Death is Not the End: The Role of Reactive Oxygen Species in Driving Apoptosis-induced Proliferation

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer growth and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial Drosophila tissues, the Caspase-9 homolog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Using a model of sustained AiP that produces a hyperplastic phenotype in Drosophila eye and head tissue, I have found that caspase-induced activation of JNK during AiP depends on extracellular reactive oxygen species (ROS) generated by the NADPH oxidase Duox. I found these ROS are produced early in the death-regeneration process by undifferentiated epithelial cells that have initiated the apoptotic cascade. I also found that reduction of these ROS by mis-expression of extracellular catalases was sufficient to reduce the frequency of overgrowth associated with our model of AiP. I further observed that extracellular ROS attract and activate Drosophila macrophages (hemocytes), which may in turn trigger JNK activity in epithelial cells by signaling through the TNF receptor Grindelwald. We propose that signaling back and forth between epithelial cells and hemocytes by extracellular ROS and Grindelwald drives compensatory proliferation within the epithelium, and that in cases of persistent signaling, such as in our sustained model of AiP, hemocytes play a tumor promoting role, driving overgrowth

Killers creating new life: caspases drive apoptosis-induced proliferation in tissue repair and disease

Apoptosis is a carefully orchestrated and tightly controlled form of cell death, conserved across metazoans. As the executioners of apoptotic cell death, cysteine-dependent aspartate-directed proteases (caspases) are critical drivers of this cellular disassembly. Early studies of genetically programmed cell death demonstrated that the selective activation of caspases induces apoptosis and the precise elimination of excess cells, thereby sculpting structures and refining tissues. However, over the past decade there has been a fundamental shift in our understanding of the roles of caspases during cell death-a shift precipitated by the revelation that apoptotic cells actively engage with their surrounding environment throughout the death process, and caspases can trigger a myriad of signals, some of which drive concurrent cell proliferation regenerating damaged structures and building up lost tissues. This caspase-driven compensatory proliferation is referred to as apoptosis-induced proliferation (AiP). Diverse mechanisms of AiP have been found across species, ranging from planaria to mammals. In this review, we summarize the current knowledge of AiP and we highlight recent advances in the field including the involvement of reactive oxygen species and macrophage-like immune cells in one form of AiP, novel regulatory mechanisms affecting caspases during AiP, and emerging clinical data demonstrating the critical importance of AiP in cancer

Genetic models of apoptosis-induced proliferation decipher activation of JNK and identify a requirement of EGFR signaling for tissue regenerative responses in Drosophila

Recent work in several model organisms has revealed that apoptotic cells are able to stimulate neighboring surviving cells to undergo additional proliferation, a phenomenon termed apoptosis-induced proliferation. This process depends critically on apoptotic caspases such as Dronc, the Caspase-9 ortholog in Drosophila, and may have important implications for tumorigenesis. While it is known that Dronc can induce the activity of Jun N-terminal kinase (JNK) for apoptosis-induced proliferation, the mechanistic details of this activation are largely unknown. It is also controversial if JNK activity occurs in dying or in surviving cells. Signaling molecules of the Wnt and BMP families have been implicated in apoptosis-induced proliferation, but it is unclear if they are the only ones. To address these questions, we have developed an efficient assay for screening and identification of genes that regulate or mediate apoptosis-induced proliferation. We have identified a subset of genes acting upstream of JNK activity including Rho1. We also demonstrate that JNK activation occurs both in apoptotic cells as well as in neighboring surviving cells. In a genetic screen, we identified signaling by the EGFR pathway as important for apoptosis-induced proliferation acting downstream of JNK signaling. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation

Detecting caspase activity in Drosophila larval imaginal discs

Caspases are a highly specialized class of cell death proteases. Since they are synthesized as inactive full-length zymogens, activation--at least of effector caspases and to some extent also of initiator caspases-requires a proteolytic cleavage event, generating a large and a small subunit, two of each forming the active caspase. The proteolytic cleavage event generates neo-epitopes at both the C-terminus of the large subunit and the N-terminus of the small subunit. The cleaved Caspase-3 (CC3) antibody was raised against the neo-epitope of the large subunit and thus detects only cleaved, but not full-length, Caspase-3. Although raised against human cleaved Caspase-3, the CC3 antibody cross-reacts in other species and detects cleaved caspases, most notably DrICE and Dcp-1, in Drosophila. This protocol describes the procedure for use of the CC3 antibody to detect caspase activity in larval imaginal discs in Drosophila

The Sound of Silence: Signaling by Apoptotic Cells

Apoptosis is a carefully choreographed process of cellular self-destruction in the absence of inflammation. During the death process, apoptotic cells actively communicate with their environment, signaling to both their immediate neighbors as well as distant sentinels. Some of these signals direct the anti-inflammatory immune response, instructing specific subsets of phagocytes to participate in the limited and careful clearance of dying cellular debris. These immunomodulatory signals can also regulate the activation state of the engulfing phagocytes. Other signals derived from apoptotic cells contribute to tissue growth control with the common goal of maintaining tissue integrity. Derangements in these growth control signals during prolonged apoptosis can lead to excessive cell loss or proliferation. Here, we highlight some of the most intriguing signals produced by apoptotic cells during the course of normal development as well as during physiological disturbances such as atherosclerosis and cancer

Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ( undead ) model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer

Deficiencies that modify the <i>ey>hid-p35</i>-induced AiP phenotype as suppressors or enhancers.

<p>The indicated chromosomal location is the smallest overlap of overlapping deficiencies. <i>Df(2L)TW137</i> is marked with a “?” because other overlapping deficiencies do not suppress AiP (see Suppl. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen.1004131.s008" target="_blank">Table S1</a>) indicating that the <i>Df(2L)TW137</i> chromosome carries a suppressor mutation independent of the deficiency.</p

Characterization of ‘genuine’ AiP in the eye imaginal disc: the <i>DE^ts</i>><i>hid</i> model.

<p><i>hid</i> expression was under control of <i>dorsal eye- (DE-)Gal4</i> and <i>tub</i>-<i>Gal80^ts</i> (<i>DE^ts</i>><i>hid</i>). A temperature shift (ts) to 30°C for 12 h during 2^nd larval stage induced <i>hid</i> expression (E). After the indicated recovery period (R), discs were labeled for GFP (to visualize the <i>DE</i> expression domain), Cas3* (the death domain) and ELAV (to outline the shape of the disc). (A–C) <i>DE^ts</i>><i>hid</i> experimental discs. <i>hid</i> expression induces a strong apoptotic response (A) causing strong tissue loss after 24 h recovery in some discs (panel B; R24 h, asterisk). After 72 h recovery (R72 h), the disc has fully recovered and has a normal photoreceptor pattern as judged by ELAV labeling (C). Please note the strong reduction of GFP intensity which suggests that most of the <i>GFP</i>⁺ cells have been replaced by new <i>GFP</i>⁻ cells. Arrows highlight a patch of cells that are moving to the center of the disc. (D) A control disc 72 h after <i>DE^ts</i>-induced GFP expression. Please note that GFP is a very stable protein that can still be detected 72 h after synthesis. (E) The protocol of the <i>DE^ts</i>><i>hid</i>-induced tissue ablation followed by recovery periods. (F,F′,F″,G,G′,G″) PH3-labeling of control (<i>DE^ts</i>><i>GFP</i>; F,F″) and experimental discs (<i>DE^ts</i>><i>hid</i>; G,G″). GFP marks the outline of the DE domain (F′,G′). (H) Quantification of the number of PH3-positive cells in dorsal and ventral compartments of control (F) and experimental discs (G). n = 40 for each genotype.</p

Requirement of <i>bsk</i> and <i>spi</i> for complete regeneration in the ‘genuine’ AiP model <i>DE^ts</i>><i>hid</i>.

<p>(A, A′) <i>DE^ts</i>><i>hid</i> discs treated following the protocol in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen-1004131-g006" target="_blank">Figure 6E</a> fully recover after 72 h (R72H). n = 30. (A′) shows the ELAV-only channel. (B, B′) About 35% of <i>DE^ts</i>><i>hid</i> discs expressing <i>UAS</i>-<i>bsk</i> RNAi do not completely recover after 72 h. n = 25. The arrow in (B′) highlights the incomplete ELAV pattern on the dorsal half of the disc indicating that the regeneration response was partially impaired by reduction of <i>bsk</i> activity. Please note that this disc has also been labeled for GFP. (C, C′) A control eye disc expressing <i>UAS</i>-<i>spi</i> RNAi under <i>DE^ts</i>-control following the protocol in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen-1004131-g006" target="_blank">Figure 6E</a>. After 72 h recovery, the obtained ELAV pattern in the dorsal half of the eye disc is largely normal (red in C, gray in C′). n = 20. (D, D′) An experimental <i>DE^ts</i>><i>hid</i> eye disc that was simultaneously treated with <i>spi</i> RNAi. The arrow in (D′) highlights the incomplete ELAV pattern on the dorsal half of the disc indicating that the regeneration response was partially impaired by reduction of <i>spi</i> activity. 30 out of 30 discs show incomplete regeneration. Please note that this disc has also been labeled for GFP. (E, E′) An experimental <i>DE^ts</i>><i>hid</i> eye disc that was heterozygous for <i>spi⁰¹⁰⁶⁸</i>. Similar to (D), the ELAV pattern is incomplete on the dorsal half of the disc (E′, arrow). n = 20. (F, F′, G, G′) <i>spi-lacZ</i> pattern in control (<i>DE^ts</i>><i>GFP</i>; red in F, grey in F′) and experimental discs (<i>DE^ts</i>><i>hid</i>; red in G, grey in G′) at 24 h after recovery. The arrow in (G′) points to the increased β-Gal pattern in the dorsal half of the disc. Blue is Cas3*. (H, H′, I, I′) <i>kek-lacZ</i> pattern in control (<i>DE^ts</i>><i>GFP</i>; red in H, grey in H′) and experimental discs (<i>DE^ts</i>><i>hid</i>; red in I, grey in I′) at 30 h recovery. The arrow in (I′) points to the increased β-Gal pattern in the dorsal half of the disc. Blue is Cas3*.</p