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. 2015 Dec 15:5:18231.
doi: 10.1038/srep18231.

A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation

Hiroaki Fujimori  1   2 , Akira Sato  1 , Sota Kikuhara  1   2   3 , Junhui Wang  1   4 , Takahisa Hirai  1   5 , Yuka Sasaki  2 , Yasufumi Murakami  3 , Ryuichi Okayasu  6 , Mitsuko Masutani  1   2   7
Affiliations

A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation

Hiroaki Fujimori et al. Sci Rep. .

Abstract

A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1β in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1β complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1β foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses.

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Figures

Figure 1
Figure 1. Comprehensive analysis identified radiosensitizing targets including DNMT3B.
(A and B) The results of a comprehensive negative screening analysis of radiosensitivity-related genes in T-REx HeLa cells transfected with a shRNA library. (C) Analysis of a functional network cluster around the genes encoding phosphatase and tensin homolog (PTEN) and glycogen synthase kinase 3β (GSK3β) (upper panel). The numbers shown in blue indicate genes whose knockdown induced radiosensitization (lower panel). (D) Confirmation of RNAi-mediated knockdown of CUL1 (a) and FBXW7 (b) expression in T-REx HeLa cells. Error bars: SE. Asterisks show P < 0.05. (E) Radiosensitization induced by CUL1 or FBXW7 knockdown in T-REx HeLa cells analyzed by colony formation assay. Mean + SE. Asterisks show P < 0.05. (F) Cluster analysis and validation of novel candidate genes. A functional network cluster of the target genes around PARP-1 (left panel). The targets whose downregulation induced radiosensitization and radioresistance are shown in blue and red, respectively (right panel).
Figure 2
Figure 2. Radiosensitization in DNMT3B knockdown cells.
(A) The DNMT3B knockdown increased radiosensitivity of HeLa cells. The cells were treated with shRNA of DNMT3B and the mRNA level and protein level of DNMT3B was detected by qRT-PCR (a) and western blot (a, inserted), respectively. The effect of DNMT3B knockdown with shRNA on the radiosensitivity of HeLa cells was analyzed by colony formation assay (b). The enhancement ratio at 10% survival was 1.2 (c-e). The knockdown levels of DNMT3B after transfection of DNMT3B specific siRNA at the mRNA (c) and protein (c, inserted) levels. The effect of DNMT3B knockdown with siRNA on the radiosensitivity of HeLa cells was analyzed by colony formation assay (e). The enhancement ratio at 10% survival was 1.3. (B and C) DNMT3B RNAi radiosensitized in A549 (B) and HCT116 (C) cell lines. The mRNA and protein levels of DNMT3B in A549 transfected with shRNA for DNMT3B (Ba with an inserted panel) and HCT116 transfected siRNA for DNMT3B (Ca with an inserted panel). Error bars: SE. The colony formation analysis was carried out (Bb and Cb). Error bars: SE. The enhancement ratio at 10% survival was 1.2 for A549 (Bb) and 1.4 for HCT116 (Cb). Asterisks show P < 0.05. The mRNA level of DNMT3B was normalized with that of GUSB (Aa, Ac, Ba and Ca).
Figure 3
Figure 3. Knockdown of DNMT3B disrupts γ-irradiation-induced formation of HP1β foci.
(A and B) Formation of HP1β foci in control and DNMT3B knockdown cells 1 h after irradiation (8 Gy). (A) Immunostaining data. Arrows: HP1β foci merged with H3K9me2/3. Arrows heads: HP1β foci not merged with H3K9me2/3. (B) Quantitative data. HP1β foci were counted at least in 50 cells. Significant differences between the number of foci in non-irradiated and irradiated cells were determined. Error bars: SE. Asterisks show P < 0.05. Scale bar: 10 μm. N.S.: P ≧ 0.05.
Figure 4
Figure 4. Knockdown of DNMT3B reduces the formation of γH2AX foci after irradiation.
(A and B) The formation of γH2AX foci 1 h and 7 h after exposure of control and DNMT3B knockdown cells to γ-irradiation (A) or carbon-ion beam irradiation (B). Failure of quick and robust γH2AX induction after γ-irradiation in DNMT3B knockdown cells (Aa); One hour after γ-irradiation, DNMT3B knockdown cells exhibited γH2AX foci with low intensity compared to that in NC cells. On the other hand, 7 h after irradiation, γH2AX signals in DNMT3B knockdown cells became more intensive compared to NC cells (Ab and Ac). Green square regions in Aa were enlarged in Ab. The γH2AX foci were counted at least in 140 cells, respectively. Asterisks show P < 0.05 (X2-test). Western blot analysis showed that carbon-ion beam irradiation also failed to the quick activation of γH2AX in DNMT3B knockdown cells (B). (C) H2AX protein levels in control and DNMT3B knockdown cells exposed to γ-irradiation. Scale bar: 20 μm. (D) H2AX mRNA levels in control and DNMT3B knockdown cells after 8 Gy γ-irradiation. The expression level of H2AX was normalized to that of GUSB. Error bars: SE.
Figure 5
Figure 5. Knockdown of DNMT3B radiosensitizes through impairment of H2AX regulation after DNA damage.
(A) H2AX overexpression suppressed radiosensitization induced by DNMT3B RNAi in HeLa cells. MTT assay was performed 4 days after γ-irradiation. Relative mRNA expression levels of DNMT3B and H2AX in indicated conditions (a). The expression level was normalized to that of GUSB. Error bars: SE. Relative growth levels of HeLa cells in each transfected condition (b). Error bars: SE. (B) Interaction between DNMT3B and HP1β or H2AX observed 1 hr after 8 Gy irradiation. Confirmation of DNMT3B-FLAG overexpression at mRNA level (a) and protein level (b). The number of PLA foci between DNMT3B and HP1β was decreased after irradiation (c), whereas that of DNMT3B and H2AX was increased after irradiation (d). Scale bar: 10 μm.
Figure 6
Figure 6. Knockdown of DNMT3B activates the P53-dependent pathway after γ-irradiation of HeLa cells.
(A) The G1, G2/M and sub-G1 populations of DNMT3B knockdown cells 8 h and 24 h after irradiation (8 Gy). Error bars: SE. Asterisks show P < 0.05. (B) The G1, G2/M and sub-G1 populations of synchronized DNMT3B knockdown cells 7 h after irradiation. The upper panel shows the scheme of synchronization experiment. Error bars: SE. Asterisks show P < 0.05. (C) The activation of P53 pathway genes in irradiated DNMT3B knockdown cells. The expression level was normalized to that of GUSB. Error bars: SE. (D) A model for DNMT3B functions in IR response. DNMT3B interacts with the DSB sensor proteins, H2AX and HP1β and protects cells from irradiation damage through regulation of damage associated responses.
Figure 7
Figure 7. Knockdown of DNMT3B enhances radiosensitivity in tumor xenograft model.
An overview of the xenograft experimental procedure (A). The temporal development of the tumor masses in animals injected with control and DNMT3B knockdown A549 cells (B). Error bars: SE. Asterisks show P < 0.05. The tumor weights at day 38 showing that knockdown of DNMT3B increased the susceptibility of the tumors to γ-irradiation. Bars show the mean tumor weight. Asterisks show P < 0.05 (C).
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