicas/metabolismo , Radiación Ionizante , Proteínas de Pez Cebra/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Androstadienos/farmacología , Animales , Apoptosis/efectos de los fármacos , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Embrión no Mamífero/efectos de la radiación , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Modelos Genéticos , Tejido Nervioso/citología , Tejido Nervioso/efectos de los fármacos , Tejido Nervioso/metabolismo , Tejido Nervioso/efectos de la radiación , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/efectos de la radiación , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Wortmanina , Pez Cebra/embriología , Proteína X Asociada a bcl-2/metabolismo
5.
J Vis Exp ; (82): e51060, 2013 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-24378359

RESUMEN

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.


Asunto(s)
Apoptosis/fisiología , Caspasa 3/metabolismo , Técnicas de Cultivo de Embriones/métodos , Técnica del Anticuerpo Fluorescente/métodos , Pez Cebra/embriología , Animales , Caspasa 3/análisis , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/citología , Activación Enzimática , Pez Cebra/metabolismo
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