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Cell Cycle
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JNK signaling is needed to tolerate chromosomal
instability
a b a ab b
Heidi Wong , Zeeshan Shaukat , Jianbin Wang , Robert Saint & Stephen L. Gregory
a
Department of Genetics; University of Melbourne; Melbourne, VIC, Australia
b
School of Molecular and Biomedical Sciences; University of Adelaide; Adelaide, SA,
Australia
Published online: 12 Dec 2013.

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To cite this article: Heidi Wong, Zeeshan Shaukat, Jianbin Wang, Robert Saint & Stephen L. Gregory (2014) JNK signaling is
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resulting in gain or loss of a chromosome section as well significant growth or survival advantage. Jianbin Wang1.27484 622 Cell Cycle Volume 13 Issue 4 . We have previously shown that if signaling through the JNK pathway is reduced. Drosophila. are normally removed before the sister chromatids cancer cells over their tolerance threshold. Stephen L Gregory. Our findings suggest that chromosomal instability represents a significant stress to dividing cells. chromosomal instability is not seen in normal dividing cells. while lengthening G2 rescues the JNK-deficient CIN cell death phenotype.edu.8 specific cell death. reactive oxygen species ©2014 Landes Bioscience.doi.4 This enhanced varia.13 This may prove to be are separated at anaphase. in an attempt to push already highly unstable spindle poles. Chromosomal instability (CIN). and the associated high mutation rate. Knoxville] at 23:04 24 December 2014 be found to specifically cause apoptosis in unstably dividing cells. clinically useful.9 Anaphase bridges can be caused by the spindle checkpoint protein Mad2. we used a Drosophila model to carry out a The structural chromosomal instability that produces trans.3. result in a bridge will be broken at some point before cytokinesis is com- wide diversity of cells across the tumor. can result in a lagging chromosome that fails to interventions that have no significant effect on normal cells. so Gain or loss of whole chromosomes in CIN tumors has been it is also a significant point of difference between normal and attributed primarily to a defect in correcting improper attach. which are usually formed as a result of non-homologous end-joining DNA damage repair. Report Cell Cycle 13:4. ROS.14 We induced chromosomal instability by knocking down bridges during anaphase. or are cleared. Email: robert. or the time available to rate in normal dividing cells. Zeeshan Shaukat2. is now recognized enated following replication or DNA damage repair.* 1 Department of Genetics.org/10. University of Adelaide. Email: stephen. Introduction dicentric chromosomes. We show that cell cycle progression timing has a strong effect on the apoptosis seen when JNK signaling is reduced in genetically unstable cells: a shortened G2 phase enhances the apoptosis. Robert Saint1. some of which may have a pleted. by which chromosomal instability (CIN) is generated. Revised: 12/09/2013. On the other hand. apoptosis. they can result from chromosomes that have not entirely decat- cant amounts of DNA with each cell division.6 Merotelic approach to exploit this difference has been to increase the rate attachments. Jun N-terminal kinase. © 2014 Landes Bioscience JNK signaling is needed to tolerate chromosomal instability Heidi W-S Wong1. cells undergo apoptosis because they lack a timely and effective response to DNA damage.au Submitted: 09/06/2013.2 These ongoing still linked when anaphase begins. Accepted: 12/10/2013. Mad2 Abbreviations: CIN. 2014. and test the involvement of p53 and DNA damage in causing apoptosis when JNK signaling is reduced in CIN cells. in which one kinetochore is connected to both of mitotic errors. and thus are to be a characteristic of many tumor types. Published Online: 12/12/2013 http://dx. but segregate at anaphase.au. Here we identify components upstream and downstream of JNK that are able to mediate this effect. Melbourne.gregory@adelaide. Alternatively.4161/cc. 622–631.10 In both cases. to aneuploid progeny. JNK. but has the drawback of increasing the mutation telic attachments form. VIC. tion could. as a DNA double-stranded break that must be repaired. the anaphase genetic changes. apoptosis is triggered in models of chromosomal instability induced by loss of the spindle checkpoint.*. University of Melbourne. explain the strong correlation between With the incidence of CIN being extremely high in some multi-drug resistance and chromosomal instability in late stage tumor types. Chromosomal instability. and Stephen L Gregory2.2.7. for example. Adelaide. and that without JNK signaling.5 As there is no effective treatment for such tumors. there nificantly worse than for non-CIN tumors. Lagging chromosomes may not only lead kill cells with chromosomal instability. as a common feature of tumors.11 and the prognosis for CIN tumors being sig- tumors. RNA mediated gene interference.saint@adelaide. SA. Ideally we would like to identify remove them. RNAi. Do not distribute.edu. chromosomal instability. One ments to the microtubule spindle during cell division. represents a potential therapeutic target if ways can Downloaded by [University of Tennessee. preliminary genetic screen for modifiers of the fate of CIN locations has been associated with the formation of chromatin cells. cancerous cells that can potentially be targeted for therapy. JNK. Australia Keywords: chromosomal instability. but potentially also to DNA damage on the To identify genes that could be targeted to induce CIN- affected chromosome. the tendency to gain or lose signifi. 2School of Molecular and Biomedical Sciences.1. February 15. Australia.12 it is plausible that has been considerable interest in understanding the mechanisms instability promotes cancer progression. Changes to the rate at which mero. which reduces the time *Correspondence to: Robert Saint.

Report Report available to correctly orient the chromosomes at metaphase15 and not affect levels of chromosomal instability in normal cells. To confirm that these effects on survival reflected CIN-specific cell death. or when half of the disc had reduced Mad2 to induce CIN (Fig. Here we identify the JNK signaling pathway that is required for CIN cell survival. In contrast.17 Cell stresses can activate an upstream sensor such as p53. In order to understand the mechanisms of CIN cell survival. Results Our previous screen of kinases and phosphatases identified JNK as a target that could be knocked down to kill cells with induced chromosomal instability. Msn. Wing discs were stained with Acridine Orange to indicate cell death. but leads to a significant rate of anaphase errors. autophagy.18 JNK can be activated by a wide range of stimuli and regulates an equally wide range of targets directly by phosphorylation or indirectly ©2014 Landes Bioscience. We observed little cell death in wild-type third instar wing discs. 1A and A'). the unmarked region (anterior compart- ment) is the control tissue. as such. Reduction of JNK or its regulators also induced little cell death in a wild-type background (Fig. Among these were Jun N-terminal apoptosis when knocked down in our induced CIN wing ima. JNKKKs (slpr and tak1). induction of CIN caused little cell death (A’). but did not cause apoptosis when knocked down in JNK. in which no RNAi constructs are expressed. including apopto- sis.com Cell Cycle 623 . Fig. and show that the resulting cell death induced when JNK is reduced in CIN cells is caspase-mediated. S1A). through transcription (eg AP1 targets). For each of these JNK pathway members. Knoxville] at 23:04 24 December 2014 CIN cells to avoid cell death. we tested a range of known mediators of JNK signaling for their effect on CIN tolerance (Table 1). For members of the JNK signaling pathway. and antioxidant produc- tion. A set of genes were identified that did implicated in many cellular responses to stress. while the dashed line shows the posterior compartment in which JNK pathway members were knocked down by RNAi expression with or without CIN induced by mad2-RNAi expression. 1B–D). with little cell death in each case. The scale bar shows 100 μm www. Do not distribute.14 In order to characterize the regulatory pathway involved. or one of the MAPKKKs.16. Tak1. the left column (B–F) shows discs in which JNK. ginal cells. DNA damage repair. has been control cells without CIN. In every disc. we therefore wished to know which of these JNK signaling processes were required in Downloaded by [University of Tennessee. We show that apoptosis in JNK-reduced CIN cells is partly p53-independent and identify a critical role for JNK signaling in G2 to avoid premature mitosis and consequent cell death. 1. we knocked down these JNK regulators in a proliferating epi- thelium (the developing wing disc) with or without inducing chromosomal instability (Fig. kinase (JNK) and some of its potential regulators.14 We tested the set were necessary for the survival of CIN cells and. gckIII). leading to signal trans- duction through kinases to produce activated JNK. We observed a significant reduction in CIN toler- ance when we knocked down several JNK regulatory kinases.landesbioscience. and Ste-20-related regulators of JNKKKs (mbt. FOXO. were of of kinases and phosphatases in Drosophila for those that caused interest for anti-CIN therapy. Knockdown of the JNK signaling pathway leads to cell death in CIN cells. originally identified as a stress response kinase. In control discs (A). ATM. including JNKK (hemipterous). reduction Figure 1. The right col- umn (B’–F’) shows discs in which CIN has been induced (mad2-RNAi) in the posterior compartment along with knockdown of the indicated JNK pathway member. or 14-3-3ζ have been reduced in the posterior com- partment (dashed). RNAi knockdown in CIN cells causes cell death that is not seen in the nor- mally dividing control cells.

we or PAK2 (mbt). Shaded boxes show results that would not be expected to have occurred by chance if there were no CIN effect (P < 0.19 In this APAF (Ark) resulted in significantly reduced levels of cell death Table 1. 2. Knockdown of the JNK targets FOXO and 14-3-3ζ also instability induced by a weakened spindle checkpoint. Do not distribute. JNKKK (tak1) somal instability rather than a specific interaction with Mad2. but not normal dividing We next examined the mechanism by which death was cells (Fig. reduction of JNK also led to a significant increase els of cell death (Fig. This cross knocks down the JNK pathway mem- ber in all cells. background. with or without also knocking down Mad2 to generate CIN. its reduction leads to significant chromosomal instability. Consistent with this. 624 Cell Cycle Volume 13 Issue 4 . adult wings showed notching conclude that signal transduction through the canonical JNK when JNK signaling was reduced in a CIN background (data not pathway is required for cells to be able to survive chromosomal shown). of JNK signaling in cells with induced CIN gave significant lev. caspase signaling by expressing p3520 was able to prevent the To confirm that the cell death observed was related to chromo. Inhibiting (data not shown). strongly induced cell death in CIN cells. Fig. suggesting that caspase-driven apoptosis was tested knockdown of JNK signaling when BubR1 was knocked involved (Fig. Survival of JNK pathway knockdowns in a CIN background Downloaded by [University of Tennessee. S1B). cell death observed for knockdown of JNK.001) using the binomial distribution (see “Materials and Methods”). Bax (Debcl). The indicated number of sibling progeny were obtained. and ©2014 Landes Bioscience. we death and consequent loss of tissue. knockdown of down. The percent survival shows the ratio of siblings surviving in the CIN background to those surviving in a wild-type background: 100% would indicate equal numbers with and without CIN. Fig. From these results. Knoxville] at 23:04 24 December 2014 Progeny Class Gene CG RNAi line Crosses kinase kinase + CIN % Survival V104569 3 6 0 - JNK bsk 5680 V34138 9 59 3 5% N-5680R-1 2 2 0 - V49413 4 116 27 23% hep 4353 V2968 3 30 10 33% JNKK N2190R-2 2 0 0 - Mkk4 9738 V26929 3 130 67 52% V33516 3 10 1 10% Slpr 2272 V33518 6 49 0 - V106449 5 107 99 93% Pk92B 4720 V34891 4 10 18 >100% JNKKK V101357 6 44 8 18% Tak1 18492 N1388R-1 1 12 4 33% Takl1 31421 V25760 4 111 86 77% V34898 8 280 184 66% Takl2 4803 V104701 6 252 134 53% Mbt 18582 v46043 5 125 10 8% V101517 5 6 0 - Ste20-related msn 16973 N16973R-1 3 1 0 - kinases V49558 5 77 14 18% GCKIII 5169 V107158 7 81 89 >100% FOXO 3134 V30556 3 0 0 - V48724 3 0 0 - JNK targets 14-3-3ζ 17870 V48725 4 85 16 19% Jra 10835 V107997 3 34 29 85% The indicated number of crosses of each RNAi line for JNK pathway members was set up with our test strain. Consistent with high levels of cell in cell death (Fig. 1 B'–D'). as did knockdown of Jun (Jra) and 14-3-3ε induced in CIN cells with reduced JNK signaling. and any of the canonical apoptotic mediators Hid. 4G and I. BubR1 is needed for the spindle assembly checkpoint. 1E–F'). S1E).

or Apaf on the amount of cell death seen in JNK-reduced CIN cells. we considered how JNK might affect the process and interact with the spin- dle assembly checkpoint defect. of either JNK or JNKKK (tak1) or PAK2 (mbt) in CIN cells Both p53 and E2F1 promote apoptosis in response to DNA gave apoptosis that was only modestly reduced by knockdown damage. (Fig. Knoxville] at 23:04 24 December 2014 sage through mitosis with a defective spindle checkpoint can be responsible for DNA damage. so we tested whether blocking cell cycle progression would prevent the DNA damage and consequent apoptosis (Fig. In control discs (A and B) CIN produced little cell death. Shortening G1 by overexpressing Cyclin E had less effect. Do not distribute. 4D). such as Pask. Hid. the unmarked region does not express RNAi constructs. Having found that cell cycle progression was needed to observe apoptosis in JNK-reduced CIN cells. high levels of cell death were observed. We conclude that the cell complete loss of the apoptosis seen with other candidates from death observed corresponds to apoptosis mediated by the canoni.landesbioscience. Bcl-2. It is known that pas- Downloaded by [University of Tennessee. Strikingly. for the CIN-specific apoptosis we had observed. The cell death observed when the JNK pathway is knocked down in CIN cells can be greatly reduced by expressing the caspase inhibitor p35 or blocking the canonical apoptosis pathway. S1F and S3). We observed some DNA dam- ineffective depletion of p53. for example by failure to resolve late-replicating DNA before an early anaphase. we generated cells with a short G2 by overexpressing the M-phase entry factor Cdc25c (string). 3. 4.7 Both of these sources of DNA damage require cells to pass through mitosis. In every disc. When JNK was reduced in CIN cells (C). however. A typical activator of apoptosis apoptosis seen in JNK-reduced CIN cells (Fig. S1D). 4J). in JNK-reduced CIN cells (Fig.23 or the late mitotic damage observed in Mad2-reduced vertebrate cells. We concluded from these experi- ments that CIN cells are particularly sensitive to an early onset of mitosis.25 To test whether CIN cells were particularly sensitive to G2 completion. This cell death was blocked by the caspase inhibitor p35 (D).21 We tested whether p53 was required lead to activation of the classical apoptotic response through p53. The same inhibition of cell death by p35 was seen in CIN cells knocked down for the upstream JNK regu- lators Tak1 (E and F) and Mbt (G and H). S1C). as this p53–RNAi construct caused age in cells with induced CIN. One possibility was that loss of JNK signaling might cause defects in G2. Wing discs were stained with Acridine Orange to show cell death. as the processes of late replication and DNA repair must be completed in G2 if cells are to avoid a catastrophe in mitosis. Elevated staining for activated apoptosis is E2F1. Figs. Fig. our screen. S1E). so we tested for the presence of double-stranded DNA of p53 (Fig. The scale bar shows 100 μm. which is a problem clinically as so from these results that reduced JNK signaling in CIN cells can many cancers lack p53. which can also activate the caspases through Caspase-3 confirmed that JNK-reduced CIN cells die by cas. Fig. 4. S2). Figure 2. Error bars indicate the 95% CI. 2I). We conclude through this pathway is p53. 5.22 Knockdown of E2F1 was also able to reduce the level of pase activation (data not shown). Knockdown but can also use a p53-independent mechanism. or reduced for JNK signaling ©2014 Landes Bioscience. cells with both CIN and reduced JNK signaling showed a significantly increased amount of DNA damage (Fig. This mild effect was not simply due to breaks in JNK-reduced CIN cells. www. The graph indicates the normalized average number of apoptotic cells per affected wing half for each genotype.14 One alternative to p53 activation of cal caspase-mediated pathway. (I) The effect of knocking down the canonical apoptosis mediators Hid.24 and we found that inducing this cell cycle arrest was able to greatly reduce the amount of apoptosis seen in JNK-reduced CIN cells (Fig. while the dashed line shows the posterior compartment affected by CIN (mad2-RNAi) and/or p35 overexpression.26 Shortening G2 in CIN cells led to an increase in the amount of DNA damage and cell death (Fig. Overexpression of the cdk2 inhibitor Dacapo leads to a G1 arrest. Fig.com Cell Cycle 625 .

. S5). as measured by γH2AX staining. about what the upstream and downstream mediators might ate a robust G2-phase arrest that could be removed by knock. we would expect that CIN-specific apoptosis would also be seen when DNA damage-sensing and repair proteins are depleted. while sis onset and that DNA damage was detected in JNK-reduced lengthening the G2 phase (but not G1 phase) suppresses the levels CIN cells before they went on to apoptose. Knoxville] at 23:04 24 December 2014 DNA was repaired. Some of the cell death observed when the JNK pathway is alone. i.31 In both cases we observed a reduction in the amount of apoptosis in JNK-reduced CIN cells (Fig. 32 and 33). while the dashed line shows reduced CIN cells corresponds to canonical caspase-mediated the area affected by CIN (induced by the expression of mad2-RNAi) and/ apoptosis. Consequently. be. E. 626 Cell Cycle Volume 13 Issue 4 . we examined CIN cells knocked down for either Grapes (Chk1) or Loki (Chk2). We show that the apoptosis observed in JNK. 5G–I. JNK has been implicated in DNA dam- age repair27 and regulates FOXO and Gadd45. Tak1.. 2 key effectors of DNA repair. Fig. for example in TNFα or heat shock arrest the cell cycle in G2 following massive externally induced responses. key mediators of DNA damage responses. To test this hypothesis we extended the length of G2 either by overexpressing the Cdk1 inhibitor Myt130 or by reducing the amount of cyclin B. with little information available in JNK-reduced cells. F. To test this. DNA damage. We conclude from these results that although JNK does not affect the ability of the DNA damage checkpoint to arrest cells in G2 following massive damage.e.29 Induction of CIN when either of these proteins was knocked down led to a significant increase in apoptosis (Fig. enhances the level of caspase in JNK-reduced CIN cells. and H) Imaginal discs showing that cell cell cycle alone is deregulated. S4). We also show that shortening the death is reduced but not eliminated by knockdown of p53 in CIN cells G2 phase (but not G1 phase) induces apoptosis in CIN cells and that are also knocked down for the indicated JNK pathway member.28 If the G2 defect is in DNA repair. Wing discs were stained signaling cascade from a MAPKKK through to the JNK targets with Acridine Orange to show cell death. refs. to trigger the DNA damage checkpoint. DNA damage. con- firming that CIN cells are particularly sensitive to defects in DNA repair. In every disc. but was signaling cascade from a MAPKKK through to the JNK tar- unaffected by JNK knockdown (Fig. or Mbt) have been knocked down in CIN cells. Having observed that CIN cells were sensitive to early mito. This kind of JNK response is induced escaping arrest. Control wings (A and B) show little cell death when CIN is induced (A) or when p53 is knocked down in CIN cells (B). We conclude that JNK was not needed to by several stresses. the unmarked FOXO and Jun. we hypothesized that of apoptosis JNK was needed to delay mitotic onset following DNA dam. The scale bar shows 100 μm. region does not express RNAi constructs. If knockdown of JNK reduces the cells’ ability to repair DNA damage in the time available in G2. (JNK. suggesting that JNK-reduced cells benefit from a longer G2 phase. way. with almost no cells gets FOXO and Jun. In most cases.14 Our analysis suggests the involvement of a typical JNK reduced in CIN cells is independent of p53.34. (D.g. Ionizing radiation was used to gener. Our analysis suggests the involvement of a typical kinase down of the DNA damage checkpoint protein Chk1. giving rise down occur in cells exhibiting CIN but not in cells in which the to high levels of cell death. the analysis of the JNK pathway with respect age. Discussion Here we report studies exploring the surprising finding that suppression of JNK activity in wing imaginal disc cells exhibiting chromosomal instability (CIN) results in highly elevated levels of cell death not observed with either CIN or JNK knockdown Figure  3. An alternative explanation for the effect of JNK knockdown on CIN cells is that JNK affects the efficiency with which Downloaded by [University of Tennessee. then delaying the onset of mitosis should be able to reduce the level of apop- tosis seen in JNK-reduced CIN cells. and that JNK-reduced CIN cells exhibit high levels of or kncockdown of p53. our data are consistent with a model in which reduced JNK signaling reduces the cell’s ability to effectively repair DNA damage before the onset of mitosis. Do not distribute. S1G). to CIN has been limited to the use of JNK mutants or inhibi- we tested whether the DNA damage checkpoint remained intact tors (e. and G) Imaginal discs in which members of the JNK signaling path- apoptosis and DNA damage phenotypes induced by JNK knock.35 ©2014 Landes Bioscience. The enhanced (C.

45 and phosphorylation of FOXO by JNK is needed for a wide range of stress survival responses.44 so clearly JNK activa- of JNK has been reported to reduce the incidence of tumors in tion is only one of many methods for triggering apoptosis.42 the apoptosis.36 Consistent with this. which has been seen when hyperploidy is induced by cen- trosome defects. some JNK is needed to activate normal stress response genes. TNFα has been shown to ©2014 Landes Bioscience. (E–J) Wing discs were stained with Acridine Orange to show cell death. (A–D) Wing discs were stained for double-stranded DNA breaks (anti-P-H2AvD/ γH2Ax).18 Nonetheless. even when CIN was induced (B). Knocking down JNK in CIN cells (I) gives considerable cell death. with no JNK activation. Do not distribute.41. JNK is needed in response to CIN. In each disc. particularly as some CIN models generate JNK- Drosophila for effective stress tolerance. It has been shown in several organisms that even relatively than 200 knockdowns that could induce Caspase 3 activation minor aneuploidy can cause proteotoxic stress. the unmarked region does not express RNAi constructs. The observation that JNK knockdown in normal cells does not result in widespread DNA damage underlines that chromosomal instability is a significant stressor. while the dashed line shows the posterior com- partment cells that are affected by CIN (mad2-RNAi) and/or JNK knockdown.38-40 In the context of cancer. We have shown that DNA damage is at least one of these alternative mechanisms: with reduced JNK signaling. cause apoptosis in normal wing discs has shown that although which leads to a high rate of anaphase errors and DNA dam.38 On the other hand. In every disc.8 Figure 4. In fact. JNK activation. but this is increased when JNK is reduced in CIN cells (D).17 again consistent with a model in which to tolerate chromosomal instability induced by spindle check. triggering JNK-dependent apoptosis. transient activation of JNK can instead promote cell question arises as to how cells that lack JNK are able to die in survival following stress. The scale bar shows 100 μm.com Cell Cycle 627 . although cell cycle arrest by overexpression of Dacapo (F) does cause some cell death. www. which is greatly reduced by overexpression of Dacapo (J). Knoxville] at 23:04 24 December 2014 knockdown of JNK sensitizes cells to CD95-mediated apoptosis. this may be partly through aneuploidy giving proteotoxic stress. Induction of CIN by reducing BubR1 (G) causes some cell death.46 These results suggest a model in which cells require appropriate levels and timing of JNK activation in response to stress.7. Too much JNK signal may be interpreted as irreparable damage.38 RNAi screening for knockdowns that our case is imposed by the weakening of the spindle checkpoint. and DNA damage sensitivity. data showing that signaling through JNK can lead to apoptosis. which is not altered by Dacapo overexpression (H).43 Similarly. DNA damage is induced when JNK is reduced in CIN cells.37 The stress involved in dependent apoptosis.landesbioscience. We have found that signaling through JNK is needed for cells several mouse models. given that activation of JNK is an essential fea- While there is no doubt that sustained activation of JNK leads to ture of some apoptotic responses such as to irradiation. so without JNK the damage will accumulate until alternative mechanisms trigger apoptosis. there were more age. there is a significant accumulation of unrepaired DNA damage in CIN cells. and the cell death induced by knockdown of the JNK pathway in CIN cells is cell cycle-dependent. Control discs (E and F) show little cell death. As noted above. including death outside the overexpressing region (also seen in H and J).39 while spindle checkpoint defects can also lead to DNA double-stranded breaks in telophase or S phase. JNK activation was common when cells died. while the dashed line shows the area affected by overexpression of Dacapo and/or reduc- tion of the indicated gene by RNAi. Knockdown of JNK also shows little DNA damage (C). loss induce JNK-independent cell death. This might appear surprising given the amount of lular transformation. signaling through JNK is necessary to tolerate the stresses of cel- point defects. the unmarked region does not express RNAi constructs. Control discs (A and B) show little DNA damage. reduced JNK signaling can even enhance cell death: Downloaded by [University of Tennessee.

so we speculate that loss of Mad2 makes pho-histone2Av in JNK-reduced CIN cells. as it suggests that repair can be performed.52 Consistent with this model. H. Cell death induced by JNK knockdown in CIN cells (G. damage repair via AP1. but that the DNA damage checkpoint does checkpoint genes such as Chk2 are.15 Consistent In support of a third alternative explanation. this worse. we found ©2014 Landes Bioscience. Do not distribute. Cell death is increased in CIN cells when G2 is shortened and decreased when G2 is lengthened. 628 Cell Cycle Volume 13 Issue 4 . and I) is reduced by lengthening G2 either by Myt1 overexpression (H) or loss of one copy of Cyclin B (I). In every disc.14 so it was plausible that loss of JNK might late unrepaired DNA damage as the cells cycle. Downloaded by [University of Tennessee. as it reduces the available time for repair. while the dashed line shows the posterior compartment that is affected by CIN (mad2-RNAi) and/or gene overexpression. Wing discs were stained with Acridine Orange to show cell death. JNK is also known is now strong evidence that sections of chromosomes are often to mediate the phosphorylation of histone 2A variants as part of still catenated even after anaphase onset. This is consistent have been disrupting the DNA damage checkpoint. Failure Figure 5. there is clear evi- with this model. at least in terms of responding to that are highly susceptible to unrepaired DNA damage. which is strongly enhanced by the induction of CIN (F).50 so the primary problem in our JNK phase bridges that must be cleared by helicases during anaphase reduced cells could have been in detecting the DNA damage. like JNK. as we detect elevated rather than reduced levels of phos- bridging and breakage.47 Treatments that increase the amount of still.48 though this study used an inhibi. which accumu- survival of CIN cells. However. Cells with a shorter G2 phase from overexpression of Cdc25 (stg-RNAi) (E and F) show some cell death. required for the not give enough time in JNK-reduced CIN cells. We do not think this contributes significantly to the phenotype. DNA damage detection.51. the unmarked region does not express RNAi constructs. The DNA damage checkpoint mitotic timing caused by the checkpoint defect results in cells thus appeared to be intact. is a remarkable result. leading to ultrafine ana. even when CIN is induced (B). This DNA damage checkpoint.49 We also know that DNA damage given enough time. the majority of anaphase defects observed in dence for a role for JNK signaling in promoting efficient DNA our Mad2 knockdown CIN model are bridges (data not shown). Cells with a shorter G1 phase from overexpression of Cyclin E (C and D) also show little cell death even when CIN is induced (D). There massive damage induced by γ-irradiation. The data reported here also support a model in which reduced in response to ionizing radiation. particularly in that simply increasing the time available in G2 was able to sig- Drosophila. to avoid breaks. catenated DNA in cells entering mitosis increase the amount of however. Knoxville] at 23:04 24 December 2014 tor of doubtful specificity. Control wings (A and B) show little cell death. The functions of JNK in G2 are still unclear. The scale bar shows 100 μm. we with a model in which the role of JNK is primarily to ensure the saw no effect of JNK knockdown on the ability of cells to arrest timely production of repair enzymes in response to stress. A study in vertebrate cells has implicated JNK in the nificantly reduce the apoptosis in JNK-reduced CIN cells.

donkey anti-mouse Dylight 649 www.53 to Cobalt-60 for a dose of 40 Gy. da-Gal4/TM6b Gal80ts) to each candidate RNAi line.30 The cyclin B allele used was CycB. been observed that disruption of the posterior compartment can potentially induce non-autonomous cell death in the anterior Acknowledgments compartment. Whether this represents a therapeutic opportunity remains All stocks were raised at 25 °C. Note that knockdown progeny numbers was assessed by assuming a fre- this is a live stain. it still has a need to man. We thank the Bloomington and VDRC stock centers.aspx?id=34929). http://www. Supplemental materials may be found here: com/ivgn/product/A11036). Quantitation of Acridine T(CIN#. goat anti-rab. with a sig- of Acridine Orange-positive cells in the wild-type anterior half nificant false-negative rate but low false-positive rate. V107158.com/Product.. Statistical analysis of product/A1301).com/ivgn/ not due to problems with parental fertility. All the binomial distribution (1-BINOMDIST(0. as JNK can induce ized by counting the number of P-H2AvD-positive cells in the apoptosis. of obtaining at least one such result by chance from the approxi- ing enough background to visualize the tissue. FALSE).com Cell Cycle 629 .landesbioscience. with the same insertion point on the second chromosome that tated by measuring the average brightness in the anterior half and should not be affected by insertion site effects were V104569.57 interaction described here. to increase both the Axioplan2 with AxioCam MRm camera. third instar larval wing discs were dissected in Table 1 produced more than 20 TM6b progeny. The explanation may be simply that although a tumor posterior wing half per 10 000 pixels and subtracting the number may be highly apoptosis resistant. CIN+NONCIN#. ensuring in PBS. Contrast. posterior half. V101517. Rabbit anti-P-H2AvD (Rockland 1:500. Because we normalized using the anterior H Richardson and L Quinn for providing fly stocks. non- expected. Quantification of DNA damage staining was normal- tumors. Do not distribute.5. 4F. Helpful comments and advice were given by Immunostainings were performed using standard protocols as M Bogoyevitch and M Murray. to repair damage before mitosis can lead to anaphase chroma. V104701. Fig. in these cases the quantification of apoptosis in University of Adelaide and the University of Melbourne for stu- the posterior compartment will be an underestimate. (Jackson 1:100). and J). Note that it has No potential conflicts of interest were disclosed. but the development of specific and effective JNK the Bloomington stock center or the Vienna Drosophila Resource inhibitors55 may permit testing in tumor cells of the JNK–CIN Center. and most were sourced from ©2014 Landes Bioscience. Ionizing radiation treatment was done by plac- tin bridges and subsequent tetraploidy or bridge-breakage-fusion ing third instar larvae on sealed agar plates. to be seen. html). http://products. rinsed briefly. A representative mately 500 lines tested in this paper and Shaukat et al. dent scholarships. Supplemental Materials bit Alexa 568 (Invitrogen 1:100.landesbioscience.17 This has been cited as surprising.2 Table 1 Downloaded by [University of Tennessee. expression of antioxidants and to ensure effective DNA repair in Drosophila stocks G2. Genes were knocked down using either engrailed- Gal4 or hedgehog-Gal4 to drive RNAi expression in the posterior Disclosure of Potential Conflicts of Interest compartment of the larval wing imaginal disc.58 Insertion of the wing from the number of Acridine Orange-positive cells site effects cannot be ruled out and may explain some of the varia- in the posterior half (marked with mCD8-GFP) using ImageJ tion between RNAi constructs targeting the same gene.cellsignal. All other images were quanti. in the anterior half per 10 000 pixels.BINOMDIS original images are available on request.14 Antibodies used were mouse anti-P-Histone3 (Cell National Health and Medical Research Council grants 525477 Signaling 1:100. and imaged in PBS. then stained for 2 min in a 1 μM Acridine Orange that when low numbers of their mutant siblings were seen. then. Additional stocks were UAS-dacapo. The level of staining in Figure 2I was normalized by subtracting the number knockdown is expected to vary between RNAi lines. Imaging was performed age high levels of DNA damage generated by CIN and ROS. Knoxville] at 23:04 24 December 2014 shows the results of crossing our test strain (UAS-Mad2RNAi/ Materials and Methods CyO. V101357. then exposing them cycles that perpetuate the damage. H.FALSE)).14 using disc was selected from at least 10 for each experiment shown. http://www. brightness and gamma levels were adjusted CIN (candidate-RNAi alone) progeny and testing the likelihood in Photoshop to most clearly reproduce the signal while retain. They were allowed to rest for Our observation that JNK signaling is needed to tolerate CIN 1 h before dissecting and staining for mitotic cells with anti-P- is consistent with the elevated expression levels of JNK seen in Histone3.500. and 1027878. and the compartment. All crosses listed described. it was solution (Invitrogen. Note that half of the progeny will carry TM6b instead of Gal4 Acridine Orange stains for cell death were performed as and hence be wild-type (not shown in Table 1).14 Briefly.invitrogen. and UAS-Myt1. http://products. This work was funded in part by described.54 on an Olympus BX60 microscope with DP71 camera or a Zeiss The JNK levels must be high enough.invitrogen. KK lines software (Cell Counter plugin).com/products/9701. and V107997. 0. candidate-RNAi) vs. so some variation in background levels is quency of 0. rockland-inc.5 for CIN (Mad2-RNAi. normalized by subtracting the average brightness of the wild-type V106449.com/journals/cc/article/27484 www..56 and this is clear in some of our genotypes (e.24 UAS-CyclinE.g.

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