centrosomal proteins such as Nek2, which is currently being pursued as a therapeutic target for cancer [34]. These results may contribute to the identification of novel targets that can specifically kill advanced, drug resistant, CIN tumor cells without harming normal cells. Results Screening for Candidate Knockdowns that Kill Cells with SAC-induced Chromosomal Instability To generate a model system in which we could induce chromosomal instability we expressed dsRNA to knock down Mad2 and thereby weaken the SAC in Drosophila. Ubiquitous expression of mad2 dsRNA in the whole organism gives ,85% reduction of Mad2 expression as compared to control lacZ dsRNA (Figure 1a). This depletion of Mad2 resulted in .25% of anaphases showing lagging chromosomes or chromosomal bridges in larval brains (Figure 1b–d), without compromising the overall viability of the organism. This survival, despite a significant rate of anaphase errors, is consistent with the viable amorphic mad2 allele described by Buffin et al. [25]. We used this background to screen a set of gene knockdowns to identify candidates that could specifically kill cells with CIN but not normal dividing cells. We screened the Drosophila kinome, testing knockdown of 397 kinase and phosphatase genes for those that were lethal only in a CIN background (Figure 1e). This screening ranked the set of genes to identify those that when depleted in the whole organism, reproducibly caused the most lethality in CIN flies compared to their non-CIN control siblings (Table S1). The siblings vary only in whether or not they have induced CIN, so a deviation from the expected 1:1 ratio of CIN: non-CIN progeny indicated a CIN- specific effect of the candidate on viability. We observed the whole range of responses from complete lethality in a CIN background through to no effect, and we prioritized those candidates with the strongest CIN-specific lethality. Screening for Cell Death We investigated the cellular phenotypes of 26 kinases from the initial screen that gave more than 75% lethality in a CIN background, that is, more than four non-CIN for every one surviving CIN sibling. This lethality could have resulted from developmental or patterning failures, so we wished to test whether our candidates generated cell death in CIN cells. First, we examined the effect on wing development when candidates were depleted with or without mad2 (Figure S1a), and these data were quantified by measuring the amount of tissue loss (notching) in the affected wing area (Figure S1b). 17 candidates resulted in significant cell loss in adult wings when Mad2 was reduced as compared to controls (Table 1). The most promising candidates identified from this screening included genes from some well characterized groups such as those involved in JNK (Jun N- terminal kinase) signaling and centrosomal regulation as well as others (e.g. PAS Kinase) with no previous connection to cell division. Acridine Orange staining of larval wing discs showed signifi- cantly elevated levels of cell death when the candidates were knocked down in CIN cells (e.g. Figure 2), consistent with the wing tissue loss being caused by cell death in the affected region. Quantification of the Acridine Orange stains was carried out by measuring the average signal per unit area in each half of the disc and subtracting the background value (from the wild type, control half) from the RNAi affected half, to show the effect of the knockdown. This gave data consistent with the adult wing tissue loss results (complete data shown in Figures S2a and S2b). Note Killing Cells with Chromosomal Instability that loss of Mad2 by itself gave little cell death (Figure 2a’; [25]), as did depletion of the candidates alone (Figure 2b, 2c), consistent with these candidates only being required for the survival of genetically unstable cells. To validate our model we induced CIN by knocking down another SAC protein, BubR1 [35], and found that our candidates also induced cell death in this CIN background (e.g. Figure S4). Interestingly, not all candidates showing a high level of cell death in a CIN background were completely depleted by the candidate-RNAi. For example, bsk and asp knockdown still left 46% (64%) and 63% (63%) respectively of the wild type RNA levels when measured by qPCR. This partial knockdown was expected for essential genes like bsk and asp, because our original screen selected for candidate RNAi lines that were not lethal in normal cells. CIN cells must be highly sensitive to dosage variations in these candidates to give such strong phenotypes following modest candidate depletion, emphasizing the significant role these candidates have in cellular responses to CIN. Apoptosis in CIN Cells To confirm that the cell death observed was a result of the activation of apoptosis, we used antibody staining for the active form of effector caspase 3 (Figure 2e). Consistent with the results of Acridine Orange staining, knockdown of either Mad2 or a candidate alone showed little apoptosis-specific cleaved caspase 3 staining. However when we knocked down both mad2 and a selected candidate (e.g. asp: abnormal spindle) we observed apoptosis in the affected area (dark staining in Fig. 2e’’). Similar caspase results were seen for other candidates (data not shown). Together, these results suggest that knockdown of the candidates from our screen does not kill normal cells but does cause cell death by apoptosis in these CIN cells. This apoptosis could result if the candidate knockdowns generated CIN themselves, as high levels of CIN can be cell-lethal [36]. To test whether loss of our candidates induced apoptosis by increasing the level of CIN over a viability threshold, we tested whether their depletion induced CIN in normal cells by scoring mitotic cells from larval brains. We did not see any significant increase in anaphase errors (Figure S5), suggesting that depletion of the candidates alone does not generate CIN. Involvement of DNA Damage Because double-stranded DNA breaks are a well known cause of anaphase errors and are implicated in p53 dependent cell death [37,38], we tested for DNA damage by anti-cH2aX antibody staining in knockdowns of selected candidates with and without Mad2 in larval wing discs (Figure 3). Depletion of Pask, bsk, loki, Nek2, CG8878, asp, mbt or CG4041 in CIN cells gave an elevated level of DNA damage, compared to the lacZ RNAi negative control (Figure S3). Other candidates such as aPKC, when depleted in CIN cells, did not show a significantly elevated level of DNA damage as compared to the negative control (Figure S3), although they showed a high level of apoptosis (Figure S2A). In contrast, DNA damage in Pask (PAS kinase) depleted CIN cells was significantly higher than aPKC and Nek2 (Figure 3 and S3), although Pask showed lower levels of apoptosis. Our results show that the CIN dependent apoptosis generated by candidate depletion was often, but not always, associated with an increase in double stranded DNA breaks. P53 Dependence for Induced Cell Death in CIN Cells Because P53 is commonly lost in tumours and has been implicated in their CIN tolerance [21,22], we tested the effect of P53 knockdown in several candidates from our screen. Specifical- PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47447