Mol. Cells

AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway

Jin Woo Park, Hana Cho, and Hyein Oh

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Abstract

Aberrations in histone modifications are being studied in mixed-lineage leukemia (MLL)-AF9-driven acute myeloid leukemia (AML). In this study, we focused on the regulation of the differentiation of the MLL-AF9 type AML cell line THP-1. We observed that, upon phorbol 12-myristate 13-acetate (PMA) treatment, THP-1 cells differentiated into monocytes by down-regulating Aurora kinase A (AURKA), resulting in a reduction in H3S10 phosphorylation. We revealed that the AURKA inhibitor alisertib accelerates the expression of the H3K27 demethylase KDM6B, thereby dissociating AURKA and YY1 from the KDM6B promoter region. Using Flow cytometry, we found that alisertib induces THP-1 differentiation into monocytes. Furthermore, we found that treatment with the KDM6B inhibitor GSK-J4 perturbed the PMA-mediated differentiation of THP-1 cells. Thus, we discovered the mechanism of AURKA–KDM6B signaling that controls the differentiation of THP-1 cells, which has implications for biotherapy for leukemia.

Keywords: AURKA, differentiation, KDM6B, MLL-AF9 AML, THP-1

INTRODUCTION

The mixed-lineage leukemia (MLL) protein fuses with more than 60 partners, ranging from transcription factors to cytoplasmic structural proteins. AF9 is the most common fusion partner, found in 30% of MLL-rearranged acute myeloid leukemia (AML) (Meyer et al., 2009; 2013). MLL-AF9 leukemia, which accounts for 10% of acute leukemia cases, is generally associated with a poor prognosis (Krivtsov and Armstrong, 2007). THP-1 cells are widely used for research on MLL-AF9-driven AML. THP-1 is a well-known acute monocytic leukemia cell line that exhibits similarities to primary human monocytes and differentiates into macrophages upon treatment with external stimuli, such as phorbol esters or vitamin D (Auwerx, 1991). Notably, prevalent MLL fusion proteins, including MLL-AF4, MLL-AF9, MLL-ENL, and MLL-ELL, cause leukemia through the dysregulation of MLL target gene expression, implying that inhibitors of MLL fusion on-coproteins, including DOT1L and BRD4 inhibitors, could be potential therapeutic agents (Daigle et al., 2013; Gilan et al., 2016). In a recent study, ATRA induced the granulocytic differentiation of acute promyelocytic leukemia (Nowak et al., 2009). Differentiation therapy forces malignant cells to undergo terminal differentiation, and then the differentiated cells can be treated. However, little is known about the differentiation mechanisms in MLL fusion protein-driven leukemic THP-1 cells.

Aurora kinase (AURK) A is a serine/threonine protein kinase that is amplified in many malignancies, including AML (Gu et al., 2007; Huang et al., 2008; Sakakura et al., 2001). The AURK family mediates mitotic phosphorylation of histone H3 serine10 (H3S10) in budding yeasts and nematodes. In particular, AURKA shows outstanding kinase activity for H3S10 (Crosio et al., 2002; Hsu et al., 2000). Remarkably, H3S10 phosphorylation is associated with mitotic chromatin condensation in early G2 phase, as well as with cell cycle progression (Hendzel et al., 1997; Van Hooser et al., 1998). A recent study suggested that AURKA can regulate the transcriptional levels of target genes through its catalytic activity (Kim et al., 2016). Alisertib (also known as MLN8237) is a novel, selective inhibitor of AURKA, which has shown remarkable anticancer effects against solid tumors and hematologic malignancies (Gorgun et al., 2010; Sehdev et al., 2012; Venkataraman et al., 2012).

Lysine (K) demethylase 6 (KDM6) family proteins possess histone H3 lysine 27 (H3K27) demethylase activity (Hong et al., 2007). Both KDM6A and KDM6B have tumor suppressive properties, such as the regulation of HOX genes, which are well-known oncogenic markers of MLL-rearranged leukemia (Agger et al., 2007). Previous studies have indicated that KDM6A is easily targeted in several types of MLL fusion leukemia cell lines with somatic mutations, and that KDM6B up-regulates the cell cycle inhibitor p21, resulting in cell cycle arrest (van Haaften et al., 2009; Zhao et al., 2013).

In this study, we investigated the role of AURKA-mediated leukemia cell differentiation via the modulation of KDM6B expression in MLL-AF9 AML cells. We demonstrated that AURKA represses KDM6B expression by interacting with YY1 and through KDM6B-dependent p21 activation. Using chromatin immunoprecipitation (ChIP) assays, we showed that AURKA and YY1 are dissociated to the KMD6B promoter in PMA-treated THP-1 cells. Furthermore, we found that alisertib induced leukemic THP-1 cell differentiation and that GSK-J4 repressed leukemia cell differentiation. The combined results of this study provide the evidence that AURKA plays a role in leukemogenesis via the repression of KDM6B expression.

MATERIALS AND METHODS

Cell culture

THP-1 cells were grown in RPMI-1640 and HEK 293T cells were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum and 0.05% penicillin–streptomycin at 37°C in a 5% CO2 atmosphere. For differentiation, THP-1 cells (2 × 107) were seeded in 100-mm plates and treated with 100 ng/ml PMA (Sigma–Aldrich) or DMSO (Duksan). After incubation for 48 h, the cells were harvested for experiments. For the inhibition of AURKA or KDM6B, THP-1 cells (4 × 106) were seeded in 60-mm plates and treated with 0.3 μM alisertib (LKT Laboratories) or 5 μM GSK-J4 (Cayman Chemical). After incubation for 24 or 48 h, the cells were collected and used in experiments.

Plasmid constructs

The plasmids pCMV3-Flag-GATA1 and -YY1 (Han et al., 2015; Son et al., 2012), pGFP-AURKA (Kim et al., 2016a), and pGL3-p21 have been described previously (Oh et al., 2014). The KDM6B promoter region was amplified from human genomic DNA using the primer pairs listed in Supplementary Table 1, then inserted into the NheI/HindIII site of the pGL3-Basic Vector (Promega). The shRNAs against human AURKA and KDM6B were designed using siRNA sequence designer software (Clontech). Double-stranded oli-gonucleotides for shRNA plasmid construction were produced using 5′-to-3′ primers ( Supplementary Table 1). The oligonucleotides were inserted into the AgeI/EcoRI site of the pLKO.1 TRC vector.

Western blot

Total proteins were isolated from the cells using RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 0.5% SDC, 1% NP-40, and 1 mM EDTA). Western blot analysis was conducted using the indicated antibodies.

Antibodies

Antibodies against GAPDH (CSB-PA00025A0Rb; Cus Ab); AURKA (sc-373856), YY1 (sc-7341), p21 (sc-397), and H3 (sc-8654) (Santa Cruz Biotechnology); H3K4me2 (07-030), H3K9me2 (07-441), H3K27me2 (07-452), and H3K36me2 (07-274) (Millipore); and H3S10ph (9701S) (Cell Signaling Technology) were used for western blot and ChIP analyses.

Histone purification

The cells were harvested and washed once with PBS, then the cell pellets were resuspended in 1 ml of TEB lysis buffer (0.5% Triton™ X-100, 2 mM phenylmethylsulfonyl fluoride, and 1× protease inhibitor cocktail) and incubated for 30 minutes at 4°C to promote hypotonic swelling. The cells were then recovered by centrifugation, resuspended in 400 μl of 0.4 N H2SO4, and incubated on a rotator overnight at 4°C. After the centrifugation, the supernatant was transferred and 132 μl of 100% TCA was added for 2 h at 4°C. The pellet was recovered by centrifugation and washed twice with acetone. The pellet was resuspended in deionized water.

Quantitative real time-PCR

Total RNA was isolated from the cells using RNAiso Plus (TaKaRa). For complementary DNA (cDNA) synthesis, we denatured 1 μg of RNA and oligo dT primers (Invitrogen) via incubation at 70°C for 5 min. cDNA was synthesized with M-MLV reverse transcriptase according to the manufacturer’s instructions (Enzynomics). Reverse transcriptase catalytic activity was inactivated by incubating the cDNA at 95°C for 30 s. The synthesized cDNA was subjected to mRNA expression analysis. Quantitative real time-PCR (qRT-PCR) was conducted using the SYBRR Green Supermix kit (TaKaRa). The amplification reaction was performed under the following conditions: 39 cycles of denaturation at 94°C, annealing at the temperatures specified for the primers in Supplementary Table 1, and extension at 72°C. The mean threshold cycle (Ct) and standard error values were calculated from the individual Ct values obtained from triplicate reactions per stage. The normalized mean Ct value was estimated as ΔCt by subtracting the mean Ct of GAPDH. The ΔΔCt value was calculated as the difference between the control ΔCt and the values obtained for each sample. The n-fold change in gene expression, relative to a control, was calculated as 2−ΔΔCt.

ChIP

THP-1 cells (1 × 107) were seeded in 100-mm plates and treated with 0.3 μM alisertib or DMSO. After 24 h, the cells were cross-linked with 1% formaldehyde, which was added to the medium for 10 min at room temperature, followed by the addition of 125 mM glycine for 5 min at room temperature, then lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris [pH 8.1]). The samples were sonicated and immunoprecipitated using the indicated antibodies. The immunoprecipitates were eluted and reverse cross-linked, after which the DNA fragments were purified using a PCR purification kit (Axygen). The precipitated DNA fragments were amplified using specific primers for the KDM6B promoter region via qRT-PCR. The following primer set was used: YY1-BS2 (forward, 5′-CTCCCTTTGGGGAAAGCTAA-3′ and reverse, 5′-TGATAAGAGTGCCCGCTACC-3′). The mean Ct and standard error values were calculated from the individual Ct values obtained from duplicates per stage. The normalized mean Ct was estimated as ΔCt by subtracting the mean Ct of the input.

Flow cytometric analysis of cell differentiation

To measure cell differentiation, THP-1 cells (1 × 106) were split into 35-mm dishes and treated with DMSO or 100 ng/ml PMA for 48 h. The cells were trypsinized, washed, and resuspended in cold PBS with 1 mM EDTA, 1% bovine serum albumin, and 10 mM sodium azide for 1 h. Before flow cytometric analysis, the cells were stained with PE-CD11b (12-0118-42) and APC-CD14 (17-0149-42) antibodies (eBioscience) for 30 min, washed using PBS with 1 mM EDTA and 1% bovine serum albumin, then subjected to flow cytometry using a BD Accuri™ C6 cytometer (BD Biosciences).

Luciferase assay

For the transcriptional activity assays, HEK 293T cells (2 × 104) were seeded in 48-well plates and co-transfected with the pGL3-p21 promoter or pGL3-KDM6B promoter reporter plasmid and the indicated DNA constructs using polyethylenimine (Polysciences), or treated with 0.1 or 0.3 μM alisertib, or treated with 2 or 5 μM GSK-J4, for 24 h. After transfection, the cells were collected and subjected to a luciferase assay (Promega). The level of β-galactosidase activity was used to normalize the reporter luciferase. The data are expressed as the means of triplicates. All results shown are representative of at least 3 independent experiments.

Statistical analysis

The data are expressed as the mean ± SEM of 3 or more independent experiments. Statistical significance (p < 0.05) was calculated using functions in Microsoft Excel. The differences between the groups were evaluated by one-way analysis of variance, followed by Student’s t-test or Bonferroni’s test, as appropriate.

RESULTS

AURK-mediated H3S10 phosphorylation levels decreased during leukemia cell differentiation

Despite the wealth of knowledge regarding the pathogenesis of MLL-rearranged AML, few studies have explored histone modification-associated leukemia cell differentiation. We first identified the epigenetic changes during the differentiation of the MLL-AF9 AML cell line THP-1 after treatment with PMA. The differentiation of the THP-1 cells was confirmed by qRT-PCR for cell surface markers of macrophages, such as CD14 and CD11b (ITGAM) (Fig. 1A). In previous study, differentiation and maturation of myeloid leukemia induces heterochromatin density (Smetana et al., 2011). Consistently, we found that the levels of H3K27me2, H3K27me3, and H3K9me2, which were closed chromatin marker, were significantly increased during the THP-1 differentiation (Fig. 1B). Interestingly, we also found that the level of H3S10 phosphorylation was significantly lower in THP-1 cells during differentiation (Fig. 1B). According to previous studies, AURK family proteins mainly regulate H3S10 phosphorylation during cell cycle progression and regulate gene expression during HL-60 differentiation (Crosio et al., 2002; Kim et al., 2016; Ota et al., 2002). We evaluated the expression levels of the AURK family members and found lower mRNA levels of AURKA and AURKB in PMA-treated THP-1 cells than in control cells (Fig. 1C). Moreover, we detected the down-regulation of AURKA protein upon PMA treatment in THP-1 cells (Fig. 1D). To investigate if the knockdown of AURKA has a role in THP-1 differentiation, we measured the effects of two different target shRNAs specific for AURKA on the differentiation of THP-1 cells. Similar to PMA treatment, knockdown of AURKA also induced increases in CD14 and CD11b levels (Fig. 1E). Consistent with previous data, we obtained similar results of the levels of CD14 and CD11b (Fig. 1F, left), and global histone modification (Fig. 1F, right) in alisertib-treated THP-1 cells. Overall, these data indicate that PMA treatment reduces AURKA expression and H3S10 phosphorylation in THP-1 cells, and that AURKA may be involved in differentiation.

AURKA regulates the transcription of KDM6B and p21 expression in PMA-treated THP-1 cells

Having determined the induction of THP-1 differentiation by alisertib treatment, we next analyzed the effect of AURKA on the transcriptional regulation of target genes. Using previously generated microarray data (Shechter et al., 2007), we found that leukemia-related gene expression changed during the differentiation of THP-1 cells by PMA. We selected 5 genes, which were up- or down-regulated by PMA treatment in THP-1 cells, and validated their expression levels by qRT-PCR. The transcription levels of KDM6B, RFX2, and CDK19 were increased, but the levels of CPT1A and SFRP1 were decreased, in PMA-treated THP-1 cells (Fig. 2A). Because we discovered that several histone modifications, including H3S10 phosphorylation and dimethylation of H3K27 and H3K9, were changed in PMA-treated THP-1 cells (Fig. 1B), we focused on the regulation of KDM6B expression in differentiated THP-1 cells. We first analyzed KDM6B mRNA levels in alisertib-treated THP-1 cells. As with PMA treatment, alisertib treatment increased KDM6B expression levels (Fig. 2B). Next, we determined if AURKA expression decreased the transcriptional activity of KDM6B. We conducted reporter assays using a KDM6B-luc construct in 293T cells. Alisertib induced transcriptional activity at the KDM6B promoter (Fig. 2C). However, AURKA reduced KDM6B promoter activity in a dose-dependent manner (Fig. 2D). A previous report showed that ectopic expression of p21 undergoes monocytic differentiation in peripheral blood monocytes (Asada et al., 1999). To determine if p21 mRNA levels were also regulated by AURKA in THP-1 cells, we performed qRT-PCR in alisertib-treated THP-1 cells. The mRNA levels of p21 were increased in alisertib-treated THP-1 cells (Fig. 2E). To investigate how AURKA regulates the p21 transcriptional level, we tested AURKA transcriptional activity at the p21 promoter in a dose-dependent manner. The promoter activity of p21 was reduced in AURKA-overexpressed 293T cells (Fig. 2F). While p21 promoter assay in HEK 293T cells treated with alisertib exhibited inactivation of AURKA enzymatic function contributes to promote p21 transcription (Fig. 2G). These results suggest that AURKA regulates KDM6B and p21 expression during leukemia cell differentiation.

AURKA repressed transcription of KDM6B via YY1 recruitment in PMA-treated THP-1 cells

In order to determine the mechanism of KDM6B down-regulation by AURKA more precisely, we next searched for transcription factors involved in AURKA-mediated transcripttional regulation. We identified GATA1- and YY1-binding regions in the KDM6B promoter using PROMO, which predicts transcription regulatory elements (Fig. 3A) (Messeguer et al., 2002). To determine if AURKA cooperates with GATA1 or YY1 in the regulation of KDM6B expression, we investigated the interaction between AURKA and YY1 using co-immunoprecipitation assays in AURKA- and YY1-overexpressing 293T cells, which showed that AURKA reciprocally interacts with YY1 (Fig. 3B). However, we did not detect an interaction between AURKA and GATA1 in the co-immunoprecipitation assay (Fig. 3C). To determine the effects of AURKA and YY1 on KDM6B transcriptional activity, we conducted reporter assays using the KDM6B-luc promoter construct in 293T cells. As expected, AURKA reduced transcriptional activity at the KDM6B promoter, and YY1 caused a further reduction in transcriptional activity (Fig. 3D). Consistent with the interaction assay, GATA1 did not affect KDM6B transcriptional activity (Fig. 3E). We next investigated if AURKA and YY1 could bind to the endogenous promoter regulatory regions of the KDM6B gene. We conducted ChIP analysis using anti-AURKA and -YY1 antibodies after PMA treatment. The ChIP analysis showed that the recruitment of AURKA and YY1 to the target gene promoters was decreased in PMA-treated cells (Fig. 3F). Because alisertib has a similar effect to PMA treatment, we tested if alisertib reduced the recruitment of YY1 to the KDM6B promoter region. The ChIP analysis suggested that the AURKA inhibitor alisertib dissociated YY1 from the KDM6B promoter sites (Fig. 3G). Taken together, these results suggest that KDM6B expression levels are induced by the disruption of the enrichment of AURKA and YY1 at the target gene promoter during leukemia cell differentiation.

AURKA regulates PMA-dependent p21 activation via KDM6B pathway during leukemia cell differentiation

A previous report showed that the H3K27 demethylase activity of KDM6B promotes p21 expression in mouse embryonic fibroblasts (Zhao et al., 2013). To gain insight into KDM6B-mediated p21 expression, we added the KDM6B inhibitor GSK-J4 in PMA-treated THP-1 cells and measured p21 expression. We identified that GSK-J4 markedly diminished p21 level compared to PMA treatment (Fig. 4A). To further determine the mechanisms underlying transcriptional regulation of p21 via KDM6B, we conducted luciferase reporter assay using p21-luciferase reporter system in HEK 293T cells. Ectopic expression of KDM6B accelerated p21 transcription (Fig. 4B), whereas KDM6B inhibition by GSK-J4 showed reduction of p21 expression (Fig. 4C). We further investigated if AURKA reduces p21 transcriptional activity via KDM6B demethylase activity. We found that the promoter activity of p21 increased in alisertib-treated THP-1 cells, but the KDM6B inhibitor GSK-J4 disrupted the alisertib-dependent induction of p21 in a dose-dependent manner (Fig. 4D). To further explore the flow of AURKA and KDM6B-dependent alteration of p21 expression, we estimated the methylation status of p21 promoter via ChIP assay. We observed that H3K27 methylation level was diminished at the p21 promoter region in THP-1 cells by alisertib treatment (Fig. 4E). These data prove that AURKA functions as a transcriptional regulator of KDM6B and the AURKA-mediated KDM6B suppression subsequently inactivates p21.

KDM6B induces THP-1 cell differentiation

To confirm the role of KDM6B in the differentiation of THP-1 cells, we used the KDM6B inhibitor GSK-J4 in THP-1 cells. The expression levels of CD11b and CD14 were slightly decreased compared to those in untreated cells upon GSK-J4 treatment (Fig. 5A). In addition, GSK-J4 strongly reduced the levels of CD11b and CD14 in PMA-treated THP-1 cells (Fig. 5A). To further confirm that knockdown of KDM6B disrupts PMA-mediated THP-1 cell differentiation, we conducted flow cytometric analysis to measure the expression of the monocyte differentiation markers CD11b and CD14. We observed increased expression of CD11b and CD14 in PMA-treated THP-1 cells compared to control cells, but the expression of CD11b and CD14 did not change after PMA treatment in THP-1 cells in which KDM6B was stably knocked down (Fig. 5B). Previously, we confirmed that alisertib induces THP-1 differentiation to monocytes. Therefore, we tested if the inhibition of KDM6B repressed alisertib-mediated THP-1 differentiation. Flow cytometric analysis suggested that the inhibition of AURKA increased the expression of CD11b and CD14 by inducing THP-1 differentiation; however, GSK-J4 slightly repressed alisertib-mediated THP-1 differentiation (Fig. 5C). Taken together, these findings suggest that KDM6B mediates the differentiation of THP-1 cells by a mechanism dependent on AURKA transcriptional regulation.

DISCUSSION

In this study, we showed that AURKA plays a role in THP-1 cell differentiation into monocytes. We found reduced AURKA expression during THP-1 cell differentiation, resulting in increased KDM6B and KDM6B-dependent p21 expression levels. We demonstrated the importance of the interaction between AURKA and YY1 for recruitment to the KDM6B promoter in PMA-treated THP-1 cells. We also defined the biological significance of AURKA-mediated KDM6B regulation in THP-1 cell differentiation. These results suggest that the loss of AURKA induces the dissociation of YY1 from the KDM6B promoter region in PMA-treated THP-1 cells and promotes KDM6B-mediated monocyte differentiation (Fig. 5D).

Previous study showed that Sox6-mediated K562 differentiation increases total hemoglobin content, but which is less than cells treated with hemin (Cantu et al., 2011). Consistently, we found that inhibitor or knockdown of AURKA slightly increases CD11b and CD14 levels (Fig. 1). A change of one factor might be not enough to fully differentiation. So, we hypothesized that AURKA mediated-THP-1 fully differentiation might be associated with regulation of other early response genes together.

AML is characterized by uncontrolled proliferation, cell cycle progression, and DNA hypermethylation (Plass et al., 2008). The DNMT family interact with HDAC1 and HDAC2, suggesting a relationship between histone acetylation and DNA methylation (Fuks et al., 2000; Rountree et al., 2000). DNMT inhibitors, such as decitabine and 5-azacitidine, induce terminal differentiation of AML, with anti-leukemic effects (Chang et al., 2016). Here, we discovered that several histone modifications are altered during the differentiation of THP-1 cells. The induction of H3 acetylation by PMA may inhibit DNA hypermethylation in AML and induce THP-1 differentiation. Previous studies showed that THP-1 cells undergo macrophage differentiation upon PMA treatment, which leads to cell cycle arrest in both G0/G1 and G2/M phase (Sugarman et al., 1995). In addition, accumulating evidence has highlighted the role of p21 in irreversible G1 arrest in MLL-fusion AML (Tonelli et al., 2006). Moreover, AURKA has kinase activity for CENP-A, TPX2, and p53 and is involved in mitotic entry and bipolar spindle formation (Gautschi et al., 2008). The loss of AURKA results in cell cycle arrest in the G2/M phase (Liu and Ruderman, 2006). Our finding indicated that the differentiation of THP-1 cells by PMA reduces AURKA expression, followed by the induction of KDM6B and p21 expression. These changes in gene expression may be responsible for the cell cycle arrest in both G0/G1 and G2/M phases.

THP-1 cells is an MLL-AF9-driven leukemia; its MLL possesses a DNA-binding domain, but has lost its H3K4 methyl-transferase activity. MLL-AF9 recruits DOT1L to the MLL target site, leading to dysregulation of target gene expression, followed by leukemia. Several studies have demonstrated that inhibitors of MLL fusion proteins, including DOT1L, BRD4, MOF, and SIRT, function as potential therapeutic agents (Chen et al., 2015; Valerio et al., 2017; Zuber et al., 2011). In addition, the differentiating agent ATRA has been co-administered with a hyperacetylating agent (an HDAC inhibitor) in attempted epigenetic therapies for AML (Warrell, 1999). Our findings indicate that the irreversible G0/G1 arrest via the KDM6B-dependent p21 pathway is accelerated during THP-1 differentiation, which is consistent with the alleviation of the leukemogenic status of THP-1 cells. In this study, we discovered that inhibition of AURKA induces differentiation and reduces cell proliferation in THP-1 cells (data not shown), and that the KDM6B inhibitor GSK-J4 disrupts the differentiation of THP-1 cells into monocytes. Our study suggests a mechanism in which AURKA–KDM6B signaling regulates the differentiation of THP-1 cells, which may help identify new therapeutic candidates for leukemia.

Supplementary data

molce-41-5-444s1.pdf

Article information

Mol. Cells.May 31, 2018; 41(5): 444-453.
Published online 2018-02-23. doi:  10.14348/molcells.2018.2311
<a href="http://www.ncbi.nlm.nih.gov/pmc/pubmed?cmd=search&amp;db=PubMed&amp;term=Jin+Woo+Park">Jin Woo Park</a>, <a href="http://www.ncbi.nlm.nih.gov/pmc/pubmed?cmd=search&amp;db=PubMed&amp;term=Hana+Cho">Hana Cho</a>, and <a href="http://www.ncbi.nlm.nih.gov/pmc/pubmed?cmd=search&amp;db=PubMed&amp;term=Hyein+Oh">Hyein Oh</a>
1Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756, Korea
*Correspondence: jykim@cau.ac.kr (JYK); sangbs@cau.ac.kr (SBS)
Received November 22, 2017; Accepted February 10, 2018.
Articles from Mol. Cells are provided here courtesy of Mol. Cells

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