Interdependence of Bad and Puma during Ionizing-Radiation-Induced Apoptosis

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

p53 is required for Bad-mediated sensitivity to IR but not wortmannin.

<p>(A) Shown are lateral views of representative tails from 27-hpf wild-type or <i>p53</i> mutant embryos injected with 50 pg of <i>mcherry</i> (cntl) or <i>hBAD</i> mRNA. Embryos were exposed (or not) to 8 Gy IR at 24 hpf and analyzed three hours later by the Casp3 assay. Apoptosis was observed in the spinal cord after <i>hBAD</i> mRNA was injected into wild-type (arrowheads), but not mutant, <i>p53</i> embryos. (B) Fluorescence intensity was measured in the spinal cords of at least 10 embryos from each group in (A). Data represent one experiment, but the experiment was independently performed three times with similar results. (C) One-cell stage wild-type or <i>p53</i> mutant embryos were injected with 50 pg of mRNA encoding either <i>mcherry</i> control (cntl) or the constitutively active mutant <i>zbad 2SA</i>. At 8 hpf, embryos were analyzed for survival (defined by a beating heart) as performed previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. Data represent one experiment, but the experiment was independently performed three times with similar results. (D) One-cell stage wild-type embryos were injected with 50 pg of mRNA encoding either zebrafish <i>bad</i> or the apoptotically-inactive <i>zbad bh3 mut</i>. At 8 hpf, embryos were treated with increasing concentrations of wortmannin, or DMSO vehicle alone. At 48 hpf, embryos were examined for survival. At least 10 embryos were analyzed per group in three independent experiments. E) Shown are lateral views of tails from <i>p53</i> wild-type (left) or mutant (right) embryos after injection with 25 pg of mRNA encoding either <i>mcherry</i> (cntl), <i>zbad</i>, or <i>zbad bh3 mut</i>. Embryos were split into two groups and treated with either 0.3 µM wortmannin or DMSO vehicle beginning at 8 hpf and analyzed at 24 hpf by the Casp3 assay. Wild-type Bad synergizes with wortmannin to induce apoptosis in multiple tissues in a <i>p53</i>-independent manner (arrowheads).</p

Puma is required for Bad-mediated radiosensitization.

<p>(A) Shown are lateral views of representative tails from 27-hpf embryos exposed to 8 Gy IR after injection at the one-cell stage with 50 pg of mRNA encoding <i>mcherry</i> (cntl) or <i>hBAD</i> in addition to either 100 nmol of <i>puma</i> MO or mismatch (mm) control MO. Analysis of Caspase 3 activity shows that <i>hBAD</i>-mediated radiosensitivity (arrowheads) is dependent on <i>puma</i> expression. (B) Fluorescence intensity was measured in the spinal cords of at least 10 embryos from each group in (A). Data represent one experiment, but the experiment was independently performed three times with similar results.</p

Bad does not augment p53 transcriptional activity.

<p>p53 transcriptional activity was analyzed in embryos injected with 50<i>mcherry</i> (cntl) or <i>hBAD</i>. Embryos were exposed to 8 Gy (or not) at 24 hpf. RNA was harvested from each group at 27 hpf and analyzed for gene expression changes by qPCR. Expression of the <i>gapdh</i> gene was measured to normalize <i>puma</i> and <i>p21</i> mRNA levels. All data was compared to unirradiated wild-type control-mRNA-injected data, which was adjusted to a value of 1. Control-injected <i>p53</i> mutant embryos irradiated at 24 hpf and harvested at 27 hpf were included as a negative control for p53-mediated transcriptional induction. Data represent one experiment, but the experiment was independently performed three times with similar results.</p

Bad is required for IR-induced apoptosis in zebrafish embryonic neural tissue.

<p>(A) Shown are lateral views of 27-hpf embryos (head is top left in each panel) either uninjected or injected with 200 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mismatch (mm) MO. Half of each group of embryos were exposed to 15 Gy IR, and all were analyzed by the Casp3 assay. In control embryos (no inj and mm), IR-induced apoptosis occurs predominantly in the brain and all along the spinal cord (white arrowheads), whereas in <i>bad</i>-deficient embryos (ATG and e2i2), residual apoptosis is only observed in the head (arrowheads). (B) Fluorescence intensity, reflecting level of Caspase 3 activity, was measured in the spinal cords of at least 10 embryos from each group in (A) as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. The fluorescence intensity in irradiated mismatch-MO-injected embryos was normalized to 1. (C) One-cell stage zebrafish embryos were injected with 100 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mm MO as indicated (“++” indicates that 200 nmol was injected to keep total concentration of MO constant between experimental groups) and irradiated and analyzed as in (A-B). Data represent one experiment, and the experiment was independently performed three times with similar results.</p

Spliceosomal components protect embryonic neurons from R-loop-mediated DNA damage and apoptosis

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

Identification of African Elephant Polyomavirus in wild elephants and the creation of a vector expressing its viral tumor antigens to transform elephant primary cells.

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