target knockdown and IR confirmed selective antagonism of G1 positivity in IR exposed cells as opposed to alteration of G1 positivity in unchallenged cells, supporting a significant role and requirement for the identified hits in IR-mediated G1 checkpoint activation. Inhibitors of canonical DSB signalling did not prevent the accumulation of cells in G1 following IR exposure, consistent with our earlier results (Figure S1) that such signalling is not involved in RB1 activation, and hence may not participate in the control of G1 checkpoint response (Figure S3). Effect of target silencing on IR-associated cell survival G1 checkpoint activation is thought to play an important role in protecting cells against IR elicited death [6]. Importantly, loss of G1 checkpoint activity was shown to exacerbate the loss of checkpoint functions in S and G2/M phase of the cell cycle, leading to radiation hypersensitivity of cells with such additional defects [48,49,50,51,52]. We thus hypothesized that ablation of the genes identified in the screen could lead to radio-sensitization and that this may be potentiated by a loss of G2 checkpoint signalling. To test these hypotheses we assessed whether silencing of the identified targets might increase the sensitivity of tumour cells to radiation, using a cell viability assay based on ATP luminescence (Figure 5, Figure S5). To assess if survival loss required (or was exacerbated) by G2 checkpoint loss we simultaneously knocked down the checkpoint kinase CHK1, a key signal transmitter in this checkpoint (Figure 5). As in previous experiments we used parallel-transfected cells for assessment of PS780 RB1 loss, to safe-guard against false negative scores caused by lack of siRNA performance (Figure 5K; Figure S5). An increased loss of viability, that scored significantly in statistical tests, was seen with all targets. Exacerbation of survival was either confined (STK4 and DYRK1A) or predominant (PRPK and HK1) in cells in which CHK1 expression was knocked down (Figure 5A–D), or equal (PRKACG and CDK4) in both CHK1 perturbed and unperturbed cells (Figure 5E, F and L; Figure S5). Knockdown of STK4, PRPK, HK1 and DYRK1A, which exacerbated survival under condition of CHK1 knockdown, mirrors the effects of RB protein knockdown, where, similarly, enhanced viability loss is dependent upon CHK1 loss (Figure S6), corroborating the prediction that G1 checkpoint control provides radioprotection under conditions of G2 checkpoint loss. We note that combined knockdown of RB1 and RBL1/p107 is required to similarly exacerbate radiation response in these experiments, in agreement with the frequently redundant functioning of these proteins in many cell lineages and signalling contexts [53]. Knockdown of PRKACG or CDK4, where increased loss of viability arises regardless of whether CHK1 function is perturbed (Figure 5L and Figures S5) in turn reflects the effects of p21CIP1/WAF1 or TP53 knockdown, or the knockdown of the DNA damage sensor ATM (Figure 5H–I and L). It is known that TP53 and its effector p21CIP1/WAF1, as well as ATM, significantly contribute to G2/M checkpoint execution [23,54,55], explaining why additional CHK1 loss does not exacerbate the loss of viability. The similarity in behaviour of PRKACG and CDK4 may indicate that these targets likewise are required in G2 checkpoint signalling. We also tested the response to knockdown of the identified targets in TP53-perturbed backgrounds (Figure S7). None of the targets yielded significantly enhanced viability loss here, (Figure S7) although target dependent sensitization was seen in parallel run assays using Mock-perturbed cells, in keeping with a signalling scenario in which TP53 plays a central role. Mathematical testing for interaction between radiation and target knockdown (Figure 5N; Figure S5) corroborate that target knockdown significantly synergized with IR treatment in reducing Mechanism of G1 Radiation Checkpoint Activation cell viability for five of the targets (Figure 5N and Figure S5). Consistent with our prior assessment knockdown of STK4, HK1 and PRPK, which yield enhanced viability loss in conjunction with CHK1 perturbation, yielded substantially enhanced sensitization in CHK1 perturbed cells as opposed to Mock-perturbed or unperturbed cells (Figure 5M, N and Figure S5). Together our experiments provide clear evidence for a modulation of radiation response in cells following perturbation the G1 checkpoint activating targets identified and highlight an interaction with G2 checkpoint competence for the majority of these. Data consolidation for derivation of a G1 checkpoint- signalling model To consolidate our data into a signalling model we entered the numerical observations from our analysis into the open access software environment for statistical and graphic data analysis, R (http://www.r-project.org/). Application of the default algorithm for unsupervised clustering analysis sorted the different targets broadly into two groups. One group containing PRPK and STK4, CDK4 and p21CIP1/WAF1 (group I) co-clustered with TP53 knockdown, a second group containing DYRK1A, PRKACG and HK1 (group II) aligned separately from the former (Figure 6A). Using the grouping information along with the features established by our experimental analysis, we assembled a signalling framework built around the known axes involving TP53 activation, consequential p21CIP1/WAF1 induction and resulting attenuation of RB1 phosphorylation (Figure 6B). According to our experi- mental results all targets within group I share the feature of being required for p21CIP1/WAF1 positivity, predicting that their knockdown either affects transcription of the p21CIP1/WAF1 gene, or the subsequent production or accumulation of p21CIP1/WAF1 protein. In contrast, group II targets are not required for accumulation of cells with p21CIP1/WAF1 positivity, suggesting a network topology whereby p21CIP1/WAF1 is required but not sufficient to attenuate RB1 phosphorylation and G1 checkpoint arrest. Discussion Accumulation of RB1 in its active, underphosphorylated form is known to arise in cells experiencing genotoxic stresses, concurrent with arrest of cell cycle progression in such cells [56]. RB1, and in some contexts its paralogues, have been shown to play a critical role in promoting survival of cells exposed to genotoxic stress, supporting a view whereby DNA damage-associated signalling leading to RB1 activation protects cells from radiation-induced death. We used a mechanism-based RNA interference screen to identify kinase signalling required for the accumulation of active RB1 in cells, and identify a group of gene products critically required for RB1 activation and radiation-associated G1 arrest. The majority of these gene products has not previously been linked to IR signalling or G1 checkpoint control. We found that several of these functions are required for the accumulation of the CDK inhibitor p21CIP1/WAF1 in IR-exposed cells. The transcription of p21CIP1/WAF1 is activated by TP53, which itself is activated by genotoxic stress [54]. Knockdown of p21CIP1/WAF1 or TP53 permits radio-resistant RB1 phosphoryla- tion and prevents G1 arrest, and p21CIP1/WAF1 was a hit in the screen, corroborating the critical contribution of this signalling. Two of the screen hits, PRPK and STK4, encode kinases previously linked to TP53 activation and p21CIP1/WAF1accumula- tion, although neither has been linked to checkpoint activation or sensitivity in IR challenged cells. PRPK has been shown to bind to TP53 in vivo and along with its yeast homologue Bud32, can PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31627