A loss of function screen identifies nine new radiation susceptibility genes

Available online at www.sciencedirect.com

Biochemical and Biophysical Research Communications 364 (2007) 695–701 www.elsevier.com/locate/ybbrc

A loss of function screen identifies nine new radiation susceptibility genes Hitomi Sudo a

a,b

, Atsushi B. Tsuji a,*, Aya Sugyo a, Takashi Imai c, Tsuneo Saga a, Yoshi-nobu Harada a

Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan b Department of Pathology and Oncology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyou-ku, Tokyo 113-8421, Japan c Radgenomics Research Group, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Received 11 October 2007 Available online 22 October 2007

Abstract Genomic instability is considered a hallmark of carcinogenesis, and dysfunction of DNA repair and cell cycle regulation in response to DNA damage caused by ionizing radiation are thought to be important factors in the early stages of genomic instability. We performed cell-based functional screening using an RNA interference library targeting 200 genes in human cells. We identified three known and nine new radiation susceptibility genes, eight of which are linked directly or potentially with cell cycle progression. Cell cycle analysis on four of the genes not previously linked to cell cycle progression demonstrated that one, ZDHHC8, was associated with the G2/M checkpoint in response to DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, DNA repair, cell death, cell growth and genomic instability, and to develop new radiation sensitizing agents for radiotherapy.  2007 Elsevier Inc. All rights reserved. Keywords: Radiation susceptibility; RNAi; Functional screening; Cell cycle; G2/M checkpoint; Palmitoylation

Carcinogenesis is thought to be a multistep process that occurs through the accumulation of mutations in multiple genes required for the maintenance of normal growth control. Genomic instability has been commonly observed in various tumor cell types and is considered the earliest cellular event in the process of carcinogenesis. Genomic instability can be induced by DNA damaging agents such as ionizing radiation or chemical compounds and is characterized by genetic changes that include chromosomal rearrangements, formation of micronuclei, cellular transformation, gene mutations and gene amplifications in the progeny of surviving cells. The molecular mechanisms underlying this process are poorly understood. Cell cycle checkpoints play a key role in cell survival following DNA damage. Dysfunction of DNA repair and cell

*

Corresponding author. Fax: +81 43 206 4138. E-mail address: [email protected] (A.B. Tsuji).

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.10.074

cycle regulation in response to DNA damage are thought to be important factors in the early stages of genomic instability. Several known genes (e.g., ATM, BRCA1, BRCA2, and NBS1) are reportedly associated not only with DNA damage repair and cell cycle regulation but also with radiation susceptibility [1]. Thus, the identification of novel radiation susceptibility genes will likely help to elucidate the molecular mechanisms underlying DNA damageinduced genomic instability, and could be potential molecular targets for chemoradiotherapy. With the completion of the human genome project, we now face the task of elucidating the functions of the identified genes. Several groups have demonstrated that RNA interference (RNAi) is a powerful tool for large-scale functional screening in mammalian cells [2–4]. We previously performed large-scale expression profiling of 15 human cell lines [5] and three mouse strains [6] that display varying degrees of susceptibility to ionizing radiation. These analyses yielded 200 genes that correlated with radiation

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susceptibility. We previously developed a high-throughput screen to identify radiation susceptibility genes using a cellbased proliferation assay and RNAi-mediated knockdown mammalian cells [7]. In the present study, we used this assay to screen for radiation susceptibility genes using an RNAi library containing vectors expressing short hairpin RNAs (shRNAs) targeted to 200 genes previously established by the large-scale expression analyses as potentially associated with radiation susceptibility [5,6]. Materials and methods Cell culture. Human embryonic kidney HEK293 cells (CRL-1573, American Type Culture Collection, Manassas, VA) were maintained in a-MEM medium (Sigma–Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (SAFC Biosciences, Kansas City, MO), 2 mM L-glutamine, 50 U/ml penicillin and 50 lg/ml streptomycin. RNAi library. The vector pcPURhU6icas encodes short hairpin-type small interfering RNAs (shRNAs) and carries the human U6 promoter [4]. Supplementary Table 1 shows the one or two independent target site(s) selected by an algorithm for the 200 genes [8]. Oligonucleotides were synthesized, annealed and ligated as described [4]. X-irradiation and radiation susceptibility assays. We screened radiation susceptibility genes by the 96-well format procedure [7] with 200 shRNA vectors (Supplementary Table 1). Briefly, HEK293 cells were seeded into 96-well plates at 1 · 105 cells/well. After 24 h, cells were transfected with 100 ng of shRNA (Supplementary Table 1) or no vector (mock) using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. At 3 days post-transfection, 1000 cells/well were seeded into each of two 96-well plates using an EDR-384S multichannel dispenser (BioTec, Tokyo, Japan), and one plate was irradiated with 4 Gy using a X-ray generator (Pantak HF320S, Shimadzu, Kyoto, Japan). After 5 days of additional culture, we assayed cell survival using the Sulforhodamine B-based Toxicology Assay (SRB) kit (Sigma–Aldrich) according to the manufacturer’s instructions. Absorbance was measured at 570 and 655 nm using an Ultramark microplate reader (Bio-Rad, Hercules, CA). For 15 genes, ARL4, ATM, ATR, CCNG1, CDKN1A, CENPE, H3F3A, IL13RA1, MAP4K2, SERP1, TRIP11, UCC1, ZDHHC8, ZNF146, and ZNF354A, we tested four additional independent shRNA transfectants for each gene in the assay (Supplementary Table 1). Cell survival was calculated as the ratio of the optical density obtained for irradiated cells to that for non-irradiated cells. Survival of non-irradiated cells was defined as 100%. The mean ± SEM were analyzed by the Student’s t-test or by ANOVA with the Dunnett multiple comparison test. Cell cycle analysis. For knockdown cells, shRNA-transfected cells were harvested at various time points (0, 4, 8, 12, 16, and 20 h) after irradiation (6 Gy), washed once with PBS, fixed with ice-cold 70% ethanol and incubated at 20 C overnight. Approximately 106 cells were resuspended in 1 ml of propidium iodide solution (PBS, 25 lg/ml propidium iodide and 100 U/ml RNase A), incubated for 30 min, and the DNA content of 104 cells was determined with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The percentage of cells in G0/G1, S, and G2/M phases was determined using the ModFit software (Verity Software House, Topsham, ME). For palmitoylation inhibitor treatment cells, 2.5 · 105 cells were seeded on 6-cm dish. After 3 days, cells were treated with 0 (vehicle only), 10, 50, and 100 lM 2-bromopalmitate (Sigma) for 24 h. The DNA content of 104 cells at various time points (1, 8, 16, and 24 h) after X-irradiation at 6 Gy was determined as described above. In addition, the percentage of cells in sub-G1 phase was measured as apoptotic population using the ModFit. Apoptosis analysis with annexin V staining. The 2.5 · 105 cells were seeded on 6-cm dish. After 3 days, cells were treated with 0 (vehicle only), 50 and 100 lM 2-BP for 24 h. Cells were harvested at 0, 8, 16, and 24 h following X-Irradiation of 6 Gy and stained with annexin V and 7-aminoactinomycin D (7-AAD) using a Guava PCA Nexin kit (Guava Tech-

nologies, Hayward, CA) according to the manufacturer’s instruction at various time points (0, 8, 16, and 24 h) after X-irradiation at 6 Gy. Two thousand five hundred events were counted, and living (annexin V-negative and 7-AAD negative) and apoptotic cells (annexin V-positive) were determined by a Guava PCA system (Guava Technologies). Real-time reverse transcription-PCR (RT-PCR). Total RNA was isolated from shRNA-transfected cells using the RNeasy Mini kit (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. The cDNA was synthesized from 1 mg total RNA using an oligo(dT) primer and the SuperScript First-Strand Synthesis system for RT-PCR (Invitrogen, Carlsbad, CA). Real-time PCR amplifications in duplication were performed with 2.5 ll cDNA (diluted 1:5) using gene-specific primer sets for target genes and b-actin and iQ SYBR Green supermix in an iCycler realtime PCR thermal cycler (Bio-Rad), according to the manufacturer’s instructions. Gene expression was quantified with reference to a calibration curve obtained from serial dilutions (107–101 molecules, in duplicate) of the control plasmid. The expression level of target genes was normalized to that of b-actin. The primers were as follows: ATM, 5 0 -AGAGGCC GGAAGATGAAACT-3 0 and 5 0 -AAGACACGTTCAGCTACTTTGTT G-3 0 ; ATR, 5 0 -CTGATGCGTGATCAGCGAGA-3 0 and 5 0 -TTCATTCA GTGGCGCTTTGG-3 0 ; CCNG1, 5 0 -AGGGTGGTTACCGCTGAGGA3 0 and 5 0 -CCACAGACCTTTGGCTGACATC-3 0 ; CDKN1A, 5 0 -ACCTT CCTCATCCACCCCA-3 0 and 5 0 -TGACTCCTTGTTCCGCTGC-3 0 ; CENPE, 5 0 -CATCACCTCATCCAGTTCGCTAT-3 0 and 5 0 -AGGACCT GGCTGAGAATCCAC-3 0 ; H3F3A, 5 0 -CTGTTATTGGTAGTTCTG AACG-3 0 and 5 0 -CCACTCGCAATCATATACTTAG-3 0 ; IL13RA1, 5 0 -C CCTAGGTCTTGGGAGCTCTTG-3 0 and 5 0 -TTACCATCCTGACACT GGGTTTG-3 0 ; MAP4K2, 5 0 -CAGCGGAGGCTACAGCAACA-3 0 and 5 0 -TGTCAGGAATCTTGGTGGACAGAG-3 0 ; UCC1, 5 0 -CAGGAGCA GATCACCGTCCA-3 0 and 5 0 -TGTATAGATGCCAATCCAGGTTTC A-3 0 ; TRIP11, 5 0 -ATTCTTGCCCAGAGTGCATCA-3 0 and 5 0 -ATTGTC TTCAGCAAGACTGTTATCC-3 0 ; ZDHHC8, 5 0 -CCCAAAGCTGTCG CCTTCA-3 0 and 5 0 -TTAACCAGCGTGTGCCGTGTA-3 0 ; ZNF146, 5 0 TGGAGATCTTCGCCAGTAACAA-3 0 and 5 0 -TCTGCTCTGCTCAA TCAATACCAC-3 0 ; ZNF354A, 5 0 -GAGAGCCTTCAGCCAGAGTG-3 0 and 5 0 -CTCCAGTATGAATGATTCGGTGTC-3 0 ; and b-actin, 5 0 -GTG CTCGCGCTACTCTCTCT-3 0 and 5 0 -TCAATGTCGGATGGATGAA A-3 0 .

Results and discussion Screening of radiation susceptibility genes We constructed an RNAi library comprised of shRNA vectors against the 200 genes potentially associated with radiation susceptibility (Supplementary Table 1). The screen was conducted by transfecting each of these vectors individually into human embryonic kidney HEK293 cells and measuring the cell survival, as determined by a sulforhodamine B-based cell proliferation (SRB) assay [7], after exposure to 4 Gy X-irradiation. A vector expressing an shRNA targeted to PRKDC, a known radiation susceptibility gene [9], was also transfected into HEK293 cells as a positive control. The fraction of mock- and PRKDC-shRNA-transfected cells that survived was 0.416 ± 0.026 and 0.259 ± 0.071, respectively. Of the 200 genes, there were 15 genes for which the fraction of shRNA-transfected cells that survived was less than 0.188 (Fig. 1A), which was one standard deviation below the mean survival for PRKDC-shRNA-transfected cells (0.259). We sought to confirm the results for the 15 genes for which RNAi transfection gave the poorest survival by assessing their susceptibility to radiation in four

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Fig. 1. Survival of cells transfected with shRNA vectors. (A) Survival of HEK293 cells individually transfected with shRNAs vectors against 200 genes (Supplementary Table 1) after 4 Gy X-irradiation, as determined by a screening SRB assay. Cell survival, represented as the fraction of cells that survived relative to non-irradiated cells, for individual vectors is indicated by black bars (<0.188, 15 genes) or gray bars (>0.188). The dashed line at 0.188 indicates one standard deviation below the mean survival for PRKDC-shRNA-transfected cells. (B) Survival at 4 Gy of HEK293 cells transfected with no vector (mock, white), pcPURhU6icas (vector, white), PRKDC-shRNA (positive control, gray) or 30 distinct shRNAs against 15 genes identified as positive by our screen in (A) (black). Data represent means ± SEM of four transfections, as analyzed by ANOVA with the Student–Newman–Keuls method multiple comparison test (vs. vector, *P < 0.01).

independent trials (Fig. 1B). To avoid off-target effect [10], knockdown of each gene was achieved by expressing each of two distinct gene-specific shRNAs individually in HEK293 cells. The shRNA-mediated knockdown of ARL4 or SERP1 did not affect cell survival. However, the knockdown of 11 genes, ATR, CCNG1, CDKN1A, CENPE, H3F3A, IL13RA1, TRIP11, UCC1, ZDHHC8, ZNF146, and ZNF354A, significantly reduced cell survival as compared with vector-transfected cells (the neg-

ative control), indicating that these genes are associated with radiation susceptibility. (Two other genes, ataxia telangiectasia mutated (ATM) and mitogen-activated protein kinase 2 (MAP4K2), are discussed below.) The expression of the target genes in these 11 shRNA-transfected cells was reduced to 4–45% compared with the negative control, as assessed by real-time RT-PCR (Supplementary Fig. 1). It suggests that the knockdown thresholds for enhancing radiation susceptibility differ

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with genes. For cells expressing ATM-shRNAs (AMTi#1 and ATMi#2), survival differed between the two different shRNA vectors. The surviving fraction of ATMi#1shRNA-transfected cells was 0.284 ± 0.011 (significantly different from the negative control), whereas the surviving fraction for the ATMi#2 shRNA was 0.431 ± 0.011 (not significantly different). The expression of ATM in ATMi#1- and ATMi#2-transfected cells was reduced to 17% and 24%, respectively, compared with the negative control (Supplementary Fig. 1). These data suggest that

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shRNA-mediated knockdown in ATMi#2-transfected cells was insufficient to significantly reduce cell survival after irradiation, despite the fact that ATM, a gene involved in cell cycle regulation, is susceptible to radiation [11]. Similar to the discrepancy between the two distinct ATM-shRNA vectors, the fraction of cells that survived irradiation for the MAP4K2-shRNA vectors (MAP4K2i#1 and MAP4K2i#2) was 0.329 ± 0.008 (significantly different from the negative control) and 0.461 ± 0.031 (not significantly different), respectively.

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DNA content Fig. 2. Cell cycle analysis of HEK293 cells transfected with two distinct shRNAs against ZDHHC8. (A) The DNA content of cells transfected with pcPURhU6icas (vector) or two distinct shRNAs directed against ZDHHC8 at 0, 4, 8, 12, 16, and 20 h after X-irradiation at 6 Gy. The DNA content of 104 cells was determined with the FACSCalibur and the percentage of cells in G0/G1, S, and G2/M phases was determined by the ModFit. Data represent the mean of three different transfected cells. (B) The percentage of cells in G0/G1, S, and G2/M phases as described in (A). Cells were transfected with either pcPURhU6icas (vector, open circles), ZDHHC8i#1 (closed squares) or ZDHHC8i#2 (gray squares). Data represent means ± SEM (n = 3).

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MAP4K2 expression in MAP4K2i#1- and MAP4Ki#2transfected cells was reduced to 20% and 40%, respectively (Supplementary Fig. 1). MAP4K2 has not been reported to be associated with radiation susceptibility, and thus our result cannot conclusively establish whether the lack of significant change in cell survival of MAP4K2i#2-shRNA-transfected cells resulted from insufficient knockdown or whether the result observed for the MAP4K2i#1-shRNA was an off-target effect [10]. Cell cycle analysis of knockdown cells Of the 12 genes detected by our screen, ATM [11], ATR [11] and CDKN1A [12] were previously identified as radiation susceptibility genes, whereas the other nine genes were newly identified as such. Interestingly, eight, ATM [11], ATR [11], CCNG1 [13], CDKN1A [12], CENPE [14], H3F3A [15], IL13RA1 [16], and TRIP11 [17], of the 12 genes have been reported to be directly or potentially associated with cell cycle progression. Thus, we focused on the role of the four remaining genes, UCC1, ZDHHC8, ZNF146, and ZNF354A, with regard

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to their possible involvement in the cell cycle. Because radiation-induced DNA damage leads to cell cycle arrest during DNA repair processes [11], we investigated cell cycle progression in knockdown cells using two distinct shRNA vectors against each of these four genes following X-irradiation. Fig. 2 shows that pcPURhU6icas-transfected cells accumulated in S and G2/M with the peak of S and G2/M accumulation observed 16 h after irradiation. Then, we performed cell cycle analysis in cells transfected with UCC1, ZDHHC8, ZNF146, and ZNF354A shRNA vectors after irradiation. We observed impairment of G2/M arrest in ZDHHC8 knockdown cells (Fig. 2), but not in UCC1, ZNF146 or ZNF354A knockdown cells (Supplementary Fig. 2), suggesting that ZDHHC8 is involved in cell cycle control. ZDHHC8 ZDHHC8 knockdown cells accumulated in S phase to the same degree as vector-transfected cells but were not arrested in G2/M phase (Fig. 2). It suggests that ZDHHC8 is involved in the G2/M checkpoint and that

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impairment of this checkpoint is responsible for the high susceptibility of ZDHHC8-knockdown cells to ionizing radiation. With regard to its clinical importance, mutant alleles of ZDHHC8 contributes to the risk of schizophrenia but its role in the pathology of the disease is poorly understood [18]. ZDHHC8 encodes a cysteine-rich domain containing a DHHC (Asp-His-His-Cys) motif, which is predicted to participate in the palmitoyltransferase activity and in protein–protein or protein–DNA interactions [19]. It is possible that ZDHHC8 protein may modify (e.g., via palmitoylation) or interact with regulatory molecule(s) of the cell cycle. To determine whether

ZDHHC8 is associated with cell cycle checkpoint via palmitoylation, we performed cell cycle analysis in cells treated with a protein palmitoylation inhibitor, 2-bromopalmitate (2-BP). First, to determine the effect of 2-BP on cell cycle progression under the non-irradiated condition, we analyzed cell cycle at 1, 8, 16, and 24 h after 2-BP treatment. Supplementary Fig. 3 shows that 2-BP has no apparent effect on cell cycle. Next, we analyzed cell cycle under the irradiated condition. Cells with 0 (vehicle), 10 and 50 lM 2-BP significantly accumulated in G2/M phase after irradiation at 6 Gy, but cells with 100 lM 2BP did not (Fig. 3A and B). G2/M checkpoint activation

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in cells with 10 and 50 lM 2-BP suggests that these doses are insufficient to inhibit palmitoylation. The sub-G1 population in cells treated with100 lM 2-BP was 1.5%, 4.5%, and 15.2% after 8, 16, and 24 h, respectively (Fig. 3B). It is also possible that apoptosis relates to the G2/M checkpoint defect in 2-BP treated cells. Although the G2/M checkpoint was already activated by 16 h after irradiation, the sub-G1 population with 100 lM 2-BP at 16 h was only 4.5% (Fig. 3B). We also measured apoptotic population using flow cytometric analysis of 2-BP treated cells stained with annexin V and 7-amino-actinomycin D (DNA staining) shown in Fig. 3C. The percentage of apoptotic population in cells with 100 lM 2-BP was not significantly different from that in cells with 0 and 50 lM. These results suggest that the palmitoylation inhibition more effective in the lack of G2/M checkpoint rather than apoptosis in this case. Interestingly, our results of the knockdown of ZDHHC8 and the palmitoylation inhibition with 2-BP suggest that ZDHHC8 controls G2/M checkpoint through palmitoylation in response to DNA damage by X-irradiation. Our present work describes a functional screening strategy whereby the expression of candidate radiation susceptibility genes identified from large-scale expression profiling was disrupted using an RNAi library. Many large-scale expression studies have been reported, and we believe that this strategy could be applied to them to identify key genes associated with various biological processes. Interestingly, we identified three known and nine new radiation susceptibility genes; one of these, ZDHHC8, is involved in G2/M checkpoint activation induced by irradiation. Further functional studies for these genes and gene pathways in response to DNA damage may provide clues to the underlying molecular mechanisms of genomic instability and carcinogenesis, and to develop new radiation sensitizing agents for radiotherapy.

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Acknowledgments This research was supported in part by a grant from the Ministry of Education, Science, Sport, and Culture of Japan, number 17790473 to A.B.T.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2007.10.074.

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