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DNA repair efficiency associated with reprogrammed osteosarcoma cells
Gene Reports 16 (2019) 100409
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Gene Reports journal homepage: www.elsevier.com/locate/genrep
DNA repair efficiency associated with reprogrammed osteosarcoma cells Pei-Feng Choong Tunku Kamarulc
a,b,⁎
b
a
b
, Hui-Xin Teh , Hoon-Koon Teoh , Han-Kiat Ong , Soon-Keng Cheong
T
a,b
,
a
National Cancer Council (MAKNA), Kuala Lumpur, Malaysia Faculty of Medicine and Health Sciences, University Tunku Abdul Rahman (UTAR), Selangor, Malaysia Tissue Engineering Group, National Orthopedic Centre of Excellence for Research and Learning, Department of Orthopedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia b c
A R T I C LE I N FO
A B S T R A C T
Keywords: DNA Damage Response DNA repair Reprogramming Osteosarcoma Nucleotide excision repair Genomic stability
Genomic instability and genetic heterogeneity are typical hallmarks of cancer, including osteosarcoma (OS), a type of bone tumor. Inactivation of DNA repair pathways may play an important role in the initiation and progression of OS pathogenesis by increasing the mutation rate and genomic instability. However, there is still a lack of information on gene alterations and mutations that may increase the risk of osteosarcoma formation. Reprogramming OS cells to a primitive stage namely induced pluripotent stem cell (iPSC) state could be a useful disease model to understand the pathogenesis of OS and to bridge the current gap of knowledge on DNA repair mechanisms in reprogrammed OS cells. By using Yamanaka factors, OS cell lines, G-292 and Saos-2, were reprogrammed to iPSC lines, respectively iG-292 and iSaos-2, both of which demonstrated pluripotency similar to embryonic stem cells. However, only iG-292 was able to form teratoma. Subsequent microarray data showed significant down-regulation of DNA Damage Response (DDR) genes expression for both iG-292 and iSaos-2. A functional assay using UV-induced DNA damage approach demonstrated efficient DNA repair mechanism in iG292. Further analysis of nucleotide excision repair (NER) genes demonstrated up-regulation of GADD45G, XPA, RPA, MNAT1, ERCC1, PCNA, and POLL, in iG-292. Up-regulation of GADD45G together with up-regulation of other NER genes synergistically repair UV damage by rapid removal of cyclobutane pyrimidine dimers. In conclusion, down-regulation of DDR genes in reprogrammed OS may render an enhanced state of genomic integrity in reprogrammed OS as compared to the parental cells. Thus, this study demonstrated for the first time DDR profile of reprogrammed OS cells and the probable involvement of GADD45G in the DNA repair mechanism of reprogrammed OS cells.
Abbreviations: ATCC, American Type Culture Collection; bFGF, basic fibroblast growth factor; CASP4, caspase 4; CASP8, caspase 8; CCNA1, cyclin A1; CDK, cyclin dependent kinase; CHK2, check point kinase 2; c-MYC, c-Myc avian myelocytomatosis viral oncogene homolog; CPD, cyclobutane pyrimidine dimer; DAPK1, deathassociated protein kinase-1; DDR, DNA Damage Response; DEG, differentially expressed gene; DSB, double-strand break; EB, embryoid body; ERCC1, excision-repair cross-complementing 1; GADD45G, growth arrest and DNA damage-inducible 45- γ; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HSC, hematopoietic stem cell; iG-292, G-292-derived induced pluripotent stem cell; IGF2, insulin-like growth factor 2; iMEF, inactivated mouse embryonic fibroblast; iPSC, induced pluripotent stem cell; iSaos-2, Saos-2-derived induced pluripotent stem cell; KLF4, Kruppel-like factor 4; MCM9, Minichromosome maintenance protein 9; MEF, mouse embryonic fibroblast; MLH1, MutL homolog 1; MNAT1, MNAT, CDK activating kinase assembly factor 1; MSC, mesenchymal stem cell; NER, nucleotide excision repair; OCT3/4, octamer-binding transcription factor 3/4; OS, osteosarcoma; OSKM, OCT4, SOX2, KLF4, c-MYC; PARP1, poly (ADP-ribose) polymerase-1; PARP3, poly (ADP-ribose) polymerase-3; PCNA, proliferating cell nuclear antigen; POLL, polymerase (DNA) lambda; REX1, ZFP42 zinc finger protein; ROCK, Rho-associated, coiled-coil containing protein kinase; RPA, replication protein A; SD, standard deviation; SEM, standard error of mean; SNP, single nucleotide polymorphism; SOX2, sex-determining region Y (SRY)-related box 2; TNFRSF1A, tumor necrosis factor receptor superfamily 1A; TP53, tumor protein 53; UV, ultraviolet; VA, valproic acid; XPA, xeroderma pigmentosum complementation group A; XPC, xeroderma pigmentosum complementation group C; XPD, xeroderma pigmentosum complementation group D; XPF, xeroderma pigmentosum complementation group F ⁎ Corresponding author at: Postgraduate Laboratory, Faculty of Medicine and Health Science, Universiti Tunku Abdul Rahman (UTAR), Bandar Sungai Long, 43000 Kajang, Malaysia. E-mail address: [email protected] (P.-F. Choong). https://doi.org/10.1016/j.genrep.2019.100409 Received 24 April 2019; Accepted 27 April 2019 Available online 01 May 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
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1. Introduction
normal cell lines and non-cancerous iPSC. However, there is no report on the profile of DNA Damage Response of reprogrammed cancer cells as well as the functionality of DNA repair pathway genes post-cancer cells reprogramming. Hence, this study was conducted to investigate the DNA Damage Response efficiency in reprogrammed OS and parental cells.
Osteosarcoma (OS), a type of bone cancer, can be considered as an ancient disease. One of the earliest cases of OS was documented to be dated between 1.6 and 1.8 million years old in the South African Journal of Science (Odes et al., 2016). This discovery implied that the pathogenesis for OS was already buried deep within the human historical evolution and has no relation to the present human lifestyle. OS is a rare tumor diagnosed prevalent in children and adolescents. Conventional treatment for OS, by combining chemotherapy and surgery, had increased the 5-year survival rates to 60%–70% (Mankin et al., 2004). However, over the years these figures remain unchanged. OS pathogenesis has been linked to genetic changes during the osteoblast differentiation process (Gokgoz et al., 2001). Genetic modifications associated with impairment of proliferation and differentiation capacity eventually increase the potential of tumorigenicity (Kenyon and Gerson, 2007). One consistent finding in all these OS studies pointed to a higher incidence of OS in individuals with mutation in genes that are involved in stabilizing the genome, such as p53 and retinoblastoma protein (RB1). Impairment of these related genes causes defective maintenance of DNA (Fuchs and Pritchard, 2002; Martin et al., 2012). OS is associated with high level of genomic instability (Selvarajah et al., 2007). This could be due to a mutation in p53 as mutant forms of p53 are significantly associated with greater genomic instability in OS (Overholtzer et al., 2003). Deregulation of TP53, a product of p53 gene, is linked with OS advancement and occurs due to a mutation of the gene locus at 17p13.1 (Martin et al., 2012). Thus, OS is often characterized as having extensive genomic instability, uncontrolled apoptosis, uncontrolled cell cycle, and lack of differentiation ability (Martin et al., 2012; Varshney et al., 2016). Development in the field of induced pluripotent stem cells (iPSC) has provided the means to study OS pathogenesis. iPSCs are pluripotent cells reprogrammed from human somatic cells through ectopic expression of OSKM transcription factors (Takahashi et al., 2007). Reprogramming of OS cell lines to a more primitive stage, however, retains the acquired genetic mutations in OS. iPSC OS cell lines thus could help to provide valuable insight into the OS pathogenesis by understanding the acquired genetic changes during the transformation process to an advanced stage. Reprogramming of OS also helps to yield a larger population of pluripotent cancer cells which could be cryopreserved and propagated for long term study (Tafani, 2012). Zhang et al. (2013) demonstrated that reprogrammed sarcoma cells lost their tumorigenicity and could dedifferentiate to mesenchymal stem cells (MSC) state. The study also showed differentiation of mature connective tissues and red blood cells from hematopoietic stem cell (HSC)-like cells, suggesting the ability of sarcoma cells to reverse back to a mutual stage of ancestor before branching out to HSC and MSC. This study showed that cancer cells could be ‘normalized’ via reprogramming despite carrying inherent cancer-causing mutations. However not much is known about the genomic stability underlying the reprogramming process in sarcoma cells. Reprogramming cancer cells into induced pluripotent cancer cells (iPCs) has been shown to reset some of the characteristics of cancer cells, and that these iPCs behave distinctly from their parental cells upon reprogramming (Allegrucci et al., 2011; Mahalingam et al., 2012). Combining molecular information collected from cancer iPSC models with the present knowledge of OS biology could assist us in gaining a deeper understanding of the pathological mechanisms triggering formation of OS. This could eventually aid in the prospective development of future OS therapies. Therefore, elucidation of individualized OS-associated gene functions to investigate the potential pathological mechanisms involved in the different stages of OS development from initiation to progression is vital for future OS detection and treatment. To date, most of the reported DNA repair studies were conducted on
2. Materials and methods 2.1. Cell lines Osteoblast cell line, hFOB, and osteosarcoma cell lines, Saos-2 and G-292, were purchased from ATCC and maintained in the following conditions; Saos-2: Dulbecco's Modified Eagle Medium, low glucose (DMEM-LG), 10% fetal bovine serum (FBS), 1% Pen-Strep (Gibco, Carlsbad, CA, USA), G-292: McCoy's 5a Medium, 10% FBS, 1% PenStrep, and hFOB: Dulbecco's Modified Eagle Medium (DMEM)/F-12, 10% fetal bovine serum (FBS), 1% Pen-Strep and Geneticin (Gibco, Carlsbad, CA, USA). Reprogrammed cells were maintained in human embryonic stem cells (hESC) medium constituted of DMEM/F12 supplemented with 20% knockout serum replacement (KOSR), 0.1 mM bmercaptoethanol (Merck, New Jersey, USA), 10 ng/ml basic fibroblast growth factor (bFGF) (Miltenyi Biotech, Germany), 1× non-essential amino-acid (NEAA), 1 mM L-glutamine and 0.1× Pen-Strep. Irradiated mouse embryonic fibroblasts (iMEF) (Global Stem, Rockville, MD, USA), were used as feeder cells and cultured in DMEM with 10% FBS. iPSC colonies were manually picked and plated on iMEF plates. All cells were cultured at 37 °C in a 5% CO2 incubator.
2.2. OS-iPSC generation Human OS-iPSC cells were produced as previously described, with slight modification (Sugii et al., 2011). Retroviral vectors for each of the transcription factors genes (pMX-c-MYC, pMX-KLF4, pMX-OCT4, or pMX-SOX2) were transfected with envelope genes (gag/pol and VSV-G) into 293FT cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). OS cells were seeded at a density of 40–60% confluency one day before transduction. Fresh retrovirus supernatants were supplemented with 8 mg/ml Polybrene (Millipore, Merck) prior to transduction. Equal amounts of supernatants containing each of the four retroviruses carrying the OCT4, SOX2, c-MYC and KLF4 genes, were mixed and transferred to the cells. The plates were centrifuged at 800g for 50 min and incubated overnight at 37 °C, 5% CO2. Fresh medium was changed on the next day and subsequently every day. Transduced cells were transferred to iMEF on day 3 post-transduction. Cells were monitored every day for the formation of colonies. Colonies were manually picked on day 15–day 20 and transferred to new iMEF. Generated OS-iPSC was characterized prior to microarray and functional assay.
2.3. Microarray analysis RNA extraction was performed using Qiagen miRNeasy ® Mini Kit (Qiagen, Germany) according to manufacturer's protocol. The RNA integrity test was done using 2100 Bioanalyzer (Agilent, Germany). Total RNA from reprogrammed OS and parental were subjected to RNA target preparation for microarray expression analysis using GeneChip ® 3′IVT Express Kit (Affymetrix, USA) according to manufacturer's protocol. After hybridization, arrays scanned using GeneChip® Scanner 3000 7G. Microarray data were imported into GeneSpring GX13.0 (Agilent, Germany) for analysis. Genes with fold change (FC) > 2 (FC > 2) and significance level, p < 0.05 were selected for validation. Raw data generated from Affymetrix microarray platform was deposited to NCBI GEO Omnibus with accession number GSE107855. 2
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expression profiling is one of the best-used methods to characterize reprogrammed population and to identify the degree of reprogramming on target cells as it offers unbiased, whole genome approach to examine both populations (Park et al., 2011; Liu et al., 2012; Medvedev et al., 2013). Upon using unsupervised hierarchical clustering analysis, the clustering demonstrated the distinctive separation of two clustered populations between the parental and the reprogrammed OS (Fig. 2a). Both reprogrammed OS showed more down-regulated differentially expressed genes (DEGs) than up-regulated DEGs (Table 1). These highly DEGs were further categorized based on their functional annotation into three categories in Gene ontology (GO), namely cellular component, molecular function and biological processes (Fig. 2b). The overall GO enrichment analysis showed diverse enrichment of GO categories between iG-292 and iSaos-2. The variability of GO enriched categories between the two populations implying the different reprogramming responses from both parental cell lines, G-292 and Saos-2. Further analyses were done on GO based on up-regulated and downregulated DEGs. In up-regulated DEGs, molecular function domain in both reprogrammed OS showed similar up-regulation in binding; iG292 (60.51%) and iSaos-2 (100%). In biological processes, single organism process was listed as top two contributors in both reprogrammed iG-292 and iSaos-2. Meanwhile, only up-regulated DEGs from iG-292 produced GO enrichment for cellular component, with the majority on cell (49.67%) and cell part (49.67%); while up-regulated DEGs from iSaos-2 did not generate any GO enrichment for cellular component (Fig. 3). For down-regulated DEGs, GO enriched category “binding” was the top in both iG-292 (77.7%) and iSaos-2 (100%) in molecular function domain. In biological processes, the top category for iG-292 was cell processes (16.6%) and single organism processes (17.92%) for iSaos-2. For cellular component domain, cell and cell part were listed top in iG292 (21.91% for both), but were listed as third in iSaos-2 (16.63% respectively). Membrane was listed top in cellular component domain for iSaos-2 (25.02%) (Fig. 3).
2.4. Taqman Gene Expression Assay for validation and NER genes expression Briefly, Taqman® Gene Expression Assay (20×), cDNA samples and Taqman® Fast Advanced Master Mix (2×) were thawed on ice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control gene in this experiment. An appropriate number of reactions were prepared according to the volumes in manufacturer's protocol. The components were mixed thoroughly, then the 0.2 ml strip-tubes were centrifuged briefly to spin down the content and eliminate any bubbles. The tubes were then placed in the thermal cycler (StepOne Plus, Thermo Fisher Scientific, USA). 2.5. Cyclobutane pyrimidine dimers (CPD)quantification Prior to UV treatment, cells grown till 80% confluency were washed and the medium was replaced with 10 ml phosphate buffered saline (PBS) with Calcium and Magnesium. Cells were irradiated at UV-dose of 40 J/m2 UVC (254 nm) with UVILite LF-206-LS (UVITEC, UK). Following treatments, cells were fed culture medium and incubated for 1 h, 6 h or 24 h to allow for DNA repair mechanism to take place. Cells were harvested for DNA extraction using Qiagen DNA extraction kit and RNA extraction using Qiagen RNA extraction kit. UV-induced DNA damage was detected by OxiSelect UV-Induced DNA damage ELISA Combo Kit (CPD quantitation) (Cell Biolabs, USA). Osteoblast cell line, hFOB, was used as a normal comparator to set the UV dosage. 3. Results 3.1. Reprogramming & characterization of reprogrammed OS cells Generation of G-292-derived iPSC, termed as iG-292, and Saos-2derived iPSC, termed as iSaos-2, was successful with a single transduction of retroviral with OSKM transcription factors. Morphologies of the selected iG-292 and iSaos-2 clones resembled ESC with clear defined borders and consisting of cells with high nucleus to cytoplasm ratio, prominent nucleoli, tightly packed cells with clear defined borders (Fig. 1a) (Thomson et al., 1998). Similar to other iPSC studies, higher expression levels of pluripotent markers, OCT3/4, SOX2, NANOG, and REX1 were also detected in both reprogrammed OS cells as compared to their parental cells (Fig. 1b). Pluripotent markers are markers expressed at higher level in pluripotent stem cells than terminally differentiated cells. Detection of these pluripotent markers distinguished pluripotent cells, such as ESC and iPSC, from somatic cells or parental cells. Embryoid body formation is another characteristic for ESC and iPSC. The ability to cluster together and form suspension body is associated with pluripotency (Li and Rana, 2012). Product of spontaneous differentiation from embryoid body showed gene expression for three germ-layers: MSX1, GATA2 and hBRACHYURY (mesoderm markers); FOXA2 and GATA4 (endoderm markers); and CDX2 (ectoderm marker) in iG-292, while iSaos-2 only expressed MSX1 and GATA2 (mesoderm); GATA6 (endoderm) and TUJ1 (ectoderm) (Fig. 1c). Teratoma/xenograft formation in vivo is another developmental characteristic of pluripotent stem cells. Pluripotency of reprogrammed OS cells was tested for teratoma formation. Histological analysis using H&E staining showed that only iG-292 managed to form teratoma in vivo. 4 out of 5 nude mice injected with iG-292 formed teratoma with the morphology of cells representing three germ layers; ectoderm, mesoderm and endoderm (Fig. 1d).
3.3. Elucidation of DNA Damage Response (DDR) pathways upon OS reprogramming Differentially expressed genes (DEGs) between reprogrammed OS and their parental cells were grouped in accordance to the pathway in which they participate in DDR system: DNA repair, cell cycle, and apoptosis (Fig. 4a–c). iG-292 displayed more DEGs in each pathway as compared to iSaos-2. There was contradictory expression between iG292 and iSaos-2 in 3 genes, CASP8, PARP3, and MLH1, where iG-292 showed down-regulation while iSaos-2 showed up-regulation of these genes. Only PARP1 was up-regulated in both iG-292 and iSaos-2 (Fig. 4a). As for cell cycle genes, 8 genes were up-regulated in iG-292 and 2 genes were up-regulated in iSaos-2. Meanwhile, 15 genes were downregulated in iG-292 and 3 genes were down-regulated in iSaos-2. Only 1 gene, MCM9, showed conflicting expression with up-regulation in iG292 but down-regulated in iSaos-2. While only CCNA1 showed upregulation in both iG-292 and iSaos-2 (Fig. 4b). 11 apoptotic genes were up-regulated in iG-292 and 5 genes in iSaos-2. Whereas, 26 genes were down-regulated in iG-292 and 5 genes in iSaos-2. In iG-292, DAPK1 was up-regulated and CASP8 was downregulated, but iSaos-2 showed the opposite expression. Only IGF2 was up-regulated in both iG-292 and iSaos-2 while CASP4, TNFRSF1A, and MYC were down-regulated in both iG-292 and iSaos-2 (Fig. 4c). 3.4. Functional assay to explicate genes related to DNA repair mechanism in reprogrammed OS
3.2. Global gene expression profile of the reprogrammed OS cells Microarray technology with global gene transcription pattern was used to measure similarity or disparity between two populations, such as reprogrammed cells against their parental cells. Global gene
Recent studies on genetic polymorphisms in nucleotide excision repair (NER) in relation to lower chemotherapy response, unfavorable 3
Fig. 1. Generation and characterization of reprogrammed OS. (a) The emergence of ESC-like colonies on Day 21 post-transduction of reprogrammed G-292 (iG-292) and reprogrammed Saos-2 (iSaos-2). (b) Expression of pluripotent genes, SOX2, OCT3/4, and NANOG, in reprogrammed OS. (c) Multilineage genes expression during EB formation, indicating attained pluripotency post reprogramming in iG-292 and iSaos-2. (d) Formation of neuronal rosette-like structures indicating ectoderm layer, capillary-sized blood vessels, adipocytes cells, and also fibrous muscle-like cells indicating mesoderm layer and columnar epithelial cells seen as lining glands, and the papillary structures indicating endoderm layers. . An asterisk (*) denotes significant level, p < 0.05.
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Fig. 2. Gene expression profile of reprogrammed OS. (a) Hierarchical clustering analysis of parental and reprogrammed OS showing distinctive clustering between parental and reprogrammed OS cells. (b) Distribution of gene ontology enriched categories for reprogrammed OS, iG-292 and iSaos-2. GO analysis was generated based on reprogrammed OS DEGs against parental DEGs as a control.
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Table 1 Differentially expressed genes between parental and reprogrammed OS with fold change ≥2 and significance level, p < 0.05.
iG-292 vs G-292 iSaos-2 vs Saos-2
Up-regulated genes
Down-regulated genes
Total differentially expressed genes
992 410
1875 730
2867 1140
parental G-292 to another phenotype of iG-292 capable of forming teratoma. Following characterization, iG-292 is believed to be fully reprogrammed in contrast to iSaos-2, which did not form teratoma in vivo. Reprogramming roadblock or resistance has been reported in a few studies using both somatic and diseased cells. There are many reasons attributed to this reprogramming roadblock. One of the hindrances in reprogramming is the expression of TGFβ1. Only Saos-2 expressed TGFβ1, which was reported previously as a roadblock to reprogramming (Li et al., 2010; Verusingam et al., 2017). Expression of c-MYC is often debated in reprogramming of both somatic and cancer cells. c-MYC is a known oncogene, playing a substantial role in cellular growth regulation and metabolism (Miller et al., 2012). Down-regulation of c-MYC expression was reported in previous sarcoma reprogramming (Zhang et al., 2013) similar to our observation in both reprogrammed OS. Though expression of c-MYC has always been reported to be up-regulated in somatic reprogramming (Aasen et al., 2008; Hester et al., 2009; Zhao et al., 2010; Park et al., 2012), this down-regulation of c-MYC in our reprogrammed OS was expected due to the involvement of c-MYC in OS progression (Broadhead et al., 2011; Han et al., 2012). Overexpression of c-MYC has been associated with increased OS invasion ability through the activation of the MEK-ERK pathway (Han et al., 2012). Previous studies have revealed that reprogramming could reduce the cancerous property of parental cancer cells (Mahalingam et al., 2012; Zhang et al., 2014; Bernhardt et al., 2017). This downregulation of c-MYC expanded the ability of reprogramming to reverse oncogenic effects in cancer cells and has been noted to be important for discovery of novel therapeutic strategies for OS. Global gene expression profiling as shown in unsupervised hierarchical clustering of DEGs was able to distinguish reprogrammed OS, iG-292, and iSaos-2, from their parental cells. The overall gene ontology (GO) enrichment analysis showed diverse enrichment of GO categories between iG-292 and iSaos-2. The variation of GO enriched categories between the two populations signifies the different reprogramming responses from both parental cell lines, G-292 and Saos-2. However, the results obtained may not represent the OS populations as the study design involves only two cell lines. None the less, the findings provide insightful information for further research on cancer derived-iPSCs or iPCs. There are a few GO terms that were enriched in the same pattern, up- or down-regulated, for both iG-292 and iSaos-2. One of the GO is the developmental process. The developmental process is a biological process that includes the developmental progression of an integrated living organism over time from an initial condition to a later condition (source: AmiGO). This is indicative that genes and gene products involved in the developmental process were differentially regulated during reprogramming of OS cells. This is consistent with a study by Liu et al. (2011) which demonstrated that iPSC generated from endoderm, ectoderm or mesoderm showed enrichment of genes associated with the developmental process. However, there were two key biological processes that were upregulated only in iG-292 dataset, which were signaling (GO: 0023052) and cellular process (GO: 0009987). Signaling (GO: 0023052) is a major GO term in biological process that triggers cellular response. Thus, the activation of both signaling and cellular process during reprogramming of G-292 into iG-292 may play a significant role in the success of reprogramming in G-292. The effect of reprogramming on the DDR and DNA repair ability in
survival of osteosarcoma and increased risk of developing OS suggested that NER plays a role in OS pathogenesis (Jin et al., 2015; Sun et al., 2015). Thus, in our functional study, we aim to study the effect of reprogramming on OS and their NER response upon UV irradiation as well as the functionality of genes related to NER in reprogrammed OS. Cyclobutane pyrimidine dimers (CPDs) are produced on the DNA of cells after UV irradiation insult. The concentration of CPDs corresponds to the level of UV damage on the DNA of affected cells and CPDs are repaired by the NER pathways. Quantification of CPDs was done using ELISA kit. Data showed a significantly lower level of CPDs in iG-292 than G-292 at two different time-points suggesting iG-292 may have more effective CPDs removal mechanism as compared to G-292 (Fig. 5a). However, similar CPDs removal pattern was observed on iSaos-2 and Saos-2 (Fig. 5a), which is consistent with the hypothesis that iSaos-2 was incompletely reprogrammed. Both data showed contrasting result as compared to osteoblast, which has limited ability to repair DNA damage. Our findings showed that reprogrammed OS, iG292 demonstrated better DNA repair response compared to the parental counterpart, G-292. After detection of CPDs, analyses of NER genes expression in reprogrammed and parental OS were performed to correlate the NER genes expression with CPDs removal. In the previous microarray dataset, GADD45G was shown to be up-regulated in iG-292 but not iSaos2 even before UV irradiation (Fig. 4a). Upon UV irradiation on reprogrammed OS, iG-292 and parental, G292, GADD45G was significantly up-regulated in all irradiated cells (Fig. 5b) indicating that UV stress activated this gene. This observation could be observed in hFOB cells after UV irradiation (Fig. 5d). Most NER genes are up-regulated in iG-292 6 h and 24 h post-irradiation while down-regulated in G-292 6 h and 24 h post-irradiation, against iG-292 and G-292 before irradiation as a control. XPA, involved in the early step of NER and responsible for DNA unwinding after initiation of repair, was significantly up-regulated in iG-292 6 h post-irradiation. This observation corresponded with the ability of iG-292 to remove CPDs adduct, as shown in Fig. 5a, at 6 h post-irradiation. On the other hand, most NER genes were down-regulated in iSaos-2 and Saos-2 (Fig. 5c) implying that UV irradiation did not activate the relevant genes. Both reprogrammed iSaos-2 and parental Saos-2 exhibited a similar pattern of NER genes expression and this arrangement corresponded with the lower CPDs removal pattern. It is postulated that this result could be due to incomplete reprogramming of Saos-2. The overall outcome of this experiment suggested that NER pathway was more efficient in fully reprogrammed cells. 4. Discussion Reprogramming of osteosarcoma (OS) offers a new approach to study the pathogenesis of OS. Traditionally, cell lines were generated from primary tumor excised from cancer patients. These primary cell lines carry phenotypes and genotypes known to the disease at the time of onset, which is normally at a terminal stage of neoplastic transformation. Any information of the disease at a more primitive or early stage of progression could not be deciphered. In this work, our reprogrammed osteosarcoma cells were able to form teratoma in vivo unlike study by Zhang X et al. that reported reduced tumorigenicity but inability to form teratoma in their reprogrammed sarcoma models (Zhang et al., 2013). Based on our in vivo results, reprogramming changed the primary cancerous property of 6
Fig. 3. Distribution of gene ontology enrichment categories based on up-regulated and down-regulated DEGs in both iG-292 and iSaos-2 respectively. Each GO domain was generated from up-regulated and downregulated DEGs in a statistically significant manner between reprogrammed OS and parental cells.
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Fig. 4. Analysis of DNA repair, cell cycle and apoptosis gene expression in both reprogrammed OS. Differentially expressed genes were obtained from reprogrammed OS against parental cells using GeneSpring GX 13.0 with fold change > 1.5 and significance level p < 0.05.
Fig. 5. Functional assay to elucidate DNA repair efficiency in reprogrammed OS. (a) Cyclobutane pyrimidine dimers (CPDs) concentration in osteoblast (hFOB), parental and reprogrammed OS at 6 h, and 24 h post UV irradiation. (b) Nucleotide excision repair (NER) genes expression after UV irradiation on G-292 and iG-292 with G-292 and iG-292 before UV irradiation as control respectively to obtain expression fold change for each gene. (c) NER genes expression after UV irradiation on Saos-2 and iSaos-2 with Saos-2 and iSaos-2 before UV irradiation as control respectively to obtain expression fold change for each gene. (d) NER genes expression after UV irradiation on osteoblasts, hFOB, serving as normal control. An asterisk (*) denotes significant level, p < 0.05.
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improved genomic stability in reprogrammed OS, thus suggesting the ability of reprogramming in reverting back OS to a primitive phenotype which is non-cancerous. In a functional assay, reprogrammed iG-292 showed better CPDs removal and DNA repair response compared to parental, G-292. Up-regulation of NER genes in iG-292 after UV irradiation is consistent with CPDs removal capability of iG-292. Since the expression of GADD45G was never reported in OS development and disease progression, reprogramming of OS enables the discovery of the role of this gene in OS formation.
reprogrammed cells was not extensively investigated. A recent study conducted by Luo et al. (2012) showed that pluripotent cells exhibit lower CPD levels than fibroblasts when exposed to equal UVC fluxes, demonstrating that pluripotent cells possess higher DNA repair capacities for NER. However, no study have been conducted on reprogrammed cancer cells. Our molecular data on reprogrammed OS demonstrated down-regulation of DNA repair, cell cycle, and apoptosis genes, all of which are associated with DDR. These observations are in contrast with the common OS characteristics such as increased DNA repair and combined cell cycle alterations and apoptosis resistance in OS, which are responsible for treatment failure and recurrence after a disease-free duration (PosthumaDeBoer et al., 2013). Differential expression of DDR genes profile in our data suggested reprogrammed OS showing more of the normal cell DDR profile rather than a cancerous DDR profile which is consistent with other OS studies (Harada et al., 2002; Li et al., 2014; Vella et al., 2016; Xu et al., 2017). The efficiency of DNA repair system is regarded as one of the most crucial mechanisms affecting patients' outcome in chemotherapy. Previous studies have linked single nucleotide polymorphisms (SNPs) of NER genes to the response of chemotherapy in osteosarcoma (Caronia et al., 2009; Biason et al., 2012; Bai et al., 2013; Jin et al., 2015). Rapid removal of CPDs which was found in iG-292 as compared to the parental counterpart suggested a more active DNA repair mechanism in iG292. On the other hand, iSaos-2 showed similar CPDs concentration pattern as the parental counterpart, Saos-2, and this could be due to failure to achieve the full reprogramming status. Up-regulation of GADD45G, XPA, and PCNA at 6 h post UV irradiation in iG-292 are consistent with the role of each gene in the NER pathway. XPA plays a role in binding to damaged DNA and facilitates assembly of repair complex at the damage site with replication protein A (RPA) (de Laat et al., 1999; Shen et al., 2014; Sugitani et al., 2017). It has been shown in previous reports that absence of XPA caused no stable pre-incision complex to form (Evans et al., 1997; Mu et al., 1997), thus NER cannot occurs. Therefore, cells deficient in XPA protein are not capable of NER and are hypersensitive to damage caused by UV radiation (Satokata et al., 1993; Köberle et al., 2006). Apart from the role in tumorigenesis, GADD45 family of genes participate in the DNA repair machinery, NER through the interaction with Proliferating Cell Nuclear Antigen (PCNA) (Tamura et al., 2012). Interaction of GADD45G and PCNA is also linked to inhibition of GADD45G as a negative regulator of cellular growth (Vairapandi et al., 2000; Azam et al., 2001). However, as both studies were conducted on non-damaged or non-irradiated cells, the role of GADD45G and PCNA in UV irradiated cells could be linked, but this remains to be defined. As GADD45G was significantly up-regulated in iG-292 before and after UV irradiation, we postulated that up-regulation of GADD45G helps to speed up the excision mechanism together with PCNA. However, as we only perform one functional assay (UV irradiation) to test the DNA repair ability of the study groups, further studies would be required to establish the exact functional role of GADD45G in DDR in reprogrammed cells. However, this study has some limitations. First, only two OS cell lines, G292 and Saos-2, were used in the study. Second, we focused on DNA Damage Response pathways and not other pathways between the groups with parental cells as the control group. A normal comparator was not used in the RNA expression study using microarray as the main focus of the study was to establish the effect of reprogramming to the differential expression of DDR genes. Nevertheless, the current findings showed an indication of an enhanced state of genomic integrity in reprogrammed OS as compared to the parental cells and further studies can be better planned to address the current limitations of this study.
Acknowledgements This research is supported by HIR-MoE Grant (Reference number UM.C/625/1/HIR/MOHE/CHAN/03, account number - A00000350001). The experiments were mainly carried out in the research laboratories at the Sungai Long Campus of the Universiti Tunku Abdul Rahman (UTAR). Competing interests The authors have declared that no competing interest exists. References Aasen, T., Raya, A., Barrero, M.J., Garreta, E., Consiglio, A., Gonzalez, F., Vassena, R., Bilić, J., Pekarik, V., Tiscornia, G., Edel, M., Boué, S., Izpisúa Belmonte, J.C., 2008. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat. Biotechnol. 26 (11), 1276–1284. https://doi.org/10.1038/nbt.1503. Allegrucci, C., Rushton, M.D., Dixon, J.E., Sottile, V., Shah, M., Kumari, R., Watson, S., Alberio, R., Johnson, A.D., 2011. Epigenetic reprogramming of breast cancer cells with oocyte extracts. Mol. Cancer 10 (1), 7. https://doi.org/10.1186/1476-4598-107. (BioMed Central Ltd). Azam, N., Vairapandi, M., Zhang, W., Hoffman, B., Liebermann, D.A., 2001. Interaction of CR6 (GADD45gamma) with proliferating cell nuclear antigen impedes negative growth control. J. Biol. Chem. 276 (4), 2766–2774. https://doi.org/10.1074/jbc. M005626200. Bai, S.-B., Chen, H.-X., Bao, Y.-X., Luo, X., Zhong, J.-J., 2013. Predictive impact of common variations in DNA repair genes on clinical outcome of osteosarcoma. Asian Pac. J. Cancer Prev. 14 (6), 3677–3680. Bernhardt, M., Novak, D., Assenov, Y., Orouji, E., Knappe, N., Weina, K., Reith, M., Larribere, L., Gebhardt, C., Plass, C., Umansky, V., Utikal, J., 2017. Melanoma-derived iPCCs show differential tumorigenicity and therapy response. Stem Cell Rep. 8 (5), 1379–1391. https://doi.org/10.1016/j.stemcr.2017.03.007. (Elsevier Company). Biason, P., Hattinger, C., Innocenti, F., Talamini, R., Alberghini, M., Scotlandi, K., Zanusso, C., Serra, M., Toffoli, G., 2012. Nucleotide excision repair gene variants and association with survival in osteosarcoma patients treated with neoadjuvant chemotherapy. Pharm. J. 12 (6), 476–483. https://doi.org/10.1038/tpj.2011.33. (Nucleotide). Broadhead, M.L., Clark, J.C.M., Myers, D.E., Dass, C.R., Choong, P.F.M., 2011. The molecular pathogenesis of osteosarcoma: a review. Sarcoma 959248. https://doi.org/10. 1155/2011/959248. Caronia, D., Patiño-García, A., Milne, R.L., Zalacain-Díez, M., Pita, G., Alonso, M.R., Moreno, L.T., Sierrasesumaga-Ariznabarreta, L., Benítez, J., González-Neira, A., 2009. Common variations in ERCC2 are associated with response to cisplatin chemotherapy and clinical outcome in osteosarcoma patients. Pharmacogenomics J. 9 (5), 347–353. https://doi.org/10.1038/tpj.2009.19. Evans, E., Moggs, J.G., Hwang, J.R., Egly, J.M., Wood, R.D., 1997. Mechanism of open complex and dual incision formation by human nucleotide excision repair factors. EMBO J. 16 (21), 6559–6573. https://doi.org/10.1093/emboj/16.21.6559. Fuchs, B., Pritchard, D.J., 2002. Etiology of osteosarcoma. Clin. Orthop. Relat. Res. 397, 40–52. https://doi.org/10.1097/00003086-200204000-00007. Gokgoz, N., Wunder, J.S., Mousses, S., Eskandarian, S., Bell, R.S., Andrulis, I.L., 2001. Comparison of p53 mutations in patients with localized osteosarcoma and metastatic osteosarcoma. Cancer 92 (8), 2181–2189. https://doi.org/10.1002/10970142(20011015)92:8<2181::AID-CNCR1561>3.0.CO;2-3. Han, G., Wang, Y., Bi, W., 2012. C-Myc overexpression promotes osteosarcoma cell invasion via activation of MEK-ERK pathway. Oncol. Res. 20 (4), 149–156. https://doi. org/10.3727/096504012X13522227232237. Harada, K., Toyooka, S., Shivapurkar, N., Maitra, A., Reddy, J.L., Matta, H., Miyajima, K., Timmons, C.F., Tomlinson, G.E., Mastrangelo, D., Hay, R.J., Chaudhary, P.M., Gazdar, A.F., 2002. Deregulation of caspase 8 and 10 expression in pediatric tumors and cell lines. Cancer Res. 62 (20), 5897–5901. Hester, M.E., Song, S., Miranda, C.J., Eagle, A., Schwartz, P.H., Kaspar, B.K., 2009. Two factor reprogramming of human neural stem cells into pluripotency. PLoS One 4 (9), e7044. https://doi.org/10.1371/journal.pone.0007044. Jin, G., Wang, M., Chen, W., Shi, W., Yin, J., Gang, W., 2015. Single nucleotide polymorphisms of nucleotide excision repair and homologous recombination repair pathways and their role in the risk of osteosarcoma. Pak. J. Med. Sci. 31 (2),
5. Conclusions The expression of down-regulation of DDR genes is consistent with 9
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DNA repair efficiency associated with reprogrammed osteosarcoma cells Pei-Feng Choong Tunku Kamarulc
a,b,⁎
b
a
b
, Hui-Xin Teh , Hoon-Koon Teoh , Han-Kiat Ong , Soon-Keng Cheong
T
a,b
,
a
National Cancer Council (MAKNA), Kuala Lumpur, Malaysia Faculty of Medicine and Health Sciences, University Tunku Abdul Rahman (UTAR), Selangor, Malaysia Tissue Engineering Group, National Orthopedic Centre of Excellence for Research and Learning, Department of Orthopedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia b c
A R T I C LE I N FO
A B S T R A C T
Keywords: DNA Damage Response DNA repair Reprogramming Osteosarcoma Nucleotide excision repair Genomic stability
Genomic instability and genetic heterogeneity are typical hallmarks of cancer, including osteosarcoma (OS), a type of bone tumor. Inactivation of DNA repair pathways may play an important role in the initiation and progression of OS pathogenesis by increasing the mutation rate and genomic instability. However, there is still a lack of information on gene alterations and mutations that may increase the risk of osteosarcoma formation. Reprogramming OS cells to a primitive stage namely induced pluripotent stem cell (iPSC) state could be a useful disease model to understand the pathogenesis of OS and to bridge the current gap of knowledge on DNA repair mechanisms in reprogrammed OS cells. By using Yamanaka factors, OS cell lines, G-292 and Saos-2, were reprogrammed to iPSC lines, respectively iG-292 and iSaos-2, both of which demonstrated pluripotency similar to embryonic stem cells. However, only iG-292 was able to form teratoma. Subsequent microarray data showed significant down-regulation of DNA Damage Response (DDR) genes expression for both iG-292 and iSaos-2. A functional assay using UV-induced DNA damage approach demonstrated efficient DNA repair mechanism in iG292. Further analysis of nucleotide excision repair (NER) genes demonstrated up-regulation of GADD45G, XPA, RPA, MNAT1, ERCC1, PCNA, and POLL, in iG-292. Up-regulation of GADD45G together with up-regulation of other NER genes synergistically repair UV damage by rapid removal of cyclobutane pyrimidine dimers. In conclusion, down-regulation of DDR genes in reprogrammed OS may render an enhanced state of genomic integrity in reprogrammed OS as compared to the parental cells. Thus, this study demonstrated for the first time DDR profile of reprogrammed OS cells and the probable involvement of GADD45G in the DNA repair mechanism of reprogrammed OS cells.
Abbreviations: ATCC, American Type Culture Collection; bFGF, basic fibroblast growth factor; CASP4, caspase 4; CASP8, caspase 8; CCNA1, cyclin A1; CDK, cyclin dependent kinase; CHK2, check point kinase 2; c-MYC, c-Myc avian myelocytomatosis viral oncogene homolog; CPD, cyclobutane pyrimidine dimer; DAPK1, deathassociated protein kinase-1; DDR, DNA Damage Response; DEG, differentially expressed gene; DSB, double-strand break; EB, embryoid body; ERCC1, excision-repair cross-complementing 1; GADD45G, growth arrest and DNA damage-inducible 45- γ; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HSC, hematopoietic stem cell; iG-292, G-292-derived induced pluripotent stem cell; IGF2, insulin-like growth factor 2; iMEF, inactivated mouse embryonic fibroblast; iPSC, induced pluripotent stem cell; iSaos-2, Saos-2-derived induced pluripotent stem cell; KLF4, Kruppel-like factor 4; MCM9, Minichromosome maintenance protein 9; MEF, mouse embryonic fibroblast; MLH1, MutL homolog 1; MNAT1, MNAT, CDK activating kinase assembly factor 1; MSC, mesenchymal stem cell; NER, nucleotide excision repair; OCT3/4, octamer-binding transcription factor 3/4; OS, osteosarcoma; OSKM, OCT4, SOX2, KLF4, c-MYC; PARP1, poly (ADP-ribose) polymerase-1; PARP3, poly (ADP-ribose) polymerase-3; PCNA, proliferating cell nuclear antigen; POLL, polymerase (DNA) lambda; REX1, ZFP42 zinc finger protein; ROCK, Rho-associated, coiled-coil containing protein kinase; RPA, replication protein A; SD, standard deviation; SEM, standard error of mean; SNP, single nucleotide polymorphism; SOX2, sex-determining region Y (SRY)-related box 2; TNFRSF1A, tumor necrosis factor receptor superfamily 1A; TP53, tumor protein 53; UV, ultraviolet; VA, valproic acid; XPA, xeroderma pigmentosum complementation group A; XPC, xeroderma pigmentosum complementation group C; XPD, xeroderma pigmentosum complementation group D; XPF, xeroderma pigmentosum complementation group F ⁎ Corresponding author at: Postgraduate Laboratory, Faculty of Medicine and Health Science, Universiti Tunku Abdul Rahman (UTAR), Bandar Sungai Long, 43000 Kajang, Malaysia. E-mail address: [email protected] (P.-F. Choong). https://doi.org/10.1016/j.genrep.2019.100409 Received 24 April 2019; Accepted 27 April 2019 Available online 01 May 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
Gene Reports 16 (2019) 100409
P.-F. Choong, et al.
1. Introduction
normal cell lines and non-cancerous iPSC. However, there is no report on the profile of DNA Damage Response of reprogrammed cancer cells as well as the functionality of DNA repair pathway genes post-cancer cells reprogramming. Hence, this study was conducted to investigate the DNA Damage Response efficiency in reprogrammed OS and parental cells.
Osteosarcoma (OS), a type of bone cancer, can be considered as an ancient disease. One of the earliest cases of OS was documented to be dated between 1.6 and 1.8 million years old in the South African Journal of Science (Odes et al., 2016). This discovery implied that the pathogenesis for OS was already buried deep within the human historical evolution and has no relation to the present human lifestyle. OS is a rare tumor diagnosed prevalent in children and adolescents. Conventional treatment for OS, by combining chemotherapy and surgery, had increased the 5-year survival rates to 60%–70% (Mankin et al., 2004). However, over the years these figures remain unchanged. OS pathogenesis has been linked to genetic changes during the osteoblast differentiation process (Gokgoz et al., 2001). Genetic modifications associated with impairment of proliferation and differentiation capacity eventually increase the potential of tumorigenicity (Kenyon and Gerson, 2007). One consistent finding in all these OS studies pointed to a higher incidence of OS in individuals with mutation in genes that are involved in stabilizing the genome, such as p53 and retinoblastoma protein (RB1). Impairment of these related genes causes defective maintenance of DNA (Fuchs and Pritchard, 2002; Martin et al., 2012). OS is associated with high level of genomic instability (Selvarajah et al., 2007). This could be due to a mutation in p53 as mutant forms of p53 are significantly associated with greater genomic instability in OS (Overholtzer et al., 2003). Deregulation of TP53, a product of p53 gene, is linked with OS advancement and occurs due to a mutation of the gene locus at 17p13.1 (Martin et al., 2012). Thus, OS is often characterized as having extensive genomic instability, uncontrolled apoptosis, uncontrolled cell cycle, and lack of differentiation ability (Martin et al., 2012; Varshney et al., 2016). Development in the field of induced pluripotent stem cells (iPSC) has provided the means to study OS pathogenesis. iPSCs are pluripotent cells reprogrammed from human somatic cells through ectopic expression of OSKM transcription factors (Takahashi et al., 2007). Reprogramming of OS cell lines to a more primitive stage, however, retains the acquired genetic mutations in OS. iPSC OS cell lines thus could help to provide valuable insight into the OS pathogenesis by understanding the acquired genetic changes during the transformation process to an advanced stage. Reprogramming of OS also helps to yield a larger population of pluripotent cancer cells which could be cryopreserved and propagated for long term study (Tafani, 2012). Zhang et al. (2013) demonstrated that reprogrammed sarcoma cells lost their tumorigenicity and could dedifferentiate to mesenchymal stem cells (MSC) state. The study also showed differentiation of mature connective tissues and red blood cells from hematopoietic stem cell (HSC)-like cells, suggesting the ability of sarcoma cells to reverse back to a mutual stage of ancestor before branching out to HSC and MSC. This study showed that cancer cells could be ‘normalized’ via reprogramming despite carrying inherent cancer-causing mutations. However not much is known about the genomic stability underlying the reprogramming process in sarcoma cells. Reprogramming cancer cells into induced pluripotent cancer cells (iPCs) has been shown to reset some of the characteristics of cancer cells, and that these iPCs behave distinctly from their parental cells upon reprogramming (Allegrucci et al., 2011; Mahalingam et al., 2012). Combining molecular information collected from cancer iPSC models with the present knowledge of OS biology could assist us in gaining a deeper understanding of the pathological mechanisms triggering formation of OS. This could eventually aid in the prospective development of future OS therapies. Therefore, elucidation of individualized OS-associated gene functions to investigate the potential pathological mechanisms involved in the different stages of OS development from initiation to progression is vital for future OS detection and treatment. To date, most of the reported DNA repair studies were conducted on
2. Materials and methods 2.1. Cell lines Osteoblast cell line, hFOB, and osteosarcoma cell lines, Saos-2 and G-292, were purchased from ATCC and maintained in the following conditions; Saos-2: Dulbecco's Modified Eagle Medium, low glucose (DMEM-LG), 10% fetal bovine serum (FBS), 1% Pen-Strep (Gibco, Carlsbad, CA, USA), G-292: McCoy's 5a Medium, 10% FBS, 1% PenStrep, and hFOB: Dulbecco's Modified Eagle Medium (DMEM)/F-12, 10% fetal bovine serum (FBS), 1% Pen-Strep and Geneticin (Gibco, Carlsbad, CA, USA). Reprogrammed cells were maintained in human embryonic stem cells (hESC) medium constituted of DMEM/F12 supplemented with 20% knockout serum replacement (KOSR), 0.1 mM bmercaptoethanol (Merck, New Jersey, USA), 10 ng/ml basic fibroblast growth factor (bFGF) (Miltenyi Biotech, Germany), 1× non-essential amino-acid (NEAA), 1 mM L-glutamine and 0.1× Pen-Strep. Irradiated mouse embryonic fibroblasts (iMEF) (Global Stem, Rockville, MD, USA), were used as feeder cells and cultured in DMEM with 10% FBS. iPSC colonies were manually picked and plated on iMEF plates. All cells were cultured at 37 °C in a 5% CO2 incubator.
2.2. OS-iPSC generation Human OS-iPSC cells were produced as previously described, with slight modification (Sugii et al., 2011). Retroviral vectors for each of the transcription factors genes (pMX-c-MYC, pMX-KLF4, pMX-OCT4, or pMX-SOX2) were transfected with envelope genes (gag/pol and VSV-G) into 293FT cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). OS cells were seeded at a density of 40–60% confluency one day before transduction. Fresh retrovirus supernatants were supplemented with 8 mg/ml Polybrene (Millipore, Merck) prior to transduction. Equal amounts of supernatants containing each of the four retroviruses carrying the OCT4, SOX2, c-MYC and KLF4 genes, were mixed and transferred to the cells. The plates were centrifuged at 800g for 50 min and incubated overnight at 37 °C, 5% CO2. Fresh medium was changed on the next day and subsequently every day. Transduced cells were transferred to iMEF on day 3 post-transduction. Cells were monitored every day for the formation of colonies. Colonies were manually picked on day 15–day 20 and transferred to new iMEF. Generated OS-iPSC was characterized prior to microarray and functional assay.
2.3. Microarray analysis RNA extraction was performed using Qiagen miRNeasy ® Mini Kit (Qiagen, Germany) according to manufacturer's protocol. The RNA integrity test was done using 2100 Bioanalyzer (Agilent, Germany). Total RNA from reprogrammed OS and parental were subjected to RNA target preparation for microarray expression analysis using GeneChip ® 3′IVT Express Kit (Affymetrix, USA) according to manufacturer's protocol. After hybridization, arrays scanned using GeneChip® Scanner 3000 7G. Microarray data were imported into GeneSpring GX13.0 (Agilent, Germany) for analysis. Genes with fold change (FC) > 2 (FC > 2) and significance level, p < 0.05 were selected for validation. Raw data generated from Affymetrix microarray platform was deposited to NCBI GEO Omnibus with accession number GSE107855. 2
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expression profiling is one of the best-used methods to characterize reprogrammed population and to identify the degree of reprogramming on target cells as it offers unbiased, whole genome approach to examine both populations (Park et al., 2011; Liu et al., 2012; Medvedev et al., 2013). Upon using unsupervised hierarchical clustering analysis, the clustering demonstrated the distinctive separation of two clustered populations between the parental and the reprogrammed OS (Fig. 2a). Both reprogrammed OS showed more down-regulated differentially expressed genes (DEGs) than up-regulated DEGs (Table 1). These highly DEGs were further categorized based on their functional annotation into three categories in Gene ontology (GO), namely cellular component, molecular function and biological processes (Fig. 2b). The overall GO enrichment analysis showed diverse enrichment of GO categories between iG-292 and iSaos-2. The variability of GO enriched categories between the two populations implying the different reprogramming responses from both parental cell lines, G-292 and Saos-2. Further analyses were done on GO based on up-regulated and downregulated DEGs. In up-regulated DEGs, molecular function domain in both reprogrammed OS showed similar up-regulation in binding; iG292 (60.51%) and iSaos-2 (100%). In biological processes, single organism process was listed as top two contributors in both reprogrammed iG-292 and iSaos-2. Meanwhile, only up-regulated DEGs from iG-292 produced GO enrichment for cellular component, with the majority on cell (49.67%) and cell part (49.67%); while up-regulated DEGs from iSaos-2 did not generate any GO enrichment for cellular component (Fig. 3). For down-regulated DEGs, GO enriched category “binding” was the top in both iG-292 (77.7%) and iSaos-2 (100%) in molecular function domain. In biological processes, the top category for iG-292 was cell processes (16.6%) and single organism processes (17.92%) for iSaos-2. For cellular component domain, cell and cell part were listed top in iG292 (21.91% for both), but were listed as third in iSaos-2 (16.63% respectively). Membrane was listed top in cellular component domain for iSaos-2 (25.02%) (Fig. 3).
2.4. Taqman Gene Expression Assay for validation and NER genes expression Briefly, Taqman® Gene Expression Assay (20×), cDNA samples and Taqman® Fast Advanced Master Mix (2×) were thawed on ice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control gene in this experiment. An appropriate number of reactions were prepared according to the volumes in manufacturer's protocol. The components were mixed thoroughly, then the 0.2 ml strip-tubes were centrifuged briefly to spin down the content and eliminate any bubbles. The tubes were then placed in the thermal cycler (StepOne Plus, Thermo Fisher Scientific, USA). 2.5. Cyclobutane pyrimidine dimers (CPD)quantification Prior to UV treatment, cells grown till 80% confluency were washed and the medium was replaced with 10 ml phosphate buffered saline (PBS) with Calcium and Magnesium. Cells were irradiated at UV-dose of 40 J/m2 UVC (254 nm) with UVILite LF-206-LS (UVITEC, UK). Following treatments, cells were fed culture medium and incubated for 1 h, 6 h or 24 h to allow for DNA repair mechanism to take place. Cells were harvested for DNA extraction using Qiagen DNA extraction kit and RNA extraction using Qiagen RNA extraction kit. UV-induced DNA damage was detected by OxiSelect UV-Induced DNA damage ELISA Combo Kit (CPD quantitation) (Cell Biolabs, USA). Osteoblast cell line, hFOB, was used as a normal comparator to set the UV dosage. 3. Results 3.1. Reprogramming & characterization of reprogrammed OS cells Generation of G-292-derived iPSC, termed as iG-292, and Saos-2derived iPSC, termed as iSaos-2, was successful with a single transduction of retroviral with OSKM transcription factors. Morphologies of the selected iG-292 and iSaos-2 clones resembled ESC with clear defined borders and consisting of cells with high nucleus to cytoplasm ratio, prominent nucleoli, tightly packed cells with clear defined borders (Fig. 1a) (Thomson et al., 1998). Similar to other iPSC studies, higher expression levels of pluripotent markers, OCT3/4, SOX2, NANOG, and REX1 were also detected in both reprogrammed OS cells as compared to their parental cells (Fig. 1b). Pluripotent markers are markers expressed at higher level in pluripotent stem cells than terminally differentiated cells. Detection of these pluripotent markers distinguished pluripotent cells, such as ESC and iPSC, from somatic cells or parental cells. Embryoid body formation is another characteristic for ESC and iPSC. The ability to cluster together and form suspension body is associated with pluripotency (Li and Rana, 2012). Product of spontaneous differentiation from embryoid body showed gene expression for three germ-layers: MSX1, GATA2 and hBRACHYURY (mesoderm markers); FOXA2 and GATA4 (endoderm markers); and CDX2 (ectoderm marker) in iG-292, while iSaos-2 only expressed MSX1 and GATA2 (mesoderm); GATA6 (endoderm) and TUJ1 (ectoderm) (Fig. 1c). Teratoma/xenograft formation in vivo is another developmental characteristic of pluripotent stem cells. Pluripotency of reprogrammed OS cells was tested for teratoma formation. Histological analysis using H&E staining showed that only iG-292 managed to form teratoma in vivo. 4 out of 5 nude mice injected with iG-292 formed teratoma with the morphology of cells representing three germ layers; ectoderm, mesoderm and endoderm (Fig. 1d).
3.3. Elucidation of DNA Damage Response (DDR) pathways upon OS reprogramming Differentially expressed genes (DEGs) between reprogrammed OS and their parental cells were grouped in accordance to the pathway in which they participate in DDR system: DNA repair, cell cycle, and apoptosis (Fig. 4a–c). iG-292 displayed more DEGs in each pathway as compared to iSaos-2. There was contradictory expression between iG292 and iSaos-2 in 3 genes, CASP8, PARP3, and MLH1, where iG-292 showed down-regulation while iSaos-2 showed up-regulation of these genes. Only PARP1 was up-regulated in both iG-292 and iSaos-2 (Fig. 4a). As for cell cycle genes, 8 genes were up-regulated in iG-292 and 2 genes were up-regulated in iSaos-2. Meanwhile, 15 genes were downregulated in iG-292 and 3 genes were down-regulated in iSaos-2. Only 1 gene, MCM9, showed conflicting expression with up-regulation in iG292 but down-regulated in iSaos-2. While only CCNA1 showed upregulation in both iG-292 and iSaos-2 (Fig. 4b). 11 apoptotic genes were up-regulated in iG-292 and 5 genes in iSaos-2. Whereas, 26 genes were down-regulated in iG-292 and 5 genes in iSaos-2. In iG-292, DAPK1 was up-regulated and CASP8 was downregulated, but iSaos-2 showed the opposite expression. Only IGF2 was up-regulated in both iG-292 and iSaos-2 while CASP4, TNFRSF1A, and MYC were down-regulated in both iG-292 and iSaos-2 (Fig. 4c). 3.4. Functional assay to explicate genes related to DNA repair mechanism in reprogrammed OS
3.2. Global gene expression profile of the reprogrammed OS cells Microarray technology with global gene transcription pattern was used to measure similarity or disparity between two populations, such as reprogrammed cells against their parental cells. Global gene
Recent studies on genetic polymorphisms in nucleotide excision repair (NER) in relation to lower chemotherapy response, unfavorable 3
Fig. 1. Generation and characterization of reprogrammed OS. (a) The emergence of ESC-like colonies on Day 21 post-transduction of reprogrammed G-292 (iG-292) and reprogrammed Saos-2 (iSaos-2). (b) Expression of pluripotent genes, SOX2, OCT3/4, and NANOG, in reprogrammed OS. (c) Multilineage genes expression during EB formation, indicating attained pluripotency post reprogramming in iG-292 and iSaos-2. (d) Formation of neuronal rosette-like structures indicating ectoderm layer, capillary-sized blood vessels, adipocytes cells, and also fibrous muscle-like cells indicating mesoderm layer and columnar epithelial cells seen as lining glands, and the papillary structures indicating endoderm layers. . An asterisk (*) denotes significant level, p < 0.05.
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Fig. 2. Gene expression profile of reprogrammed OS. (a) Hierarchical clustering analysis of parental and reprogrammed OS showing distinctive clustering between parental and reprogrammed OS cells. (b) Distribution of gene ontology enriched categories for reprogrammed OS, iG-292 and iSaos-2. GO analysis was generated based on reprogrammed OS DEGs against parental DEGs as a control.
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Table 1 Differentially expressed genes between parental and reprogrammed OS with fold change ≥2 and significance level, p < 0.05.
iG-292 vs G-292 iSaos-2 vs Saos-2
Up-regulated genes
Down-regulated genes
Total differentially expressed genes
992 410
1875 730
2867 1140
parental G-292 to another phenotype of iG-292 capable of forming teratoma. Following characterization, iG-292 is believed to be fully reprogrammed in contrast to iSaos-2, which did not form teratoma in vivo. Reprogramming roadblock or resistance has been reported in a few studies using both somatic and diseased cells. There are many reasons attributed to this reprogramming roadblock. One of the hindrances in reprogramming is the expression of TGFβ1. Only Saos-2 expressed TGFβ1, which was reported previously as a roadblock to reprogramming (Li et al., 2010; Verusingam et al., 2017). Expression of c-MYC is often debated in reprogramming of both somatic and cancer cells. c-MYC is a known oncogene, playing a substantial role in cellular growth regulation and metabolism (Miller et al., 2012). Down-regulation of c-MYC expression was reported in previous sarcoma reprogramming (Zhang et al., 2013) similar to our observation in both reprogrammed OS. Though expression of c-MYC has always been reported to be up-regulated in somatic reprogramming (Aasen et al., 2008; Hester et al., 2009; Zhao et al., 2010; Park et al., 2012), this down-regulation of c-MYC in our reprogrammed OS was expected due to the involvement of c-MYC in OS progression (Broadhead et al., 2011; Han et al., 2012). Overexpression of c-MYC has been associated with increased OS invasion ability through the activation of the MEK-ERK pathway (Han et al., 2012). Previous studies have revealed that reprogramming could reduce the cancerous property of parental cancer cells (Mahalingam et al., 2012; Zhang et al., 2014; Bernhardt et al., 2017). This downregulation of c-MYC expanded the ability of reprogramming to reverse oncogenic effects in cancer cells and has been noted to be important for discovery of novel therapeutic strategies for OS. Global gene expression profiling as shown in unsupervised hierarchical clustering of DEGs was able to distinguish reprogrammed OS, iG-292, and iSaos-2, from their parental cells. The overall gene ontology (GO) enrichment analysis showed diverse enrichment of GO categories between iG-292 and iSaos-2. The variation of GO enriched categories between the two populations signifies the different reprogramming responses from both parental cell lines, G-292 and Saos-2. However, the results obtained may not represent the OS populations as the study design involves only two cell lines. None the less, the findings provide insightful information for further research on cancer derived-iPSCs or iPCs. There are a few GO terms that were enriched in the same pattern, up- or down-regulated, for both iG-292 and iSaos-2. One of the GO is the developmental process. The developmental process is a biological process that includes the developmental progression of an integrated living organism over time from an initial condition to a later condition (source: AmiGO). This is indicative that genes and gene products involved in the developmental process were differentially regulated during reprogramming of OS cells. This is consistent with a study by Liu et al. (2011) which demonstrated that iPSC generated from endoderm, ectoderm or mesoderm showed enrichment of genes associated with the developmental process. However, there were two key biological processes that were upregulated only in iG-292 dataset, which were signaling (GO: 0023052) and cellular process (GO: 0009987). Signaling (GO: 0023052) is a major GO term in biological process that triggers cellular response. Thus, the activation of both signaling and cellular process during reprogramming of G-292 into iG-292 may play a significant role in the success of reprogramming in G-292. The effect of reprogramming on the DDR and DNA repair ability in
survival of osteosarcoma and increased risk of developing OS suggested that NER plays a role in OS pathogenesis (Jin et al., 2015; Sun et al., 2015). Thus, in our functional study, we aim to study the effect of reprogramming on OS and their NER response upon UV irradiation as well as the functionality of genes related to NER in reprogrammed OS. Cyclobutane pyrimidine dimers (CPDs) are produced on the DNA of cells after UV irradiation insult. The concentration of CPDs corresponds to the level of UV damage on the DNA of affected cells and CPDs are repaired by the NER pathways. Quantification of CPDs was done using ELISA kit. Data showed a significantly lower level of CPDs in iG-292 than G-292 at two different time-points suggesting iG-292 may have more effective CPDs removal mechanism as compared to G-292 (Fig. 5a). However, similar CPDs removal pattern was observed on iSaos-2 and Saos-2 (Fig. 5a), which is consistent with the hypothesis that iSaos-2 was incompletely reprogrammed. Both data showed contrasting result as compared to osteoblast, which has limited ability to repair DNA damage. Our findings showed that reprogrammed OS, iG292 demonstrated better DNA repair response compared to the parental counterpart, G-292. After detection of CPDs, analyses of NER genes expression in reprogrammed and parental OS were performed to correlate the NER genes expression with CPDs removal. In the previous microarray dataset, GADD45G was shown to be up-regulated in iG-292 but not iSaos2 even before UV irradiation (Fig. 4a). Upon UV irradiation on reprogrammed OS, iG-292 and parental, G292, GADD45G was significantly up-regulated in all irradiated cells (Fig. 5b) indicating that UV stress activated this gene. This observation could be observed in hFOB cells after UV irradiation (Fig. 5d). Most NER genes are up-regulated in iG-292 6 h and 24 h post-irradiation while down-regulated in G-292 6 h and 24 h post-irradiation, against iG-292 and G-292 before irradiation as a control. XPA, involved in the early step of NER and responsible for DNA unwinding after initiation of repair, was significantly up-regulated in iG-292 6 h post-irradiation. This observation corresponded with the ability of iG-292 to remove CPDs adduct, as shown in Fig. 5a, at 6 h post-irradiation. On the other hand, most NER genes were down-regulated in iSaos-2 and Saos-2 (Fig. 5c) implying that UV irradiation did not activate the relevant genes. Both reprogrammed iSaos-2 and parental Saos-2 exhibited a similar pattern of NER genes expression and this arrangement corresponded with the lower CPDs removal pattern. It is postulated that this result could be due to incomplete reprogramming of Saos-2. The overall outcome of this experiment suggested that NER pathway was more efficient in fully reprogrammed cells. 4. Discussion Reprogramming of osteosarcoma (OS) offers a new approach to study the pathogenesis of OS. Traditionally, cell lines were generated from primary tumor excised from cancer patients. These primary cell lines carry phenotypes and genotypes known to the disease at the time of onset, which is normally at a terminal stage of neoplastic transformation. Any information of the disease at a more primitive or early stage of progression could not be deciphered. In this work, our reprogrammed osteosarcoma cells were able to form teratoma in vivo unlike study by Zhang X et al. that reported reduced tumorigenicity but inability to form teratoma in their reprogrammed sarcoma models (Zhang et al., 2013). Based on our in vivo results, reprogramming changed the primary cancerous property of 6
Fig. 3. Distribution of gene ontology enrichment categories based on up-regulated and down-regulated DEGs in both iG-292 and iSaos-2 respectively. Each GO domain was generated from up-regulated and downregulated DEGs in a statistically significant manner between reprogrammed OS and parental cells.
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Fig. 4. Analysis of DNA repair, cell cycle and apoptosis gene expression in both reprogrammed OS. Differentially expressed genes were obtained from reprogrammed OS against parental cells using GeneSpring GX 13.0 with fold change > 1.5 and significance level p < 0.05.
Fig. 5. Functional assay to elucidate DNA repair efficiency in reprogrammed OS. (a) Cyclobutane pyrimidine dimers (CPDs) concentration in osteoblast (hFOB), parental and reprogrammed OS at 6 h, and 24 h post UV irradiation. (b) Nucleotide excision repair (NER) genes expression after UV irradiation on G-292 and iG-292 with G-292 and iG-292 before UV irradiation as control respectively to obtain expression fold change for each gene. (c) NER genes expression after UV irradiation on Saos-2 and iSaos-2 with Saos-2 and iSaos-2 before UV irradiation as control respectively to obtain expression fold change for each gene. (d) NER genes expression after UV irradiation on osteoblasts, hFOB, serving as normal control. An asterisk (*) denotes significant level, p < 0.05.
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improved genomic stability in reprogrammed OS, thus suggesting the ability of reprogramming in reverting back OS to a primitive phenotype which is non-cancerous. In a functional assay, reprogrammed iG-292 showed better CPDs removal and DNA repair response compared to parental, G-292. Up-regulation of NER genes in iG-292 after UV irradiation is consistent with CPDs removal capability of iG-292. Since the expression of GADD45G was never reported in OS development and disease progression, reprogramming of OS enables the discovery of the role of this gene in OS formation.
reprogrammed cells was not extensively investigated. A recent study conducted by Luo et al. (2012) showed that pluripotent cells exhibit lower CPD levels than fibroblasts when exposed to equal UVC fluxes, demonstrating that pluripotent cells possess higher DNA repair capacities for NER. However, no study have been conducted on reprogrammed cancer cells. Our molecular data on reprogrammed OS demonstrated down-regulation of DNA repair, cell cycle, and apoptosis genes, all of which are associated with DDR. These observations are in contrast with the common OS characteristics such as increased DNA repair and combined cell cycle alterations and apoptosis resistance in OS, which are responsible for treatment failure and recurrence after a disease-free duration (PosthumaDeBoer et al., 2013). Differential expression of DDR genes profile in our data suggested reprogrammed OS showing more of the normal cell DDR profile rather than a cancerous DDR profile which is consistent with other OS studies (Harada et al., 2002; Li et al., 2014; Vella et al., 2016; Xu et al., 2017). The efficiency of DNA repair system is regarded as one of the most crucial mechanisms affecting patients' outcome in chemotherapy. Previous studies have linked single nucleotide polymorphisms (SNPs) of NER genes to the response of chemotherapy in osteosarcoma (Caronia et al., 2009; Biason et al., 2012; Bai et al., 2013; Jin et al., 2015). Rapid removal of CPDs which was found in iG-292 as compared to the parental counterpart suggested a more active DNA repair mechanism in iG292. On the other hand, iSaos-2 showed similar CPDs concentration pattern as the parental counterpart, Saos-2, and this could be due to failure to achieve the full reprogramming status. Up-regulation of GADD45G, XPA, and PCNA at 6 h post UV irradiation in iG-292 are consistent with the role of each gene in the NER pathway. XPA plays a role in binding to damaged DNA and facilitates assembly of repair complex at the damage site with replication protein A (RPA) (de Laat et al., 1999; Shen et al., 2014; Sugitani et al., 2017). It has been shown in previous reports that absence of XPA caused no stable pre-incision complex to form (Evans et al., 1997; Mu et al., 1997), thus NER cannot occurs. Therefore, cells deficient in XPA protein are not capable of NER and are hypersensitive to damage caused by UV radiation (Satokata et al., 1993; Köberle et al., 2006). Apart from the role in tumorigenesis, GADD45 family of genes participate in the DNA repair machinery, NER through the interaction with Proliferating Cell Nuclear Antigen (PCNA) (Tamura et al., 2012). Interaction of GADD45G and PCNA is also linked to inhibition of GADD45G as a negative regulator of cellular growth (Vairapandi et al., 2000; Azam et al., 2001). However, as both studies were conducted on non-damaged or non-irradiated cells, the role of GADD45G and PCNA in UV irradiated cells could be linked, but this remains to be defined. As GADD45G was significantly up-regulated in iG-292 before and after UV irradiation, we postulated that up-regulation of GADD45G helps to speed up the excision mechanism together with PCNA. However, as we only perform one functional assay (UV irradiation) to test the DNA repair ability of the study groups, further studies would be required to establish the exact functional role of GADD45G in DDR in reprogrammed cells. However, this study has some limitations. First, only two OS cell lines, G292 and Saos-2, were used in the study. Second, we focused on DNA Damage Response pathways and not other pathways between the groups with parental cells as the control group. A normal comparator was not used in the RNA expression study using microarray as the main focus of the study was to establish the effect of reprogramming to the differential expression of DDR genes. Nevertheless, the current findings showed an indication of an enhanced state of genomic integrity in reprogrammed OS as compared to the parental cells and further studies can be better planned to address the current limitations of this study.
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5. Conclusions The expression of down-regulation of DDR genes is consistent with 9
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