Killing Cells with Chromosomal Instability Figure 1. Establishment of a screening strategy using an induced-CIN model. (a) Reverse transcriptase-qPCR shows that the ubiquitous expression of UAS-mad2 RNAi resulted in ,85% knocked down of mad2 expression level (black bar) which is significantly less than the mad2 level in UAS-LacZ RNAi control (grey bar). Error bars represent SD. P-values are calculated by two-tailed Student’s t-test: p,0.001 = www. (b–c) Third instar larval brain cells stained with Hoechst 33342 to label DNA. (b) Normal segregation in a wild type anaphase. (c) Defective anaphase in an induced-CIN brain cell (da.mad2) resulting in a lagging chromosome (arrowed). (d) The fraction of defective anaphases (lagging chromosomes or bridges) observed in mad2 knocked down (black bar) brain squashes and wild type controls (grey bar). Error bars represent 95% CIs. P-values are calculated by two-tailed Fisher’s exact test: p,0.001 = www. (e) Diagrammatic representation of viability screen crosses. Males with Kinase-RNAi (UAS-kinasedsRNA) were crossed with females carrying the CIN background (UAS-mad2dsRNA; da-Gal4). Progeny were double knockdown (A: mad2 and kinase) or single knockdown (B: kinase only). The ratio of viable progeny A/B was used to rank candidates for further analysis. doi:10.1371/journal.pone.0047447.g001 drug resistance and other poor clinical outcomes, which may increase the efficacy of current therapies. Here we have used depletion of the SAC to induce CIN. We have then carried out a systematic genome-wide screen for kinase and phosphatase genes that, when depleted, can trigger apoptosis only in these genetically unstable cells, but not in normal cells. Our rationale is that for a therapy to be effective, cell death should be restricted to the tumour, i.e. the cells that exhibit CIN. We set up an assay system using Drosophila melanogaster in which we induced tissue-specific chromosomal instability in a wild type organism. We generated CIN by knocking down the SAC protein Mad2, which shortens metaphase, giving cells less time to correctly orient their chromosomes before the onset of anaphase [25], resulting in chromosomal bridges and lagging chromosomes. While there may be numerous defects that lead to CIN in a tumour, loss of Mad2 is found as a contributing factor in a range of CIN cancers [26,27] and more than 80% of colorectal cancers carry APC mutations that have been shown to sequester Mad2 [28] and BubR1 [29] at least in some cell lines [30]. Kinases and phosphatases are key regulatory enzymes control- ling vital processes such as cell growth, differentiation, and survival [31]. Alteration in levels of these proteins can lead to abnormal cell growth and cell death mechanisms, which can result in tumori- genesis [32]. Many kinases and phosphatases are approved as good drug targets and currently they are the main focus of drug discovery efforts against cancer [33]. Our screening of kinase and phosphatase genes in a CIN background gave a set of potential candidates that reproducibly caused significant lethality via apoptosis in CIN flies compared to their non-CIN control siblings. The screen identified several groups of candidates including PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47447