OPA1 gene. Our previous investigation identified the c.1198C > G (p.P400A) mutation in the OPA1 in a large Han Chinese family with ADOA. In this report, we performed a functional characterization using lymphoblostoid cell lines derived from affected members of this family and control subjects. Mutant cell lines exhibited the aberrant mitochondrial morphology. A ~24.6% decrease in the mitochondrial DNA (mtDNA) copy number was observed in mutant cell lines, as compared with controls. Western blotting analysis revealed the variable reductions (~45.7%) in four mtDNA-encoded polypeptides in mutant cell lines. The impaired mitochondrial translation caused defects in respiratory capacity. Furthermore, defects in mitochondrial ATP synthesis and mitochondrial membrane potential (ΔΨm) were observed in mutant cell lines. These abnormalities resulted in the accumulation of oxidative damage and increasing of apoptosis in the mutant cell lines, as compared with controls. All those alterations may cause the primary degeneration of RGCs and subsequent visual loss. These data provided the direct evidence for c.1198C > G mutation leading to ADOA. Our findings may provide new insights into the understanding of pathophysiology of ADOA.Autosomal dominant optic atrophy (ADOA) is a dominantly inherited optic neuropathy, affecting the specific loss of retinal ganglion cells (RGCs). The majority of affected cases of ADOA are associated with mutations in  This disorder is characterized by an insidious loss of visual acuity in early childhood with color vision deficits, central scotoma (blind spot) and temporal or diffuse pallor of the optic disc3. The typical features in this disorder involve the primary degeneration of RGCs, and accompanied by ascending optic atrophy5. The clear autosomal dominant pattern of inheritance and congenital nature of ADOA can be distinguished it from Leber hereditary optic atrophy (LHON), which exhibited the maternal inheritance and late-onset of optic atrophy and was caused by mutations in mitochondrial DNA (mtDNA)8. Mutations among five genes OPA1, OPA3, OPA4, OPA5 and OPA8 have been identified to be associated with ADOA13. Of these, the OPA1 gene, encoding a mitochondrial dynamin-like GTPase protein, is the most prevalently causative gene for ADOA14. About 350 variants of OPA1 gene, consisting of 30 exons, were identified from various ethnic origins (http://mitodyn.org/home.php)14. The majority of OPA1 mutations are located at the GTPase domain (Exons 8–15) and dynamin central regions (Exons 16–23)14. The primary defects of OPA1 were the altered mitochondrial fusion and morphology17. These mutations also affected the mitochondrial genome maintenance19. As a result, mutations in OPA1 gene caused the deficient oxidative phosphorylation (OXPHOS) and sensitivity to apoptosis24.Autosomal dominant optic atrophy  is the most common inherited optic neuropathies, affecting one individual per 12,000 to 35,0008. In our previous study, we reported a large four-generation Han Chinese family with ADOA25. Twelve of 44 family members were affected in this pedigree, and the ratio of affected male and female relatives was 1:1. The affected family members exhibited the early-onset and progressive visual impairment, ranging from mild to profound loss of visual acuity. The average age-at-onset was 14 years. The sequencing analysis of entire OPA1 gene identified a novel c.1198C > G (p.P400A) heterozygous mutation at the exon 12 in all affected members of this family. The p.P400A mutation located at a highly conserved residue of GTPase domain of this protein. To further investigate the pathogenic mechanism of the c.1198C > G (p.P400A) mutation, lymphoblastoid cell lines were generated from 3 affected members carrying the c.1198C > G mutation and 3 control subjects lacking any of OPA1 mutation. These cell lines were assessed for the effects of the OPA1 mutation on mitochondrial morphology, mtDNA copy number, oxygen consumption ratio (OCR), ATP production, mitochondrial membrane potential, generation of reactive oxygen species (ROS) and sensitivity to apoptosis.Despite progress made in the identification of the genetic defects associated with ADOA in European and North American populations, less has been known about the pathogenesis of ADOA in the Asians, especially in the Chinese population. With aim to identify the molecular basis of ADOA in Chinese population, we recruited a large cohort of Chinese subjects with ADOA and LHONImmortalized lymphoblastoid cell lines used for biochemical assays were derived from three affected members carrying the c.1198C > G mutation and one vision normal member lacking the mutation of the family, and two genetically unrelated control individuals with the same mitochondrial background  and one vision normal subject (III-12) were stained with mitochondrial-specific dye MitoTracker RedTM and with DNA specific dye DAPI and analyzed with a confocal microscope system. As shown in Fig. OPA1 mutant cells (III-11) displayed a distinct punctate pattern, as compared with a balanced mitochondrial network in control cells (III-12). The mitochondria morphology from mutant and control lymphoblastoid cell lines was further examined by using transmission electron microscopy. As shown in Fig. To assess the effects of the OPA1 affected the copy number of mtDNA18. Here, we examined the mtDNA copy number by quantitative PCR using total genomic DNA as template. For this purpose, we made standard curve of three pairs of primers , as shown in the Supplemental Figure P = 0.0056) and using fragments spanning MT-ND1 gene, from 79.1% to 83.5%, with an average of 81.5% (P = 0.0103), respectively, related to average values of control cell lines.It was anticipated that the alteration in maintenance of mitochondrial genome caused by mutation in P = 0.0002) of the man value measured in three control cell lines. Notably, the average levels of p.MT-ND5, p.MT-Cytb, p.MT-CO2 and p.MT-ATP6 in the mutant cell lines were 48.4%, 38.7%, 60.4% and 35.2% of the average values relative to the mean value measured in the control cell lines, respectively  relative to the mean value measured in three control cell lines. To investigate which of the enzyme complexes of the respiratory chain was affected in the mutants, OCR were measured after the sequential addition of oligomycin (inhibit the ATP synthase), FCCP , rotenone (to inhibit complex I) and antimycin A (to inhibit complex III)28. The difference between the basal OCR and the drug-insensitive OCR yields the amount of ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR. As shown in Fig. P = 0.0114, 0.9606, 0.0036, 0.0219 and 0.1465), respectively.To evaluate if the c.1198C > G heterozygous mutation alters cellular bioenergetics, we measured the OCR of three mutant and three control cell lines using a Seahorse Bioscience XF-96 Extracellular Flux Analyzer. As shown in Fig. 29. The levels of ATP production in mutant cell lines in the presence of glucose  or 2-DG with pyruvate to inhibit the glycolysis (oxidative phosphorylation) were comparable with those measured in the control cell lines. By contrast, total cellular levels of ATP have no difference between mutant and control cell lines   with pyruvatenes Fig. , while t71) Fig. .Figure 5P = 0.0156) of the mean value measured in three control cell lines. By contrast, the levels of ΔΨm in mutant cell lines in the presence of FCCP were comparable with those measured in the control cell lines (P = 0.5986).The mitochondrial membrane potential (ΔΨm) alterations were measured using a fluorescence probe JC-10 assay system. The ratio of fluorescence intensities Ex/Em = 490/590 and 490/525 nm (FL590/FL525) were recorded to delineate the ΔΨm level of each sample. The relative ratios of FL590/FL525 geometric mean between mutant and control cell lines were calculated to represent the level of ΔΨm. As shown in Fig. 2O2 stimulation30. Geometric mean intensity was recorded to measure the rate of ROS in each sample. The ratio of geometric mean intensity between unstimulated and stimulated cell lines with H2O2 was calculated to delineate the reaction upon increasing level of ROS under oxidative stress. As shown in Fig. P = 0.0090) of the mean value measured in three control cell lines. Furthermore, the levels of mitochondrial ROS (mito-ROS) among lymphoblastoid cell lines were determined using a MitoSOX assay via flow cytometry32. As show in Fig. P = 0.0142) of the man value measured in three control cell lines.The levels of the ROS generation in the vital cells derived from three affected relatives carrying the c.1198C > G mutation and three control individuals lacking this mutation were measured with flow cytometry under normal and HP = 0.0497, 0.0050, 0.0136 and 0.0489).To evaluate if the c.1198C > G mutation affects the apoptotic process, we examined the apoptotic state of mutant and control lymphoblastoid cell lines by using immunocytostaining with the antibodies and analyzed with a confocal microscope system and Western blotting analysis. Figure OPA1 gene encoding a mitochondrial dynamin-like GTPase protein is the most important causative gene for ADOA. Indeed, OPA1 is a critical factor connecting the mitochondrial morphology and energetics. In this investigation, we characterized the novel c.1198C > G (p. P400A) mutation in OPA1 gene using lymphoblastoid cell lines generated from members of a large Chinese family with ADOA. The p. P400A mutation was located at a highly conserved residue of GTPase domain where the majority of pathogenic mutations in OPA1 gene cluster14. The G401 residue is one of the key residues in the G3 (DxxG) motif and may form a hydrogen bond with the γ-phosphate of GTP15. Furthermore, the p.P400A mutation may also cause misalignment of GTP in the GTP-binding pocket of the GTPase domain of OPA1, resulting in mitochondrial dysfunction25. It was anticipated that the p.P400A mutation caused the mitochondrial fragmentation, instability of mtDNA, and OXPHOS defect. The primary defects in the mutations of OPA1 gene appeared to alter the mitochondrial morphology and fusion36. In the present study, mutant lymphoblastoid cell lines carrying the heterozygous c.1198C > G mutation revealed the altered mitochondrial dynamics and led to the fragmented network, compared with control cell lines. In particular, the mutant cells exhibited the perturbed mitochondrial morphology including mitochondrial fragmentation, swollen and cristae structure and spotted distribution of mitochondria, as in the case of cell lines carrying the other OPA1 mutations36. In fact, the altered fusion activity and the increase in mitochondrial network fragmentation in mutant cells carrying the c.1198C > G mutation may be due to the dominant negative effect17.The 18. The mutations in OPA1 gene led to the depletion of mtDNA in cells and skeletal muscular tissues derived from ADOA subjects38. In the present study, 24.6% decreases in mtDNA content observed in mutant cells were due to the unstable maintenance of mtDNA caused by the OPA1 mutation. The unstablity of mtDNA caused by the OPA1 mutation contributed to the deficient mitochondrial translation. In the present study, a markedly decreased level of mitochondrial proteins (an average decrease of ~45.7%) was observed in mutant cells, as compared to the average levels in control cells. The reduced level of variable mitochondrial proteins detected in lymphoblastoid cell lines using a Western blot analysis was comparable with the reduced rate of mitochondrial protein synthesis observed in cell lines carrying mtDNA mutations31. The impairment of mitochondrial translation then resulted in the respiration defects. In particular, alteration in mitochondrial protein synthesis was apparently responsible for the significant reduced rates in the basal OCR, ATP-linked OCR, maximal OCR and reserve capacity OCR in the control and mutant cell lines. This correlation is clearly consistent with the importance that OPA1 mutation plays a critical role in producing their respiration defects, as in the cases of cells both carrying the homozygous YARS2 c.572G > T and LHON-associated m.11778G > A mutations28. The respiratory deficiency then affects the efficiency of mitochondrial ATP synthesis. Our study showed that these lymphoblastoid cell lines carrying the OPA1 mutation had 33.2% decrease in the mitochondrial ATP production. These results were consistent with other report, showing significant reductions in the ATP content in cell lines carrying the other OPA1 mutations18. Furthermore, our previous investigations revealed the marked decreases in mitochondrial ATP production in lymphoblastoid cell lines derived from Chinese pedigrees with LHON29. Alternatively, the reduction in mitochondrial ATP production in mutant cells was likely a consequence of the decrease in the proton electrochemical potential gradient of mutant mitochondria39. As a result, RGC cells carrying the OPA1 mutation may be particularly sensitive to decreased ATP demand. Furthermore, the deficient activities of respiratory chain complexes often alter mitochondrial membrane potentials, which indicated the viability of cells40. In this investigation, 24% reduction in mitochondrial membrane potential in lymphoblastoid cell lines carrying the OPA1 mutation was observed, which was lower than those in cell lines carrying the m.12201T > C mutation in the tRNAHis gene 27. The defects in mitochondrial membrane potential may be due to strongly decreased efficiency of respiratory chain mediated proto extrusion for the matrix. The impairment of OXPHOS can lead to more electron leakage from electron transport chain, and in turn, elevate the production of ROS in mutant cells41. In the present study, the levels of ROS generation in the lymphoblastoid cell lines derived from three affected individuals carrying this mutation were 21.4% higher than control cell lines, and mitochondrial ROS generation increased 16.7% in the mutants, while 47% increase of ROS production in cells carrying both m.11778G > A and homozygous YARS2 c.572G > T mutations was the consequence of the altered activities of complexes I and IV28. The overproduction of ROS can establish a vicious cycle of oxidative stress in the mitochondria, thereby damaging mitochondrial and cellular proteins, lipids and nuclear acids42.The OPA1/MGM1 are required for the maintenance of mitochondrial genomes44. At a molecular level, apoptosis is regulated by two protein families: the Bcl-2 family which is responsible for the initiation phase, and the caspase family of proteases are that are involved in the execution phase of apoptosis46. Mitochondrial cytochrome c plays an important role in the propagation of many pro-apoptotic signals through acting as a co-factor for a caspase-activating complex in the cytoplasm, called the apoptosome. As a result, release of cytochrome c from the mitochondrial intermembrane space (IMS) represents an important checkpoint in apoptosis47. Downregulation of OPA1 leads to aberrant cristae remodeling and the release of cytochrome c which is normally sequestered within the tight cristae junctions23. In the present investigation, immunocytostaining on cytosolic and mitochondrial fractions showed that the levels of cytochrome c in mutant cell lines was higher than in control cell lines. Furthermore, Western blot analysis demonstrated that levels of the cytochrome c mutants exhibited markedly higher than those in control cell lines. In addition, the levels of apoptosis-related proteins: PARP, as well as caspases 3, 7 and 9 in the mutant cell lines were significantly higher those in control cell lines. These data demonstrated that c.1198G > C mutation in the OPA1 gene increased apoptosis, especially in the RGCs carrying c.1198C > G mutation. As a result, mitochondrial dysfunction caused by the c.1198G > C mutation in the OPA1 gene produced a phenotype of visual loss in this Chinese family.Apoptosis is the final common pathway leading to RGCs loss in ADOA and cell death is likely being complex, being triggered by a combination of several interacting factors. The two best-understood activation mechanisms are the intrinsic pathway  and the extrinsic pathwayOPA1 gene. The c.1198C > G mutation alters the mitochondrial morphology and mitochondrial genome maintenance. These defects impaired mitochondrial translation and respiration. As a result, this respiratory deficiency reduced mitochondrial ATP production, increased the production of oxidative reactive species, elevated apoptosis of RGCs and subsequently produced a phenotype of visual loss. Thus, our findings may provide new insights into the understanding of pathophysiology of ADOA.In summary, our findings suggest the pathogenic mechanism leading to an impaired oxidative phosphorylation in lymphoblastoid cell lines cell lines carrying the c.1198C > G mutation in the OPA1 c.1198C > G mutation, vision normal member (III-12) lacking the c.1198C > G mutation25, and from two genetically unrelated control individuals (WZ11 and WZ12) lacking any OPA1 mutation but belonging to the same mtDNA haplogroup were immortalized by transformation with the Epstein-Barr virus, as described previously48. Cell lines were grown in RPMI1640 medium (Invitrogen), supplemented with 10% fetal bovine serum.This study was in compliance with the Declaration of Helsinki. Informed consent, blood samples, and clinical evaluations were obtained from all participating family members, under protocols approved by the Ethic Committees of Zhejiang University and the Wenzhou Medical University. Lymphoblastoid cell lines derived from four members of this Chinese ADOA family 3 vision-impaired members  carrying the TM (Invitrogen) and 4′, 6-diamidino-2-phenylindole  were employed. The cells grown on cover slips were incubated in growth medium supplemented with 80 nM MitoTracker RedTM for 30 min, stained with DAPI for 15 minutes, and then washed in warm fresh medium, mounted. Cells were examined using a confocal fluorescence microscope  with two lasers (Ex/Em = 579/599 nm and 358/461 nm). Mitochondrial morphology was determined in 100 individual cells.Cells were cultured on collagen-coated cover glass slips (Thermo Fisher) for 24 hours in the presence of serum. To visualize the mitochondrial network, MitoTracker RedTriple fluorescent staining was performed to detect translocase of outer mitochondrial membrane 20 , cytochrome C (Abcam) and nuclei, using 4′, 6-diamidino-2-phenylindole . Cells were cultured on collagen-coated cover glass slips (Thermo Fisher), fixed in 4% formaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100, blocked with 5% Fetal Bovine Serum (FBS) for 1 hour, and immunostained with an anti-TOMM20 antibody and anti-cytochrome C overnight at 4 °C. The cells were then incubated with Alex Fluor 594 goat anti-rabbit IgG (H + L) and Alex Fluor 488 goat anti-mouse IgG (H + L) (Thermo Fisher), stained with DAPI for 15 minutes, and mounted with Fluoromount (Sigma-Aldrich). Cells were examined using a confocal fluorescence microscope  with three lasers .Specimens were fixed with 2.5% glutaraldehyde in phosphate buffer  for more than 4 hours, followed by 1% osmium tetroxide in phosphate buffer  for 1–2 hours. Specimens were then dehydrated by a graded series of ethanol, infiltration, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and alkaline lead citrate for 5–10 min respectively and viewed with Hitachi Model H-7650 TEM.49.Total genomic DNAs were extracted with the MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa) from mutant and control cell lines. 50 ng of DNA was used in each QPCR reaction using Universal SYBR Green Master  with two pairs of primers for mtDNA (300 nM each) in a 7900 HT Fast Real-time PCR System (Applied Biosystems) with one pair of primers for β-Actin for normalization. The following primers were used: MT-D-loop region: 5′-TCACCCTATTAACCACTCA-3′ (sense) and 5′-AGACAGATACTGCGACATA-3′ (anti-sense); MT-ND1: 5′-CACCCAAGAACAGGGTTTGT-3′ (sense) and 5′-TGGCCATGGGTATGTTGTTAA-3′ (anti-sense); β-Actin: 5′-TCACCCACACTGTGCCCATCTACGA-3′ (sense) and 5′-CAGCGGAACCGCTCATTGCCAATGG-3′ (antisense). mtDNA contents were calculated using the ΔΔCt method whereby all MT-D-loop and MT-ND1 (mtDNA target) Ct values were first normalized to β-Actin. Data from multiple experiments were analyzed using the procedure as described elsewhere28. Twenty micrograms of cellular proteins obtained from lysed cells were denatured and loaded on sodium dodecyl sulfate (SDS) polyacrylamide gels. Afterward, the gels were electroblotted onto a polyvinylidene difluoride (PVDF) membrane for hybridization. The antibodies used for this investigation were from Abcam , Santa Cruz [Anti-Cytb]; CST . Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was performed as detailed previously28.Western blotting analysis was performed as detailed previously27. XF96 creates a transient, 7 μl-chamber in specialized microplates that allows for the determination of oxygen and proton concentrations in real time. To allow comparison between different experiments, data are expressed as the rate of oxygen consumption in pmol/min or the rate of extracellular acidification in mpH/min, normalized to cell protein in individual wells determined by the Bradford protein assay (Bio-Rad). A density of 1 × 105 cells per well in 96-well plate was coated with Cell-TakTM adhesive. The rates of O2 were determined under basal condition and the addition of oligomycin (1 μM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μM), rotenone (1 μM) and antimycin A (1 μM), as detailed elsewhere27.The rates of oxygen consumption in lymphoblastoid cell lines were measured with a Seahorse Bioscience XF-96 Extracellular Flux Analyzer (Seahorse Bioscience), as detailed previously28. In brief, samples of 2 × 106 cells were incubated for 2 hours in the record solution  with 10 mM glucose, 10 mM glucose plus 2.5 μg/mL oligomycin (glycolytic ATP generation), or 5 mM 2-deoxy-D-glucose plus 5 mM pyruvate (oxidative ATP production). Cells were lysed and then incubated with the luciferin/luciferase reagents. After 10 min incubation in room temperature, the luminescences were read on a microplate reader .The levels of cellular and mitochondrial ATP levels were measured using the ATP Bioluminescence Assay Kit HS II  according to the manufacturer’s instruction27. In brief, cells were plated onto 96-well cell culture plate overnight in growth medium. JC-10 dyeloading solution was added for 30 min at 37 °C, 5% CO2. Alternatively, plated cells were preincubated with 10 μM of the protonophore uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 30 min at 37 °C, 5% CO2 prior to staining with JC-10 dye. The fluorescent intensities for both J-aggregates and monomeric forms of JC-10 were analyzed by BD-LSR II flow cytometer system  at Ex/Em = 490/525 and 490/590 nm. Ten thousand events were analyzed in each sample.Mitochondrial membrane potential was assessed with JC-10 Assay Kit Flow Cytometry (Abcam) following general manufacturer’s recommendations with some modifications50. Briefly, approximate 2 × 106 cells of each cell line were harvested, resuspended in PBS supplemented with 100 μM of 2′,7′- dichlorodihydrofluorescein diacetate (DCFH-DA) and then incubated at 37 °C for 20 min. After washing with PBS twice, cells were resuspended in PBS in the presence of 2 mM freshly prepared H2O2 and 2% FBS and then incubated at room temperature for another 45 min. Cells were further washed with PBS and resuspended with 1 ml of PBS with 0.5% paraformaldehyde. Samples with or without H2O2 stimulation were analyzed by BD-LSR II flow cytometer system , with an excitation at 488 nm and emission at 529 nm. Ten thousand events were analyzed in each sample. The levels of ROS generation by mitochondria in living cells were analyzed using the mitochondrial superoxide indictor MitoSOX-Red (Invitrogen), as detailed elsewhere31.ROS measurements were performed following the procedures detailed previouslyP value < 0.05.All statistical analyses were performed using Prism software (GraphPad Prism). Significance between two sets of experiments was determined using a Student’s t-test whereas group sets were analyzed using ANOVA. Differences were considered statistically significant when Suppelmental inforamtion


KRASG12D and TP53R167H transgenes, allowing for STS modeling in a spatial and temporal manner. This study utilized Oncopig STS cell line (fibroblast) and tumor (leiomyosarcoma) RNA-seq data to compare Oncopig and human STS expression profiles. Altered expression of 3,360 and 7,652 genes was identified in Oncopig STS cell lines and leiomyosarcomas, respectively. Transcriptional hallmarks of human STS were observed in Oncopig STS, including altered TP53 signaling, Wnt signaling activation, and evidence of epigenetic reprogramming. Furthermore, master regulators of Oncopig STS expression were identified, including FOSL1, which was previously identified as a potential human STS therapeutic target. These results demonstrate the Oncopig STS model’s ability to mimic human STS transcriptional profiles, providing a valuable resource for sarcoma research and cell line development.Human soft-tissue sarcomas (STS) are rare mesenchymal tumors with a 5-year survival rate of 50%, highlighting the need for further STS research. Research has been hampered by limited human sarcoma cell line availability and the large number of STS subtypes, making development of STS cell lines and animal models representative of the diverse human STS subtypes critical. Pigs represent ideal human disease models due to their similar size, anatomy, metabolism, and genetics compared to humans. The Oncopig encodes inducible  STS arises from a number of tissue types, including muscle, fat, nerves, and blood vessels. As they are relatively uncommon, STS represent an understudied cancer type and the underlying molecular mechanisms leading to tumor formation are poorly understood. Consequently, a low 5-year survival rate of 50% is currently observed2, a rate that has remained unchanged for decades, highlighting the need for further research into this rare cancer type. Although a number of gene fusions are implicated in STS formation, few highly recurrent driver genes have been identified to date3. This has resulted in a lack of disease-specific treatments for sarcoma patients, with therapies primarily limited to surgery, radio-, and chemotherapy4, with additional targeted and combinational trials currently underway2. Although many STS patients would benefit from further research into STS diagnosis and treatment, limited STS sample availability is one of the main factors limiting their inclusion in large scale genomic studies3. This represents a significant challenge for STS research, as STS classifications are constantly evolving, and diagnosis of many subtypes requires molecular tests and cooperation between geneticists and pathologists2. Therefore, there is a need to develop STS models to deepen our understanding of sarcoma formation, detection, progression, and prognosis.Soft-tissue sarcomas (STS) are aggressive, often lethal mesenchymal tumors that represent roughly 1% of adult cancers and comprise approximately 50 subtypes5. These cell lines must be representative of the vast genomic variability observed in the human sarcoma population, as this variability is responsible for observed differences in treatment responses. Unfortunately, the limited availability of human sarcoma cell lines, as well as the complex genomic structures associated with the large number of STS subtypes, including chromosomal translocations and complex unstable karyotypes, has hampered sarcoma research6. Therefore, there is a need to develop model STS cell lines representative of the diverse human sarcoma subtypes. Production of such cell lines represents a crucial step in understanding the mechanisms behind the variable responses to targeted therapies observed across STS subtypes6.Human tumor-derived cell lines play an important role in the discovery and development of new cancer therapiesin vivo models are also needed to translate in vitro treatment developments and discoveries to clinical settings. While xenografts produced by injecting human STS cell lines into immunocompromised mice are frequently used to model human sarcomas, potential treatments identified in these model systems have not translated to clinical settings3. This is due to a number of factors, including introduction of new mutations in culture, difficulties in modeling interactions between tumor cells and host microenvironment, and the inability to study early tumor formation events. Alterative genetically engineered mouse models that reproduce characteristics of human STS are also commonly used in sarcoma research. These include transgenic murine models containing Pten7 or Kras and Trp538 mutations that can develop a number of STS subtypes. While genetically engineered small animal models may be useful for a variety of studies including biomarker discovery and therapeutic testing, animal models that more closely mimic humans in terms of anatomy, physiology, genetics, and epigenetics are needed to address a range of unmet clinical cancer needs, including improving diagnostic imaging and locoregional combination therapies.In addition to cell lines, 10. Pigs are less expensive and ethically more acceptable than canines and non-human primates, and porcine studies are more predictive of therapeutic treatments in humans than rodents11. The recent production of a transgenic porcine cancer model, referred to as the Oncopig12, provides the opportunity to study biomarker discovery, treatment development/response, functional genomics, and provide surgical training in a model highly similar to humans. The Oncopig model encodes Cre recombinase inducible porcine transgenes encoding KRASG12D and TP53R167H, which represent a commonly mutated oncogene and tumor suppressor in human cancers, respectively12. This allows the Oncopig to model a wide variety of human sarcomas in an inducible, temporal manner. Although KRAS is rarely mutated in human STS3, TP53 is among the most frequently mutated genes identified14, and deregulation of both TP53 and KRAS signaling pathways is commonly utilized to produce sarcoma animal models14. This strategy has been used to develop a porcine model that produces spontaneous osteosarcomas due to constitutive TP53R167H expression15. Unlike this model, the Oncopig allows for spatial and temporal induction of STS cell lines and tumors originating from a wide variety of cell types, helping fill the gap created by the lack of relevant human sarcoma-derived cell lines and tissue samples available.Pigs represent an ideal large animal models for human diseases due to their similar size, anatomy, metabolism, genetics and epigenetics compared to humansThis study validates the Oncopig as a suitable model for human STS studies through the identification of similar alterations in transcriptional regulation in Oncopig and human STS. Towards this end, commonly identified alterations in gene expression and pathway regulation in human STS, including altered TP53 signaling, activation of Wnt signaling, and epigenetic reprogramming were investigated in Oncopig STS cell lines (fibroblast) and tumors (leiomyosarcoma). The results presented here demonstrate the validity of the Oncopig model to mimic human STS on the transcriptomic level, providing further validation for the use of the Oncopig in sarcoma research.in vitro12. The primary fibroblasts isolated from four Oncopigs stained positively with vimentin and negatively for cytokeratin, confirming their mesenchymal origin  were characterized by loss of contact inhibition, increased cell migration, and reduced cell cycle length compared to primary fibroblasts. In contrast to the spindle shaped, tightly adherent primary fibroblasts, STS cells were smaller, rounder, and less adherent. These phenotypic changes are characteristic of cellular dedifferentiation and transformation, which, in addition to tumor formation following injection in immunocompromised mice, support their tumorigenic nature12.This study sought to validate the Oncopig STS model as a suitable model for preclinical STS research by identifying similar alterations in transcriptional regulation in Oncopig and human STS. Transformed Oncopig fibroblast cell lines from a previously published study were utilized to profile Oncopig STS gene expression in vivo12. Briefly, Oncopig STS tumors were produced via intramuscular injection of AdCre into the Oncopig hind legs, resulting in the formation of tumor masses visible by ultrasound within 10 days. Their mesenchymal origin was confirmed through blinded pathological analysis and positive vimentin staining, resulting in the classification of the tumors as leiomyosarcomas. The Oncopig leiomyosarcomas contained densely cellular, non-encapsulated, and locally infiltrative neoplasm, and cells were arranged in bundles supported by a fibrous stroma. The neoplastic tumor cells were pleomorphic with single to multiple nuclei, with areas of necrosis and inflammation identified throughout the tumors. Together, these samples were used to identify altered transcriptional regulation in the Oncopig STS model both in vitro and in vivo.In addition, previously produced RNA-seq data from three Oncopig skeletal muscle and four STS tumor samples were utilized to profile Oncopig STS gene expression KRASG12D and TP53R167H expression in vitro , and significantly lower WT KRAS and TP53 expression  were observed in the STS cell lines compared to primary fibroblasts  had no effect on total, wild type (WT), or mutant tro Fig. , increassts Fig. . IncreasKRAS or TP53 expression, both primary and AdGFP treated fibroblasts were used as controls in comparisons with STS cell lines. A total of 3,360 differentially expressed genes (DEGs) were detected between the STS and control cell lines, with 1,792 and 1,568 DEGs displaying elevated and reduced expression in the STS lines, respectively  was compared to the expression profile of the same line after culturing for 99 passages to investigate the temporal stability of Oncopig STS expression profiles in vitro. The expression profiles of the two time points were highly correlated when comparing the 22,499 genes with expression information for both time points  and D (CCND1) expression was observed in both Oncopig leiomyosarcomas  and STS cell lines . In addition, increased CCND2 expression (log2 fold change 2.14) was observed in the Oncopig leiomyosarcomas. The increased cyclin expression coincided with reduced expression of the cyclin B inhibitors GADD45B and GADD45G in both Oncopig leiomyosarcomas  and STS cell lines , in addition to the cyclin B inhibitor SFN (log2 fold change −2.36) in the STS cells lines. Together, these results provide a mechanism for the previously observed reduced cell cycle length in Oncopig STS cell lines12, and highlight fundamental similarities in cell cycle deregulation between human and Oncopig STS. In addition, GADD45B and GADD45G are also important for TP53 dependent DNA repair and damage prevention22. Thus, reduced expression of these genes, in addition to SESN1 in both Oncopig STS cell lines and leiomyosarcomas  also suggests reduced TP53 dependent DNA repair and damage prevention in Oncopig STS. Therefore, investigation into the accumulation of somatic mutations and chromosomal instability may be of interest for future Oncopig STS studies.Reduced cell cycle length was previously reported in the Oncopig STS cell linesTP53 signaling is the induction of apoptosis, with evasion of apoptosis via altered TP53 signaling commonly observed in human STS23. This occurs through increased expression of GTSE1 in leiomyosarcomas21, a negative regulator of TP53 involved in the repression of TP53 induced apoptosis, as well as reduced expression of the proapoptotic factors IGFBP324 and CASP3 in osteosarcomas25. Consistent with observations in human STS, GTSE1 expression was elevated in the Oncopig leiomyosarcomas and STS cell lines , in addition to reduced IGFBP3 and CASP3 expression in the Oncopig STS cell lines , suggesting evasion of TP53 dependent apoptosis in Oncopig STS. However, although reduced apoptotic activity is associated with cancer progression, apoptotic cells are still present in tumors with up to 15% reported to undergo apoptosis in human STS26. This addresses why expression of some TP53 dependent proapoptotic factors including BAX are commonly observed in human leiomyosarcomas27. Indeed, increased BAX expression was observed in the Oncopig leiomyosarcomas (log2 fold change 1.93), in addition to a number of other TP53 dependent proapoptotic factors, including PMAIP1 (log2 fold change 1.59), PERP (log2 fold change 1.76), BID (log2 fold change 1.37), TP53I3 (log2 fold change 2.69), SHISA5 (log2 fold change 0.91), IGFBP3 (log2 fold change 4.26), APAF1 (log2 fold change 1.11), CASP9 (log2 fold change 0.95), and CASP3 (log2 fold change 1.83). The expression of these proapoptotic factors in the Oncopig leiomyosarcomas suggests high levels of apoptotic cells were present in the samples collected for sequencing. This theory is supported by the pathology reports identifying areas of necrosis and chronic inflammation throughout the tumors12 and also explain why elevated expression of these factors was not observed in the Oncopig STS cell lines. Together, these results demonstrate similar deregulation of TP53 dependent signaling between Oncopig and human STS suggestive of altered cell cycle and apoptotic regulation.Another important role of 28. Although human STS with upregulated Wnt signaling display a variety of Wnt expression profiles, each expresses elevated levels of at least one Wnt gene, with downregulation of Wnt signaling leading to inhibition of sarcoma cell proliferation28. These results suggest Wnt signaling plays an essential role in driving human STS development, and therefore altered Wnt signaling is expected to occur during Oncopig STS development. As expected, increased Wnt gene expression was observed during Oncopig STS development, with 4 and 3 Wnt genes displaying elevated expression in the Oncopig leiomyosarcomas and STS cell lines, respectively 29, TCF7L1 (log2 fold change −3.56)30, DKK3 (log2 fold change −1.88)31, CTNND1 (log2 fold change −1.15)32, and TCF7L2 (log2 fold change −0.85)30. Of particular interest is SFRP2 (log2 fold change −7.61), a Wnt pathway inhibitor whose expression was the most severely reduced of the DEGs in the Wnt signaling pathway. This gene is epigenetically silenced in human sarcomas, resulting in activation of Wnt signaling33. In addition, a number of Wnt receptor signaling genes commonly expressed at high levels in human STS also displayed elevated expression in the Oncopig STS cell lines, including TLE4 (log2 fold change 0.86)34, LEF1 (log2 fold change 1.34)35, and TCF7 (log2 fold change 1.44)36.In addition to the altered Wnt gene expression profiles, the Wnt receptor signaling pathway was significantly enriched for DEGs in the Oncopig STS cell lines Fig. . This inFRZB (log2 fold change −4.32)37, as well as DKK2 (log2 fold change −1.39), which is epigenetically silenced in a number of human STS subtypes including leiomyosarcomas28. In addition, elevated expression observed in the Oncopig leiomyosarcomas is also consistent with human STS, including increased SOX4 (log2 fold change 1.19) and SOX2 expression (log2 fold change 2.5), which promote proliferation and invasion in human osteosarcomas39, LEF1 (log2 fold change 3.2), whose expression is associated with a number of human STS subtypes35, and the previously mentioned CCND1 (log2 fold change 2.1). Together, the observed similarities in Wnt signaling activation between Oncopig and human STS suggests the Oncopig may be an ideal model to test novel STS therapies targeting the Wnt signaling pathway.Although the Wnt receptor signaling pathway was not enriched for DEGs in the Oncopig leiomyosarcomas, enrichment of the regulation of Wnt receptor signaling pathway was observed Fig. . The obs40. One of the most well studied forms of epigenetic regulation is DNA methylation, which is maintained by DNA methyltransferases (DNMT) and altered in a wide variety of human STS including leiomyosarcomas41. Although DNA methylation analysis was not performed in this study, altered expression of DNMTs is linked to altered genome-wide DNA methylation patterns42. Therefore, the expression levels of epigenetic regulators were investigated to provide indirect evidence of epigenetic reprogramming in Oncopig STS. As expected, altered DNMT expression was observed in the Oncopig STS model, with increased DNMT1 (log2 fold change 0.7) expression in the Oncopig STS cell lines, and increased DNMT1 and DNMT3L , as well as decreased DNMT3A (log2 fold change −1.35) expression in the Oncopig leiomyosarcomas. These results suggest that as in human STS, genome-wide alterations in DNA methylation patterns exist in Oncopig STS.The role of genome-wide epigenetic reprogramming in the development of human cancers is well established, and epigenetic regulators are emerging as promising therapeutic targets for STSin vitro and in vivo control and tumorigenic samples, resulting in modeling of different STS subtypes, as well as differences in in vitro and in vivo microenvironments. The effects of the differential in vitro and in vivo microenvironments on Oncopig STS transcriptional profiles is evidenced by the above mentioned areas of necrosis and chronic inflammation throughout the leiomyosarcoma tumors12 that were not present in the STS cell line cultures.The lack of differential expression of the same DNA methyltransferases in the Oncopig leiomyosarcomas and STS cell lines, as well as differences in the above-mentioned pathways is likely due to a number of factors, including differential cell origin of both the 40. The histone methyltransferase EZH2 methylates histone H3 lysine 2743, and has been identified as an oncogene in human STS, with elevated expression observed across subtypes40. Furthermore, knockdown of EZH2 induces cell death and inhibits tumor growth44, suggesting that EZH2 may be an effective therapeutic target for STS. Consistent with observations in human STS, elevated EZH2 expression was observed in both the Oncopig leiomyosarcomas and STS cell lines , suggesting the presence of altered histone modification patterns in Oncopig STS. Together these results, in addition to the above mentioned reduced expression of genes known to be epigenetically silenced in human STS, provide evidence for similar genome-wide epigenetic reprogramming in Oncopig and human STS, suggesting the Oncopig may be an ideal model to test therapies targeting epigenetic regulators. However, further epigenetic studies are required to confirm altered epigenetic regulator expression results in genome-wide epigenetic reprogramming in Oncopig STS, as well as their similarity to alterations observed in human STS subtypes.In addition to DNA methylation, histone modifications represent another crucial form of gene regulation, and therapeutics targeting histone modifying enzymes are also being investigated for STS treatmentsFOSL1, which was predicted to regulate 49% of the genes with elevated expression in the Oncopig STS cell lines , sample processing and storage , and the relatively few number of studies focused on molecular profiling of leiomyosarcomas, which tend to include a mixture of STS subtypesin vitro and in vivo sample comparisons  primary fibroblasts, AdGFP treated, and AdCre treated cell cultures from a previously published study were the source of RNA for this experiment12. All animal work was conducted according to relevant national and international guidelines, and approved by The University of Illinois Institutional Animal Care and Use Committee . Untreated (control), AdGFP, and AdCre cell lines were maintained separately for an average of 8, 16, and 31 passages after treatment, respectively  for vimentin and cytokeratin immunostaining to determine cell origins.Total RNA was extracted from each cell line using the AllPrep DNA/RNA Mini Kit  following the manufacturer’s protocol. A NanoDrop spectrophotometer was used to determine RNA concentrations and RNA integrity and the presence of genomic DNA was determined using an Agilent 2100 Bioanalyzer using an RNA Nano bioanalyzer chip by the Carver High-Throughput DNA Sequencing and Genotyping Unit . RNA samples were utilized for sequencing if their RNA integrity number was greater than 7.TruSeq Stranded RNA-seq libraries  were produced from high-quality RNA (1 μg) by the HTS lab  following standard protocols. Qubit  was used to quantify libraries, and an Agilent bioanalyzer DNA7500 DNA chip  was used to determine the average library size, which was diluted to 10 nM. Finally, library dilutions were quantitated using an ABI 1900 to ensure high accuracy quantification for consistent pooling and cluster maximization on the Illumina flowcell.RNA-seq was performed on an Illumina HiSeq2500. The libraries were paired-end sequenced to a total read length of 100 bp.http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used to trim raw reads for adapter contamination, A-tails, minimum quality score (20), and minimum length (20 bp) by setting the–stringency option to 6. A minimum length of 35 bp was used to retain unpaired reads. Trimmed reads were aligned to the swine reference genome (Sscrofa10.2)58 using Tophat v.2.2.1059 using the –M, –G, and fr-firststrand options, setting the –g option to 20, the –read-realign-edit-dist option to 0, and the –mate-std-dev option to 450. Previously aligned RNA-seq bam files for control Oncopig skeletal muscle (3) and leiomyosarcoma tumors (4) produced via injection of AdCre into the skeletal muscle were downloaded from the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3382. The bam files were aligned in the same manner as described above12, and the number of reads/sample is listed in Supplementary Table in vitro and in vivo experiments were assessed for differential gene expression using cufflinks v.2.2.160. Transcripts were assembled for each sample using the fr-firststrand option and merged together with the reference transcripts using Cuffmerge. Gene expression levels were pre-computed using Cuffquant with the –u and fr-firststrand options. Normalized gene expression levels were calculated using Cuffnorm using the fr-firststrand option and with the–library-norm-method set to geometric. Finally, differential expression was assessed using Cuffdiff including the –u and fr-firststrand options. Genes were considered differentially expressed with a q-value < 0.05. Samtools mpileup61 was used to determine WT:mutant TP53 and KRAS expression ratios based on the reads overlapping the mutation site. The Shapiro-Wilk normality test was used to test normality, and equality of variance between groups was determined using an F-test. A two-way Student’s t-test was used to identify significant differences between groups for normally distributed data, while the Wilcoxon signed-rank test was used for non-normally distributed data. Correlations were performed using a Spearman’s rank correlation.An average of 34.7 million raw stranded paired-end reads were produced for each sample, ranging from 29.9 to 39.9 million  > 4 were considered enriched.Master regulators responsible for genome-wide increases and decreases in gene expression identified using the cytoscape plugin iRegulon v1.3Supplementary TablesSupplementary Figures


Melanoma has two key features, an over-representation of UV-induced mutations and resistance to DNA damaging chemotherapy agents. Both of these features may result from dysfunction of the nucleotide excision repair pathway, in particular the DNA damage detection branch, global genome repair (GGR). The key GGR component XPC does not respond to DNA damage in melanoma, the cause of this lack of response has not been investigated. In this study, we investigated the role of methylation in reduced XPC in melanoma.To reduce methylation and induce DNA-damage, melanoma cell lines were treated with decitabine and carboplatin, individually and sequentially. Global DNA methylation levels, XPC mRNA and protein expression and methylation of the XPC promoter were examined. Apoptosis, cell proliferation and senescence were also quantified. XPC siRNA was used to determine that the responses seen were reliant on XPC induction.XPC mRNA expression. Lower, clinically relevant dose of decitabine also resulted in global demethylation including the CpG island shores and induced XPC in 50% of cell lines. Decitabine followed by DNA-damaging carboplatin treatment led to significantly higher XPC expression in 75% of melanoma cell lines tested. Combined sequential treatment also resulted in a greater apoptotic response in 75% of cell lines compared to carboplatin alone, and significantly slowed cell proliferation, with some melanoma cell lines going into senescence. Inhibiting the increased XPC using siRNA had a small but significant negative effect, indicating that XPC plays a partial role in the response to sequential decitabine and carboplatin.Treatment with high-dose decitabine resulted in global demethylation, including the the shores of the XPC CpG island and significantly increased Demethylation using decitabine increased XPC and apoptosis after sequential carboplatin. These results confirm that sequential decitabine and carboplatin requires further investigation as a combination treatment for melanoma.The online version of this article (10.1186/s12885-018-4010-9) contains supplementary material, which is available to authorized users. There are two features of melanoma that suggest a defect in DNA repair, an extremely high mutation load, indicative of unrepaired UV-induced DNA damage , and innThe NER pathway consists of approximately 30 proteins that remove helix distorting lesions through for steps: a) damage recognition, b) unwinding of the DNA locally around damage, c) incision of damaged DNA by endonucleases, and d) DNA resynthesis and ligation . There aSeveral previous studies have found an association between high or low levels of NER protein and mRNA levels and platinum chemoresistance (reviewed in ). The NEXPC expression have significantly shorter survival [In addition to the evidence supporting ERCC1 as a biomarker of platinum chemoresistance, our previous research has shown that the GGR damage detection branch of NER, does not function correctly in melanoma. We have found that the three GGR components XPC, DDB1 and DDB2 do not respond to UV treatment in melanoma cell lines, resulting in reduced repair of UV-induced DNA damage , 19. We survival . The funsurvival  and coulsurvival , 22. We survival .A role for GGR in melanoma development and chemotherapy resistance may come from the broad functions it has in controlling the DNA damage response. Damage recognition by XPC and DDB2 leads to activation of other pathways that control cell cycle and apoptosis, in addition to NER. XPC and DDB2 are involved the activation of the checkpoint signalling protein ATR in response to UV-induced DNA damage . Both prThe underlying mechanism that is responsible for the GGR deficiency seen in melanoma is yet to be identified. To date, somatic mutations in XPC, DDB1 and DDB2 have rarely been reported in melanoma tumours. We reported that upstream regulators of GGR including p53, BRCA1 and PCNA are not responsible , 23, 29.V600E and p14ARF−/− background developed melanoma in response to UV radiation, with impaired DNA repair capacity due to reduced XPC expression from promoter hypermethylation [To date there has only been only one study to investigate the methylation of XPC in melanoma. A mouse model found that melanocytes with BRAFhylation . HoweverMore recently, the importance of CpG island shore methylation altering the expression of genes in cancer , has beeAs there is evidence of silencing of XPC by methylation in melanoma the aim of this study was to investigate the methylation pattern of the XPC promoter region, including the CpG island and flanking shores, and its effect on gene expression in our melanoma cell lines that display reduced GGR. We also examined if methylation patterns could be altered by demethylation and restore XPC function, therefore reinstating platinum chemotherapy sensitivity.2 .Four melanoma cell lines were used in this study: MM200, Sk-mel-28, Me4405 and Mel-RM. The source, tumour status and p53 status of each cell line has been previously described –39. A hu2O at 10 mg/mL and 1 mM respectively, with decitabine stored at − 80 °C. For treatment decitabine was diluted in cell culture medium to either 10 μM or 0.26 μM where indicated. Cell lines were treated with decitabine for 72 h with cell culture media replaced every 24 h with fresh media and decitabine. Carboplatin was diluted in cell culture medium to 8 μg/mL and cells treated for 48 h. For combination treatment cell lines were treated first with 0.26 μM decitabine for 72 h followed by 48 h of 8 μg/mL carboplatin. These doses were chosen as they were based on plasma concentrations of each drug when used as chemotherapy agents [Carboplatin (Sigma-Aldrich) and decitabine (5-aza-2′-deoxycytidine) (Sigma-Aldrich) were resuspended in MilliQ Hy agents , 41.Global DNA methylation levels were quantified using a 5-mC DNA ELISA Kit (Zymo Research) as per manufacturer’s instructions. DNA from melanoma cell lines before and after decitabine treatment was extracted by Quick-DNA Universal Kit (Zymo Research). 100 ng of genomic DNA and methylated standards were bound to an ELISA plate and methylated DNA was detected with antibodies to 5-methylcytosine, quantified by colorimetric analysis.XPC was measured in triplicate and normalised to the geometric mean of three housekeeping genes, GAPDH, ACTB and 18S rRNA using TaqMan gene expression assays and a Viia7 system (Applied Biosystems). Relative expression was calculated using 2-ΔCt.Before and following treatment at specified time points RNA was collected by phenol chloroform extraction. RNA (1 μg) was reverse transcribed in triplicate using the High Capacity Reverse Transcription Kit (Thermo Fisher Scientific) and the resultant cDNA was diluted 1:20 as previously described . Relativw/v) glycerol, 0.02% bromophenol blue, 15% beta-mercaptoethanol) and denatured by boiling for 5 min. Samples were loaded onto 4-20% TGX precast polyacrylamide gels (Bio-Rad Laboratories) and run at 150 V (constant voltage) in Tris-Glycine buffer . Proteins were transferred onto nitrocellulose using the TransBlot Turbo system  and visualised using Ponceau S (0.1% (w/v) Ponceau S in 5% acetic acid; Sigma-Aldrich). Following transfer, blots were blocked in 5% skim milk for 1 h at room temp. XPC was detected using anti-XPC rabbit polyclonal antibody (H-300)  and anti-TATA binding protein (TATA-BP)  was used as a nuclear loading control. Primary antibodies were incubated at 4 degrees overnight. Blots were washed three times for 5 min in PBS-T then incubated for 1 h at room temperature with HRP-conjugated secondary antibodies . Blots were washed as done previously then proteins detected by chemiluminescence using Clarity Western ECL reagent (Bio-Rad) and imaged using the ChemiDoc MP system (Bio-Rad Laboratories). Image processing and densitometry analysis was performed on all blots using ImageJ. Data was normalised to TATA-BP and expressed as fold induction from baseline.Nuclear protein fractions were obtained using the NucBuster protein extraction kit (Merck Millipore). Protein lysate (40 μg) was added to 4X SDS-sample loading buffer  and the primers  according to manufacturer’s instructions. The CpG island and surrounding shores of 6 cells before and after treatment were washed and stained with 7-AAD and PE conjugated Annexin-V for 15 min in the dark. Data was analysed on FlowJo v10 . Apoptotic cells were quantified as the percentage of cells that stained positive for Annexin-V and double Annexin/7-AAD positive cells.After treatment with 0.26 μM decitabine, 8 μg/ml carboplatin and in combination, both attached and detached cells were collected. Apoptotic cells were quantified after drug treatment using an Annexin V Apoptosis Detection Kit (BD Biosciences) following manufacturers instruction performed on a BD FACSCanto II flow cytometer (BD Biosciences). 1 × 103 cells per well overnight before drug treatment and luminescence measured on a Cytation 3 plate reader (BioTek). After combination treatment senescence was measured by β-galactosidase staining using an Abcam Senescence Detection Kit (Abcam). Cells were plated and fixed in 6-well plates after combination treatment and stained with X-gal. Positively stained cells were identified under a light microscope.Cellular proliferation after treatment was measured using a CellTitre-Glo Luminescent Cell Viability Assay kit (Promega) according to manufacturer’s instructions. Cells were seeded in 96-well plates at 5 × 10XPC was knocked down after decitabine treatment using siRNA purchased from Dharmacon . Transfections were carried out in the last 24 h of decitabine treatment with 25 nM of XPC siRNA in OptiMEM medium (Gibco) using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s instruction. An NTC siRNA  was used as a control for transfection in identical conditions to XPC siRNA.The expression of p-value of < 0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). Non-parametric Mann-Whitney tests were used to assess differences between groups. A XPC relative expression (RE) were quantified in response  reduced methylation levels in all melanoma cell lines with an average reduction of 38.22% ±4.98  increases in XPC mRNA expression in all cell lines (1.27-7.93 fold increase)  and .98 Fig. . This cose) Fig. .Fig. 1Glp = 0.005, Sk-mel-28 p = 0.03, Me4405 p = 0.002, Mel-RM p = 0.001)    was also tested. Lower doses of decitabine limits the formation of DNA damage and cytotoxicity but can still demethylate . 0.26 μM01) Fig.  with an 01) Fig. , which wXPC mRNA expression in melanoma, the promoter region of XPC, containing the CpG island and adjacent shores, was bisulfite sequenced in all melanoma cell lines before and after decitabine treatment to identify if promoter methylation is responsible for reduced expression. The XPC promoter region was sequenced by next generation bisulfite sequencing allowing for quantification of methylation at base resolution  shore showed high levels of methylation (average 91.8%) with the exception of the four CpG sites closest to the island which were methylated approximately 0-5%. The downstream (3′) shore showed a similar pattern, of high methylation, to the upstream shore. While most sites in the shores were consistently highly methylated, several sites varied in methylation levels across the melanoma cell lines. For example, the CpG site 1849 bp from the TSS displayed methylation between 28 and 61% in melanoma cell lines. Similarly, the last four CpG sites in the shore had reduced methylation in Me4405 where methylation was as low as 11%  which contains methylation data for 470 melanoma tumours. Although the TCGA dataset data set was collected using the Infinium HumanMethylation450 array it only covers 21 CpG sites across the XPC CpG island and 2 within one shore. Each of these sites showed similar methylation pattern as our sequencing data.These methylation patterns are consistent with data from the Cancer Genome Atlas (TCGA)  and carboplatin (8 μg/mL), both individually and in sequential combination and the expression of XPC was measured . When decitabine is used to demethylate before carboplatin treatment, the increase in XPC expression is significantly greater, increasing the fold change from 1.52-3.86  to 1.49-7.55 fold increase (decitabine and carboplatin). With the exception of Sk-mel-28, the level of XPC expression after combination treatment was significantly higher than carboplatin alone . This suggests that demethylation, while not consistently affecting baseline expression of XPC, can lead to a greater induction of XPC in response to DNA damaging agents such as carboplatin.As the lower dose (0.26 μM) still demethylated but did not increase red Fig. . CarboplThe increased expression of XPC in response to combination treatment was confirmed at the protein level Fig. . Three oTo investigate if the increase in XPC expression following demethylation has a functional consequence on the cytotoxic response to carboplatin, apoptosis was quantified following single and combination treatment Fig. . Figure The cytotoxic potential of combined decitabine and carboplatin treatment is seen where combined treatment resulted in significantly higher levels of cell death in three out of the four cell lines  when compared to carboplatin alone , to a level similar to baseline. This is also reflected in the expression of XPC protein  decrease in the number of apoptotic cells in both Me4405 and Mel-RM (1.09 fold) after combination treatment  after treatment compared to NTC control  showing that the XPC CpG island shores is responsible for increased XPC expression in response to decitabine. It is possible that demethylation of the shores allows for some other elements such transcription factors access to XPC which may result in the increased expression in response to decitabine. One possible explanation is that an upstream regulator of XPC expression is also being demethylated by decitabine and can induce XPC expression.Methylation within the CpG island shores was present in melanocytes and the 4 melanoma cell lines in this study. The methylated regions were partially demethylated by decitabine in the melanoma cell lines. As melanocytes have low replication rates in vitro and do not replicate in vivo the blocking of methylation does not occur to the same extent as in melanoma cells, therefore the effect of decitabine is not seen in melanocytes. More research is needed to confirm that demthylation of the XPC when compared with 0.26 μM. This may have been due to the fact that high doses of decitabine can induce DNA damage by prolonged binding of DNMT1 leading to double stranded breaks [XPC upregulation. The lower dose of 0.26 μM should not have induced as great of levels of damage and as such could explain why only two cell lines Mel-RM and Me4405 increased XPC expression after 0.26 μM decitabine.Two doses of decitabine were used in this study. The first (10 μM) was chosen as it is a high dose that would ensure demethylation across the genome, while the second (0.26 μM) represented a pharmacological dose . 10 μM dd breaks . It coulXPC following demethylation, we treated the cells with carboplatin following demethylation with 0.26 μM decitabine. Combined treatment resulted in a significantly greater XPC response in 3 of 4 cell lines. While low dose decitabine was not enough to increase XPC expression alone it may demethylate a particular region of XPC or an upstream target that then allows XPC to respond to the DNA damage signal caused by carboplatin.To identify if platinum-induced DNA damage would induce HOX10 gene has CpG island shore methylation that is associated with transcriptional repression in breast cancer [CAV1) gene [CAV1 shore methylation and expression in breast cancer. This relationship has been found further in other genes in other cancers [CpG island shores have been gaining more consideration in the past few years after being confirmed as one of the major regions for differential methylation in cancer , 48. Altt cancer . MethylaV1) gene . This st cancers , 50. AllXPC demethylation and expression, this study revealed some exciting results with translational potential. Demethylation with decitabine increased the sensitivity of melanoma cells to the growth inhibitory and apoptotic effects of carboplatin, which is typically ineffective in melanoma [XPC or apoptosis in response to decitabine and carboplatin. Sk-mel-28 carries a mutation that results in constitutively activated p53 [Regardless of the dynamics of melanoma . The celated p53  which maXPC in the response to combined treatment was investigated. When XPC was knocked down with siRNA during decitabine treatment minor changes were seen in apoptosis and proliferation. Given the effect of XPC knockdown on apoptosis was minor, despite robust attenuation of XPC, the effect of exogenous overexpression of XPC expression is required to further clarify if the effects that are being observed are indeed related to XPC expression.The importance of XPC is not the only driver of the responses seen. This could be expected as there are numerous and complex pathways that are involved in cell death and proliferation, which combined with the fact decitabine demethylates globally, may lead to many changes in various pathways. It is unlikely that one single demethylated target will drive the response alone. The melanocyte-lineage specific master regulator, MITF may additionally contribute to the apoptotic response to decitabine and carboplatin. Although this requires further investigation, MITF is involved in melanoma proliferation and survival  and hasiewed in . RegardlCDKN2A. Differentiation was driven by demethylation of the melanocyte differentiation driver SOX9 promoter and subsequent increased expression. Exit from the cell cycle and reduced proliferation was a result of upregulated p21 (CDKN1A), and p27 (CDKN1B). Whole transcriptome analysis in response to decitabine [While some studies have examined the effect of decitabine on melanoma growth and apoptosis, ours is the first study to examine the combination of demethylation with platinum therapy within melanoma. A study in 2011 showed that decitabine has a cytotoxic effect on melanoma that is independent of apoptosis . While acitabine  confirmsHOXA10 and RASSF1A correlated with progression free survival. Similar results have been found in neuroblastoma [While a new idea in melanoma, combining demethylation treatment with platinum chemotherapies has been studied in other cancer types with positive results. One particular study examining multiple combinations found that decitabine had the greatest combination potential with platinum compounds, out of a screen of 16 drugs . Decitabblastoma , renal cblastoma  and non-blastoma . This maThe benefits of a combination decitabine and carboplatin treatment are not limited to DNA damaging and apoptotic mechanisms. One major challenge in cancer treatment is the ability of tumours to escape from immune detection and prevent efficient T-cell response to cancer cells . EmerginPlatinum chemotherapy compounds including carboplatin can induce a combination of stress and cell death that can initiate a tumour-specific immune response. Firstly, platinum therapy induces endoplasmic reticulum (ER) stress that results in calreticulin exposure on the cell surface, acting as a signal for dendritic cells . SecondlXPC induction in response to DNA damage. Demethylation of CpG island shores is likely to be responsible for this, but further research is needed to confirm this finding. Regardless, we have discovered the potential of a combination treatment for melanoma using decitabine to re-instate carboplatin sensitivity resulting in greatly increased apoptosis and decreased cellular proliferation. Further research into how this combination affects methylation genome wide in melanoma is needed to completely elucidate the mechanisms leading to this outcome.Taken together the results of this study indicate treatment of melanoma with decitabine can sensitise cells to sequential carboplatin treatment. Demethylation can restore Additional file 1: Table S1.XPC bisulfite promoter primers for PCR. (DOCX 14 kb)Additional file 2: Figure S1.Representative bright-field microscopy images of senescence associated β-galactosidase staining in all four melanoma cell lines after combined decitabine and carboplatin treatment. Arrows indicate regions of positive staining, bar = 100 μm. (TIFF 42085 kb)Additional file 3: Figure S2.DNA methylation pattern of the XPC CpG island in melanocytes and melanoma. Methylation levels in melanocytes (black) and each melanoma cell line at baseline (grey) was quantified by bisulfite sequencing. CpG position is shown relative to XPC transcription start site (TSS). Upstream (5′) shore = position − 2341 to − 423, CpG island = position − 364 to 568, Downstream (3′) shore = position 714 to 2596. (TIFF 603 kb)


Met C4467A mutation in a Han Chinese family with hypertension. The maternal members presented with increased glucose, total cholesterol, low-density lipoprotein, and serum sodium as well as decreased potassium compared with non-maternal members (P < 0.05). Segregation analysis showed this mutation was maternally inherited. We analyzed lymphocyte cell lines derived from three maternal and three non-maternal family members. Reactive oxygen species production in the mutant cell lines was 114.5% higher compared with that in controls (P < 0.05) while ATP was 26.4% lower. The mitochondrial membrane potential of the mutated cell lines was 26.2% lower than that in controls (P < 0.05). Oxygen consumption rates were decreased in the mutant cell lines (P < 0.05). The activation of caspase-3/7 was 104.1% higher in the mutant cell lines compared with controls (P < 0.05). The expression of voltage-dependent anion channel (VDAC), Bax and apoptosis-inducing factor (AIF) in the mutant cell lines was higher compared with that in controls, with the increased colocalization of VDAC and Bax. Therefore, this mutation contributes to oxidative stress and mitochondrial biogenesis dysfunction, which may be involved in the pathogenesis of hypertension.To investigate the relationship between mitochondrial DNA (mtDNA) and hypertension as well as the mechanism involved in mitochondrial metabolic dysfunction. We identified a novel tRNA Essential hypertension is known as a multifactorial disease with both genetic and environmental factors affecting its onset and severity. Estimates of genetic variance range from 20–50%4, and both maternal and paternal patterns have been reported7. Significant blood pressure (BP) correlations between excess maternal transmission and mother-and-offspring hypertension have been observed8. Indeed, we have previously reported the excess maternal transmission of hypertension in hypertensive families14 with the identification of several mitochondrial DNA (mtDNA) mutations, including T3308C in ND19, A4435G in transfer tRNAMet11, A4263G in tRNAIle13, and A4401G in tRNAMet and tRNAGln12. We showed that these mutations lead to a decrease in tRNAs and several polypeptides encoded by mtDNA13. However, the relationship between mtDNA mutations and high BP remains unclear.Hypertension is a common risk factor for many cardiovascular diseases, including coronary heart disease, heart failure, stroke and renal disease14. Here, we reported on a Han Chinese family with maternally inherited hypertension and the novel mutation tRNAMet C4467A. We carried out a systematic investigation of changes in oxidative stress and mitochondrial biogenesis that are associated with the tRNAMet C4467A mutation to further explore the possible mechanisms of mtDNA mutation pathogenesis in maternally inherited hypertension.To investigate the effect of mtDNA mutations on hypertension pathogenesis, we performed a systematic mtDNA mutational screening in a large cohort of hypertensive subjects in the Geriatric Cardiology Clinic at the Chinese PLA General Hospital in ChinaThe proband III-10), a 42-year-old male, underwent a health evaluation at the clinic and was found to have borderline hypertension with a BP of 140/90 mmHg. He did not smoke or drink alcohol and did not have a history of coronary artery disease, diabetes, or hyperlipidemia. The pedigree originated from Shan Xi Province, China , low-density lipoprotein (LDL), blood glucose and serum sodium as well as a decrease in potassium compared with non-maternal relatives  at the aminoacyl acceptor stem of tRNAMet.Because the transmission pattern of hypertension in this family appeared to be maternal inheritance, mtDNA may be involved in the disease pathogenesis. We therefore analyzed the whole mitochondrial genome of members in this family using PCR amplification and subsequent sequence analysis of the PCR fragments. The tRNAArtibeus jamaicensis, the wild boar Sus scrofa, mice15, cows16, and Xenopus laevis17 .DCFH-DA probe-labeled cells were used to detect the effect of the mitochondrial tRNA3, 2 × 103, 4 × 103, and 8 × 103 cells presented with a linear relationship with ATP concentrations both for the cells carrying the tRNAMet C4467A mutation and the control cell lines  and three controls  to evaluate whether the tRNAMet C4467A mutation affects cellular bioenergetics. The basal OCR in mutant cell lines decreased 50.2% , FCCP , rotenone (a complex I inhibitor), or antimycin A (a complex III inhibitor). ATP-linked OCR, proton-leak OCR, maximal OCR, reserve capacity, and non-mitochondrial OCR that were associated with the basal OCR and the drug-insensitive OCR were compared between the mutant and non-mutant cell lines. These values decreased in mutant cell lines by 54.7%, 61.6%, 65.9%, 58.6%, and 62.6%, respectively, compared with the control cell lines  of lymphocyte cell lines that were derived from three tRNA3, 2 × 103, 4 × 103, and 8 × 103 cells presented a linear relationship with caspase-3/caspase-7 activity both in tRNAMet C4467A-mutated and control cell lines. Caspase-3/7 activity in the lymphocytes with the tRNAMet C4467A mutation was significantly increased by 57.1% in the 2 × 103 cell group, 93.7% in the 4 × 103 cell group, and 104.1% in the 8 × 103 cell group compared with the control cell lines (P < 0.05).Cells were harvested, and caspase-3/7 activity was compared between the mutant and non-mutant cell lines. Activation of apoptosis was also represented by the activity of caspase-3/caspase-7. As shown in Fig. In control and mutated cell lines, confocal microscopy was used to observe the colocalization of voltage-dependent anion channel , Bax (red fluorescence) and apoptosis-inducing factor . There was no evidence of yellow fluorescence in the control group. In contrast, the mutated cells presented with yellow fluorescence see Fig.  and showP < 0.05, Fig. The protein expression of VDAC, Bax and AIF in the mutated cell line was higher than that in the control cell line . Another study by Wilson et al.22 documented a large family of metabolic defects caused by a mitochondrial tRNAIle 4291 T→C mutation. Additionally, we previously also identified excess maternal transmission of hypertension in several hypertensive families14. By far, the most widely studied mitochondrial diseases are those affecting single tRNA genes. Recently, Pro. Guan et al.23 studied the pathophysiology of the tRNAAla 5655 A→G mutation in hypertension. A failure in tRNAAla metabolism was shown to contribute to different reductions in six polypeptides encoded by mtDNA. The activity of the mitochondrial respiratory chain was decreased in the mutant cell lines, which was accompanied by decreases in ATP levels and mitochondrial membrane potential and an increase in the production of ROS. Therefore, the tRNAAla 5655A→G mutation may contribute to hypertension. However, more detailed effects of tRNA mutations on high BP are still unclear, partly because of ethical limitations on human material.In 2001, Watson Met 4467C→A mutation in a Chinese family with maternally inherited hypertension. Biochemical testing showed a significant increase in blood glucose, TC, and LDL-c, and serum sodium as well as a decrease of potassium in maternal relatives compared with non-maternal relatives. Similarly, Wilson reported a large family with metabolic defects that were associated with the tRNAIle 4291 T→C mutation, in which maternal members presented with increased incidence of hypertension, hypercholesterolemia, and hypomagnesemia compared with non-maternal members. In this study, we also found that the average onset age tended to be earlier and that genetic factors contribute to the early onset age phenomenon.In this study, we are the first to report a novel tRNAMet in a Chinese family. The results strongly suggest that this novel mutation may be involved in the pathogenesis of hypertension, as it is absent from 366 Chinese controls. The acceptor arm of the tRNAMet is a 7-bp stem that is generated by the pairing of the 3′-terminal nucleotide with the 5′-terminal nucleotide. The 4467C→A mutation is in the second position from the 3′-terminal nucleotide. The CCA 3′-terminus is able to carry the amino acid. A subset of nucleotides is post-transcriptionally modified with a CCA triplet attached to the pre-tRNA 3′ end. In addition, the correctly folded and mature tRNAs are esterified by the corresponding aminoacyl-tRNA synthetase at the 3′ end with the cognate amino acid. Finally, protein synthesis is initiated after these tRNAs are transported to the ribosome by the translation elongation factor (EF)-Tu. The mutation may impair the cleavage of polycistronic mtRNA transcripts into mature mt-tRNA species at the tRNAase Z cleavage site. Thus, the point mutation located at the 3′ end is important for both tRNA synthesis and function24. Several studies have shown that pathogenesis-associated mutations in tRNAs could reduce the efficiency of precursor 3′-end cleavage using wild-type and pathogenesis-associated mutations in tRNASer(UCN), tRNAIle and tRNALeu(UUR), which were at a similar position as tRNAMet4467C>A28. In the present study, phylogenetic analysis also demonstrated that the 4467C→A mutation site is highly evolutionarily conserved among species.In this study, the sequence analysis of the whole mitochondrial genome identified a novel mutation 4467C→A in tRNAWe established cell lines from three maternal and three non-maternal members of a Chinese family to study the effect of the 4467C→A mutation on mitochondrial function. ROS production in cells carrying the mutation was increased by 114.5% compared with that in control cell lines, while ATP production was decreased by 26.4%, and the mean mitochondrial membrane potential was decreased by 26.2%.Met C4467A mutation.The mitochondrial electron transport system (ETS) was investigated by measuring OCR. As we know, the ATP-linked OCR and the reserve capacity reflect a “mitochondrial stress test,” which is evaluated based on the OCR in response to drugs, including oligomycin and FCCP. The basal OCR decreased by 50.2% in the mutant lymphocyte cell lines compared with controls, while ATP-linked OCR decreased by 54.7% in the mutant cell lines, which reflects the OCR of the coupling of mitochondrial oxidative phosphorylation. The maximal OCR, proton leak OCR, and reserve capacity OCR decreased by 61.6%, 65.9%, and 58.6%, respectively, in the mutant cell line, reflecting mitochondrial oxygen biogenesis dysfunction. Finally, significantly increased activation of caspase-3/7 was observed in the mutant cell lines, which is associated with the increased apoptosis of cells carrying the tRNA29. Bax is a pro-apoptotic protein. Studies have shown that the binding of Bax to VDAC could lead to a change in mitochondrial membrane permeability, which significantly decreases the mitochondrial membrane potential, resulting in the release of apoptosis related factor (AIF) and cytochrome c, which promote apoptosis30. Our previous studies have also showed that the increased expression and colocalization of VDAC and Bax contribute to apoptosis31.VDAC is the main channel by which ATP and other metabolites translocate between the mitochondria and the cytoplasm, and it plays an important role in the regulation of mitochondrial metabolism and cell growth; VDAC is located in the mitochondrial outer membraneDue to the limitations of ethical human material and transmitochondrial animal models, we can only get peripheral blood lymphocytes from patients with mtDNA mutations, which is a challenge for all mtDNA-related diseases. Mitochondrial diseases presented with heteroplasmy, which indicates that both normal and mutant mtDNA coexist in one cell. In addition, the percentage of mutant mtDNA is different among different tissues, and different tissues show clinical symptoms at different thresholds. The percentage of mutant mtDNA in cardiomyocytes, neuronal cells and vascular endothelial cells are much higher than that in peripheral blood lymphocytes. However, the thresholds in these cells for the presentation of clinical symptoms are lower than that in lymphocytes. Therefore, if mtDNA mutations in lymphocytes could lead to increased ROS synthesis, decreased ATP synthesis and increased caspase3/7 apoptosis-associated activity, we could speculate that these changes would be more obvious in endothelial cells.Met C4467A mutation in a Han Chinese family with hypertension. Segregation analysis and pedigree mtDNA sequence analysis showed that this family presented with a maternally inherited mutation pattern. Functional analysis of lymphocyte cell lines carrying the tRNAMet C4467A mutation showed that it contributes to mitochondrial dysfunction, including increased oxygen free radicals and cell apoptosis as well as decreased ATP synthesis and mitochondrial membrane potential, thereby causing mitochondrial oxygen consumption disorders. The expression of VDAC, Bax and AIF in mutant cell lines increased compared with that in control cell lines; additionally, there was an increase of VDAC and Bax colocalization, which contributes to cell apoptosis. Despite our findings that mitochondrial biogenesis dysfunction is associated with the tRNAMet C4467A mutation, the exact mechanism remains unclear and should be examined in future studies.In conclusion, the present study identified a novel tRNA32. This Han Chinese family with maternally inherited hypertension was identified in the Cardiology Department of the Chinese PLA General Hospital. As part of a genetic screening program for hypertension, the protocol has been previously described in detail33.The protocols were approved by the ethics committee of the Chinese PLA General Hospital and performed in accordance with the approved JNC 8 guidelines and clinical regulationsOne physician measured the systolic and diastolic blood pressures of all subjects using a mercury column sphygmomanometer. The first and the fifth Korotkoff sounds were indicative of systolic and diastolic blood pressure, respectively. The average of three measured blood pressure readings was taken as the examination blood pressure. Hypertension was defined according to the guidelines of the Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure (JNC VI) and the World Health Organization-International Society of Hypertension as a systolic blood pressure of 140 mmHg or higher and/or a diastolic blood pressure of 90 mmHg or greater or with a history of hypertension and under antihypertensive drug treatment.A series of tests were conducted to exclude secondary hypertension. Bilateral renal and adrenal ultrasound and renal artery ultrasound were performed in all of the hypertensive patients in this study. The blood levels of renin, angiotensin, and aldosterone as well as the cortisol secretion rhythm were also evaluated.In all cases, venous blood samples were drawn upon admission for biochemical examination. In addition, DNA samples of the maternal members from this family and 366 healthy controls were acquired for sequence analysis. Written informed consent was obtained from all participating subjects.36.Genomic DNA was isolated using the Puregene DNA Isolation Kits according to the kit instructions . The entire mtDNA of all subjects, including hypertensive and control individuals, was PCR amplified in 24 overlapping fragments, with more detailed information described elsewhere37. Lymphoblastoid cell were cultured in RPMI 1640 medium  supplemented with 15% fetal bovine serum.Lymphocyte cell lines were derived from three maternal members  and three non-maternal members  of this family  liposoluble probe following the manufacturer’s instructions . The probe is hydrolyzed to DCFH, which is oxidized by ROS to fluorescent DCF. The fluorescence intensity is proportional to the level of ROS and was detected at 488 nm and 528 nm by flow cytometry .3, 2 × 103, 4 × 103, and 8 × 103 lymphocytes from each group were homogenized in a protein extraction solution and centrifuged at 8,000 × g for 10 min; then, the supernatant was collected for ATP measurement using a bioluminescence-based cell viability assay (Promega Co.). Light emitted from the luciferase-mediated reaction was detected by a tube luminometer .Samples of 1 × 1038.Lymphoblastoid cells were incubated with 5 μmol/L JC-10  for 10 min at 37 °C. Fluorescence intensities of JC-10 monomers and aggregates were measured by the FL1 (530/30 nm) and FL2 (585/42 nm) detectors of the flow cytometer, respectively. The JC-10 aggregate/monomer ratio is associated with the mitochondrial membrane potential intensity as previously described2 were determined under basal conditions and with various substrates and inhibitors, including oligomycin (1.5 μM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μM), rotenone (1 μM), and antimycin A (1 μM), as detailed elsewhere39.The rates of oxygen consumption in lymphocyte cell lines were evaluated with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience). The rates of OThe level of apoptosis after treatment was assessed with the Caspase-Glo 3/7 assay kit . A protein sample (1 mL) was diluted in 24 μL of assay buffer, and then, the manufacturer’s protocol was followed.40.Studies have shown that the binding of Bax to VDAC could lead to a change in mitochondrial membrane permeability, which promotes the release of AIF from the mitochondria to the nucleus. Therefore, we evaluated the expression of VDAC-1, Bax and AIF. Immunostaining was used to assess the co-localization of AIF, VDAC-1 and Bax polypeptides. Specimens were fixed  and then blocked with 5% BSA for 15 min. Lymphocytes were incubated in blocking solution (10% BSA in PBS) for 1 h at 37 °C followed by polyclonal monoclonal anti-Bax (1:100), anti-VDAC (1:200), and monoclonal anti-AIF (1:200) primary antibody incubation overnight at 4 °C. The cells were washed and then incubated with rhodamine 123, FITC green and AMCA for 60 min at 37 °C, respectively. Then, slides were imaged using a confocal laser scanning system. Excitation-emission involved the use of an Argon 488 nm laser in conjunction with a 505–525-nm filter and a 350-nm filter41. Equivalent amounts of protein were separated by SDS-PAGE, transferred to a membrane, and then incubated with anti-VDAC  or anti-Bax  or anti-AIF  primary antibodies, followed by goat anti-mouse, rabbit or goat IgG/HRP  secondary antibodies. Quantity One software  was used to calculate the gray value and gray area of the protein bands. Each cell sample was measured three times, and the average value was recorded.The cells were isolated for protein extraction, and the protein concentrations were determined as previously describedt-test was used to compare the control and mutated cell lines. The statistical software GraphPad Prism  was used for data analysis. All analyses were performed using SPSS 18.0 statistical software. P ≤ 0.05 was considered statistically significant.The data are represented as the mean ± SEM. The independent samples Supplementary Info File #1


Xenograft studies in NOD/SCID mice demonstrated efficient tumorigenesis from low cell numbers of ALDH1-positive and ALDH1-negative subpopulations. Targeting ALDH1 with Diethylaminobenzaldehyde (DEAB) and Disulfiram, significantly re-sensitized resistant lung cancer cells to the cytotoxic effects of cisplatin. Our data demonstrate the existence of a lung CSC population and suggest a role for targeting ALDH1 as a potential therapeutic strategy in re-sensitizing NSCLC cells to the cytotoxic effects of cisplatin.Non-small cell lung cancer (NSCLC) accounts for a large proportion of cancer deaths and is characterized by low treatment response rates and poor overall prognosis. In the absence of specific treatable mutations, cisplatin-based chemotherapy plays an important role in the treatment of this disease. Unfortunately, the development of resistance has become a major therapeutic challenge in the use of this cytotoxic drug. Elucidating the mechanisms underlying this resistance phenotype, may result in the development of novel agents that enhance sensitivity to cisplatin in lung cancer patients. In this study, targeting the cancer stem cell activity of aldehyde dehydrogenase 1 (ALDH1) was investigated as a strategy to overcome chemoresistance in NSCLC. Tumors from NSCLC patients showed an increase in their profile of pluripotent stemness genes. Cisplatin exposure induced the emergence or expansion of an ALDH1-positive subpopulation in cisplatin sensitive and resistant NSCLC cell lines, respectively, further enhancing cisplatin resistance. Using the Aldefluor assay and FACS analysis, ALDH1 subpopulations were isolated and evaluated in terms of stem cell characteristics. Only ALDH1-positive cells exhibited asymmetric division, cisplatin resistance and increased expression of stem cell factors  With more than one million cases of lung cancer diagnosed each year, it has become the leading cause of cancer-related death worldwide. Lung cancer is classified into two main subtypes; non-small cell lung cancer (NSCLC) which accounts for 85% of cases, while the remaining 15% consists of small-cell lung cancer (SCLC) . NSCLC iSince its introduction into clinical trials in 1971 and subsequent FDA-approval in 1978, cis-diammine-dichloro-platinum II (cisplatin) represents a major landmark in the history of successful anti-cancer therapeutics . It has The cancer stem cell (CSC) hypothesis suggests that a rare population of cells exist within tumors which display stem-like characteristics such as multipotency, increased expression of stemness-associated markers and the ability to self-renew and differentiate, both of which are essential for tumor initiation, maintenance, progression and metastasis , 7. CellMembers of the aldehyde dehydrogenase (ALDH) family of cytosolic isoenzymes are responsible for oxidising intracellular aldehydes and play a role in the oxidation of retinol to retinoic acid in early stem cell differentiation . HaematoNANOG, OCT-4, SOX-2, KLF4 and C-MYC), in addition to the CSC-specific markers, CD133 and ALDH1, in a cohort of matched normal and tumor lung tissues from NSCLC patients (n=20). Expression of the stemness-associated gene profile was significantly altered in both adenocarcinoma , KLF4 (p<0.05) and C-MYC (p<0.001). Similarly, gene expression of the CSC markers, CD133 (p<0.01) and ALDH1 (p<0.001) were significantly altered in tumor tissues. A similar, but more significant increase in the number of cancer stemness genes was also found in squamous cell carcinoma patients, NANOG (p<0.01), OCT-4 (p<0.05), SOX-2 (p<0.01), C-MYC (p<0.05). CD133 (p<0.01) and ALDH1 (p<0.001) mRNA was significantly up-regulated in squamous cell tumors. These data imply a greater stem-like population in NSCLC tumors relative to their matched normal tissues.RT-PCR and densitometric quantification analysis was used to quantify mRNA expression of a panel of stemness genes  cell fractions to examine the CSC potential of these subpopulations of cells. Cell fractions (ALDH1+ve and ALDH1-ve) were treated with increasing concentrations of cisplatin to assess their proliferative capacity Figure . The ALDStem cells have the unique ability to asymmetrically divide, where a single stem-like cell has the potential to give rise to a heterogenic population. To examine the potential ability of ALDH1+ve cells to asymmetrically divide, single cells were isolated, plated and their ability to give rise to a heterogenic ALDH1 population was assessed by flow cytometry Figure . Clones NANOG and OCT-4 gene expression relative to their ALDH1-ve counterparts , SOX-2 expression was significantly increased in ALDH1+ve fractions from all three CisR sublines . Similarly, the CSC marker, CD133, was significantly up-regulated in H460 ALDH1+ve cells  and SKMES-1 ALDH1+ve cells  relative to their ALDH1-ve controls. To assess the tumor initiating capacity of ALDH1+ve subpopulations in vivo, ALDH1+ve and ALDH1-ve cells (1×103) isolated from the H460 CisR subline, were subcutaneously injected into the right flank of NOD/SCID mice and tumor growth was measured over time  and post-treatment (week 2)  and following chronic treatment (week 2) to determine the proliferative effects and resistance of this chemotherapeutic drug on these cells. Chronic exposure to cisplatin resulted in an increase in drug resistance, as demonstrated by differences in IC) Figure .50 concentration of cisplatin. Similarly, chronic exposure of the H460 CisR subline resulted in a significant expansion of the ALDH1+ve subset . This increase in ALDH1 was associated with a corresponding increase in the IC50 concentration of cisplatin, from 30.16μM (week 0) to 34.78μM (week 2). In H1299 cells, cisplatin exposure promoted the emergence of a highly significant ALDH1+ve CSC subset in PT cells  and significantly enriched the ALDH1+ve subpopulation in the corresponding CisR subline from 10.5±1.02% to 19.17±2.43% (p<0.05). These observed increases in ALDH1+ve cell subsets were also associated with increased cisplatin IC50 concentrations and resistance to cisplatin . Similar expansion of the ALDH1+ve subset was observed in SKMES-1 PT and CisR cells, where cisplatin significantly enhanced the presence of the ALDH1+ve subpopulations from 1.53±0.03% to 11.31±1.42% (p<0.01) in the SKMES-1 PT cell line and from 17.06±0.96% to 25.46±0.77% (p<0.01) in the SKMES-1 CisR subline. This in turn, corresponded with an increase in cisplatin IC50 concentrations .The presence of the ALDH1+ve subpopulations were investigated following chronic exposure of the cell lines for two weeks, relative to controls (week 0) using the Aldefluor assay and flow cytometry analysis Figure . ChronicNANOG (p<0.05) and SOX-2 (p<0.05) in the H460 PT cell line, while NANOG (p<0.05), OCT-4 (p<0.01), SOX-2 (p<0.05), in addition to the CSC-specific markers, CD133 (p<0.001) and ALDH1 (p<0.01), were significantly up-regulated in its CisR counterpart. In the H1299 PT cell line, cisplatin induced a significant induction of OCT-4 (p<0.05), SOX-2 (p<0.05), CD133 (p<0.05) and ALDH1 gene expression (p<0.05). In the CisR subline, there was a significant up-regulation of all markers, NANOG (p<0.01), OCT-4 (p<0.01), SOX-2 (p<0.05) and KLF4 (p<0.05), CD133 (p<0.01) and ALDH1 (p<0.05), with the exception of C-MYC. While chronic treatment of the SKMES-1 PT cells with cisplatin significantly increased OCT-4 (p<0.001), SOX-2 (p<0.05), CD133 (p<0.001) and ALDH1 (p<0.05) mRNA, only CD133 (p<0.01) was significantly altered in the the CisR subline relative to expression at baseline (week 0).Based on our observations that ALDH1+ve cell subsets show increased expression of stemness markers relative to ALDH1-ve subsets, the stemness-associated mRNA gene profile of H460, H1299 and SKMES-1 cell lines following prolonged cisplatin exposure was examined. An up-regulation of both stemness and CSC-associated genes was observed Figure . CisplatThese data support our hypothesis that lung cancer cells exposed to cisplatin enrich for a subpopulation of ALDH1+ve cells with stem-like characteristics which may, at least in part, account for the resistance phenotype exhibited by these cells.To assess whether inhibition of ALDH1 can overcome cisplatin resistance, CisR sublines were treated with the ALDH1 inhibitor, DEAB, for 72hrs and ALDH activity was reassessed by flow cytometry. A significant reduction in the ALDH1+ve cell subset was observed across all three cisplatin resistant sublines Figure  relativein vitro.To further assess the functional effects of ALDH1 inhibition in cisplatin resistant NSCLC, cisplatin resistant sublines were treated with increasing concentrations of cisplatin (0-100μM) alone, and in combination with DEAB 15μM). The functional effects of inhibiting ALDH1 activity in combination with cisplatin treatment in CisR NSCLC sublines were examined in terms of proliferative capacity, clonogenic survival and apoptotic cell death relative to cisplatin-only treated cells . The effect of Disulfiram has previously been shown to be potentiated by copper when used in combination in vitro and as such, all experiments carried out as part of this study employed Disulfiram in a complex with copper chloride, and from herein, is referred to as DSF   . Experimin vivo data was carried out using the non-parametric Mann-Whitney test. All data were analysed using GraphPad PrismTM (version 5) statistical software.Analysis between groups was carried out using analysis of variance (ANOVA). Statistical comparison of two means was carried out using an unpaired, two-tailed Student's t-test. Significance was defined as p≤0.05. Data is graphically represented as mean ± standard error of the mean (SEM). Statistical analysis of in vitro and in vivo models and to further explore CSC-mediated mechanisms of resistance. While our data demonstrate an important role for ALDH1 as a stemness marker in chemoresistant lung cancer cells, inhibition of its activity and subsequent re-sensitization to the cytotoxic effects of cisplatin via chemical and pharmacological inhibition using DEAB and Disulfiram, respectively, demonstrates the potential use of these agents as a future strategy in targeting subsets of CSCs in cisplatin resistant lung tumors. Further molecular studies characterizing lung CSCs will yield additional information regarding the behaviour of these stem cell subsets, in addition to identifying novel targeted therapies. Of interest, ALDH1A1−/− mice are viable [This study set out to investigate the potential targeting of ALDH1 in cisplatin resistant NSCLC using e viable , suggest