Communicated by: Fumio Hanaoka
Over-expression of ATR causes autophagic cell death
Article first published online: 25 JAN 2013
© 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
Genes to Cells
Volume 18, Issue 4, pages 278–287, April 2013
How to Cite
Mori, C., Yamaguchi, Y., Teranishi, M., Takanami, T., Nagase, T., Kanno, S., Yasui, A. and Higashitani, A. (2013), Over-expression of ATR causes autophagic cell death. Genes to Cells, 18: 278–287. doi: 10.1111/gtc.12034
- Issue published online: 25 MAR 2013
- Article first published online: 25 JAN 2013
- Manuscript Accepted: 17 DEC 2012
- Manuscript Received: 1 MAY 2012
ATR is highly conserved in all eukaryotes and functions as a cell-cycle checkpoint kinase to stabilize the nuclear genome. In addition, knockout mouse models indicate that ATR is essential for viability. Here, it is shown that moderate overproduction of ATR, but not of the other phosphatidylinositol 3′ kinase-related kinases, ataxia-telangiectasia-mutated, mTOR and SMG-1, and a downstream target p53, resulted in cell death. ATR over-expression induced cellular vacuolization from 12 to 48 h after transfection, before cell death progression. A series of deletion analyses showed that overproduction of the N-terminal HEAT repeat segments of ATR was sufficient for the induction of the vacuolization. Moreover, post-transcriptional modification of LC3, a marker of autophagy, and autophagosomes with double membranes were evident in ATR-overproducing cells. The vacuolization was also suppressed in autophagy-deficient MCF7 cells. In addition, both cellular vacuolization and cell death were reduced by inhibition of Ras activity using farnesyl thiosalicylic acid. Conversely, neither inhibition of mTOR nor activation of the checkpoint system could be observed in the vacuolated cells. These results suggest that the Ras signaling pathway is involved in the autophagic response caused by ATR overproduction, and tight regulation of ATR protein expression is crucial for cell viability.
Of the ataxia-telangiectasia-mutated (ATM) kinase family, ATM and ATR are members of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) superfamily, which are key signal transducers that regulate the status of nutrient supply, genome stability and general transcription. The mammalian genome has three other PIKKs, mTOR (mammalian target of rapamycin, Schmelzle & Hall 2000); DNA-PKcs (DNA-dependent protein kinase catalytic subunit, Ma et al. 2002); and SMG-1 (suppressor with morphological effect on genitalia, Yamashita et al. 2001). PIKKs are large proteins (270–470 kDa) with a C-terminal PI3K-like kinase domain and a FATC (FRAP, ATM, TRRAP, C-terminal) domain. In addition, the N-termini of ATR, ATM, mTOR and DNA-PKcs contain the α-helical HEAT (huntingtin, elongation factor 3, a subunit of PP2A, TOR1) repeat segments (Perry & Kleckner 2003; Brewerton et al. 2004).
ATR is an essential gene whose complete absence results in early embryonic lethality (O'Driscoll 2009), and a hypomorphic mutation with reduced ATR expression levels causes a complex human disease known as Seckel syndrome (MIM 210600: O'Driscoll et al. 2003). Seckel syndrome is characterized by intrauterine growth retardation, dwarfism and microcephaly with mental retardation (Abou-Zahr et al. 1999). Recently, a hypomorphic mouse, in which ATR levels are nearly undetectable but still sufficient for viability, has been created (Murga et al. 2009). These ATR-Seckel mice showed accelerated aging and phenotypes similar to those of human Seckel syndrome. Replicative stress is increased in ATR-Seckel mice, which suggests that some manifestations of ATR-Seckel syndrome may be explained by chromosomal instability due to the loss of checkpoint function. In addition, Li et al. (2009) reported that ATR, in neurons, exists in cytoplasm and physically associates with cytoplasmic ATM and synaptic vesicle proteins, VAMP2 and synapsin-I. The neurological manifestations of ATR-Seckel syndrome possibly result from defective non-nuclear ATR functions; however, there are few studies focusing on cytoplasmic ATR.
Recently, several reports indicate that inhibition of the mTOR pathway and/or Ras-induced pathway activates autophagy. mTOR is a key component that regulates the balance between growth and autophagy in response to nutrient and growth factor status. Nutrient starvation and reduction in growth factors alarm eukaryotic cells to adjust metabolism to survive, and induce autophagy (reviewed in Jung et al. 2010). It was also shown that Ras activation inhibits autophagy by stimulating the target of mTOR pathway (Furuta et al. 2004). However, Ras also induces autophagy via a parallel pathway through an increased expression of autophagy-related proteins, ATG5 and Beclin-1 (Byun et al. 2009; Elgendy et al. 2011). The over-expression of oncogenic Ras causes cellular vacuolization and cell death accompanied by processing of microtubule-associated protein 1 light chain 3 (LC3) (Chi et al. 1999). LC3 is a mammalian homologue of yeast Atg8 (Liang et al. 1999) and exists in two forms: a lower-mobility LC3-I and a higher-mobility LC3-II, which is associated with the formation of autophagosomes. These reports show that accumulated LC3-II may cause cellular vacuolization-mediated death.
Here, we report for the first time that cell death and cellular vacuolization with autophagy, including post-transcriptional modification of LC3, are caused by ATR over-expression (ATR-OX). A series of deletion and amino acid substitution analyses of ATR showed that the N-terminal α-helical HEAT repeats (Perry & Kleckner 2003) are sufficient to induce cellular vacuolization. Our results show that over-expression of ATR HEAT repeats activates the LC3 pathway following cellular vacuolization death. We also studied the effect of pre-treatment of farnesyl thiosalicylic acid (FTA), an inhibitor of Ras, on vacuolization and monitored alterations in mTOR and checkpoint signaling caused by ATR overproduction.
ATR-OX caused cellular vacuolization and cell death
ATR is essential for cell-cycle progression, whose disruption causes defects in cell growth via the DNA-replication checkpoint function (Cortez et al. 2001). To study the effect of ATR protein levels, GFP-fused ATR proteins were overproduced in U2OS cells by the transfection of a pEGFP-ATR plasmid. The recombinant EGFP-ATR was detectable 12 h after transfection, and the expression ratios of EGFP-ATR to endogenous ATR were estimated as 0.7 at 12 h and 2.6 at 24 h, respectively (Fig. 1A). Calculating the expression levels with the transfection efficiency at approximately 39 ± 1.0% (SE), exogenous ATR over-expression increased ATR concentrations 2- to 6-fold over control cells. In cells transfected with pEGFP-ATR, recombinant EGFP signals first appeared 12 h after transfection (Fig. 1B). Interestingly, abnormal cellular vacuolization was also observed in these cells (Fig. 1B). At 24 h after transfection, the frequency of vacuolated cells reached almost 50% among EGFP-positive cells (Fig. 1C).
In addition to cellular vacuolization, abortion of cytokinesis was observed in the transfected cells. Abnormal binuclear cells were observed with a frequency of 6.8% (Fig. 1D). Moreover, the number of dead cells increased gradually to 4.0% at 24 h, 14.6% at 48 h and 28.5% at 72 h after transfection, whereas transfection with a vector control did not induce cell death (Fig. 1E). In addition to EGFP-ATR, over-expression of N-terminal or C-terminal HaloTag-fused ATR resulted in similar patterns of vacuolization (Fig. 1F). These results indicate that moderate over-expression of ATR causes cell death with cellular vacuolization and suggest that ATR protein levels are strictly controlled for normal cell growth.
Vacuolization caused by over-expression of HEAT repeats
To define which region of ATR is involved in cellular vacuolization and cell death, a series of deletion mutants and amino acid substitutions in ATR were constructed (Fig. 2A). Over-expression of the C-terminal 1314-amino-acid-containing the kinase domain (1331–2644) failed to produce cellular vacuolization (Fig. 2B). However, over-expression of a kinase-inactive ATR containing the amino acid substitution D2494E (Asp at 2494 to Glu) and a C-terminal deletion ranging from the kinase domain to the FATC domain retained vacuolization activity (Fig. 2B). Moreover, cells that expressed only the N-terminal 1330 amino acids of the HEAT repeat segments resulted in cellular vacuolization (Fig. 2B). Surprisingly, each 500 amino acid sequence of N-terminal ATR, 1–480, 481–960 or 961–1330, all of which contain HEAT repeats, showed vacuolization activity at a frequency of approximately 50% at 24 h after each transfection (Fig. 2B). Because ATR is phosphorylated at serine 428 in response to DNA damage (Bhattacharya et al. 2009), the contribution of the phosphorylation was also examined. Both amino acid substitutions of Ser to Asp (S428D) and Ala (S428A), which mimic the phosphorylated and nonphosphorylated forms of ATR, respectively, did not affect autophagic vacuolization activity (Fig. 2B). These results clearly indicate that the expression of ATR HEAT repeat segments exerts the cellular vacuolization.
Cellular vacuolization accompanying autophagy
To investigate whether an autophagic response occurs during cellular vacuolization mediated by ATR-OX, we measured the rates of cellular vacuolization following transfection of ATR HEAT repeats clone (481–960) in autophagy-deficient MCF7 human breast carcinoma cells (Liang et al. 1999; Furuya et al. 2005). The results shown in Figure 3 indicate that the frequency of vacuolated cells was significantly reduced in MCF7 compared with U2OS and HeLa cells.
In addition, we monitored LC3 transcriptional and post-transcriptional regulation. Western blotting analysis showed that the conversion of the higher-mobility form of a recombinant GFP-LC3 occurred following ATR-OX (Fig. 4A). LC3B gene expression, which was predominant in U2OS cells compared to the three isoforms of mammalian LC3 genes, LC3A, LC3B and LC3C (He et al. 2003), was up-regulated by ATR-OX (Fig. 4B). Furthermore, in HeLa cells stably expressing mRFP-tagged LC3, the signals of mRFP-LC3 increased and were widely localized around the vacuoles by ATR-OX (Fig. 4C). Cells were then fixed 24 h after transfection and examined by electron microscopy. The results showed autophagosomes and vacuoles identified by their characteristic double membrane and engulfed cytoplasmic components in cells transfected with ATR, but not in cells transfected with the vector control (Fig. 4D).
Possible role for Ras signaling in the effect of ATR-OX
Similar cell death accompanied by vacuolization has been observed following the over-expression of oncogenic Ras (Chi et al. 1999). Therefore, the effect of pre-treatment with an inhibitor of Ras, farnesyl thiosalicylic acid (FTS: Marom et al. 1995), on ATR-OX-mediated vacuolization was investigated. The results shown in Fig. 5A–C indicate that FTS inhibited cellular vacuolization as well as cell death, regardless of the level of GFP-fluorescent intensity in the transfected cells. These results indicate that Ras signaling pathway is involved in the cellular vacuolization and cell death induced by ATR-OX. In contrast, ATR-OX with only HEAT repeats (481–960) neither activated ATR-dependent checkpoint signaling with phosphorylation of Chk1 at Ser-317 nor inhibited mTOR/Akt pathway with phosphorylation of 4E-BP1 at Thr37/46 (Fig. 6A,B).
Cellular vacuolization occurred in ATR-specific over-expression
Vacuolization was also evident in HeLa and HCT116 cells transfected with pEGFP-ATR at almost the same frequency observed in U2OS cells (Fig. 7A). Moreover, in p53-deficient HCT116 cells, ATR-OX caused vacuolization in the same manner as HCT116 parental cells with functional p53 (Fig. 7A). However, the over-expression of other PIKKs: EGFP-ATM; HaloTag-mTOR; and HaloTag-SMG1 fusion proteins, and of p53-EGFP, exhibited neither cellular vacuolization nor cell death (Fig. 7B), indicating that cellular vacuolization occurred in an ATR-specific manner.
Activation of ATM family kinases, ATM and ATR, results in cell-cycle arrest, DNA repair or programmed cell death (PCD). In most cases, DNA damage causes type I cell death, apoptosis, which is mediated by the activation of the caspase cascade and release of mitochondrial cytochrome c via the Bax/Bak channel and is characterized by the condensation of the nucleus and cytoplasm and a ladder-like pattern of DNA fragmentation (Taylor et al. 2008; Tait & Green 2010). Conversely, type II PCD was shown to be caspase-independent cell death. By inhibiting caspase activity in mammalian apoptotic systems, apoptotic morphology could be prevented, but cell death still occurred (Miller et al. 1997; Kitanaka & Kuchino 1999; Hetz et al. 2005). Type II cell death exhibits dramatic cellular vacuolization and only partial chromatin condensation, which is also referred to as autophagic cell death.
Recently, several reports indicate that inhibition of the mTOR pathway and/or Ras-induced pathway activates autophagy. mTOR is a key component that regulates the balance between growth and autophagy in response to nutrient and growth factor status. Nutrient starvation and reduction in growth factors alarm eukaryotic cells to adjust metabolism to survive, and induce autophagy (reviewed in Jung et al. 2010). It was also shown that Ras activation inhibits autophagy by stimulating components of the mTOR pathway (Furuta et al. 2004). However, deregulated H-Ras activity can lead to caspase-independent cell death with features of autophagy (Elgendy et al. 2011). Ras-induced autophagy is associated with increased expression of certain autophagy-related proteins, including ATG5 and Beclin-1 (Byun et al. 2009; Elgendy et al. 2011). In addition, over-expression of oncogenic Ras causes cellular vacuolization and cell death accompanied by processing of microtubule-associated protein 1 LC3 (Chi et al. 1999).
In this report, a novel effect of ATR-OX on cellular vacuolization with features of autophagy such as a post-transcriptional modification of LC3 and vacuoles with double membranes and following cell death was identified (Figs 1, 4). Furthermore, ATR protein N-terminal HEAT repeat over-expression alone is sufficient to induce this response (Fig. 2). The vacuolization was repressed in autophagy-deficient MCF7 cells (Fig. 3), in which expression levels of endogenous Beclin-1 are low or undetectable (Liang et al. 1999; Furuya et al. 2005). ATR-OX-induced vacuolization and cell death were inhibited by an inhibitor of Ras (FTS; Fig. 5), but neither activated checkpoint response to DNA damage nor repressed mTOR signaling (Fig. 6). Moreover, the vacuolization occurred in a p53-independent manner (Fig. 7). These results clearly suggest that ATR-OX causes autophagic type II cell death through the activation of Ras signaling.
Based on immunoblotting and cytochemical analyses, cellular vacuolization and cell death were present in the cells overproducing ATR by 2- to 3-fold over basal levels (Fig. 1). In addition, the expression of a kinase-inactivated ATR, ATR D2494E, also induced cell death (Fig. 2). These findings are in contrast to previous studies in which cell lines stably overexpressed doxycycline-inducible wild-type or kinase-inactivated ATR (Cliby et al. 1998; Wright et al. 1998). The previous results indicated that the induction of ATRkd induced UV and radiation sensitivity and cell-cycle checkpoint defects. However, the reports do not explain the similar phenomena of cell death and cellular vacuolization. This divergence with previous results may be due to the use of a stable cell line and/or shorter experimental duration.
Kumamoto-Yonezawa et al. (2009) also reported increased ATR protein levels in response to treatment with cEPA, an inhibitor of DNA polymerases including pol α, δ and ε, which are able to inhibit DNA replication without accumulating DNA damage, indicating that the stalled replication fork resulted in increased ATR levels. It will be interesting to study whether doxycycline-inducible over-expression in stable transformed lines and cEPA treatment induce cell death, cellular vacuolization and autophagy over longer time periods. Wang et al. (2008) showed that embryonic fibroblasts from knockout mice that lack a gene essential for macroautophagy became sensitive to the Fas/TNF-α mitochondrial death pathway but resistant to death from UV light. This suggests that the autophagic system may function as a survival mechanism against the mitochondrial death pathway and promote UV-induced cell death. It is well known that UV light causes DNA damage and, following the stalled replication fork, results in the activation of ATR. Therefore, the induction of ATR by cEPA treatment and UV irradiation may cause autophagic cell death.
The HEAT repeat domain in ATR is arranged in 45 tandem repeats (Perry & Kleckner 2003). In this study, expression of the eight N-terminal repeats was sufficient for cellular vacuolization, whereas the C-terminal 1314 amino acids did not cause vacuolization, regardless of which HEAT repeats were included (Fig. 2). Although they consist of a consensus pattern of conserved hydrophobic residues, together with highly conserved proline, aspartate and arginine residues at repeat positions 11, 19 and 25, respectively, entire amino acid sequences of HEAT repeats are degenerate. One HEAT repeat consists of 37–43 amino-acid residues and forms a hairpin structure with two α-helices (Perry & Kleckner 2003). In the other PIKKs, ATM, mTOR and DNA-PKCs, the HEAT repeat domain was conserved in each N-terminal region but the amino acid sequences differed among family members, even within ATR and ATM (Perry & Kleckner 2003; Sibanda et al. 2010). Therefore, it appears that specific functions and the specificity of binding partners are determined through the N-terminal region in each PIKK. Recently, Takai et al. (2007, 2010) found that Tel2 regulates the stability of all PIKKs through Hsp90-dependent maturation of each PIKK complex, for example, mTOR, TORC1 and TORC2, and ATR and ATRIP. Tel2 directly binds to the HEAT repeat segments of PIKKs. Whereas the down-regulation of Tel2 results in the reduction of function in all PIKKs, over-expression of ATR did not alter PIKK activity, at least of the mTOR complex (Fig. 6). Because the expression of Tel2 protein is abundant, while those of other PIKKs are low (Takai et al. 2010), moderate over-expression of the HEAT repeats of ATR or GFP- or HaloTag-fused ATR might not be sufficient to effectively titrate Tel2 protein. Moreover, over-expression of ATM and mTOR proteins (harboring their HEAT repeats domain) did not affect cell death with cellular vacuolization (Fig. 7). These results suggest that the over-expression of ATR containing the N-terminal HEAT repeat segments affects ATR-specific functions, such as the maturation of the ATR–ATRIP complex and interaction of other unknown factors with the complex. In conclusion, this study shows the importance of regulating ATR protein levels and that the over-expression of ATR results in autophagic cell death. A growing understanding of ATM function in cytoplasmic vesicles and ATR-OX-mediated autophagy may help to resolve some of the diverse symptoms of neurological phenotypes in ATM patients and those with ATR-Seckel syndrome.
Cell culture and transfection with recombinant ATRs
HeLa and U2OS cells were cultured in DMEM (Sigma) supplemented with 8 mm glutamine (Wako) and 10% fetal bovine serum (FBS) (BioWest, France). HCT116 p53 +/+ and −/− cells were obtained from Dr B. Vogelstein (Johns Hopkins Kimmel Cancer Center). HeLa cells stably expressing GFP-tagged LC3 and mRFP-tagged LC3 were obtained from Dr T. Yoshimori (Osaka University). These lines were maintained in McCoy's 5A modified medium with glutamine and FBS. All cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
Full-length cDNA clones of human ATR, ATM and p53 in the pEGFP-C1 vector (Clontech); human ATR and mTOR in the pFN21A HaloTag CMV Flexi Vector (Promega); and human ATR in the pFC14A HaloTag CMV Flexi Vector (Promega) were constructed in this study. Amino acid substitutions and deletions of the ATR gene were introduced with the GeneTailor site-directed mutagenesis system (Invitrogen). For transfection, cells (1.5 × 105) were seeded in 30-mm dishes 1 day before transfection and transfected with 2 μg of each DNA plasmid using FuGene HD (Roche) according to the manufacturer's instructions. The transfection efficiency was estimated by the measurement of GFP-positive cells with a flow cytometer (BD Biosciences). For visualization, HaloTag-fusion proteins were labeled with HaloTag-diAcFAM ligand (Promega). Farnesyl thiosalicylic acid (FTS; Cayman Chemical) and bleomycin (Enzo Life Sciences) were dissolved in DMSO. Torin 1 (Tocris Bioscience) was dissolved in sterile water. Each transfected cell was observed with LSM 710 confocal fluorescence microscope under the same exposure and excitation conditions (Carl Zeiss).
Antibodies and Western blot analysis
For immunoblotting, total proteins extracted from cells were boiled for 5 min in 2× SDS-loading buffer (63 mm Tris–HCl [pH 6.8], 4% SDS, 5% β-mercaptoethanol, 20% glycerol) and then homogenized by sonication for 30 s. Anti-ATR rabbit polyclonal antibody (1 : 1000 dilution; Thermo Fisher Scientific), anti-LC3 (4E12) mouse monoclonal antibody (1 : 500 dilution; MBL), anti-phospho Chk1 (Phospho-Chk1 at Ser-317) antibody (1 : 1000; Cell Signaling Technology), anti-Chk1 antibody (1 : 1000; Cell Signaling Technology), anti-phospho 4E-BP1 (Phospho-4E-BP1 at Thr-37/46: 236B4) (1 : 1000; Cell Signaling Technology), anti-4E-BP1 (53H11) (1 : 1000; Cell Signaling Technology), anti-mTOR (7C10) (1 : 1000; Cell Signaling Technology), peroxidase-coupled sheep anti-mouse IgG and donkey anti-rabbit IgG secondary antibodies (1 : 5000 dilution; GE Healthcare) were used for immunoblotting. Signals were visualized using the ECL Plus Western Blotting Detection System (GE Healthcare) and quantified with a Fuji LAS 1000 digital image analyzer (Fuji film).
For ultrastructural analysis, cells were fixed for 24 h at 4 °C in a solution of 2% (w/v) glutaraldehyde and 2% paraformaldehyde in 30 mm HEPES buffer (pH 7.5). Following three washes in phosphate buffer, cells were post-fixed by incubation for 2 h at 4 °C in 1% (w/v) osmic acid in 0.1 m cacodylate buffer (pH 7.4). They were then dehydrated in a series of alcohol washes of 30 min each (50%–100%) and embedded in EPON812 resin at 60 °C. Ultrathin sections (t = 80–90 nm) were cut with a diamond knife on an ultramicrotome, mounted on mesh copper grids, stained with uranyl acetate and lead citrate and viewed on a JEOL 1200 EX transmission electron microscope (JEOL) at 80 kV.
Quantification of cell death and cellular vacuolization
Cell death was measured with propidium iodide (PI), a membrane-impermeant dye that stains DNA of cells with a disrupted plasma membrane. At the indicated time after transfection, cells were harvested by trypsinization, washed once in phosphate-buffered saline (PBS) and incubated in PBS with 2.5 μg/mL PI for 5 min at room temperature. Cells were then washed twice with PBS and analyzed on a FACS Array (Becton Dickinson; 10 000 cells per sample). Data were collected and analyzed using software provided by the Becton Dickenson FACS Array system.
U2OS, HeLa and HCT116 cells (1.5 × 105) adhered to cover slips in 30-mm dishes were transfected with plasmid DNAs with or without inhibitor pre-treatment. At 24 h after transfection, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, followed by washing. GFP-fluorescence signals were visualized with a microscope CCD camera (Olympus DP70) using the same exposure conditions for each sample. The fluorescence intensities were quantified using ImageJ software. GFP-positive cells were analyzed to quantify the rates of cellular vacuolization. For each experiment, 80–250 GFP-positive cells were analyzed, and the data represent the average of three independent experiments.
Expression analyses using quantitative RT-PCR
Total RNA was isolated from cells harvested 24 h after transfection with TRIzol reagent (Invitrogen), and cDNA was synthesized with the PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa Bio). To measure expression differences in each gene, real-time quantitative RT-PCR was performed using MiniOpticon (Bio-Rad Laboratories) with a SYBER Premix Ex Taq (TaKaRa Bio) using the primer sets shown below. The expression level of beta-2-microglobulin (B2M) was used as an internal standard, and the relative ratio of expression of each gene was calculated. Data represent the average of three independent experiments.
- B2M FW: 5′- GGC TAT CCA GCG TAC TCC AAA G-3′
- B2M RV: 5′- CGG ATG GAT GAA ACC CAG AC-3′
- MAP1LC3B FW: 5′- AGC AGC ATC CAA CCA AAA TC-3′
- MAP1LC3B RV: 5′- CTG TGT CCG TTC ACC AAC AG-3′
We thank the following: Professors Tamotsu Yoshimori of Osaka University and Bert Vogelstein of Johns Hopkins University for providing HeLa cells expressing EGFP-LC3, mRFP-LC3 and p53−/− cells, and Dr Timothy Etheridge for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from the Japan Society for the Promotion of Science (JSPS). Part of this work was carried out under the Cooperative Research Project Program of the Institute of Development, Aging and Cancer, Tohoku University.
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