Abstract
Ionizing radiation (IR) is of clinical importance for glioblastoma therapy; however, the recurrence of glioma characterized by radiation resistance remains a therapeutic challenge. Research on irradiation-induced transcription in glioblastomas can contribute to the understanding of radioresistance mechanisms. In this study, by using the total mRNA sequencing (RNA-seq) analysis, we assayed the global gene expression in a human glioma cell line U251 MG at various time points after exposure to a growth arrest dose of γ-rays. We identified 1656 genes with obvious changes at the transcriptional level in response to irradiation, and these genes were dynamically enriched in various biological processes or pathways, including cell cycle arrest, DNA replication, DNA repair and apoptosis. Interestingly, the results showed that cell death was not induced even many proapoptotic molecules, including death receptor 5 (DR5) and caspases were activated after radiation. The RNA-seq data analysis further revealed that both proapoptosis and antiapoptosis genes were affected by irradiation. Namely, most proapoptosis genes were early continually responsive, whereas antiapoptosis genes were responsive at later stages. Moreover, HMGB1, HMGB2 and TOP2A involved in the positive regulation of DNA fragmentation during apoptosis showed early continual downregulation due to irradiation. Furthermore, targeting of the TRAIL/DR5 pathway after irradiation led to significant apoptotic cell death, accompanied by the recovered gene expression of HMGB1, HMGB2 and TOP2A. Taken together, these results revealed that inactivation of proapoptotic signaling molecules in the nucleus and late activation of antiapoptotic genes may contribute to the radioresistance of gliomas. Overall, this study provided novel insights into not only the underlying mechanisms of radioresistance in glioblastomas but also the screening of multiple targets for radiotherapy.
Similar content being viewed by others
Main
Glioblastoma is the most frequent and aggressive primary brain tumor.1 Despite optimal treatment with a combination of surgical resection, radiotherapy and chemotherapy, patients with glioblastoma have a median survival only of 12–15 months.2 Although ionizing radiation (IR) is considered as the gold-standard adjuvant treatment for prolonging survival in patients,3 glioblastomas are often characterized by radiation resistance; therefore, universal recurrence remains a therapeutic challenge.4 Comprehensive understanding of the response of glioblastomas to IR and detailed radioresistance analysis of gliomas may help to identify radiosensitizing agents for combination treatment of the disease.
In the past decade, many advances have been made in understanding IR-induced DNA damage response (DDR), such as the activation of checkpoint pathways, repair and cell death. A previous study showed that preferential activation of the DNA damage checkpoint response and increased DNA repair capacity contributed to glioma radioresistance.5 Furthermore, targeting of the DDR signaling network in gliomas was also found to sensitize tumors to radiation therapy and reverse therapeutic resistance.6 For example, inhibition of PARP1, which is actively involved in the single-stranded DNA (ssDNA) excision repair process, can disturb the cellular repair of radiation-induced ssDNA, and therefore, PARP1 inhibitors have been incorporated into radiation therapy for glioblastomas.7 Furthermore, inhibition of WEE1 or MYT kinases has also been shown to abrogate the G2/M checkpoint and increase radiosensitivity.8, 9 Moreover, it has been found that glioma cells underwent apoptotic cell death in response to IR via both p53-dependent and p53-independent pathways and that evasion of apoptosis led to radioresistance in glioblastomas.10, 11 IR has also been reported to activate NF-κB, which transactivates its target genes, including cox-2, bcl-2, bcl-xL, XIAP and survivin,12 and the expression of these antiapoptotic genes disrupts apoptosis signaling, thereby mediating radioresistance. Vellanki et al.13 demonstrated that therapeutic targeting of XIAP increased the radiosensitivity of glioblastomas by promoting apoptosis. Furthermore, other mechanisms and pathways, including aberrant p21 regulation,14 the Notch signaling pathway,15 the Wnt pathway,16 radiation-induced Akt activation,17 as well as abnormal p53 function,18 have been correlated with radioresistance in human glioblastoma cells.
Although multiple mechanisms have been proposed for radioresistance in glioblastoma cells, the analysis of molecular signaling events is still not comprehensive. To date, advances in high-throughput sequencing methodology have provided a large amount of information regarding gene expression at the transcriptome level, as well as the underlying molecular events in response to irradiation.
In this work, we used the RNA-seq technique to investigate irradiation-responsive genes and the dynamic DDR in the glioma cell line U251 MG. We compared the genome-wide expression between the irradiation group and the corresponding control at 6, 12, 24 and 48 h after irradiation. Functional categories of differentially expressed genes (DEGs) were also analyzed based on gene ontology. Finally, we focused on the apoptosis pathway activated by irradiation and investigated the significance of proapoptosis and antiapoptosis genes in glioblastoma radioresistance. Together, these should provide prime information for research on radiotherapy of glioblastomas.
Results
Characteristics of human glioma cells treated with γ-irradiation
U251 MG cells were treated with a series of γ-irradiation doses from 0 to 30 Gy, followed by the survival assay. On the basis of the results shown in Figures 1a and b, 7 Gy, the half lethal dose at 24 h, was selected as the transition dose for further studies. After treatment with 7 Gy, the cell number remained almost unchanged until 96 h after γ-irradiation (Figure 1c). Furthermore, irradiated cells seemed to lose the proliferative activity at 48 h, with observable morphological changes, such as increased granularity (Figure 1d). Therefore, the time points before 48 h were chosen to capture the gene network controlling the growth arrest and survival.
Global changes in gene expression in response to irradiation
Genes with twofold changes or greater (P<0.0005) at 6, 12, 24 and 48 h were defined as DEGs, as shown in the Supplementary Information (Supplementary Dataset 1). Ten DEGs were confirmed by quantitative real-time PCR (Q-RT-PCR) for independent validation and all showed a good similarity between RNA-seq and Q-RT-PCR results. We presented six genes in Figure 2a, indicating the good quality of our data. The dissimilarities among different samples on a global scale are also demonstrated by the scatter plot and principal component analysis (Supplementary Figures S1a and b). The results of PCA analysis are consistent with the number of DEGs that show altered expression in each treatment, relative to the control (Supplementary Dataset 1). Taken together, these observations indicate that there existed much more changed genes in sample at 48 h after irradiation (R48 h) compared with control (C48 h).
As shown in Figure 2b, the number of DEGs induced by irradiation increased with time (180, 365, 698 and 1460 at 6, 12, 24 and 48 h, respectively), and the majority of them were either upregulated or downregulated after 24 h. Venn diagrams were then used to illustrate these radiation-affected genes (Figure 2c). Totally, 1656 genes were determined as radiation-affected genes, with 546 genes upregulated (33%) and 1110 genes downregulated (67%). Among 546 upregulated genes, 81 were upregulated at 6 h, 131 at 12 h, 144 at 24 h and 418 at 48 h; Among 1110 downregulated genes, 99 were downregulated at 6 h, 234 at 12 h, 554 at 24 h and 1042 genes at 48 h. Meanwhile, it was in particular that most of radiation-affected genes were activated exclusively at the 48 h time point, 319 out of 546 upregulated genes (58.42%) compared with only 16 (2.93%), 36 (6.59%) and 43 (7.88%) at 6, 12 and 24 h, similarly, 529 out of 1110 downregulated genes (47.66%) compared with only 7 (0.63%), 10 (0.9%) and 47 (4.23%) at 6, 12 and 24 h, respectively. To better distinguish dynamic regulation and response under radiation stress, 6, 12 and 24 h were chosen as early responsive time points, with 48 h as the late responsive time point. Genes with transcriptional expression changes at any of the time points of 6, 12 and 24 h were designated as early and transiently upregulated or downregulated genes (ETU or ETD, respectively marked as yellow or pink). Genes with expression changes at all time points among 6, 12, 24 and 48 h were grouped as early and continually upregulated or downregulated genes (ECU or ECD, respectively, marked as gray or orange). Furthermore, genes that showed altered expression only at 48 h were considered as late upregulated or downregulated genes (LU or LD, respectively, marked as light or dark green). According to this, it was calculated that among early radiation-affected genes (297), 158 (53.2%) or 139 (46.8%) genes were upregulated (ETU+ECU) or downregulated (ETD+ECD), respectively; among late radiation-affected genes (848), 319 (31.6%) or 529 (62.4%) genes were upregulated (LU) or downregulated (LD), respectively.
Overall, these observations indicate that the time points before 24 and 48 h can be considered as the early and late phases, respectively, of the continuous dynamic process and that both upregulation and downregulation have a role in early radiation responses, whereas downregulation may constitute a major part of the late radiation response in glioma cells under the test conditions.
Dynamic cellular damage responses induced by irradiation
The gene ontology analysis was conducted to analyze these early transiently, early continually and late genes separately (Table 1). The ETU genes were most enriched for genes involved in the induction of apoptosis, p53 signaling pathway and response to radiation, indicating the early stress responses of cells and the activation of related pathways upon radiation exposure. As for the ECU genes, they were most enriched in the p53 signaling pathway, positive regulation of apoptosis and cell cycle arrest, indicating a mechanism of radiation-induced cell growth arrest. During the late response, many genes involved in cell–cell adhesion and various metabolic processes were enriched (LU). Interestingly, many genes involved in the negative regulation of apoptosis were LU, suggesting the counterbalance between antiapoptosis- and proapoptosis-related genes in response to irradiation.
Most radiation-downregulated genes were ECD genes and LD genes, with only 68 (6.13% of all downregulated genes) being ETD genes that were mostly enriched in the regulation of transcription. ECD genes were highly enriched for genes involved in cell cycle phase, DNA replication and repair, suggesting the regulation of cell cycle and disorders in the DNA metabolic process. During the late response, the most significant events were RNA splicing, indicating radiation-induced alterations in the post-transcriptional regulation of gene expression.
Taken together, we observed that genes involved in cell cycle progression, DNA replication and DNA repair were highly enriched at early time points after irradiation, whereas irradiation-induced proapoptosis-related genes were enriched at the early stage and antiapoptosis-related genes at the later stage.
Irradiation induces G2/M DNA damage checkpoint
It has been demonstrated that DNA damage induced by IR caused cell cycle arrest in proliferating mammalian cells.19, 20 Here, our flow cytometry analysis showed that the cell populations in the G2/M and S phases were significantly higher and lower, respectively, at 24, 48 and 72 h, compared with 0 h control (Figures 3a and b), indicative of cell cycle arrest at the G2 phase. Consistent with previous report,21 we show that irradiation induced the downregulation of many genes related to DNA replication (Supplementary Table S1), thereby inhibiting the process of DNA replication and cell cycle progression. Next, genes associated with the G2/M checkpoint were selectively analyzed. CDKN1A (p21) and GADD45A, two downstream target genes of p53 in the G2 checkpoint, were found to be increased at the transcriptional level at 6 h after irradiation, whereas the expression levels of CHEK1, WEE1, E2F1, E2F2, CDK1, CCNB1 and CDC25C were decreased in a time-dependent manner (Figure 3c). Previous studies have reported that increased p21 expression led to the repression of cyclin B1 and Cdc2 promoters and that increased GADD45A expression inhibits Cdc2 activity, thereby mediating G2/M arrest.22, 23 The G2/M checkpoint protects cell viability by allowing time for DNA repair.24, 25, 26, 27 Interestingly, according to our analysis (Supplementary Table S2), genes involved in the DNA repair pathways, including recombinational repair, base excision repair, mismatch repair and nucleotide excision repair, showed systematic repression, indicating the inefficiency of DNA repair during a certain period after irradiation.
Irradiation induces the counteraction of proapoptosis and antiapoptosis
The inactivation or inefficiency of DNA repair may accelerate cell death, and it is known that cell death by apoptosis has a pivotal role in γ-irradiation-induced cytotoxicity.28, 29 In this study, the activities of caspase cascades in irradiated cell lysates were investigated. We observed that the caspase-3 zymogen was cleaved into active fragments (17/19 kDa) after irradiation (Figure 4a). The enzymatic activities of caspase-3/7 and caspase-8 were also higher than the control after irradiation (Figure 4b). However, annexin V-FITC and PI staining, followed by the flow cytometric analysis, indicate that irradiation did not exert significant apoptotic effects on cells with time (Figure 4c), suggesting the existence of antiapoptosis mechanisms in response to irradiation.
On the basis of the gene ontology analysis, we identified 83 genes that associated with apoptosis, of which 43 and 27 genes may function as positive and negative regulators, respectively, and the other 13 genes may regulate apoptosis either in both ways or in an unclear manner, according to the reported information (Supplementary Table S3). Nevertheless, the expression changes shown by the heat map revealed that radiation tended to induce proapoptosis genes (ETU and ECU) at early phases, whereas antiapoptosis genes (LU) were induced at late phases (Figure 4d).
Among proapoptosis genes, p53-dependent target genes, including TP53I3, BBC3, AEN, CYFIP2 and PHLDA3, were induced by irradiation at early time points. In contrast, death receptor genes (FAS and TNFRSF10B), as well as Bcl-2 family proapoptosis genes (BAX and MAGED1, as inhibitors of the IAP family members), were upregulated early or late after irradiation. Furthermore, antiapoptotic genes, TRAF2, MAP3K14, and members of the IAP family (such as BCL2 and BIRC3) were LD (Supplementary Figure S2).
Although the altered expression of genes described above could bring about apoptosis, genes involved in antiapoptosis were significantly induced at a late time point by irradiation. For example, the upregulation of PTGS2, NOTCH1,and BNIP3 may have important roles in radioresistance or antiapoptosis of glioma cells.30, 31, 32, 33, 34, 35
Downregulation of genes related to nuclear DNA fragmentation after irradiation
Table 2 summarizes a selective list of potential target genes whose upregulation or downregulation may promote radioresistance of glioma cells by inhibiting apoptotic cell death after irradiation, according to the reports. We found that three genes, HMGB1, HMGB2 and TOP2A were repressed which were involved in the regulation of apoptotic DNA fragmentation.36 HMGB1 and HMGB2 have been reported to activate the DFF40/CAD nuclease activity during apoptosis,37, 38, 39 whereas TOP2A (encoding topoII) has been found to have a role in the formation of DNA fragments at advanced nuclear apoptotic stages.40 On the basis of these findings, repression of these genes may result in the inhibition of apoptosis downstream of the caspase cascade. That is to say, although there have been upregulated apoptosis signals in the cytoplasm after irradiation, negative feedback of nucleic apoptosis signals might interrupt signal transmission downstream of caspase cascades to the nucleus and trigger the antiapoptosis effects. The significantly downregulated expression of these kinds of genes may reflect the cellular antiapoptosis mechanism induced by irradiation in the nuclear apoptotic stage (Supplementary Figure S2a).
Targeting of the TRAIL/death receptor 5 pathway increases the radiosensitivity of glioma cells
Our RNA-seq data revealed that TNFRSF10B (the gene encoding Death Receptor 5, DR5) was induced by irradiation (Supplementary Table S3), which was further confirmed by RT-PCR and western blotting (Figures 5a and b). As DR5 is known to be primarily expressed in malignant glioma cells,41, 42 we next investigated whether combination treatment with tumor-related apoptosis-induced ligand (TRAIL), a DR5 ligand, and irradiation destroys the balance between pro- and antiapoptotic factors by activating the TRAIL/DR5 apoptotic pathway. We found that after the cells were treated with TRAIL (20 ng/ml) after irradiation, the number of cells that survived was significantly lower than that for either treatment alone (Figure 5c). The FACS assay results also reflected that the early apoptotic population was increased significantly in a time-dependent manner after the combination treatment (Figure 5d; 24 h, 34.88%±3.13%; 48 h, 45.69%±2.72%), compared with the irradiation alone group (24 h, 3.4%±1.05%; 48 h, 5.73%±0.97%). Markedly increased catalytic activities of caspase-8 and caspase-3/7 were also observed in the early apoptotic population (Figure 5e). Interestingly, we found that radiation-induced downregulation of HMGB1, HMGB2 and TOP2A was significantly attenuated after combination treatment (Figure 5f), suggesting that these genes (involved in DNA fragmentation) may have important roles in the apoptotic process of caspase cascades transmission to the nucleus. Together, these results indicate that the activation of the TRAIL/DR5 signaling pathway in response to irradiation is efficacious for killing radioresistant glioma cells.
Discussion
DDR induced by IR was validated to be dose- and time-dependent. Understanding these complex cellular responses requires the detailed analysis of global molecular expression profiling. Here, by using high-throughput RNA sequencing, the dynamic gene expression network in glioma cells induced by irradiation was explored. We identified 1656 genes that displayed significant expression changes at or before 48 h after irradiation. The results also revealed that genes related to DNA repair, DNA replication and cell cycle arrest were transcriptionally modulated and highly enriched at early time points, whereas proapoptosis-related genes were enriched at the early stage and antiapoptosis-related genes were enriched at the later stage after irradiation.
First, our studies demonstrated irradiation-induced repression of numerous genes associated with DNA replication (Supplementary Table S1) and G2 phase arrest. The G2/M checkpoint which may be explained by transcriptional expression changes of some crucial factors involved (Figure 3) was reported to have a protective role against DNA damage-induced cytotoxicity by allowing time for damage repair, the abrogation of which may increase radiosensitivity.43, 44 In our study, U87 also sustained G2/M arrest (data not shown). Collectively, it reflected that cell cycle arrest and increased DNA repair ability may contribute to the radioresistance of glioma cells.
Second, we detected apoptotic effects induced by irradiation. Analysis of apoptosis genes subsequently revealed that the progress of apoptosis was accompanied by changes in both proapoptosis and antiapoptosis gene expression, consistent with the observation in other irradiated glioblastoma cell lines.44 Interestingly, the dynamic gene expression pattern, as shown in Figure 4d, indicated that proapoptosis genes showed early upregulation, and were followed by the late upregulation of antiapoptosis genes. Meanwhile, based on the high-throughput RNA sequencing, we explored the relation of the dynamic gene expression network to radioresisitance in different glioma cells, such as U87 at the dose of 10 Gy (which has worse radiosensitivity than U251 cell line). As shown in Supplementary Figure S2c, most of the proapoptosis genes showed early upregulation (ECU), followed by the similar early upregulation of antiapoptosis genes (ECU). These changes meant that the activation of antiapoptosis genes in U87 cell line was earlier than that in U251 cell line, which might contribute to the stronger radioresistance of U87 glioma cells.
In addition, our data indicated that p53 signaling pathway genes were mainly enriched at the early stage, although they were involved in wide range of columns, including ECU, ETU and ECD (Table 1), and associated with pro- or antiapoptosis responses to DNA damage (Supplementary Figure S2). It seemed like that the radioresistance of glioma cells was mainly due to the regulation at the later stage. Therefore, in spite of the fact that a significant status of p53 regulation in the radioresistance mechanism,8, 9, 10, 11, 14, 18, 24, 45 more studies are needed.
Furthermore, we analyzed factors whose gene expression alterations may contribute to the radioresistance of glioma cells with a focus on the upregulated antiapoptosis genes and the downregulated proapoptosis genes. First, among the upregulated antiapoptosis genes, PTGS2, encoding COX-2, has been reported to increase the radiosensitivity of glioblastoma cells by regulating Ku expression30, 31 and activating the PI3K and PKA pathways.46 Although BNIP3 is a pro-cell death member of the Bcl-2 family, it was found to be localized in the nucleus of glioblastoma cells and repress the gene expression of apoptosis-inducing factor (AIF), thereby preventing cell death.34, 35 In addition, NOTCH1 could activate gene expression programs via the translocation of the intracellular NOTCH domain (NICD).33, 47 Thus, the upregulated gene expression of these three molecules may inhibit the apoptotic effect caused by irradiation and thereby increases the radioresistance of glioma cells. Second, among the downregulated proapoptosis genes, p73 (encoded by TP73), with functions similar to those of p53, was reported to participate in the apoptotic response to DNA damage and had an important role in cellular radiosensitivity.45, 48, 49 Moreover, according to the fact that the downregulation of HMGB1, HMGB2 and TOP2A transcriptional levels was attenuated significantly after combination treatment of TRAIL and irradiation (Figure 5f), we further studied the changes of these three proteins. As shown in Supplementary Figure S3a, HMGB1 indeed decreased at 48 h in a dose-dependent manner (Supplementary Figure S3b), and no significant changes for the other two proteins (data not shown). HMGB1 restored after 15 Gy, which was consistent with the significant activation of caspase-3 at 48 h (Supplementary Figure S3c). Although these results indicate the corresponding signaling cascade from outer space to inter space of the nucleus (Supplementary Figure S3d), it is still unknown whether this type of regulatory network is a key mechanism of the apoptosis-resistance and cell survival in glioma cells after irradiation. We propose that these three genes may be potential targets for increasing radiosensitivity.
On the other hand, p53-targeting genes (PUMA, AEN, BAX and MAGED1) and death receptor genes (FAS and TNFRSF10B) involved in glioma cell death progress were upregulated. The enhancement of these proapoptosis signaling may also increase its radiosensitivity. In particular, the TRAIL/DR5 signaling pathway has been confirmed in glioma cells.50 We found that irradiation could induce significant upregulation of DR5 (encoded by TNFRSF10B) and the combination treatment with TRAIL and irradiation obviously reduced the number of surviving cells and activated caspase cascades, compared with that of irradiation treatment alone (Figures 5c–e). This meant that the activation of TRAIL/DR5 signaling could increase the radiosensitivity and cell-killing efficacy of glioma cells, and the combination of radiation and TRAIL led to additive effects and low synergy in apoptosis induction only, consistent with the results reported by Kuijlen et al.51
Meanwhile, it is noticeable that many genes involved in RNA splicing (based on gene ontology, Table 1) were significantly downregulated during late phase responses. It was reported that γ-irradiation-induced stress affected the splicing of many genes by repressing and redistributing splicing factors and other components of the transcription machinery, which might be used by cells as a survival mechanism to adapt to the stress environment by producing specific mRNA variants.52, 53, 54, 55, 56
In summary, we first identified the radiation-induced dynamic gene expression network involved in various DDRs at a dose that induced cell growth arrest. In particular, apoptotic response was counteracted by dynamic changes in proapoptosis and antiapoptosis gene expression, as well as by the complex interactions between signaling molecules in the cytoplasm and nucleus. These provide important references and sources for exploring radiosensitizing targets for glioblastoma therapy. Studies on the dynamic network of molecules involved in cell death at different irradiation doses are under way to clarify the regulatory mechanisms in glioma cells after irradiation and identify the candidate targets.
Materials and Methods
Cell culture, irradiation and cell proliferation assay
The human glioma cell line U251 MG and U87 was purchased from Cell Center of Peiking Union Medical College (Beijing, China) and cultured in MEM supplemented with 10% fetal bovine serum (Hyclone, Beijing, China), 100 units/ml penicillin-100 μg/ml streptomycin (Beijing Solarbio Science & Technology Co., Beijing, China). The culture was maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were irradiated using γ-ray of Co-60 source under atmospheric pressure and ambient temperature (Peking University, Beijing, China). The dose rate of 1 Gy/min was used. Cells were seeded in T-25 flask (Corning, NY, USA) with a density of 2.4 × 105 or 4.3 × 105 cells per flask. For combination treatment, cells treated with 20 ng/ml TRAIL (PeproTech, Rocky Hill, NJ, USA) immediately after irradiation. Cell proliferation and diameter were assessed by counting live cells after enzyme digestion using Scepter hand-held automated cell counter (Milipore, Billerica, MA, USA).
MTT (dimethyl thiazolyl diphenyl tetrazolium) assays
MTT assay was used to evaluate cell survival rate after irradiation. U251 cells were seeded at 5.0 × 103 cells per well of 96-well plates with at least three replicate wells, and then were treated with different irradiation dose at 24 h or irradiated with 7 Gy at the time points of 24, 48, 72 and 96 h. Twenty microliters MTT (Sigma, St. Louis, MO, USA) reagent was added to each well, and then cells were incubated for 4 h. After that, the supernatant was removed and 100 μl dimethyl sulfoxide (DMSO) was added. OD value of each sample was measured at a wavelength of 570 nm. The cells without irradiation were used as control at different time point. All experiments were repeated at least three times and finally the survival rate was calculated as following:
Cell survival rate (%)=OD(the experimental group)/OD (control) × 100%
RT-PCR and quantitative real-time PCR (Q-RT-PCR)
Total RNA from cells treated with or without irradiation was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA integrity was detected by analyzing 18S and 28S bands on a 1% agarose gel. Purity of all RNA samples were determined by the OD260/OD280 rations using a Spectrophotometer (Eppendorf, Hamburg, Germany). Two micrograms RNA from different samples was reverse transcribed to cDNA for gene expression detection by reverse transcriptase PCR (RT-PCR) or Q-RT-PCR. The relatively quantitative PCR reaction was set up in a total volume of 15 μl containing 3 μl of diluted cDNA, 7.5 μl of 2 × SYBR Green Real-time PCR Master mix (Toyobo Co. Ltd., Osaka, Japan), 0.25 μl 10 μmol/l of each primer and 4 μl ddH2O. Q-RT-PCR runs were performed on MX3000P instrument (Stratagene, La Jolla, CA, USA). GAPDH levels were used to for normalization and non-reversed transcribed RNA was used to correct for the presence of genomic DNA. The relative expression of genes was calculated as follows: fold-change=2 ^ (Treatment (Tc target−Tc GAPDH)−control (Tc target−Tc GAPDH)). Primer pairs for 13 genes are detailed in Supplementary Dataset 2.
Illumina sequencing and bioinformatics analysis
Total mRNA of eight samples (including samples at 6, 12, 24 and 48 h after radiation and their corresponding controls) were sent to whole transcriptome sequencing by Illumina HiSeq 2000. The gene expression level was calculated using RPKM (reads per kilobase per million reads). If there is more than one transcript for a gene, the longest one is used to calculate RPKM for the whole genes. DEG were identified by combination of fold-change (≥2) and FDR (false discovery rate; ≤0.001) (Supplementary Dataset 1). The Gene Ontology enrichment analysis was performed using DAVID Bioinformatics Resources 6.7, NIAIS/NIH (http://david.abcc.ncifcrf.gov/). The heat map illustrating dynamic changes of apoptosis-related genes (Figure 4d) was performed using Mev. The scatter plot and PCA analysis (Supplementary Figure S1) were performed via R project.
Analysis of cell cycle progression
Cells were collected after irradiation at different time point (0, 12, 24, 48, 72 and 96 h), and washed with PBS twice, then fixed in 1 ml 70% cold ethanol in test tubes and incubated at −20 °C overnight. After incubation, cells were centrifuged at 1000 r.p.m. for 5 min and the cell pellets were resuspended in 500 μl propidium iodide (PI)/Triton X-100 staining solution containing 50 μg/ml PI (Roche, Penzberg, Germany), 0.1% (v/v) Triton X-100 (Sigma) and 100 μg/ml RNase (Sigma). Then cells were incubated on ice for 30 min and given assay by FC 500 MCL. Cell cycle distribution was calculated from 10 000 cells with ModFit LT software (Becton Dickinson, San Jose, CA, USA).
Annexin V-FITC/PI FACS apoptotic assay
According to the instruction of FITC-Annexin V Apoptosis Detection Kit (Pharmingen BD, San Diego, CA, USA), cells were trypsinized and washed twice with cold PBS, then resuspended in 200 μl 1 × binding buffer. Hundred microliters of cell suspension was transferred to a 5 ml culture tube and incubated with 5 μl of FITC-Annexin V and 10 μl of PI (10 μg/ml). Gently vortex the cells and incubate for 15 min at RT in the dark. Five hundred microliters 1 × binding buffer was added to each tube and the cells were analyzed with flow cytometry FC 500 MCL within 1 h.
Western blotting
Cells were washed twice in cold PBS, and lysed in ice-cold lysis buffer containing 150 mM NaCl, 1.0% Nonidet-P40 and 50 mM Tris-Cl (pH 8.0). Hundred micrograms of the whole-cell protein were electrophoresed by SDS-PAGE and then blotted to an Immobilon-P 0.22 μm membrane (Millipore, Billerica, MA, USA), followed by blocked with 5% nonfat milk in 1% TBST and incubated with primary antibodies overnight. The secondary antibody was conjugated to horseradish peroxidase (CWBIC, Beijing, China), and antigen–antibody complexes were detected by the chemiluminescence (Millipore). Both the primary and secondary antibodies were diluted in 5% nonfat milk in TBST. The primary antibodies were anti-caspase-3 (Abcam, Cambridge, MA, USA), anti-DR5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-actin (Sigma).
Caspase activity assays
The caspase-3/7 and caspase-8 activation was performed using Apo-One homogenous caspase-3/7 assay and caspase-8 assay (Promega, Madison, WI, USA), respectively, according to the manufacturer’s instructions. Briefly, 1 × 104 cells (treated with or without irradiation) were collected at different time point (0, 12, 24, 48, 72 and 96 h) and lysed in the manufacturer-provided homogeneous caspase-3/7 or caspase-8 reagent. The lysates were incubated at room temperature for 1.5 h before reading in a fluorometer at 485/530 nm. The relative caspase activity was given to evaluate the fold-changes of samples at 6, 12, 24, 48, 72 and 96 h (compared with sample at 0 h).
Statistical analyses (excluding Illumina data)
All experiments were performed at least three times and each time was done in triplicate. The results are shown as the mean values±standard deviation (S.D.), and statistical significance was evaluated by Student’s t-test and ANOVA assay; P values were considered to be statistically significant when less than 0.05.
Abbreviations
- IR:
-
ionizing radiation
- DEGs:
-
differentially expressed genes
- Q-RT-PCR:
-
quantitative real-time PCR
- ETU:
-
early and transiently upregulated
- ETD:
-
early and transiently downregulated
- ECU:
-
early and continually upregulated
- ECD:
-
early and continually downregulated
- LU:
-
late upregulated
- LD:
-
late downregulated
- DR5:
-
death receptor 5
- TRAIL:
-
tumor-related apoptosis-induced ligand
References
Wen PY . New developments in targeted molecular therapies for glioblastoma. Expert Rev Anticancer Ther 2009; 9: 7–10.
Houillier C, Lejeune J, Benouaich-Amiel A, Laigle-Donadey F, Criniere E, Mokhtari K et al. Prognostic impact of molecular markers in a series of 220 primary glioblastomas. Cancer 2006; 106: 2218–2223.
Stupp R, Mason WP, Van Den Bent MJ, Weller M, Fisher B, Taphoorn MJB et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005; 352: 987–996.
Taghian A, Suit H, Pardo F, Gioioso D, Tomkinson K, DuBois W et al. In vitro intrinsic radiation sensitivity of glioblastoma multiforme. Int J of Radiat Oncol Biol Phys 1992; 23: 55–62.
Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006; 444: 756–760.
Lord CJ, Ashworth A . The DNA damage response and cancer therapy. Nature 2012; 481: 287–294.
Russo AL, Kwon HC, Burgan WE, Carter D, Beam K, Weizheng X et al. In vitro and in vivo radiosensitization of glioblastoma cells by the poly (ADP-ribose) polymerase inhibitor E7016. Clin Cancer Res 2009; 15: 607–612.
Wang Y, Li J, Booher RN, Kraker A, Lawrence T, Leopold WR et al. Radiosensitization of p53 mutant cells by PD0166285, a novel G2 checkpoint abrogator. Cancer Res 2001; 61: 8211–8217.
Jin P, Gu Y, Morgan DO . Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells. J Cell Biol 1996; 134: 963–970.
Afshar G, Jelluma N, Yang X, Basila D, Arvold ND, Karlsson A et al. Radiation-induced caspase-8 mediates p53-independent apoptosis in glioma cells. Cancer Res 2006; 66: 4223–4232.
Hara S, Nakashima S, Kiyono T, Sawada M, Yoshimura S, Iwama T et al. p53-independent ceramide formation in human glioma cells during γ-radiation-induced apoptosis. Cell Death Differ 2004; 11: 853–861.
Li F, Sethi G . Targeting transcription factor NF-κB to overcome chemoresistance and radioresistance in cancer therapy. Biochim Biophys Acta 2010; 1805: 167–180.
Vellanki SHK, Grabrucker A, Liebau S, Proepper C, Eramo A, Braun V et al. Small-molecule XIAP inhibitors enhance γ-irradiation-induced apoptosis in glioblastoma. Neoplasia 2009; 11: 743–752.
Kraus A, Gross MW, Knuechel R, Münkel K, Neff F, Schlegel J . Aberrant p21 regulation in radioresistant primary glioblastoma multiforme cells bearing wild-type p53. J Neurosurg 2000; 93: 863–872.
Wang J, Wakeman TP, Lathia JD, Hjelmeland AB, Wang XF, White RR et al. Notch promotes radioresistance of glioma stem cells. Stem Cells 2009; 28: 17–28.
Kim Y, Kim KH, Lee J, Lee YA, Kim M, Lee SJ et al. Wnt activation is implicated in glioblastoma radioresistance. Lab Invest 2011; 92: 466–473.
Li HF, Kim JS, Waldman T . Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells. Radiat Oncol 2009; 4: 43.
Yount GL, Haas-Kogan DA, Vidair CA, Haas M, Dewey WC, Israel MA . Cell cycle synchrony unmasks the influence of p53 function on radiosensitivity of human glioblastoma cells. Cancer Res 1996; 56: 500–506.
Gartner A, Milstein S, Ahmed S, Hodgkin J, Hengartner MOA . Conserved checkpoint pathway mediates dna damage–induced apoptosis and cell cycle arrest in C. elegans. Mol Cell 2000; 5: 435–443.
Wang X, McGowan CH, Zhao M, He L, Downey JS, Fearns C et al. Involvement of the MKK6-p38γ cascade in γ-radiation-induced cell cycle arrest. Mol Cell Biol 2000; 20: 4543–4552.
Aziz K, Nowsheen S, Pantelias G, Iliakis G, Gorgoulis VG, Georgakilas AG . Targeting DNA damage and repair: embracing the pharmacological era for successful cancer therapy. Pharmacol Ther 2012; 3: 334–350.
Jin S, Antinore MJ, Lung FDT, Dong X, Zhao H, Fan F et al. The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression. J Biol Chem 2000; 275: 16602–16608.
Yang Q, Manicone A, Coursen JD, Linke SP, Nagashima M, Forgues M et al. Identification of a functional domain in a GADD45-mediated G2/M checkpoint. J Biol Chem 2000; 275: 36892–36898.
Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O'Connor PM . UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J Natl Cancer Inst 1996; 88: 956–965.
Kastan MB, Bartek J . Cell-cycle checkpoints and cancer. Nature 2004; 432: 316–323.
Lu X, Yang C, Hill R, Yin C, Hollander MC, Fornace AJ et al. Inactivation of gadd45a sensitizes epithelial cancer cells to ionizing radiation in vivo resulting in prolonged survival. Cancer Res 2008; 68: 3579–3583.
Wouters BG, Giaccia AJ, Denko NC, Brown JM . Loss of p21Waf1/Cip1 sensitizes tumors to radiation by an apoptosis-independent mechanism. Cancer Res 1997; 57: 4703–4706.
Giagkousiklidis S, Vogler M, Westhoff MA, Kasperczyk H, Debatin KM, Fulda S . Sensitization for γ-irradiation–induced apoptosis by second mitochondria-derived activator of caspase. Cancer Res 2005; 65: 10502–10513.
Zhou L, Yuan R, Lanata S . Molecular mechanisms of irradiation-induced apoptosis. Front Biosci 2003; 8: d9–19.
Karim A, Mccarthy K, Jawahar A, Smith D, Willis B, Nanda A . Differential cyclooxygenase-2 enzyme expression in radiosensitive versus radioresistant glioblastoma multiforme cell lines. Anticancer Res 2005; 25: 675–679.
Chang HW, Roh JL, Jeong EJ, Lee S, Kim SW, Choi SH et al. Wnt signaling controls radiosensitivity via cyclooxygenase-2-mediated Ku expression in head and neck cancer. Int J Cancer 2007; 122: 100–107.
Fassl A, Tagscherer K, Richter J, Diaz MB, Llaguno SRA, Campos B et al. Notch1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1. Oncogene 2012; 31: 4698–4708.
Purow BW, Haque RM, Noel MW, Su Q, Burdick MJ, Lee J et al. Expression of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma cell survival and proliferation. Cancer Res 2005; 65: 2353–2363.
Burton TR, Henson ES, Baijal P, Eisenstat DD, Gibson SB . The pro-cell death Bcl-2 family member, BNIP3, is localized to the nucleus of human glial cells: Implications for glioblastoma multiforme tumor cell survival under hypoxia. Int J Cancer 2005; 118: 1660–1669.
Burton TR, Eisenstat DD, Gibson SB . BNIP3 (Bcl-2 19 kDa interacting protein) acts as transcriptional repressor of apoptosis-inducing factor expression preventing cell death in human malignant gliomas. J Neurosci 2009; 29: 4189–4199.
Zhang J, Xu M . Apoptotic DNA fragmentation and tissue homeostasis. Trends cell Biol 2002; 12: 84–89.
Kalinowska-Herok M, Widlak P . High mobility group proteins stimulate DNA cleavage by apoptotic endonuclease DFF40/CAD due to HMG-box interactions with DNA. Acta Biochim Pol 2008; 55: 21–26.
Trisciuoglio L, Bianchi ME . Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway. PLoS One 2009; 4: e6234.
Widlak P, Garrard WT . Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. J Cell Biochem 2005; 94: 1078–1087.
Solovyan VT, Bezvenyuk ZA, Salminen A, Austin CA, Courtney MJ . The role of topoisomerase II in the excision of DNA loop domains during apoptosis. J Biol Chem 2002; 277: 21458–21467.
Hao C, Beguinot F, Condorelli G, Trencia A, Van Meir EG, Yong VW et al. Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells. Cancer Res 2001; 61: 1162–1170.
Song JH, Song DK, Pyrzynska B, Petruk KC, Meir EG, Hao C . TRAIL triggers apoptosis in human malignant glioma cells through extrinsic and intrinsic pathways. Brain Pathol 2003; 13: 539–553.
Russell KJ, Wiens LW, Demers GW, Galloway DA, Plon SE, Groudine M . Abrogation of the G2 checkpoint results in differential radiosensitization of G1 checkpoint-deficient and G1 checkpoint-competent cells. Cancer Res 1995; 55: 1639–1642.
Alexander BM, Pinnell N, Wen PY, D’Andrea A, Targeting DNA . Repair and the cell cycle in glioblastoma. J Neurooncol 2012; 107: 463–477.
Wakatsuki M, Ohno T, Iwakawa M, Ishikawa H, Noda S, Ohta T et al. p73 protein expression correlates with radiation-induced apoptosis in the lack of p53 response to radiation therapy for cervical cancer. Int J of Radiat Oncol Biol Phys 2008; 70: 1189–1194.
Park MK, Kang YJ, Ha YM, Jeong JJ, Kim HJ, Seo HG et al. EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells. Biochem Biophys Res Commun 2009; 379: 1043–1047.
Kanamori M, Kawaguchi T, Nigro JM, Feuerstein BG, Berger MS, Miele L et al. Contribution of Notch signaling activation to human glioblastoma multiforme. J Neurosurg 2007; 106: 417–427.
Liu SS, Leung RCY, Chan KYK, Chiu PM, Cheung ANY, Tam KF et al. p73 expression is associated with the cellular radiosensitivity in cervical cancer after radiotherapy. Clin Cancer Res 2004; 10: 3309–3316.
Shinoura N, Muramatsu Y, Asai A, Han S, Horii A, Kirino T et al. Degree of apoptosis induced by adenovirus-mediated transduction of p53 or p73α depends on the p53 status of glioma cells. Cancer Lett 2000; 160: 67–73.
Ciusani E, Croci D, Gelati M, Calatozzolo C, Sciacca F, Fumagalli L et al. In vitro effects of topotecan and ionizing radiation on TRAIL/Apo2L-mediated apoptosis in malignant glioma. J Neurooncol 2005; 71: 19–25.
Kuijlen JMA, Van Steenbergen W, Den Dunnen WFA, Mooij JJA, Kampinga HH . Effect of γ-radiation on rhTRAIL efficacy in glioblastoma multiforme cells In: Kuijlen JMA, (ed.) On TRAIL for Glioma Therapy?. University Library Groningen: Groningen, The Netherlands, 2010 pp 140–153.
Biamonti G, Caceres JF . Cellular stress and RNA splicing. Trends Biochem Sci 2009; 34: 146–153.
Ip JY, Schmidt D, Pan Q, Ramani AK, Fraser AG, Odom DT et al. Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation. Genome Res 2011; 21: 390–401.
Busà R, Sette C . An emerging role for nuclear RNA-mediated responses to genotoxic stress. RNA Biol 2010; 7: 390–396.
Muñoz MJ, Santangelo M, Paronetto MP, de la Mata M, Pelisch F, Boireau S et al. DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation. Cell 2009; 137: 708–720.
Bassi C, Mello S, Cardoso R, Godoy P, Fachin A, Junta C et al. Transcriptional changes in U343 MG-a glioblastoma cell line exposed to ionizing radiation. Hum Exp Toxicol 2008; 27: 919–929.
Acknowledgements
We thank the support from the Ministry of Science and Technology, China (No. 2012YQ040140), the National Natural Science Foundation of China (No. 31200636) and the opening foundation of the State Key Laboratory of Space Medicine Fundamentals and Application, Chinese Astronaut Research and Training Center (No. SMFA10K05).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Edited by A Verkhratsky
Supplementary Information accompanies this paper on Cell Death and Disease website
Supplementary information
Rights and permissions
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
About this article
Cite this article
Ma, H., Rao, L., Wang, H. et al. Transcriptome analysis of glioma cells for the dynamic response to γ-irradiation and dual regulation of apoptosis genes: a new insight into radiotherapy for glioblastomas. Cell Death Dis 4, e895 (2013). https://doi.org/10.1038/cddis.2013.412
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/cddis.2013.412
Keywords
This article is cited by
-
Nuclear respiratory factor 1 transcriptomic signatures as prognostic indicators of recurring aggressive mesenchymal glioblastoma and resistance to therapy in White American females
Journal of Cancer Research and Clinical Oncology (2022)
-
Genomic analyses of early responses to radiation in glioblastoma reveal new alterations at transcription, splicing, and translation levels
Scientific Reports (2020)
-
Inhibition of TAZ contributes radiation-induced senescence and growth arrest in glioma cells
Oncogene (2019)
-
Effects of ionizing radiation on the viability and proliferative behavior of the human glioblastoma T98G cell line
BMC Research Notes (2018)
-
Immune microenvironment of experimental rat C6 gliomas resembles human glioblastomas
Scientific Reports (2017)