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Articles| March 31 2003
Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response
Gary D. Kao,
Gary D. Kao
1Department of Radiation Oncology, Diabetes, and Metabolism, Department of Medicine
W. Gillies McKenna,
W. Gillies McKenna
Matthew G. Guenther,
Matthew G. Guenther
2Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine
Ruth J. Muschel,
Ruth J. Muschel
3Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
Mitchell A. Lazar,
Mitchell A. Lazar
Tim J. Yen
4Fox Chase Cancer Center, Philadelphia, PA 19111
Address correspondence to Gary D. Kao, Hospital of the University of Pennsylvania, Department of Radiation Oncology, 3400 Spruce St. 2 Donner, Philadelphia, PA 19104. Tel.: (215) 573-5503. Fax: (215) 349-0090. E-mail: kao@xrt.upenn.edu
The online version of this article includes supplemental material.
Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; DNA-PK, DNA protein kinase; HDAC, histone deacetylase; IR, irradiation; NBS, Nijmegen breakage syndrome; RNAi, RNA interference; siRNA, short interfering RNA; TSA, trichostatin A.
Revision Received: January 28 2003
The Rockefeller University Press
J Cell Biol (2003) 160 (7): 1017–1027.
https://doi.org/10.1083/jcb.200209065
Revision Received:
Gary D. Kao, W. Gillies McKenna, Matthew G. Guenther, Ruth J. Muschel, Mitchell A. Lazar, Tim J. Yen; Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response . J Cell Biol 31 March 2003; 160 (7): 1017–1027. doi: https://doi.org/10.1083/jcb.200209065
Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage–induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage–induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.
A number of proteins, including 53BP1 (Schultz et al., 2000; Anderson et al., 2001; Rappold et al., 2001; Xia et al., 2001), Rad51, γH2AX, NBS1/Mre11/Rad50 complex (Maser et al., 1997; Paull et al., 2000), and BRCA1 (Scully et al., 1997; Zhong et al., 1999) have been observed to accumulate at multiple foci in the nucleus in response to DNA damage. 53BP1 was first identified in a yeast two-hybrid screen as one of two proteins that interacted with the transactivation domain of p53. Recently it was shown that 53BP1 participates in the phosphorylation of p53 and Chk2 and in the maintenance of S and G2 cell cycle checkpoints after DNA damage (DiTullio et al., 2002; Fernandez-Capetillo et al., 2002; Wang et al., 2002). 53BP1 foci are detectable within minutes after exposure to ionizing radiation, the number of foci increases with increasing dose, and the foci colocalize with that of γH2AX, suggesting an early role in the DNA damage response for 53BP1, as perhaps a marker of unrepaired double-strand breaks. Recent studies of cell lines derived from H2AX knockout mice show that this gene mediates radiation resistance and is essential for various components of the DNA damage response pathway to form foci (Bassing et al., 2002; Celeste et al., 2002). Although these data point to a role of chromatin in the DNA damage response, it remains unclear to what extent these foci represent alterations of the underlying chromatin.
Chromatin undergoes expansion and compaction in the course of many fundamental cellular processes, including gene expression, differentiation, and cell cycle progression. These alterations of the chromatin are largely mediated by histone acetylases and histone deacetylases (HDACs).*HDACs act on key acetylated lysine residues of core histones to induce chromatin compaction, which in turn results in gene silencing and heterochromatin formation (Taunton et al., 1996; Yang et al., 1996; Fischle et al., 1999; Grozinger et al., 1999). HDACs have been categorized into three classes that are based on sequence homology and domain organization. Class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) are similar to the yeast deacetylase Rpd3 (Yang et al., 1996; Dangond et al., 1998; Emiliani et al., 1998; Buggy et al., 2000; Hu et al., 2000). Class II HDACs (HDAC4, HDAC5, HDAC6, and HDAC7) possess catalytic domains homologous to that of yeast Hda1 (Rundlett et al., 1996; Fischle et al., 1999; Grozinger et al., 1999; Miska et al., 1999; Verdel and Khochbin, 1999; Wang et al., 1999; Kao et al., 2000). Proteins similar to the yeast NAD1-dependent deacetylase Sir2 (Frye, 2000; Imai et al., 2000; Landry et al., 2000; Smith et al., 2000) compose the third class of HDACs. Class I and II HDACs have been found to function as corepressors recruited for transcriptional repression, whereas the class III HDACs are important for gene silencing at telomeres and HM (mating type) loci in yeast (Sherman et al., 1999).
Although HDACs are most prominently linked with transcriptional repression, there are indications that HDACs may play broader roles in regulating cellular processes that affect survival after exposure to DNA-damaging agents. For example, p53 has been found to be deacetylated and inactivated by human Sir2α in mammalian cells, leading to reduced apoptosis and increased survival after exposure to ionizing radiation or etoposide. Conversely, expression of catalytically inactive Sir2α leads to increased apoptosis and radiosensitization in mammalian cells (Luo et al., 2001; Vaziri et al., 2001). Effects of the class III HDACs may extend beyond effects on p53. In budding yeast, members of the SIR2 family of HDACs appear to be important components of the DNA damage pathway. Whereas SIR2 appears to be static, SIR3 and SIR4 are mobilized to sites of DNA breaks (Gottschling et al., 1990; Tsukamoto et al., 1997; Martin et al., 1999; Moazed, 2001). SIR2, SIR3, and SIR4 are required for the efficient recircularization of linear plasmids by nonhomologous end joining, and mutations in these genes resulted in increased sensitivity to γ-radiation in fission yeast (Boulton and Jackson, 1998; Tsukamoto et al., 1997). A role in the DNA damage response has not yet been described for the class I and II HDACs, but is suggested by the observation that treatment with the class I and II HDAC inhibitor trichostatin A (TSA) leads to significant radiosensitization of human cells (Biade et al., 2001) (the class III Sir2 family deacetylases are resistant to TSA [Bernstein et al., 2000]). The mechanism of how TSA might lead to radiosensitization has likewise been unclear.
To assess for a role for class I and II HDACs in the DNA damage response in human cells, we examined several members for their ability to form foci in response to DNA damage. After exposure to double-strand DNA damage in a variety of human cells, we found that human HDAC4 is recruited to nuclear foci with kinetics similar to 53BP1. We found that HDAC4 and 53BP1 colocalize at foci and could be coimmunoprecipitated. HDAC4 foci formation was seen in a wide variety of cell lines, which included radiosensitive cell lines lacking the gene products of ATM (ataxia telangiectasia mutated), Nibrin, and DNA protein kinase (DNA-PK). HDAC4 foci disappeared after several hours in repair-proficient cells that were exposed to nonlethal doses of radiation. However, HDAC4 foci failed to resolve in radiosensitive cell lines or in normal cells that were exposed to a lethal dose of radiation. Silencing of HDAC4 resulted in decreased 53BP1 protein levels and did not markedly affect cell cycle distribution in unirradiated cells, but markedly abrogated the G2 checkpoint in radiosensitized cells after DNA damage. These results together suggest that HDAC4 is a critical component of the DNA damage response pathway and suggests an additional role for this protein beyond transcriptional silencing.
HDAC4 is recruited to nuclear foci after exposure to double-strand DNA damage
To investigate a potential role of HDACs in the DNA damage response in human cells, we studied the response of HDAC2, 4, and 6 to DNA damage. HeLa cells exposed to γ-irradiation (IR) or etoposide exhibited distinct foci of HDAC4 in their nucleus (Fig. 1, B and D), whereas unirradiated (Fig. 1 A) or UV exposure did not have this effect (Fig. 1 E). In contrast, DNA damage did not induce foci formation by HDAC2 (Fig. 1 I) or HDAC6 (Fig. 1 J) or noticeably alter their intracellular localization. We observed IR-induced HDAC4 foci in both transformed and untransformed cell lines, including the breast cancer cell lines MCF7, PA1, SKBR, and MO59J and K (both of gliomatous origin), the sarcoma cell lines U20S, HT29, and HT1080 (latter two are colon cancer lines), and Wi38 and WSC (normal human fibroblasts) (unpublished data). Thus, foci formation by HDAC4 in response to DNA damage appears to be a general cellular response that does not appear to be dependent on p53.
The formation of HDAC4 foci after exposure to DNA damage was not accompanied by significant changes in HDAC4 deacetylase activity or protein levels (Fig. 2, A and B). We noted however a dose-dependent increase in the average number of HDAC4 foci per cell (Fig. 2 C). IR resulted in approximately 30 foci per Gy up to 3 Gy, beyond which the increase in the number of foci with increasing dose was difficult to discern. Kinetics of foci formation was the same between cells exposed to nonlethal (1 Gy) and lethal (8 Gy) doses (Fig. 2 D). HDAC4 foci were evident within 5 min after IR and reached a maximum by 1 h. Interestingly, the number of foci in cells irradiated with 1 Gy progressively decreased to background levels by 24 h after IR. In contrast, the majority (87 ± 7%) of HeLa cells irradiated with 8 Gy retained their HDAC4 foci as long as 48 h after IR. Foci were evident beyond 48 h when there was a high level of cell death (unpublished data). The persistence of HDAC4 foci therefore appears to correlate with lethal doses of IR, which presumably result in accumulation of unrepaired DNA.
HDAC4 interacts with 53BP1
DNA damage induces 53BP1 to form foci within minutes after DNA damage, suggesting that they participate in the early steps of the DNA damage response pathway. In contrast, Rad50 and Rad51 foci appear hours after DNA damage (Paull et al., 2000). We found that HDAC4 formed foci with kinetics very similar to 53BP1. The average number of foci containing either 53BP1 or HDAC4 reached maximal levels by 1 h after 3 Gy and then progressively decreased to near baseline by 36 h (Fig. 2 E). The similarity in the kinetics of HDAC4 and 53BP1 foci formation motivated us to look further for interactions between HDAC4 and 53BP1. Immunoprecipitation experiments showed that HDAC4 and 53BP1 formed a complex in HeLa cells, but the formation of this complex was unaffected by DNA damage (Fig. 2 F). However, immunocytochemical data clearly showed that HDAC4 and 53BP1 colocalized at DNA damage–induced foci (Fig. 2 G). At later times after radiation when Rad51 foci began to accumulate, the numbers of HDAC4 and 53BP1 foci were reduced. The majority of Rad51 foci did not colocalize with HDAC4, although it may be possible that Rad51 was localized to sites that had previously been occupied by HDAC4 (see Figs. S1 and S2).
HDAC4 foci induced by IR persist in radiosensitive cells
We next examined in a range of human cell lines the genetic determinants that specified HDAC4 foci formation. We found that HDAC4 foci formation did not depend on DNA damage response genes, i.e., ATM (ataxia telangiectasia mutated), Nibrin, or DNA-PK, as cell lines defective for these genes formed foci with similar kinetics as HeLa cells (Fig. 3). However, the repair-deficient cell lines differed from HeLa (and other repair-proficient) cells in the persistence of HDAC4 foci after exposure to low doses of IR. For example, HDAC4 foci readily formed 1 h after radiation in the ATM-deficient FT169 cell line, as well as in its isogenic derivatives Y25 (in which ATM is restored by expression of a full-length cDNA) and PEB (expressing empty vector and hence remaining ATM deficient). 24 h after IR, HDAC4 foci were significantly reduced only in the ATM-positive Y25 cells (Fig. 3, A–D). A similar difference in the resolution of HDAC4 foci was observed between DNA-PK–deficient MO59J cells and DNA-PK–positive MO59K cells. Although MO59K cells did not completely resolve their HDAC4 foci, the average number of foci was less than in the MO59J cells (Fig. 3, E–G). We believe that MO59K cells did not efficiently resolve HDAC4 foci because of their inherent radiosensitivity relative to HeLa cells (Wang et al., 1997; unpublished data), which efficiently resolves foci at low doses of IR (Fig. 3 J). Lastly, we examined HDAC4 foci formation in the radiosensitive Nijmegen breakage syndrome (NBS) mutant cell lines and found that they too retained high levels of foci 24 h after IR (Fig. 3, H and I). We found that foci formation by HDAC4 in HeLa cells was unimpeded by TSA. However, the resolution of HDAC4 foci in HeLa cells was partially inhibited by TSA (Fig. 3, H and K).
HDAC4 foci are detected in interphase cells
HDAC4 foci were induced by DNA damage in nearly all cells, suggesting it was not limited to a specific phase of the cell cycle. We sought to confirm this in both asynchronous and synchronized HeLa cells (Fig. 4 A). Using cyclin E expression to distinguish G1–S from G2 cells, we found IR-induced HDAC4 foci in both. Next, we irradiated synchronized cells in S phase and then harvested when cells were beginning to progress into G2 (Fig. 4 B) or mitosis (Fig. 4 C). The average number of foci per cell was not decreased in the G2 cells but, interestingly, was decreased in prophase cells and not evident in mitotic cells (Fig. 4 D). These observations, however, do not permit us to distinguish whether HDAC4 foci form less efficiently in mitotic cells or whether the foci resolve as cells enter mitosis.
Silencing of HDAC4 expression via RNA interference (RNAi) results in decreased 53BP1 protein
To assess further the functions of HDAC4, we silenced its expression by RNA interference (RNAi) (Elbashir et al., 2001). We found that short interfering RNA (siRNA) directed against two regions of HDAC4 efficiently repressed levels of HDAC4 protein at 48 h after transfection. A control siRNA that was unrelated to HDAC4 had no effect (Fig. 5 A). We conducted a time course experiment to determine the optimal time when HDAC4 expression was at its lowest after siRNA treatment. Considerable amounts of HDAC4 protein were detectable 12 h after transfection, but the level of protein progressively decreased such that very little HDAC4 protein was detectable by 36 h, indicating that it is optimal to assess silencing after 24 h (Fig. 5 B). We next examined the effect of the HDAC4 siRNA on foci formation after IR. As expected, HDAC4 foci formation after IR was substantially reduced with both HDAC4 siRNAs, whereas the control siRNA had no effect (Fig. 5, C and D).
We next explored the effect of silencing HDAC4 expression on 53BP1 and were surprised to find that 53BP1 levels were reduced in cells transfected with HDAC4 siRNA (Fig. 5 E). We conducted the converse experiment and found that silencing 53BP1 also led to a reduction of HDAC4. These effects were not seen with a control siRNA. We conducted a time course experiment with both HDAC4 and 53BP1 siRNA and found that levels of either target protein were substantially decreased 24 h after treatment with either siRNA, whereas levels of an unrelated protein (tubulin) were unaffected (Fig. 5 F). To exclude the possibility that the HDAC4 and 53BP1 siRNAs inadvertently precipitated the degradation of 53BP1 and HDAC4 mRNAs, respectively, we assessed the levels of endogenous HDAC4 and 53BP1 mRNA after siRNA treatment. We found that each siRNA reduced only its target mRNA (Fig. 5 G). Thus, the stabilities of HDAC4 and 53BP1 proteins are dependent on each other. Consistent with the observed reduction in protein expression, cells treated with 53BP1 or HDAC4 siRNA showed a dramatic reduction in 53BP1 foci in response to DNA damage (Fig. 5, H and I). Together, these experiments further suggest that HDAC4 interacts in the DNA damage response pathway that involves 53BP1, perhaps as components of the same protein complex.
Silencing of HDAC4 expression via RNAi results in decreased cell viability
We further studied the effects of silencing HDAC4 on cell cycle distribution and cellular viability after DNA damage. Treatment with HDAC4 siRNA did not appear to have a major effect in altering the cell cycle distribution of unirradiated HeLa cells, compared with cells treated with control siRNA (Fig. 6, A and B) or untreated cells (unpublished data). IR of HeLa cells treated with control siRNA resulted in the expected accumulation of cells in G2, characteristic of the IR-induced checkpoint. In contrast, treatment with HDAC4 siRNA markedly decreased the proportion of cells with G2/M DNA content but was accompanied by an increase in cells with DNA content less than 2N (sub-G1) (Fig. 6, A and B). This reduction in G2/M cells was not due to an S-phase delay because HDAC4 siRNA did not affect normal S-phase progression, as determined by FACS® analysis and BrdU incorporation (unpublished data). Flow cytometry based on DNA content alone does not distinguish between cells in G2 and mitosis. We therefore performed immunofluorescence on irradiated cells that were transfected with control and HDAC4 siRNA. Control cells were blocked in G2 after IR, as determined by high cyclin B1 levels in the cytoplasm, CENP-F distributed uniformly in the nucleus, and uncondensed chromatin (Fig. 6, C and D), as described previously (Liao et al., 1995; Kao et al., 2001). In contrast, few of the HDAC4 siRNA–treated cells appeared to be in G2. Although many of these cells still retained cyclin B1 and CENP-F staining, the cyclin B1 was often not uniformly cytoplasmic, the CENP-F staining of the nuclei was frequently uneven, and many of nuclei were condensed or fragmented. Many of these condensed cells (>70%), in fact, showed high levels of phosphohistone H3 staining, along with cyclin B1, suggesting progression into mitosis (see Fig. S3). These observations together suggest that cells silenced for HDAC4 do not maintain a uniform G2 delay after IR. One notes, however, that as 53BP1 has been established to mediate the damage-induced G2 checkpoint, the abrogation of this checkpoint by silencing HDAC4 may be mediated, at least in part, through decreasing 53BP1 protein (DiTullio et al., 2002; Fernandez-Capetillo et al., 2002; Wang et al., 2002).
To further explore the effects on cellular viability of silencing HDAC4 and 53BP1, we performed clonogenic assays to assess the effect on the survival of HeLa cells after DNA damage. Silencing of HDAC4 and 53BP1 had a dramatic effect on the plating efficiency of the cells (Fig. 6 E), indicating that HDAC4 and 53BP1 are likely key determinants of cell proliferation. However, even after correcting for the reduced plating efficiency of these cells, silencing of HDAC4 and 53BP1 further radiosensitized these cells (Fig. 6 F), with 53BP1 having the larger effect of the two. Together, these results suggest that HDAC4 and 53BP1 are required for cells to maintain in G2 after ionizing radiation; when either protein expression is silenced, cells do not block in G2, but instead rapidly lose viability.
Lastly, we examined whether abrogation of the G2 delay occurs when HDACs other than HDAC4 are silenced. Silencing of HDAC2 and HDAC6 did not interfere with the cell's ability to arrest in G2 in response to DNA damage (Fig. 7, A and B). Only silencing of HDAC4 appreciably diminished the proportion of cells delayed in G2 after IR.
Our studies have revealed a new role for a HDAC in the DNA damage response pathway. In response to double-stranded DNA breaks, HDAC4, but not HDAC2 or 6, rapidly formed foci that are coincident with the DNA damage response protein 53BP1. Foci formation by HDAC4 occurred throughout interphase and was observed in a wide panel of tumor and normal cell lines. HDAC4 foci formation did not depend on DNA damage response genes ATM, DNA-PK, or NBS, but their resolution was greatly delayed or blocked in these mutant cell lines. This is similar to the finding that ATM is not required for foci formation by 53BP1. When HeLa cells were exposed to a nonlethal dose of IR, where cells are presumably able to repair damaged DNA, HDAC4 foci were efficiently resolved. However, at lethal doses, where cells are presumably unable to effectively repair all of the damaged DNA, the HDAC4 foci persisted. We believe that the efficiency of resolution of HDAC4 foci correlates with cellular radiosensitivity. DNA-PK–defective MO59J cells are highly radiosensitive and were much less efficient at resolving their HDAC4 foci than DNA-PK–positive MO59K cells. MO59K cells, more radiosensitive than HeLa cells, were in turn less efficient at resolving foci than HeLa cells. The failure to resolve HDAC4 foci may reflect the radiosensitizing properties of TSA. These observations together suggest that HDAC4 foci formation is a general cellular response to DNA damage, and conversely, impaired resolution of HDAC4 foci may be correlated with radiosensitivity.
These data suggest that HDAC4 foci may facilitate recruitment of, or stabilize, repair factors such as 53BP1, which in turn may help maintain damage-induced cell cycle checkpoints. The interaction between HDAC4 and 53BP1 is established here by the findings that the two proteins colocalize and coimmunoprecipitate, as well as the unexpected findings that siRNA-mediated silencing of HDAC4 leads to reduced levels of 53BP1 protein and vice-versa. Thus, the stability of each protein appears to depend on the stability of the other, or on being associated in a common complex. Our findings that silencing of HDAC4 expression abrogates the damage-induced G2 delay and increases radiosensitivity support the notion that the checkpoint determines the ability of the cell to survive radiation damage. Along with 53BP1, localization of HDAC4 to unrepaired damage may signal the cell to stall at a checkpoint to allow the repair to be completed, while possibly serving as a marker to facilitate repair. These results, therefore, may be consistent with the model proposed by Fernandez-Capetillo et al. (2002) in which 53BP1 and other factors capable of signal transduction may accumulate into a chromatin microenvironment within megabases of an actual DNA double-strand break. This localized concentration of protein complexes may then generate the amplification of signal sufficient to invoke the G2 checkpoint.
These findings do not exclude roles for HDAC4 in mediating other cellular functions. HDAC4 is an established component of complexes mediating transcriptional repression (Guenther et al., 2001; Fischle et al., 2002). Although transcription is globally decreased during mitosis (Spencer et al., 2000; Pflumm, 2002), it appears unlikely that the role of HDAC4 in the DNA damage response is restricted to transcriptional repression. Radiation, at the doses used in this study, does not globally repress transcription (Maity et al., 1995), and drugs that globally inhibit transcription, such as α-amanitin, do not reverse the effects of silencing HDAC4 (unpublished data). The radiation dose dependency of the number of HDAC4 and 53BP1 foci does not exclude, but does reduce, the likelihood that foci are mere storage sites for these proteins.
The recruitment of HDACs to sites of DNA damage may be at least partially evolutionarily conserved. Members of the SIR2 family of HDACs have been implicated in the DNA response in budding yeast, contributing either directly or indirectly to the repair of double-strand breaks (Astrom et al., 1999; Bennett et al., 2001). Whereas SIR2 has been most prominently implicated in the silencing of chromatin at the mating type loci and at telomeres, SIR3 is recruited to sites of DNA damage to form foci that are visible at the cytological level. It has been proposed that the accumulation of some of these deacetylases may induce a repressed chromatin state to facilitate repair or protect unrepaired broken DNA ends (Tsukamoto et al., 1997; Martin et al., 1999). Although HDAC4 does not belong to the same family of deacetylases, we propose that HDAC4 may perform an analogous function in mammalian cells. As a component of protein complexes that mediate transcriptional repression by inducing heterochromatin formation, HDAC4 likely serves a similar role in processing chromatin in the DNA damage response. Recruitment of HDAC4 to foci after DNA damage might reflect its role in silencing the chromatin near the site of damage. The silenced chromatin would prevent processes such as DNA replication and transcription from passing through damaged DNA. In addition, the silenced chromatin might also reduce nonspecific end joining of broken DNA ends or unwanted recombination events.
In linking a HDAC with the DNA damage response to agents inducing double-strand breaks, the results presented here may also have clinical implications for treating patients with cancer. Inhibitors of HDAC inhibitors have entered clinical trials with reports of efficacy in certain tumors (Piekarz et al., 2001; Sandor et al., 2002). These protocols have largely entailed administering the HDAC inhibitor as a single agent. With further development and confirmation, the observations reported here suggest that it may be fruitful to pursue strategies to block HDAC function in combination with standard treatments, such as radiation and chemotherapy, to maximize the killing of cancer cells.
Polyclonal antibodies were generated by using full-length human HDAC2, 4, and 6 expressed in bacteria to immunize rabbits. For each antigen, the antibody was purified from at least two rabbits and confirmed to have equivalent specificity. The serum was affinity purified using protein immobilized on Affigel beads (Bio-Rad Laboratories). Specificity of the antibody was further confirmed by preabsorbing purified antibody with the immunizing antigen (Fig. S4). Anti-53BP1 antibody was generated against the corresponding domain in human 53BP1 encoded by the 1.5-kb HindIII–EcoRI fragment of XL53BP1 (the 53BP1 cDNA was a gift from Y. Adachi, University of Edinburgh, Edinburgh, UK). Anti-HDAC4, anti-53BP1, anti-Mad1 (gift from M. Campbell, Yen lab), anti–CENP-F (Yen lab), anti–α tubulin (Sigma-Aldrich), and anti–cyclin B1 (BD Biosciences) were used at 1:1,000 for staining and/or immunoblotting. Anti–cyclin E (HE12, sc-247; Santa Cruz Biotechnology, Inc.) and anti-Rad51 antibody (rabbit polyclonal, catalog no. PC130, Oncogene Research Products; mouse monoclonal, RAD51–14B4, GeneTex) was used at 1:500. The anti–phosphohistone H3 antibody recognizes histone H3 phosphorylated on serine 10 (no. 06–570; Upstate Biotechnology) and was used at 1:1,000. Cells were fixed with methanol/acetone 50:50 at the indicated times after IR before staining with the respective antibodies.
All cells were grown in DME medium supplemented with 20% FBS at 37°C with 5% CO2. The ATM-deficient FT169A (ATM−), ATM-restored YZ5 cells (ATM+, consisting of FT169A transfected with and stably expressing full-length ATM), and ATM-deficient vector-control PEB cells (ATM control, consisting of FT169A cells transfected with the parental vector only) were provided by Y. Shiloh (Tel Aviv University, Ramat Aviv, Israel). Nibrin-deficient human cells were obtained from the American Type Culture Collection. Cells grown on coverslips were irradiated with cesium-137 γ rays from a JL Shepherd and Associates 81–14R panoramic irradiator at a dose rate of 1.35 Gy/min. UV IR was delivered in a single pulse (50 J/m2) using a Stratalinker UV source (Stratagene). Before UV IR, the culture medium was removed, and the medium was replaced immediately after IR. All cells were returned to the incubator for recovery and harvested at the indicated times. Etoposide was used at 20 μg/ml for 20 min. To block cells in mitosis, cells were exposed to 0.04 μg/ml of nocodazole for 15 h.
RNAi was performed with siRNA that was commercially synthesized (Dharmacon) and used as described in protocols provided by the manufacturer. Cells were treated with siRNA to a final concentration of 10 μM. siRNA against HDAC6 was applied twice on consecutive days, whereas all other siRNAs were applied once and harvested as described for each experiment. Paired siRNA sequences targeting each protein were as follows: HDAC4, GACGGGCCAGUGGUCACUG and CAGUGACCACUGGCCCGUC; 53BP1, CACACAGAUUGAGGAUACG and CGUAUCCUCAAUCUGUGUG; HDAC2, GCCUCAUAGAAUCCGCAUG and CAUGCGGAUUCUAUGAGGC; HDAC6, CCAGCCAGCGAAGAAGUAG and CUACUUCUUCGCUGCCUGG. Control siRNA consisted of the unannealed single-strand RNA and siRNA targeted against luciferase (both of which did not affect levels of endogenous proteins).
HDAC assays were performed as previously described (Huang et al., 2000). In brief, [3H]acetylated histones purified from HeLa cells (25,000 cpm/10 μg) were incubated with enzymes at 37°C for 15 min. The reaction was stopped by the addition of concentrated HCl, extracted with 1 ml of ethylacetate, and the amount of radioactivity released into the organic layer was quantitated using a scintillation counter. In experiments in which HDAC4 deacetylase activity was inhibited, HeLa cells were pretreated with 1 μM TSA. For immunoprecipitations, for each sample, 0.1 μg of antibody or 10 μl of preimmune rabbit serum was incubated with cell lysate from 1.0 × 106 cells and incubated in the presence of ethidium bromide (10 μg/ml) to exclude nonspecific protein–DNA associations (Lai and Herr, 1992). Cell preparation, image acquisition and processing, and FACS® analysis were as previously described (Kao et al., 1997, 2001). Endogenous mRNA was isolated using Trizol reagent (GIBCO BRL), as per the manufacturer's instructions, and assessed via RT-PCR. The Titan One Tube RT-PCR System (Roche) was used with the following primers: HDAC4, CAAGAACAAGGAGAAGGGCAAAG and GGACTCTGGTCAAGGGAACTG; 53BP1, AGGTGGGTGTTCTTTGGCTTCC and TTGGTGTTGAGGCTTGTGGTGATAC; glyceraldehyde-3-phosphate dehydrogenase, CAACTTTGGTATCGTGGAAGGACTC and AGGGATGATGTT-CTGGAGAGCC (specific details regarding the PCR parameters used are available from the authors).
Plating efficiency was defined as the proportion of cells that remained viable 8 h after trypsinization and replating in fresh media. Cell viability was assessed by trypan exclusion. Clonogenic survival assays were performed as previously described (Biade et al., 2001), except that cells were counted and plated 48 h after treatment with siRNA and colonies of at least 50 cells were counted 14 d after plating. Statistical analyses were performed with SPSS for Windows Release 10 and Microsoft Excel (Office 2000).
Online supplemental material
Additional data (Figs. S1–S4) regarding antibody specificity, Rad51 localization after IR, and phosphorylated histone H3 (Pi-Histone H3) labeling of siRNA-treated cells are available as supplemental material.
We are grateful to Dr. T. Halazonetis for helpful comments. Drs. G.K.T. Chan and S.T. Liu, Mr. James Hittle, and Ms. Jennifer DeVirgilis in the Yen lab provided technical advice and assistance. Dr. Fang Liu and Ms. Kimberly Clay of the Kao lab assisted in the protein assays and RT-PCR.
G.D. Kao is a recipient of an award from the WW Smith Charitable Trust. T.J. Yen, R.J. Muschel, and W.G. McKenna are supported by PO1-CA75138-05. T.J. Yen was also supported by grants from the National Institutes of Health, PO1 core grant CA06927, and an Appropriation from the Commonwealth of Pennsylvania.
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Figure S1
HeLa cells were irradiated and 6 h later costained with (a) rabbit anti-HDAC4 and (b) mouse anti-Rad51. (c) Merged images show partial colocalization of foci while many others are not. Arrows point to areas of overlapping HDAC4 and Rad51 staining. The nuclei are outlined in blue. Bar, 5 µm.
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Time course of HDAC4 and Rad51 foci after irradiation. HeLa cells were irradiated (3 Gy), fixed at the indicated times, and costained for HDAC4 (left) and Rad51 (right). Identical cells appear in both columns. Cell nuclei are outlined in blue. Bar, 5 µm.
Phosphohistone H3 is detectable in irradiated cells silenced for HDAC4. HeLa cells were transfected with control or HDAC4 siRNA, followed 36 h later by 3-Gy irradiation. 8 h after irradiation, cells were fixed and stained for cyclin B1 (top), phosphohistone H3 (middle), or DAPI (bottom). The panels of each column are of identical cells, and representative cells are shown. Arrow in left column (cells treated with control siRNA) indicates a cell that has progressed into mitosis, with chromatin beginning to condense (DAPI), coincident with cyclin B1, and strongly expressing phosphohistone H3. Most of the control cells, however, were more similar to the two cells above the indicated cell, which in contrast remain in G2, with cytoplasmic cyclin B1, uncondensed chromatin, and no detectable phosphohistone H3. In contrast, many of the cells treated with HDAC4 siRNA have condensed chromatin, in which phosphohistone H3 is detectable and which coincides with cyclin B1. Bar, 5 µm.
Specificity of HDAC4 antibody. 50 µg of HeLa cell lysate was incubated for 2 h under agitation with affinity-purified rabbit anti-HDAC4 antibody, an identical amount of the antibody preabsorbed on membranes on which the bacterially expressed immunizing antigen had been transferred, or rabbit serum prior to immunization. 15 µl of protein A agarose beads was then added to each sample and further incubated for 1 h. The beads were pelleted and washed extensively five times with NET-N buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, 0.5% NP-40) before separation on 7.5% SDS-PAGE and transfer onto nitrocellulose. The transferred membranes were finally probed with anti-HDAC4 and a dominant band detected on ECL chemi-illuminescence.
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Recruitment of HDAC4 to nuclear foci after double-strand DNA damage. HeLa cells that were (A) unirradiated, (B) irradiated with 2 Gy, (C) irradiated and probed with secondary antibody only, (D) irradiated and probed with primary antibody (anti-HDAC4) immunodepleted with the immunizing antigen. (E) HeLa cells exposed to UV (50 J/m2) and (F) 20 μM etoposide were fixed after 1 h and stained with HDAC4 antibodies. (G and H) DAPI stain of cells in E and F, respectively, were merged with HDAC4. Cells irradiated with 2 Gy were stained for (I) HDAC2 and (J) HDAC6. All staining of irradiated cells was performed 1 h after IR. Bar, 5 μm.
Dose and time dependency of DNA damage–induced HDAC4 foci. (A) HDAC4-associated deacetylase activity is not appreciably changed by IR. HDAC4 or Ncor was immunoprecipitated from HeLa lysates and assayed for deacetylase activity. Ncor and HDAC4 treated with TSA serve as positive and negative controls, respectively, for the deacetylase assays. (B) HDAC4 levels do not change after IR. Equal amounts of total protein (25 μg) from HeLa cells were separated via 4–20% gradient SDS-PAGE, transferred to nitrocellulose, and probed with rabbit preimmune serum (left), anti-HDAC4 (right), or anti–α-tubulin antibody (loading control) (bottom). Mock-irradiated (−IR) or irradiated (+IR) cells were harvested 1 h after 3 Gy. (C) HDAC4 foci formation is dependent on the dose of IR. HeLa cells exposed to increasing doses of IR were fixed after 1 h and stained for HDAC4 foci. At least 100 cells were counted for each dose. Error bars represent the SD, and each point represents the average of three experiments. (D) Kinetics of HDAC4 foci formation. HeLa cells irradiated with 1 and 8 Gy were fixed at various times, and the average number of cells showing induced HDAC4 foci was counted. Time is depicted on a log scale. (E) Comparison of the kinetics of foci formation amongst HDAC4, 53BP1, and Rad51. HeLa cells were irradiated (3 Gy), fixed at the indicated times, and separately stained for HDAC4, 53BP1, and Rad51 foci. Error bars represent the SD, and each point represents the average of three experiments. (F) Coimmunoprecipitation of HDAC4 and 53BP1. HeLa cells were lysed in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, 0.5% NP-40) and, for each sample, 50 μg of lysate from unirradiated (IR−) or irradiated cells (IR+) was incubated with the indicated antibodies or preimmune serum (IP). The immunoprecipitates were separated on 7.5% SDS-PAGE gels and probed for 53BP1 (top) and HDAC4 (bottom) (IB). Total cell lysate (25 μg, Whole cell) served as a positive control for the immunoblots. (G) Colocalization of HDAC4 foci relative to 53BP1 and Rad51 foci. HeLa cells were irradiated with 3 Gy and 1 h later costained with (a) rabbit anti-HDAC4 and (b) rat anti-53BP1. (c) Images from A and B were merged to reveal that most foci were coincident. The nuclei are outlined in blue. Bar, 5 μm.
DNA repair–deficient cell lines are unable to efficiently resolve HDAC4 foci. Cells deficient for ATM, DNA-PK, and Nibrin were exposed to 1 Gy of IR and fixed and stained for HDAC4 1 and 24 h after IR. The average percentage of each cell line showing IR-induced HDAC4 was determined and the data presented in a histogram. Representative images of the HDAC4 staining pattern in the respective cell lines 24 h after IR are presented in the right panels. (A) 1 h after IR (1 Gy), respectively 95 ± 4.3% and 89 ± 4.0% of ATM-deficient FT169A and ATM-restored Y25 cells showed induced foci, a difference that was statistically insignificant. In contrast, 24 h after IR, respectively 82 ± 3.1% and 33 ± 5.9% of FT169A and Y25 cells showed induced foci (P < 0.001). PEB cells, derived from FT169A cells via stably transfecting with the empty parental vector (and which remain deficient for ATM protein and radiosensitive) continued to show high levels of induced HDAC4 foci after IR. Representative images of (B) FT169A (ATM−), (C) Y25 (ATM+), and (D) PEB (ATM control) cell lines 24 h after IR. (E) 1 h after IR, respectively 93 ± 6.7% and 95 ± 3.4% of DNA-PK–deficient MO69J and DNA-PK–proficient MO59K cells showed induced foci, a difference that was not significant. In contrast, 24 h after IR, respectively 86 ± 10.5% and 40 ± 9.6% of MO59J and MO59K showed induced foci (P < 0.001). Representative images of (F) MO59J (DNAPK−) and (G) MO59K (DNAPK+) cells 24 h after IR. In the MO59K cells with persistent foci, the number of foci per cell was also consistently fewer than in the MO59J cells (average of 11 ± 3.1 vs. 45 ± 3.7 foci, respectively, per MO59K vs. MO59J cell; P < 0.001). (H) Nibrin-deficient cells (NBS−) showed high levels of HDAC4 foci at both 1 (95 ± 5.4% of cells) and 24 h (83 ± 2.6%) after IR. In contrast, respectively 89 ± 2.4% and 9 ± 3.1% of HeLa cells (HeLa) showed foci. TSA treatment of HeLa cells (HeLa + TSA) did not prevent foci formation, but inhibited their resolution (93 ± 4.5% and 31 ± 14.5% of HeLa cells pretreated with TSA showed foci at 1 and 24 h after IR). Representative images of (I) Nibrin-deficient (NBS−), (J) HeLa (HeLa), and (K) HeLa cells pretreated with TSA (HeLa + TSA) 24 h after IR. Bar, 5 μm. Data represent the average of three experiments. The nuclei are outlined in blue. Error bars indicate the SD.
HDAC4 foci are induced by IR in interphase cells. (A) Asynchronous HeLa cells were irradiated with 2 Gy IR and 1 h later were fixed and stained for HDAC4, cyclin E, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin E and DAPI. HDAC4 foci are apparent in cells in late G1/early S phase (G1–S) as well as in those not expressing cyclin E and presumably in G2 phase (G2). (B) Cells synchronized and mock irradiated or irradiated (2 Gy) in late S phase were fixed and stained 3 h later for HDAC4, cyclin B1, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin B1 and DAPI. After mock IR (No IR), S-phase cells have not yet expressed cyclin B1 (S), whereas cyclin B1 is cytoplasmic in G2 cells (G2) and coincides with the condensed chromatin in mitotic cells (M) (Kao et al., 1997; Hagting et al., 1999). After IR (IR, 3 h), HDAC4 foci are seen in most cells, including S phase (S) and those that have progressed to and blocked in G2 phase (G2). (C) Synchronized cells were irradiated in late S phase as in B, but fixed 5 h after IR (IR, 5 h). Most cells remain in G2, as indicated by cytoplasmic cyclin B1. However, a few cells have progressed into prophase, as indicated by the entry of cyclin B1 into the nucleus (arrowhead); in these cells, the HDAC4 foci are fewer and less distinct. Bar, 5 μm. (D) Average number of IR-induced HDAC4 foci at each position of the cell cycle. At least 100 cells were counted for each data point, and the experiment was repeated three times with similar results. Error bars represent the SD.
RNAi efficiently silences HDAC4 and 53BP1 protein expression. (A) HeLa cells were transfected with siRNA targeting two different sequences in HDAC4 or control siRNA, harvested 36 h later, and immunoblotted for HDAC4 and α-tubulin (loading control). Whole cell lysates from untransfected cells serve as a positive control. (B) Time course of HDAC4 siRNA–mediated silencing of protein expression. Parallel plates of HeLa cells were treated with HDAC4 siRNA and harvested at the times indicated. All lysates were separated on 7.5% SDS-PAGE and immunoblotted for HDAC4 or α-tubulin, indicating maximal silencing of the targeted HDAC4 protein by 36 h. (C and D) HDAC4 siRNA diminishes foci formation after IR. HeLa cells were treated with oligofectamine only, control siRNA, or HDAC4 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed and immunofluorescence for HDAC4 was performed. Representative cells are shown in C. The number of HDAC4 foci found in each cell was counted. At least 300 cells were counted for each treatment group. (D) The average number of HDAC4 foci per cell after IR for each treatment group is displayed in the histograms. (E) HDAC4 and 53BP1 siRNA silence expression of their targeted protein, as well as each other. HeLa cells were transfected with HDAC4 or 53BP1 siRNA and harvested 48 h later. Lysates were separated on SDS-PAGE and immunoblotted for 53BP1, HDAC4, and α-tubulin. (F) siRNA-mediated silencing of 53BP1 and HDAC4 protein expression is achieved by 24 h after treatment. Parallel plates of HeLa cells were transfected with 53BP1 siRNA and harvested at the indicated times after transfection. Cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-53BP1, HDAC4, and α-tubulin antibodies. (G) HDAC4 and 53BP1 siRNA reduce only their target mRNA. RT-PCR was performed on total mRNA extracted from HeLa cells 48 h after treatment with control, HDAC4, or 53BP1 siRNA. RT-PCR was performed in parallel under identical conditions using primer pairs targeting 53BP1, HDAC4, or GADPH (as control), and the final product was separated via electrophoresis in ethidium bromide–labeled agarose and photographed under UV illumination. Each RT-PCR reaction yielded a single band as shown. (H and I) HDAC4 siRNA diminishes 53BP1 foci formation after IR. HeLa cells were transfected with oligofectamine control, HDAC4 siRNA, control siRNA, or 53BP1 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed, immunofluorescence for 53BP1 was performed, and the average number of foci per cell was determined and displayed in the histograms in H. Representative cells are shown in I.
Silencing of HDAC4 expression abrogates the DNA damage–induced G2 checkpoint and decreases cell viability. HeLa cells were transfected with control or HDAC4 siRNA, followed 36 h later by mock (No IR) or 5-Gy IR (IR). 8 h after IR, parallel plates of cells were harvested, and cell cycle distribution was analyzed via flow cytometry (A and B) or immunofluorescence (C and D). (A) Silencing of HDAC4 does not have a large effect on unirradiated cells, but it reduced the proportion of irradiated cells with G2/M DNA content. Cells with DNA content less than G1 cells (sub-G1) likely represent apoptotic cells/cell debris, and is especially apparent in cells silenced for HDAC4 and irradiated. (B) Histograms showing cell cycle distribution data from A, indicating the percentage of cells in each part of the cell cycle. The experiment was repeated three times with similar results. (C and D) To better distinguish the proportions of the total population of G2 cells, cells were stained for cyclin B1, CENP-F, and DAPI and analyzed via immunofluorescence. G2-phase cells show cytoplasmic cyclin B1 staining, diffuse nuclear CENP-F staining, and uncondensed chromatin (stained with DAPI) (Liao et al., 1995; Kao et al., 2001). (C) For each pair of images, the left panel shows cyclin B1 or CENP-F staining, and the right panel shows the same cells stained with DAPI. Cells treated with control siRNA show cytoplasmic cyclin B1 and nuclear CENP-F staining, indicative of the IR-induced G2 checkpoint. In contrast, few HDAC4 siRNA–treated cells were blocked in G2, but instead showed fragmented and condensed chromatin that retained cyclin B1 and CENP-F staining. (D) Histogram showing the distribution of CENP-F staining cells with uncondensed (G2 blocked) versus condensed or fragmented chromatin after IR of cells treated with control or HDAC4 siRNA. At least 300 cells were counted for each experiment. (E) Histogram showing plating efficiency of untreated controls or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were plated onto fresh dishes with fresh media 48 h after siRNA treatment, and the proportion of surviving cells capable of excluding Trypan blue was determined 12 h later. The experiment was repeated three times with similar results. Error bars indicate the SD. (F) Survival curves indicating the radiosensitivity of control cells or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were prepared as in E and irradiated with the doses indicated, and the proportion of surviving colonies was determined 2 wk later. All values were corrected for the plating efficiency. Error bars indicate the SD.
Silencing of HDAC2 and HDAC6 does not abrogate the G2 checkpoint. Parallel plates of cells were treated with control, HDAC2, HDAC4, or HDAC6 siRNA. 36 h after siRNA treatment, cells were irradiated (5 Gy) and harvested 8 h later for immunoblotting (A) and cell cycle analysis by FACS® (B). (A) Immunoblot of cell lysates from cells treated with siRNA. Cell lysates were separated on 7.5% SDS-PAGE, transferred onto nitrocellulose, and the membranes were probed with anti-HDAC4, HDAC6, HDAC2, Mad1, or α-tubulin antibodies. These show efficient silencing of the target proteins by the HDAC2, 4, and 6 siRNA. Mad1 and α-tubulin are not affected by the specific siRNA treatments and serve as loading controls. (B) Cell cycle distribution of treated cells. Cell cycle analysis shows that the silencing of HDAC4 protein had the most conspicuous effect on abrogation of the G2 checkpoint after IR, relative to silencing of HDAC2 and HDAC6.
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Perspectives of tumor treatment: Researchers investigate combination of carbon ion and immunotherapy
Photo: Adrian Rodríguez Rodríguez/GSI
The work with cell cultures is an important part of tumor research on the GSI/FAIR campus, in the photo Dr. Alexander Helm from the Department of Biophysics.
It is still a glance into the future: The combination of carbon ion and immune therapy could become an effective tool in the fight against cancer. Promising results for the potential benefit of this treatment combination have now been published in the renowned radiation therapy journal "International Journal of Radiation Oncology, Biology, Physics" of the American Society for Therapeutic Radiation Oncology (ASTRO). The authors are an international team of researchers, led by the Department of Biophysics at the GSI Helmholtzzentrum für Schwerionenforschung in Darmstadt, and including the Parthenope University of Naples and the Japanese National Institute for Radiological Sciences (NIRS-QST) in Chiba.
Even though the results are only preclinical and the way to clinical application is still long, the current findings point into a promising direction: It was shown that carbon ions, as used in the cancer therapy developed at GSI, can be very effective when used in combination with specific molecules, called checkpoint blockers, that stimulate the immune system against the tumor metastases.
The aim of the published research work was to compare the efficacy of conventional radiation therapy (high-energy X-rays) and carbon ion therapy in combination with immunotherapy. The immune system plays an important role in the prevention of cancer. Usually, it recognizes degenerated cells and can "sort them out". At the same time, it is equipped with highly complex control mechanisms to avoid overreactions. This is exactly what cancer cells can sometimes use to their advantage and to down-regulate immune surveillance. They disappear from the radar, so to speak. Immunotherapy can reactivate the immune system in the fight against cancer. It is now frequently used for advanced malignancies and metastatic patients, but unfortunately is effective only in some tumor types.
In the other cases, conventional radiation therapy is added as a second component, which under certain conditions can release such brakes on the immune system. The radiation-induced triggering of an immune response and its amplification by immunotherapy can lead to good results especially in the control of metastases, such as a slowdown of growth. But only parts of the patients respond to this combination of therapies.
Can radiotherapy with carbon ions, which was developed very successfully at GSI and is now in clinical application in Heidelberg and Marburg and in other nine centers worldwide, for certain types of tumors, open up new perspectives and help to control metastasis? It is possible that this form of therapy is more immunogenic, i.e. it could trigger an even stronger immune response than conventional radiation therapy and, together with immunotherapy, might result in more patients responding to this therapy combination. Based on such considerations, the team with lead author Dr. Alexander Helm (GSI) in the current experiment, conducted at the accelerator in Chiba, Japan, for the first time has directly compared carbon ions with conventional X-rays in a mouse model.
In the control of the primary tumor (here osteosarcoma, a bone tumor), carbon ions and X-rays, each combined with immunotherapy, produced similar results. However, when looking at the growth of metastases, the results show that metastases are significantly reduced if the primary tumor is treated with carbon ion radiation and a following immunotherapy. The researchers were able to demonstrate that carbon ions plus immunotherapy is more effective in controlling lung metastases than both therapies alone and more effective than X-rays plus immunotherapy.
In order to better assess this potential, further research has to be conducted and finally, together with international partners, the application in clinical studies must be tested. Professor Marco Durante, head of the GSI Biophysics Research Department, explained the future research: "At GSI/FAIR, the focus of our research is on understanding the cellular and molecular mechanisms that trigger a strong immune response. The goal is to answer the central question: How should irradiation be applied to achieve the most efficient, the best immune response in the fight against cancer."
The possibilities of combining cutting-edge molecular biology with high-energy heavy ion physics at the GSI/FAIR campus and the future accelerator center FAIR promise unique scientific knowledge. The Scientific Director of GSI and FAIR, Professor Paolo Giubellino, emphasized "The present results show the great potential of carbon ion therapy, which is far from being exhausted. Together with our national and international partners, we will continue to conduct research on this highly relevant topic in the coming years. The first stage of the FAIR experimental program, FAIR Phase 0, already offers outstanding opportunities in this field". (BP)
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Updating our abilities
This could probably be filed in the "should have done this sooner" drawer. We've just finished an analysis of our many and various abilities. We've copied all the abilities into a single spreadsheet so that we have everything in one place.
And now that we can see all 500+ abilities in one place, we're marveling at some of the contradictions.
For example, we found eight different variations on the Not so Fast! ability. We found some abilities that had different wordings in the same game. We found some abilities that have different effects in different games.
A sample ability
All this leads to inconsistency – we don't want you to have to relearn the abilities every time you play. Once you've learned Not so Fast! you shouldn't have to re-learn it.
This has crept up on us over time. Our approach to abilities has changed over time. As an example, an early version of Sudden Insight states "After talking for five minutes with any person, you realise that they revealed more than they intended. They must show you everything on their "Information" card." Feedback from players suggested that some players were timing themselves to the second so that they could play the ability. That wasn't really what we intended – we just wanted them to talk for a short while. So later versions said "After talking briefly to …" or even just "After talking to…". But we didn't then go back and change the original abilities.
So we've agreed on some standardised wordings for our "standard" abilities, the ones we use time and again. For example, Sudden Insight will now say: "After talking to another player, you realise that they have revealed more than they intended. They must must show you their Clue." (As discussed previously, Clue is our new term for Information.)
And we're going to sort out all our old games and bring them up to standard. (This isn't going to happen overnight, obviously.) So when you see Sudden Insight in Court in the Act, you know that it's going to have the same effect as Sudden Insight in Casino Fatale.
The only things that may change are flavour text and restrictions. We sometimes use flavour text to give the abilities a bit of colour and make them fit the character. And in some cases we put a restriction on an ability (in Happy Birthday R.J. the Harrington Stock cannot be pickpocketed, for example). However, neither flavour text or restrictions change the basic effect of the ability.
We will also ensure that the balance of abilities is about right. For example, Hollywood Lies currently has over 20 versions of "I love talking to people – I never know what I'm going to learn!" We're going to replace some of those with abilities that do slightly different things so that there's a good variety.
First games to get the new abilities will be Lord and Lady Westing's Will (due 15th April) and Death on the Gambia
.Steve Hatherley
This entry was posted in Design, Rules on March 25, 2013 by Steve Hatherley.
Writing an optional character for a FFG murder mystery game
Sometimes more guests attend a murder mystery party than you originally envisaged. While some of our games have additional character packs that allow you to add up to 10 extra characters, most don't. In that situation you may need to write an additional character (or two).
We provide templates so that you can create your characters in the right format so that they don't stand out too much from the "real" characters. And we will also award anyone with a free game if they create a character that we like and publish on our site. But what we haven't done is provide advice on how to write those extra characters. Until now
For the purposes of this article, we're going to create a couple of imaginary optional characters for an imaginary murder mystery party: Murder at the Ball. Murder at the Ball is a murder mystery set in the world of fairytales – characters include Prince Charming, Cinderella, Snow White, the Wicked Witch, Sleeping Beauty, Beauty, the Beast and other familiar characters. The setting is the grand ball, where all the plots will unfold.
Two extra characters
We've realised that we need two extra characters as we have two more guests coming than originally expected.
Here are some typical types of extra characters that we find fairly easy to add:
Assistants, deputies, underlings.
Rivals and enemies.
Family – close relatives (sons, daughters, husbands, wives, brothers and sisters).
Characters linked to plots that involve lots of people.
Investigator-types – reporters, extra detectives.
Spies or diplomat in a national diplomacy or espionage games.
So the first thing to do is to decide what additional characters to add. Having read through the game we've decided to add Fairy Tulip, an assistant to the Fairy Godmother, and Jack the Giant Killer (from Jack and the Beanstalk). We've decided to pick these two because we believe the Fairy Godmother already has plenty to do and Murder at the Ball has a plot all about electing a new Mayor, which involves a lot of people and we can fit Jack into.
We usually start our character backgrounds with a brief summary of who the character is and why they are present in the game:
"You are Fairy Tulip and you are here to help the Fairy Godmother."
"You are Jack the Giant Killer and you are here because you want to become Mayor."
Then we explain any background information that the characters know (including links to other plots), along with what they need to achieve. So for Fairy Tulip, this would include some of the things that the Fairy Godmother is working on that she can help with. We would refer to the Fairy Godmother's character sheet for examples – but we wouldn't copy it word for word, but instead write it from Fairy Tulip's perspective.
For Jack, we would look at other Mayoral candidates and use their description of how the election works in Jack's background.
Do the same with other plots. For example, we would normally tie them into the murder plot. For example, it could be that Fairy Tulip was a close friend of the victim, and is therefore motivated to find the murderer. With Jack, on the other hand, we might decide that some of the clues that point to the murderer also point to Jack – turning him into a suspect.
Where possible we try to include conflict in our plots. So Fairy Tulip might not agree with all of the Fairy Godmother's plans, and might actually be working to foil one or two. Similarly, Jack could be a rival to one of the other Mayoral candidates.
The goals section of our character sheets are a reminder and clarification of that character's objectives. They shouldn't add anything new that isn't already covered by the background. The number of goals varies from game to game and character to character, but four goals is a good number to aim for.
We use the Other People section of the character sheet to add useful snippets of information about other characters in the game that our new characters know. We try not to repeat the information on the cast list as that doesn't add anything new. Instead, we prefer to add something that lets our character strike up a conversation, either with that person or someone else.
Our new characters should need no more than five or six Other People entries, so they won't need an entry for everyone.
We find it useful to look at the Other People sections of the original characters. In many cases those entries can be copied word for word as they often reflect things that they have seen or have heard stories about – and our new characters might have seen those same things or heard those same stories.
Prince Charming: You met Prince Charming earlier and he told you that he was looking for a glass slipper.
Snow White: You've heard a rumour that Snow White has been living in the forest with some dwarves.
Tips for beginners consist of two actions that the person playing your new character can do at the start of the game. The idea is to give them something to get them going, to start the game with a bang. We don't tend to include actions that will directly solve their goals – the idea is to get a new player started, not solve their goals for them.
In our example, Fairy Tulip might have an action to report to the Fairy Godmother and ask if there is anything she can to do help, while one of Jack's actions might be to talk to Prince Charming about the glass slipper.
The easiest way to give abilities to our new characters is simply to copy some abilities from the other characters. If you've played one of our other games, you could also copy an ability from that game.
We normally give each character three abilities (some of our older games don't follow this rule, however – do whatever suits the game, but three is about right).
Secret and Information/Clue
Most characters have a guilty secret that they don't want anyone else to know – that's what goes in their Secret. This is usually pretty incriminating, although we don't put the identity of the murderer in Secrets because we want the murder to be solved by using deduction rather than abilities!
Information/Clue contains a piece of information that the character knows. This relates to one of the plots, often the murder plot. We put key information here to ensure that it circulates around the game (to those that need to know) via the use of abilities.
Items and money
Our new characters may need items that are both needed for their backstory and help with other plots. For example, Fairy Tulip ought to have a wand and Jack the Giant Killer might have some magic beans, a golden harp and (perhaps) and axe.
Information for the original characters
Once we've finished our characters we need to link them to the original characters. If we don't do that then our new characters may struggle to interact with the original characters.
We first decide who needs to know these new characters. Where we have created an assistant or a family member then they will need to know who this new character is, but there will also be other characters who will know something about your new characters.
In our example, clearly the Fairy Godmother will need some information to introduce Fairy Tulip, and Fairy Tulip may well be known by other characters as well.
Where we've told one of our new characters that they have interacted previously with an original character, then it is important to tell that character of the interaction. For example, we added to one of our characters that Prince Charming had told them that he was looking for a glass slipper (see Other People, above). It's worth telling Prince Charming that he has asked Jack the Giant Killer about the glass slipper but that Jack didn't know where it was.
When we give our characters guilty secrets, we make sure that another character either knows their secret already, or has clues that they aren't what they seem. For example, suppose we decide that Jack the Giant Killer is a fraud and didn't actually climb the beanstalk and kill the giant. In this case we might give Rapunzel some additional information that she recognises that Jack is really Rumplestiltskin.
Send your characters to us!
Once you've written your characters and used them in your games, please send them to us! If we like them we'll upload them to our site (after we edit them a bit) and give you a free game in return. (Note that any characters you send to us become our property and copyright of Freeform Games, and we may use them in future releases, although we will credit you as author.)
Note on intellectual properties: We're very happy if you want to include characters based on existing intellectual properties (such as Captain Jack Sparrow for A Dead Man's Chest, or Harry Potter for Spellbound). However, if you write those characters up we will change their identities to avoid infringement.
Steve Hatherley
This entry was posted in Design, General on March 18, 2013 by Steve Hatherley.
Telling lies in our murder mystery games
In some murder mystery party games you are instructed not to lie about the details in your background. This is because to solve the murder everyone needs all the clues – but unfortunately my experience is that some people can't just help themselves, and they don't always tell the truth.
We, on the other hand, are absolutely fine with people lying and fibbing in a Freeform Games murder mystery party. We tell our players up front what awful deeds their characters have committed and we don't expect them to tell the complete truth in front of the other players.
Of course we do realize that if everyone lied all the time, that would cause problems for other people trying to fulfill their goals. That's why we include in the game Information/Clues and Secrets. These can't be lied about, because they're printed. If another player uses an Ability to make you reveal your Information/Clue or Secret, then you must do so. Then the other player can see written down in black and white something that you might otherwise have wanted to lie about.
Secrets – not lies!
The other balancing mechanism we use is that each player has incentives to be honest and open with at least some of the other players, if they're to try and get their own goals achieved. So in general, you'll tell the truth to your friends and allies, but might be dishonest to your enemies. Then by making connections, third-party characters will be able to piece the clues together.
Note that there's a difference between your character telling a lie (which is fine) and you as a player lying (which absolutely isn't). So for example, if someone asks you "Did you murder so-and-so?", you're quite free to say "No I didn't." But if they use an Ability to make you show your Secret, you mustn't lie and say that you don't have a Secret to show. It might sound like a subtle distinction when written down like this, but in practice players are able to understand that they must be honest 'as players' even if their characters aren't always honest.
This entry was posted in General on March 11, 2013 by Steve Hatherley.
Interviewing absent characters
Here's an idea that you may wish to include in your game. (Note that if you've not played our games before, we suggest that you stick with the basic rules – but if you're an old hand, go crazy!)
An investigation during Death on the Gambia
Interviewing Absent Characters: Sometimes it can be useful for the detective characters to interview absent characters to eliminate them from their inquiries.
When you're preparing the game, print out all the characters – even if you know that you haven't got all the roles filled.
Also print out the items and other handouts for that character – and put it all in that character's envelope.
When the game is running, if one of the players wants to ask a question of one of the missing players, you've got all the information in one place so that you can answer the query. (This is easy if the detective has the "I'd like to ask you a few questions" ability as you can simply show them each characters' Information/Clue.)
An advantage of bringing the complete characters to the party is that if you do get someone arriving at the last minute, you can easily get them involved by giving one of the spare characters to them.
This entry was posted in Options, Rules on March 4, 2013 by Steve Hatherley.
Freeform Games in 2020
Casting a Freeform Games murder mystery game
Using Discord for online murder mystery games
Hosting and Playing Death in Venice
Meanwhile, elsewhere… – Four Letters at Random on Big Money
Meanwhile, elsewhere… – Four Letters at Random on Looking back at 2018
Corinne on Lockdown Way out West
2020 in games – Four Letters at Random on Freeform Games in 2020
Using Discord for online murder mystery games | Freeform Games murder mystery blog on Tips for hosting our games online
From our mailbag
Lockdown stories
Peaky
Tips for hosts


Pillows instantly make a room feel cozy and inviting, Don't you think? One of my favorite things to decorate a room with are throw pillows. There are so many options and they can really pull a room together.
You can never have enough throw pillows!
First I am going to show you some amazing styled Living Rooms with farmhouse pillows and then I am going to share where you can find some of those pillows on Amazon. How awesome is that!
First up is Liz from LizMarieBlog. She fills her sun room couches to the brim with pillows.
Doesn't it just looks amazingly cozy!
Next up is Julie from The Design Twins and her adorable entry way that just looks so inviting and has such a great homey feel to it!
Then Courtney from 12th and White and her beautiful cozy couch. It just looks like a great place to sit with some friends and hang out and talk over coffee or tea.
So now that you have seen some gorgeous spaces with some comfy pillows its time to show where to snag these Farmhouse pillows on Amazon!
There are some great farmhouse pillows on Amazon to get you started on your pillow collection or add to what you have already in your home!
Now to share one of my decorating secrets!
Its not that big of a secret, because I bet some of you already do this, but here it is.
I do not own a lot of pillows, but I have different pillows for each season.
Instead of buying a new pillow for each season I exchange the covers on my pillows. This keeps my pillow count low but my decorating options wide open. It also helps save some money by only buying the pillow covers. Now if you see a pillow that you love at the store and don't think you can find a cover similar to it then go for it!
Do you switch out your pillow covers to change your decor or do you have a mountain of pillows stashed somewhere?
My teenagers tend to throw the "Throw Pillows" on the floor- or just leave them in awkward arrangements on the couch! So, I limit the number of pillows out at any given time! I can only take so much! However, I cannot be totally comfortable on the couch without at least a couple pillows behind me! I do have pillow love too!
I love your blog and I love these ideas! Thank you for the inspiration! I mentioned this post in my most recent blog post about where to find affordable throw pillows. I also grew up in Metro Detroit and my in-laws live close to Traverse City. It's beautiful up there!
Thank you Leah! I will be sure to stop by your blog and check it out! What a small world! I Love it up here in Traverse City! It really is a beautiful place!
Awesome! Thanks for joining Home Sweet Home!
I was running into that problem as well! Glad to be of some help!
These are all fantastic options! I also am in love with pretty much every interior photo you have included. My goodness, those are gorgeous rooms! Thanks for sharing your round-up with us at Merry Monday this week!
Me too Marie! They are definitely on my living room goals list!
These are beautiful! You can never have too many Pillows.


Kane Landscapes is proud to announce that Amanda Hamilton, Designer, has officially passed the Landscape Industry Certified Technician-Exterior test. The Landscape Industry Certified Technician Test is a national, hands-on testing program administered by the Landscape Contractors Association (LCA). It recognizes proficiency in the landscape workforce, upgrades the status of the landscape professional, and provides the public with a means of identifying qualified landscape professionals.
Amanda is now among an elite group of landscape professionals, including Josh Kane, Head Designer, who have earned the designation of Landscape Industry Certified Technician. Congratulations to Amanda for proving her ability and dedication to the industry. We are so proud to have her on board at Kane Landscapes. She is a both a talented designer and hardworking, team player.


There will be a Scholastic Bowl meeting for all interested students TODAY in the new gym at 12:30.
All Key Club members, please meet in Mrs. Jones room TODAY during lunch for a meeting.
If you are in FFA and wanting to paint pumpkins have your $2 to Ms. Meisner by FRIDAY!
Last night FFA Members competed in the Section 16 Ag Sales, Greenhand Quiz Bowl, Grain Fair and SAE Expo and had a successful event. The Ag Sales team was 6th overall with Megan Schumacher, Katie Cole, Gracie Myers-Nichols, and Abby Bolen making up the team. Abby placed 10th overall individual. The Greenhand Quiz Bowl team won 1st place overall with team members including Henry Mitchell, Sami Boogren, Tray Hardesty, Anthony Thompson, and Tori Wingett. Sami was 1st overall individual and the team will have the opportunity to advance to state and compete in June.
Jaton Shaffer, Devyn Dumas, Daisy Miller, and Wyatt Summers competed in the Section 16 FFA Grain Fair earning "A" ratings for their projects and Jaton and Gracie both were 2nd in their classes during the SAE Expo. Congrats to all of our FFA Members for a job well done last night!
Tonight the Lady Knights Volleyball Team plays Cerro Gordo at HOME and it is Senior Night. The JV begins at 6:00 and the Varsity shortly after. Good Luck Knights!


Fridays are tagged for Recipes From the Ranch. However, after mulling over a reader's comment on contests I decided to post my reflection as I believe it has value for all writers, especially those considering contests as a means to build a writing presence.
Entering into the traditional book publishing industry is a new river for me to navigate (as if I were Huck Finn). There's so much advice out there in the form of industry news, author interviews, publisher insights and, of course, tons of writers like me who are forging dreams of publishable novels. Entering your writing into contests is one of those venues for getting noticed if you are seeking an agent or traditional publisher.
Sylvestermouse posted a comment on my post, "A Question of Contests." Her discussion is based on her own experiences and realizations. She was wise and confident enough at a young age to recognize the slippery slope of contests. The danger is that the scrutiny of judges, which is only the opinions of a few, can devastate budding creativity. After reading her comment, I sighed relief. She made me realize the discontent that had come over me. Instead of enjoying the productivity of my fellow contestants, I became hypercritical–of my writing, their writing, comments, the whole thing. And that is not what inspires me as a writer.
Each story selected, mine included, was a submission deemed worthy of publication in a contest by an editor. In that sense, all stories accepted are equalized as "winners." But once the competition began, anxiety churned my gut. I'd rather read what writers craft than develop a pretentious sense of what is best. Different writers have different voices; different readers resonate to different stories. Best is subjective.
That isn't to say that contests aren't for me, but I need to be more self-aware of how they can create inner turmoil for me and stay objective, but above all, kind. Writing is a business, but business professionals do not have to "win" to be successful at what they do. In fact, as professionals, we need to connect and learn from one another without creating barriers. When entering contests, keep a balanced perspective and acknowledge the accomplishment of being selected in the first place. You don't have to wave the blue ribbon to have a solid portfolio piece.
In one form or another, contests will continue to be a part of the literary scene–you can even conclude that making the New York Best Seller's List is a contest. For marketing and profitability, success will continue to have markers that seem like trophies. But for true success as a writer, I still say that finding your own voice and writing into your own truth is the greatest gift you can share with readers.
To any writers reading this, keep in mind that contests are a way to get noticed in the industry and a viable way to build credibility and a portfolio. However, it is merely one avenue. If you write, please continue to write beyond competition outcomes and develop your own special voice, discover your own inner truths and practice (practice, practice) your craft. Do not let a contest be the final judge on your work; do not succumb to discouragement or haughtiness. Be you. And write.
And if you are George R. R. Martin and by the very slim chance happen to be reading my blog…I'm waiting for the next installment of "Song of Fire and Ice." Write, GRRM, write.
By Charli Mills in Tips for Writers on February 1, 2014 .
Good for you, Charli, in discovering the 'truth' within your discontent… and finding your way back to your own 'special voice'. My daughter is an artist and (I believe) a very talented one. But nearly every piece of artwork she creates she becomes critical about it and thinks her artist friends' work is so much better. Until she stops herself and remembers that she has her own special style and there is no comparison needed.
My friend, we have not known each other long. It seems odd to consider that it has only been a few weeks. But, as I have read your posts and articles, I feel as if I have known you for many years. Your writing has a quality that embraces the reader and wraps them in a warmth that is so clearly you.
I am but one inconsequential judge, but I think you're the best! May your voice be forever strong and your passion for writing be forever inextinguishable.
Honest reflections, Charli. Thank you for sharing. I get where you are coming from.
I'd like to talk about my poetry contest that I've hosted since 2010–four years. This 2014 would have been my fifth contest. Not sure if it'll happen. Kinda doubt it.
My poetry contest grew from an online poetry group, Mindful Poetry, that I hosted at Gather.com. That social media site is not running as usual and I've put my time there on hiatus.
The annual, month-long (April) contest worked, however, due to our group's cohesiveness and camaraderie. Together and growing since September, 2008, we'd built up a level of trust, support, and willingness. We learned and practiced and triumph together.
So once the contest began, we all were in it together. Sometimes outsiders would submit a daily poem, but mostly, submissions came from our group of over 200 members. There was not rivalry as one might experience with strangers.
The contest was difficult–less than 24 hours to create a poem with very tight specifications–in other words, we were all in the same boat. The person who could create a shining poem under those conditions was recognized not only by me, the judge, but by the other contestants.
Contests where the competition does not know one another sets itself up for more raw emotions. Players have more to lose personally if their work is not accepted–face. And less to lose if they play dirty–anonymity drives them and absolves them (so they think).
Oh, yes, loosing face and the ugliness that anonymity can bring out in those suffering from their own raw emotional state. Good point, that contests between strangers can increase anxiety. If the moderators can control any "trolling" that can help. Also, if the contest organizers are professional and fair, that can keep the contestants elevated to a professional level, too.
What you have done to create a supportive community among poets is amazing. I've tackled a portion of your contest (April in 2012) and learned so many new poetry structures. I am not a poet, yet I felt so welcome among your members to be able to learn and practice during that contest. I think reading and practicing poetry makes me a more creative writer. I hope that your contest evolves into something new for poetry because I believe in the power of "mindful poetry" as it can also heal hearts, connect people and improve access to the creative mind.
Like you, I'm hoping to build a community of creative writers by developing a flash fiction blog hop that may grow into a monthly contest. I need to talk to you about best practices for such an endeavor.
Thanks for all your thoughtful inclusions to this discussion!
The only contest worth winning, in my humble opinion, is that of living authentically. Before we can write our truth, we must live our truth. The truth will set us free: free of disillusionment; free of the fear of not measuring up; and free of the need for outside affirmation of our worthiness. As one who is very competitive, I have found that contests sometimes rob me of peace, of joy, and of the sense that I am enough. Yet, I still feel compelled to enter into that arena where I contend with the best that I can be. I so appreciate your willingness to share the vulnerability we all know as writers who put ourselves out there every day.
You mirror my own beliefs about truth and writing. Readers know right away when a writer is not authentic. Living authentically and writing into our truths is hugely a vulnerable act. Add the pressure of a contest on top and it can be an anxious situation. Personally, I'm not competitive, but I harbor this huge flaw that "I have to be right." Contests play into that flaw and "best" soon equates to "being right." But the beauty of being a truth-writer is that we tend to be reflective and can work through the stuff that rises, editing inappropriate emotional attachments as if they were unnecessary words. Again, I think personal integrity has to be balanced with professional credibility and we can carefully select the best contests to match our career goals without letting results rule our psyches. Thanks for joining in the discussion with your insights!


Abigail - Ajax,Ontario : I'm a Motivational Coach who is excited about helping you learn how to live a healthier life! I'll help you discover your personal strengths and skills that'll enable you to make lasting changes!
I am a Graphic Designer and I have experience as a Personal Trainer. I have experience in coaching my clients to find the motivation to lose weight and feel better about their body. Which also boosts their self esteem.


text:
Beauty and The Beast – Christmas Pantomime
8th December - 1st January
Malvern Theatres 2022 Christmas Pantomime is BEAUTY AND THE BEAST Join Belle, The Beast, Fairy Bon Bon, larger than life Polly La Plonk and her cheeky chappy son Louis La…
Robin Hood Pantomime at The Regal
14th December - 31st December
Regal Tenbury's Annual Pantomime Performances: Wed 14 Dec: 6:30pm Thurs 15 Dec: 6:30pm Fri 16 Dec: 6:30pm Sat 17 Dec: 2pm & 6:30pm Sun 18 Dec: 2pm & 6:30pm Tues…
7:00 pm Recurring
27th December Recurring
Malvern Theatres Stage Company presents A Christmas Carol by Charles Dickens A Seasonal treat for all the family. A Christmas Carol recounts the story of Ebenezer Scrooge, an elderly miser who is visited by…
CBSO Viennese New Year
FEATURING Christoph Altstaedt – Conductor Anna Devin – Soprano PROGRAMME Reznicek Donna Diana: Overture, 5′ Strauss Telephone Polka, 3′ Menotti The Telephone, 4′ Lumbye Copenhagen Steam Railway , 4′ Waldteufel The Skaters Waltz , 5′ Strauss Pizzicato Polka, 4′ Gounod Romeo and Juliet: Je veux vivre , 4′ Johann…
MET: Fedora
Live Broadcast An opera by Umberto Giordano. Soprano Sonya Yoncheva, one of today's most riveting artists, sings the title role of the 19th-century Russian princess who falls in love with her…
Like Water for Chocolate: ROH Broadcast
Broadcast from The Royal Ballet A co-production between The Royal Ballet and American Ballet Theatre. The Royal Ballet's new production is inspired by Laura Esquivel's novel. It is a family…
ROH Broadcast: Like Water for Chocolate
The Royal Ballet's new production is inspired by Laura Esquivel's novel. It is a family saga, a modern Mexican classic. The central character's emotions spill out through cooking to influence…
Shakespeare's Fool
Tortive Theatre presents Shakespeare's Fool, starring Robin Leetham, written by T G Hofman and directed by Ben Humphrey. Join 'Cavaliero' Kempe on the streets of London as he gives his final…
Jason Fox – Life at the Limit
Star of SAS: Who Dares Wins and ex-special forces soldier Jason Fox comes to Malvern. He brings to the stage his remarkable story of his various daring exploits in a…
Wodehouse in Wonderland
26th January - 28th January
P.G. Wodehouse, or Plum as he's known to his nearest and dearest, is trying to write the latest book in the Jeeves and Wooster series. However, a would-be biographer, his…
NT Live – The Crucible
The Crucible by Arthur Miler directed by Lyndsey Turner A witch hunt is beginning in Arthur Miller's captivating parable of power with Erin Doherty (The Crown) and Brendan Cowell (Yerma).…
NT Live: The Crucible
Filmed at the National Theatre for screening in cinemas, a contemporary new staging of Arthur Miller's classic play, directed by Lyndsey Turner and designed by Tony Award-winner Es Devlin A…
The Story of Guitar Heroes
This awe-inspiring, dynamic show, allows the audience to travel from the 1950s to the present day, enjoying the work of such guitar legends as Chuck Berry, Hank Marvin, Jimi Hendrix,…
Agatha Christie's The Mousetrap
30th January - 4th February Recurring
Agatha Christie's The Mousetrap - 70th Anniversary Tour Have you seen the world's longest-running play? As news spreads of a murder in London, a group of seven strangers find themselves…
1st February
Tap Factory
2nd February
Following more than 500 shows across five continents, Tap Factory is thrilled to come back to the UK for a 10th anniversary tour! Take twelve oil barrels, one stepladder, four…
The Tree Rings by Strange Futures
3rd February
Trees hold stories, they hold memories and myths. Strange Futures theatre group bring their quirky humour and physical theatre to Malvern Cube. A story of a boy's relationship with a…
Jon Boden & The Remnant Kings
Folk music from performer, Jon Boden, whose repertoire extends beyond the traditional boundaries of the genre. He was the leader singer of the band Bellowhead, with impressive album sales, and…
Verdi Aida Senbla presents Opera International's award-winning Ellen Kent Production featuring the Ukrainian Opera & Ballet Theatre Kyiv, with international soloists, highly-praised chorus and full orchestra. Aida is an opera in four acts by…


text:
using System.Collections.Generic;
using System.Linq;
using System.Threading.Tasks;

namespace LinFx.Extensions.Setting
{
    public class GlobalSettingValueProvider : SettingValueProvider
    {
        public const string ProviderName = "G";

        public override string Name => ProviderName;

        public GlobalSettingValueProvider(ISettingStore settingStore)
            : base(settingStore)
        {
        }

        public override Task<string> GetOrNullAsync(SettingDefinition setting)
        {
            return SettingStore.GetOrNullAsync(setting.Name, Name, null);
        }

        public override Task<List<SettingValue>> GetAllAsync(SettingDefinition[] settings)
        {
            return SettingStore.GetAllAsync(settings.Select(x => x.Name).ToArray(), Name, null);
        }
    }
}

redpajama_set_name: RedPajamaGithub