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.

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.

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.

No abstract

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.

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.

The goal of our study was to determine if elephant TP53 (EP53) proteins contributing to increased apoptosis and possible cancer resistance in elephants could translate into human cancer cells as a future effective cancer treatment. We previously reported that elephants have a lower than expected rate of cancer, 20 copies of TP53 (1 ancestral gene with introns [EP53-anc] and 19 retrogenes [EP53-retro1-19]), and increased p53-mediated apoptosis induced by DNA damage in elephant cells compared to human cells (Abegglen JAMA 2015). For the current study, we expressed various EP53 proteins in human cancer cells with different p53 status, including osteosarcoma (U2-OS, Saos-2), glioblastoma (T98G), and breast cancer (MCF7). Western blot analysis confirmed EP53 expression. We compared apoptosis in the human cancer cells transfected/transduced with negative control vectors vs. epitope or protein-tagged EP53 exposed to doxorubicin (to induce DNA damage). Apoptosis was measured by cell viability, caspase activity, Propidium Iodide/Annexin V staining, and fluorescence microscopy. We observed a significant increase in caspase activity (normalized to cell viability) of U2-OS and T98G cells expressing EP53 compared to negative control treated cells as shown in Table 1, and apoptosis with p21 restoration in Saos-2. In U2-OS, which overexpress MDM2, EP53 was more effective at inducing apoptosis compared to human TP53. Taken together, we found that EP53-anc restored p53-mediated apoptosis and EP53-anc / EP53-retro9 enhanced p53-mediated apoptosis. These data suggest for the first time that EP53 functions in human cancer cells to promote cell death. Ongoing efforts are exploring the EP53 mechanism of action that leads to increased apoptosis, including expression of EP53 in additional cancer types (lung, melanoma, colon, prostate, and others) with a variety of genetic backgrounds to characterize its functional context. These results support the further exploration of EP53-based cancer therapeutics. Table 1:Increase in apoptosis with EP53 expression relative to EP53 empty vector control cellsCell TypeTP53 StatusEP53-ancEP53-retro9ConstructAssay ResultsNo Treatment (fold difference)P-value1uM doxorubicin (fold difference)P-valueU-2 OS (osteosarcoma)WTApoptosisApoptosismyc-EP53-retro9Increase in caspase relative to control1.730.00911.791.7x10^-5eGFP-EP53-retro9Increase in caspase relative to control1.686.7x10^-52.590.0041eGFP-EP53-retro9Percent decrease in GFP positive cells23% percent decrease4.8x10^-5--dyk-EP53-ancIncrease in caspase relative to control14.225.4x10^-73.311.0x10^-5dyk-huTP53Increase in caspase relative to control10.91.2x10^-72.475.8x10^-5MCF7 (breast cancer)WT-ApoptosiseGFP-EP53-retro9Cell death by Propidium Iodide staining0.15% GFP/surface area0.011--T98G (glioblastoma)MUTApoptosisNo Apoptosisdyk-EP53-ancIncrease in caspase relative to control3.077.9x10^-91.552.6x10^-5myc-EP53-ancIncrease in caspase relative to control3.460.000151.526.8x10^-5dyk-huTP53Increase in caspase relative to control2.821.2x10^-51.88.6x10^-5 Citation Format: Lisa M. Abegglen, Lauren N. Donovan, Genevieve Couldwell, Rosann Robinson, Cristhian Toruno, Mor Goldfeder, Wendy K. Kiso, Dennis L. Schmitt, Aleah F. Caulin, Katrin P. Guillen, Bryan E. Welm, Carlo C. Maley, Avi Schroeder, Joshua D. Schiffman. Elephant p53 (EP53) expression induces apoptosis of human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2153. doi:10.1158/1538-7445.AM2017-2153

Amplification of elephant p53 (EP53) was recently described as a potential mechanism for cancer resistance in elephants. Osteosarcoma is the most common pediatric bone tumor and also occurs frequently in pet dogs. Both human and canine osteosarcoma contain a very high rate of TP53 alterations leading to genomic instability. The purpose of our study was to determine if EP53 could enhance and/or restore p53 function in osteosarcoma and trigger p53-mediated cell death. We expressed various EP53 proteins in canine and human osteosarcoma cell lines (OSCA-40, U-2 OS, and Saos-2) by transfection or viral transduction. Expression of EP53 was confirmed by Western blot. Apoptosis of cells transfected/transduced with EP53 was compared to cells transfected/transduced with negative control vectors. Apoptosis was measured by fluorescence microscopy and caspase activity. We observed a significant increase in caspase activity (normalized to cell viability) of U-2 OS (TP53-wild type) cells expressing EP53 compared to negative control cells (p<0.0001). Saos-2 (TP53-null) and OSCA-40 cells underwent apoptosis as visualized by time-lapse fluorescence microscopy. Experiments to confirm these results with caspase activity assays are currently under way. Increased expression of p21, a direct p53 target, was observed in Saos-2 cells by Western blot. Taken together, our results indicate that EP53 enhances and restores p53-mediated apoptosis in osteosarcoma. These data suggest for the first time that EP53 can function in both human and canine osteosarcoma cells to promote cell death. Efforts to define the mechanism of action of EP53 in osteosarcoma cells are ongoing. Our results support further exploration of EP53-based osteosarcoma therapeutics for both humans and dogs. Citation Format: Lisa M. Abegglen, Cristhian Toruno, Lauren N. Donovan, Rosann Robinson, Mor Goldfeder, Genevieve Couldwell, Wendy K. Kiso, Dennis L. Schmitt, Aleah F. Caulin, Carlo C. Maley, Avi Schroeder, Joshua D. Schiffman. Elephant p53 (EP53) enhances and restores p53-mediated apoptosis in human and canine osteosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A25.