Grp/DChk1 is required for G(2)-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-dependent pathways in Drosophila are not understood in detail. Here, we report the function of these checkpoint kinases, referred to as Grp/DChk1 and Dmnk/DChk2 in Drosophila Schneider's cells, and identify an upstream regulator as well as downstream targets of Grp/DChk1. First, we demonstrate that S2 cells are a suitable model for G(2)/M checkpoint studies. S2 cells display Grp/DChk1-dependent and Dmnk/DChk2-independent cell-cycle-checkpoint activation in response to hydroxyurea and ionizing radiation. S2 cells depleted for Grp/DChk1 using RNA interference enter mitosis in the presence of impaired DNA integrity, resulting in prolonged mitosis and mitotic catastrophe. Grp/DChk1 is phosphorylated in a Mei-41/DATR-dependent manner in response to hydroxyurea and ionizing radiation, indicating that Mei-41/ATR is an upstream component in the Grp/DChk1 DNA replication and DNA-damage-response pathways. The level of Cdc25(Stg) and phosphorylation status of Cdc2 are modulated in a Grp/DChk1-dependent manner in response to hydroxyurea and irradiation, indicating that these cell-cycle regulators are downstream targets of the Grp/DChk1-dependent DNA replication and DNA-damage responses. By contrast, depletion of Dmnk/DChk2 by RNA interference had little effect on checkpoint responses to hydroxyurea and irradiation. We conclude that Grp/DChk1, and not Dmnk/DChk2, is the main effector kinase involved in G2/M checkpoint control in Drosophila cells

Genome-Wide Expression Analysis Identifies a Modulator of Ionizing Radiation-Induced p53-Independent Apoptosis in Drosophila melanogaster

Tumor suppressor p53 plays a key role in DNA damage responses in metazoa, yet more than half of human tumors show p53 deficiencies. Therefore, understanding how therapeutic genotoxins such as ionizing radiation (IR) can elicit DNA damage responses in a p53-independent manner is of clinical importance. Drosophila has been a good model to study the effects of IR because DNA damage responses as well as underlying genes are conserved in this model, and because streamlined gene families make loss-of-function analyses feasible. Indeed, Drosophila is the only genetically tractable model for IR-induced, p53-independent apoptosis and for tissue regeneration and homeostasis after radiation damage. While these phenomenon occur only in the larvae, all genome-wide gene expression analyses after irradiation to date have been in embryos. We report here the first analysis of IR-induced, genome-wide gene expression changes in wild type and p53 mutant Drosophila larvae. Key data from microarrays were confirmed by quantitative RT-PCR. The results solidify the central role of p53 in IR-induced transcriptome changes, but also show that nearly all changes are made of both p53-dependent and p53-independent components. p53 is found to be necessary not just for the induction of but also for the repression of transcript levels for many genes in response to IR. Furthermore, Functional analysis of one of the top-changing genes, EF1a-100E, implicates it in repression of IR-induced p53-independent apoptosis. These and other results support the emerging notion that there is not a single dominant mechanism but that both positive and negative inputs collaborate to induce p53-independent apoptosis in response to IR in Drosophila larvae

E2F1 and E2F2 have opposite effects on radiation-induced p53-independent apoptosis in Drosophila

AbstractThe ability of ionizing radiation (IR) to induce apoptosis independent of p53 is crucial for successful therapy of cancers bearing p53 mutations. p53-independent apoptosis, however, remains poorly understood relative to p53-dependent apoptosis. IR induces both p53-dependent and p53-independent apoptoses in Drosophila melanogaster, making studies of both modes of cell death possible in a genetically tractable model. Previous studies have found that Drosophila E2F proteins are generally pro-death or neutral with regard to p53-dependent apoptosis. We report here that dE2F1 promotes IR-induced p53-independent apoptosis in larval imaginal discs. Using transcriptional reporters, we provide evidence that, when p53 is mutated, dE2F1 becomes necessary for the transcriptional induction of the pro-apoptotic gene hid after irradiation. In contrast, the second E2F homolog, dE2F2, as well as the net E2F activity, which can be depleted by mutating the common cofactor, dDp, is inhibitory for p53-independent apoptosis. We conclude that p53-dependent and p53-independent apoptoses show differential reliance on E2F activity in Drosophila

Force-activated DNA substrates for probing individual proteins interacting with single-stranded DNA

Single-molecule force spectroscopy provides insight into how proteins bind to and move along DNA. Such studies often embed a single-stranded (ss) DNA region within a longer double-stranded (ds) DNA molecule. Yet, producing these substrates remains laborious and inefficient, particularly when using the traditional three-way hybridization. Here, we developed a force-activated substrate that yields an internal 1000 nucleotide (nt) ssDNA region when pulled partially into the overstretching transition (∼65 pN) by engineering a 50%-GC segment to have no adjacent GC base pairs. Once the template was made, these substrates were efficiently prepared by polymerase chain reaction amplification followed by site-specific nicking. We also generated a more complex structure used in high-resolution helicase studies, a DNA hairpin adjacent to 33 nt of ssDNA. The temporally defined generation of individual hairpin substrates in the presence of RecQ helicase and saturating adenine triphosphate let us deduce that RecQ binds to ssDNA via a near diffusion-limited reaction. More broadly, these substrates enable the precise initiation of an important class of protein-DNA interactions

<i>tie</i> is needed for IR-induced changes in GFP <i>ban</i> sensor.

<p>(A–I) Third instar larvae were irradiated at 96±2 hr after egg deposition with 0R (-IR) or 4000R (+IR) of X-rays and wing discs imaged live 24 h later. Images were acquired and treated identically. The mean GFP signal was quantified for each disc using Image J, and the averages are shown for each genotype/treatment. Age-matched sensor only controls (i.e. wild type for <i>tie</i>) were included in each experiment (images not shown here but see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004220#pgen.1004220.s001" target="_blank">Fig. S1</a>). (A–C) Wing discs from homozygotes for Df(3L)Exel2098 that removes only <i>tie</i> (‘<i>tie</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004220#pgen.1004220-Huang1" target="_blank">[<i>17</i>]</a>’). N = 31, 28, 31 and 31 discs (left to right) in three independent experiments. (D–F) Wing imaginal discs from homozygotes for <i>tie^e03394</i>. N = 26, 27, 26 and 25 discs (left to right) in three independent experiments. (G–I) Wing imaginal discs from trans-heterozygotes <i>tie^e03394/</i>Df(3L)Exel2098. N = 29, 29, 30 and 30 discs (left to right) in three independent experiments. (J–L) Wing imaginal discs from Df(3L)Exel2098 homozygotes that carry one copy of a <i>UAS-tie</i> transgene. The data are from 5 discs per sample in two independent experiments. The larvae carried two copies of the GFP sensor in A-I and one copy in J-L. Error bar =  ±1SEM. p-values reflect a comparison of two samples at the ends of each bracket. (M) <i>tie</i> is needed for IR-induced changes in <i>ban</i> levels. Quantitative RT-PCR was used to quantify mature <i>ban</i> miRNA. The values were normalized to an internal a-tubulin control, and expressed as fold change from un-irradiated <i>w¹¹¹⁸</i> controls (‘w2-’ set at 1). ‘-‘  =  no IR; ‘+’  =  4000R. ‘w’  =  <i>w¹¹¹⁸</i>; Df’  =  homozygotes of Df(3L)Exel2098 that removes only <i>tie</i>; ‘3394’  =  <i>tie^e03394</i> homozygotes. *  =  difference compared to w2-. ** and *** =  significance compared to w2+. *  =  p <0.01; **  =  p <0.001; ***  =  p < 10⁻⁶. Student's t-test was used to determine significance.</p

Protection by Hid/Rpr-induced cell death is sensitive to <i>ban</i> gene dosage.

<p>Wing imaginal discs from larvae carrying one copy each of <i>ptc4-GAL4</i>, <i>UAS-hid, UAS-rpr</i> and <i>tub-GAL80^ts</i> were fixed and stained for DNA (A, C, E) and cleaved Caspase 3 (B, D, F) at 4 h after irradiation with 0 (-IR) or 4000R (+IR) of X-rays. Additional genotypes were as indicated. (G) shows the timeline followed. In otherwise wild type background (D), areas outside the <i>ptc</i> domain showed reduced caspase staining reflecting protection in response to <i>ptc4>Hid/Rpr</i> (arrows). In contrast, the corresponding areas in <i>ban</i>/+ discs showed caspase activity (F, arrows).</p

Dying Cells Protect Survivors from Radiation-Induced Cell Death in <i>Drosophila</i>

<div><p>We report a phenomenon wherein induction of cell death by a variety of means in wing imaginal discs of <i>Drosophila</i> larvae resulted in the activation of an anti-apoptotic microRNA, <i>bantam</i>. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR)-induced apoptosis. Both <i>ban</i> activation and increased protection from IR required receptor tyrosine kinase Tie, which we identified in a genetic screen for modifiers of <i>ban</i>. <i>tie</i> mutants were hypersensitive to radiation, and radiation sensitivity of <i>tie</i> mutants was rescued by increased <i>ban</i> gene dosage. We propose that dying cells activate <i>ban</i> in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect we report differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in <i>Drosophila</i> larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy.</p></div

Cell death and protection in discs with clonal Hid/Rpr expression.

<p>The larvae were generated as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004220#pgen-1004220-g001" target="_blank">Figure 1</a> and irradiated with 4000R of X-rays after de-repression of GAL4. Wing imaginal discs were extirpated 4 h after irradiation and fixed and stained for DNA (A, E) and cleaved active Caspase 3 (B, F). RFP clonal marker (C, G) was used to mark the clonal boundaries in the images. (D, H) show merged images. (I) shows the timeline followed. * shows areas outside the clone that display robust caspase activation in RFP only discs (top row) but not in RFP, Hid/Rpr discs (bottom row).</p

Mean fluorescence in the wing pouch (Anterior normalized to Posterior).

<p>N  =  number of discs examined. ‘PE3’  =  <i>ptc-GAL4>UAS-dsRNA</i> against dE2F1.</p