L. These data provide confirmation of EZH2 in determining cell fate following DNA damage and propose a novel therapeutic strategy for patients with aggressive treatment-resistant B-cell lymphoma.Reactivation of apoptotic pathways is an attractive strategy for patients with treatment-resistant B-cell lymphoma. The tumor suppressor, p53 is central for apoptotic response to multiple DNA damaging agents used to treat aggressive B-cell lymphomas, including etoposide. It has been demonstrated that etoposide induced DNA damage and therapeutic efficacy is enhanced by combination with inhibitors of the histone methyltransferase, enhancer of zeste homolog 2 (EZH2). Further, EZH2 was identified to regulate cell fate decisions in response to DNA damage. Using B-cell lymphoma cell lines resistant to etoposide induced cell death; we show that p53 is dramatically down regulated and MDMX, a negative regulator of p53, is significantly up regulated. However, these cell lines remain responsive to etoposide mediated DNA damage and exhibit cell cycle inhibition and induction of senescence. Furthermore, chemical inhibition of EZH2 directs DNA damage to a predominant p53 dependent apoptotic response associated with loss of MDMX and BCL-X TP53 (p53) is inactivated or mutated in multiple cancers, including B-cell lymphoma [The tumor suppressor lymphoma . P53 actlymphoma . GenotoxFailure to achieve clinical responses in patients using genotoxic chemotherapy in part maybe attributed by induction of senescence rather than apoptosis by p53. Multiple studies indicate that p53-mediated senescence impairs the apoptotic response to chemotherapy in various cancers , resultiBRG1 or EGFR mutations [Previously, our investigations have demonstrated significant combinatorial effect of etoposide with EZH2 inhibition that resulted in increased cellular apoptosis associated with sustained DNA damage in models of lethal prostate cancer . Also, iutations .This study investigated the p53 response of two independent multidrug resistant B-cell lymphoma models to etoposide. We demonstrate that etoposide treatment of multidrug resistant B-cell lymphomas results in p53 activation and DNA damage associated with induction of cell cycle arrest and senescence. Inhibition of EZH2 in multidrug resistant B-cell lymphomas directed etoposide mediated DNA damage response towards a p53 dependent apoptotic response associated with the loss of MDMX and BCL-XL expression. These data broaden the relevance and utility of etoposide and EZH2 inhibitor combination as a novel therapeutic strategy for aggressive B-cell lymphomas.It was previously demonstrated that repeated exposure of B-cell lymphoma cell lines RL and Raji to rituximab induced a rituximab-resistant phenotype (RL-4RH and Raji-4RH) . SurprisWhile RL-4RH and Raji-4RH cell lines were resistant to etoposide-mediated apoptosis, it was unclear if p53 was still functional and if resistance to all p53 mediated mechanisms including cell cycle arrest and senescence were abrogated. Treatment for 72 hours with 10μM etoposide showed marked increase of p53 mRNA and the p53 target gene, p21 in Raji-4RH cells Figure . RL-4RH Recently, EZH2 has become a high priority therapeutic target in multiple cancers, including aggressive B-cell lymphomas. In addition, EZH2 is demonstrated to dictate cell fate in response to DNA damage and combination of etoposide with EZH2 inhibitors elicits potent anti-tumor activity , 10. TrePreviously, our work and others have demonstrated the greater anti-tumor activity of etoposide combined with EZH2 inhibitors , 10. It As shown in figures myc B-cell lymphoma cell lines [myc lymphomas exposed over 3 days were highly sensitive to etoposide-induced apoptosis, both as a single agent and in combination with GSK126. GSK126 single agent activity had minimal effect with inducing apoptosis in Eμ-myc lymphomas  retain wild type p53, though its expression is dampened due increased p53 proteasome degradation by ubiquitin ligase activity [Arf−/−myc/p19 lymphomas following 3-day drug exposure  epigenetic mechanism. Apoptotic response was restored following de-repression of miR-377 with concurrent loss of BCL-XL expression [L protein expression associated with increased apoptotic response to combination treatment, adding that EZH2 potentially regulates BCL-XL via epigenetic mechanisms.Previously, it had been shown that RL- and Raji- 4RH multi-drug resistant cell lines displayed aberrations involving the intrinsic apoptotic pathway . ResistaL . All thrrd et al  also indpression . Our datMost strikingly, we observed a significant increase of MDMX protein expression in RL- and Raji-4RH cell lines compared to their respective parental lines. MDMX is a part of the MDM family that function as critical inhibitors of p53 expression and function . Upon DNL and MDMX, important regulators of apoptotic and p53 signaling respectively, are in part epigenetically regulated by EZH2. Ultimately, these results provide exciting potential for the combination of genotoxic therapy with inhibitors of EZH2 as a novel therapeutic strategy for patients with aggressive treatment resistant B-cell lymphomas.Because of the toxic side effects that accompany chemotherapy, utility of a combination therapy approach that directs DNA damage response towards apoptosis and allows for lower chemotherapy dosing would provide great benefit to patients. Overall, we demonstrate that inhibition of histone methyltranferase EZH2 regulates cell fate decisions in response to genotoxic chemotherapy in multi-drug resistant models of B-cell lymphoma. Further, this data suggests novel information that BCL-Xmyc, Eμ-myc/p53−/−, and Eμ-Arf−/−myc/p19 B-cell lymphomas were kindly provided by Dr. Ricky Johnstone. Eμ-myc lymphomas were cultured in 6-well plates in high-glucose DMEM (Dulbecco modified Eagle medium) supplemented with 10% fetal calf serum, penicillin/streptomycin, 0.1 mM L-asparagine and 50 μM 2-mercaptoethanol in a 37°C, 10% CO2 humidified incubator. Human B-cell lymphoma cell lines (RAJI-4RH and RL-4RH) were kindly provided by Dr. Francisco Hernandez-Ilizaliturri and cultured under standard conditions  in RPMI  1640 medium supplemented with 10% FBS and penicillin/streptomycin. Etoposide (Sigma-Aldrich) and GSK126 (Xcess Biosciences Inc.) were maintained in DMSO at 1mM and 10mM stock concentrations respectively.Eμ-4 cells/well. The day after, the selected compounds were added at the indicated concentrations. Viability tests were performed by propidium iodide (PI) uptake (Sigma Aldich), Annexin V staining (BD Pharmigen), cell-cycle analysis or Tetramethylrhodamine ethyl ester (TMRE) staining (Sigma Aldrich) according to the manufacturer “s instructions. Proliferation assays were performed using the MTS reagent (Promega) prepared according to manufacturer's instructions. Senescence was determined using a senescence detection kit as per manufactures instructions (Biovisions Incorporated).Cells were seeded in 24-well plates to a final concentration of 1.5×102O, and reagents (5X iScript reaction mix + iScript reverse transcriptase) from the kit in a total volume of 20μl. cDNA was diluted 1:4 prior to qRT-PCR. PCR primers were designed with NCBI's primer blast tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), with a melting temperature 57-63°C and a resulting product size of 75-200bp. Primers were obtained from Integrated DNA technologies . Primer sequences were MDMX , TP53 , P21  and GAPDH . Each PCR reaction was carried out in technical triplicates in a 10μl volume utilizing SYBR Green Master Mix . GAPDH was used a control gene. The resulting Ct-values for each gene were normalized to the expression values of GAPDH.RNA isolation from cell lines was performed by TRIzol extraction. Synthesis of cDNA was performed according to iScript cDNA Synthesis Kit . 1μg of RNA was added to a master mix containing nuclease free HWhole Cell Lysate Preparation (WCL): Cells were harvested and lysed with RIPA buffer  + 1X P-STOP + 1X PIC (Roche) for 30 minutes on ice. Eppendorf tubes were vortexed every ten minutes for 10 seconds. After cell lysis tubes were centrifuged at 13,000 rpm for 15 minutes at 4°C. Supernatant of each tube was collected and transferred to a new tube.Histone Extraction: Histone extractions were performed using the Epigentek  histone extraction kit.Protein concentrations of whole cell lysates (WCL) and histone extractions were measured by the bradford protein assay (Bio-Rad laboratories). Protein lysates  where separation using 4-15% by SDS-PAGE gels (Bio-Rad). The proteins were transferred from the SDS-PAGE gel onto nitrocellulose membrane (0.2 μm)  via the semi-dry method  for 35 minutes at 15V. Membranes were blocked in either 5% skim milk or BSA in 0.1% tween-PBS (tPBS) for 1-hour at RT. Membranes were washed briefly 3x with tPBS prior to primary antibody incubation at 4°C over night. Membranes were then washed 3×10 minutes before the addition of secondary horseradish peroxidase (HRP)-conjugated antibodies  diluted in tPBS. After incubation at RT for 1-hour with agitation the membranes were washed 3×10 minutes in tPBS. The immunoreactive bands were visualized by enhanced chemiluminescence with ECL detection reagents . The blots were exposed to Bio film for 1 second-10 minutes. The films were then developed in a Kodak film developer. To estimate molecular weight of bands a pre-stained protein ladder was used . The following primary antibodies were used for Western blotting: Tp53 , MDMX (ProteinTech), EZH2 , H3K27me3 , Total Histone H3 , GAPDH . Each western blot data has been confirmed in at least two biological independent experiments.


The patient was administered two cycles of CHOP chemotherapy for 56 days, however, ultimately succumbed as a result of disease progression. Therefore, PBL represents a diagnostic and therapeutic challenge. PBL must be considered in the differential diagnosis of gastrointestinal tumors in daily practice, even in immunocompetent patients. Furthermore, CHOP does not appear to be an optimal treatment regimen and more intensive regimens are required.Plasmablastic lymphoma (PBL) is a rare and recently described entity of large B-cell lymphoma. It predominantly occurs in the oral cavity of human immunodeficiency virus (HIV)-positive patients and exhibits a highly aggressive clinical behavior without effective treatment. Recently, sporadic cases describing PBL in extraoral locations of HIV-negative patients have been reported; frequently in patients with underlying immunosuppressive states. To develop the understanding of PBL, the current study reports the unusual presentation of duodenal PBL and reviews the pathogenesis, immunohistochemical features, clinical and differential diagnoses, as well as the treatment of PBL as described in previous studies. The case of a 75-year-old female with duodenal PBL without definite immunosuppression is presented in the current report. The tumor was composed of large B-cell-like cells, and was positive for cluster of differentiation 138 and melanoma ubiquitous mutated-1, with ~80% of the tumor cells positive for Ki-67. The features of the tumor were as follows: Extraoral location, HIV-negative, immunoglobulin M λ-type M protein expression, light chain restriction  and Epstein-Barr virus-encoded small RNA-negative, which are considered to be unusual for PBL. These unusual features complicate the differentiation of PBL from other plasma cell diseases. To the best of our knowledge, this is the first study to report a case of duodenal PBL in an immunocompetent patient. To date, the standard treatment of PBL remains elusive, however, the most commonly administered chemotherapy treatments are CHOP [intravenous cyclophosphamide (750 mg/m Plasmablastic lymphoma (PBL) has been recently recognized as an aggressive non-Hodgkin’s B-cell neoplasma, which exhibits diffuse proliferation of large neoplastic cells that resemble B immunoblasts with an immunophenotype of plasma cells . PBL wasPBL typically presents clinically with rapid progressive growth and is associated with poor outcomes . It may A 75-year-old female was admitted to the outpatient clinic of the West China Hospital  in July 2009. The patient had a history of epigastric distension and abdominal pain that had persisted for more than one month. During physical examination, an abdominal mass was palpable in the epigastrium without lymphadenopathy or hepatosplenomegaly.Helicobacter pylori (HP) was detected by light microscopy  with Giemsa staining. The bone marrow was free of lymphoma and no radiological evidence of a lytic bone lesion was found.The laboratory abnormalities identified included mild anemia, an elevated lactate dehydrogenase level of 1,087 U/l , and the detection of monoclonal immunoglobulin M (IgM)-λ-type M protein and λ-type Bence Jones protein. The differential white blood cell and platelet counts, hepatic and renal function tests, serum carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 199 and CA125 levels, and immunological detection reflecting T/B-lymphocyte function were normal. In addition, serology was negative for HIV, hepatitis B virus (HBV), hepatitis C virus, EBV and the HHV8 infection. The esophagogastroduodenoscopy revealed an ulcer in the duodenal bulb, measuring 1.2×1.5 cm in size, as well as mild chronic non-specific inflammation of the gastric antrum. An enhanced computed tomography scan of the abdomen  showed m1, granzyme B, B-cell lymphoma (BCL)-6, CD30, CEA, anaplastic lymphoma kinase (ALK), EBV-encoded small RNA (EBER) and HHV8  and frequent mitotic figures. Upon immunostaining, the neoplastic cells were positive for cluster of differentiation (CD) 138, melanoma ubiquitous mutated-1 (MUM-1), endomysial antibodies (EMA), IgM and Igλ; weakly positive for CD45 and CD10; and negative for CD19, CD20, CD3, CD5, CD79a, CD56, a B-cell-specific activator protein (PAX5), cyclin Dand HHV8 . Monoclo2, day 1), pegylated liposomal doxorubicin  vincristine  and prednisone  (CHOP) chemotherapy was administered, which lasted for 56 days. The abdominal mass continued to enlarge with extensive lymph node metastasis , positivity for plasma cell markers  and variable expression of the leukocyte common antigen CD45) . The maj5 . The mDifferentiating PBL from other malignant diseases, including MM, DLBCL, poorly differentiated carcinomas, malignant melanoma, gastrointestinal stromal tumors (GIST), Burkitt’s lymphoma and anaplastic large cell lymphoma (ALCL), is diagnostically difficult. Furthermore, the treatments for these diseases are significantly different; therefore, differentiating between these entities is critical. CD20 negativity facilitates with distinguishing PBL from DLBCL and Burkitt’s lymphoma, while malignant melanoma can be excluded by observation of S-100 protein and HMB45 antigen negativity. Cytokeratin positivity aids with differentiating PBL from poorly differentiated carcinomas, and GIST is a common mesenchymal tumor of the small intestine, which is usually characterized by CD34 and CD117 expression. Furthermore, tumor cells in ALCL are consistently immunoreactive for CD30 and usually immunoreactive for CD3 and ALK. The distinction between PBL and MM is frequently dependent on their clinical presentations as PBL exhibits an identical immunophenotypic profile and an overlapping morphological spectrum with MM (multiple myeloma) . DetectiThe outcomes of PBL are usually poor, with an annual mortality rate of ~60% . HIV-negIn conclusion, PBL represents a diagnostic and therapeutic challenge. Its predominant presentation in males, a predilection for the oral cavity, history of HIV infection and EBER expression are features that may aid with its differential diagnosis. However, an extraoral location, and HIV- and EBER-negative female patients hinder the clinical differentiation. Therefore, PBL must be considered when unexplained duodenal ulcers are exhibited, even in immunocompetent individuals that do not demonstrate EBER expression.


Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines , yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. In some cases, even the only susceptible cell available to propagate specific viruses for which vaccines are needed could be tumor cells. Therefore, currently several tumor cells lines are being explored for their capacity to propagate viral vectors, like the Madin-Darby canine kidney (MDCK) cell line [Vaccines are pharmacological formulations that incorporate the disease-causing agent or an antigen derived from this agent, which are capable of inducing an immune response once administered to a healthy individual, without causing the disease itself. Licensed vaccines can be divided into viral and bacterial vaccines, and viral vaccines can be further classified into four categories: live attenuated viruses, inactivated viruses, subunit vaccines, and virus-like particles. For the production of the first two categories, large amounts of viral particles are needed, and most of these viral vaccines are produced by infecting susceptible cell lines. Since there is no standard cell line that can be used for the replication of every virus, a whole panel of different cell lines has been used for vaccine production processes throughout the years. Cell lines that have historically often been used for the production of viral vaccines are MRC-5 and WI-38 , 2. Thesell line , HeLa ceell line , and theell line . At the ell line –26.Overall, it can thus be stated that the regulatory opinion on cell lines that are used for the production of viral vaccines has changed radically at the last 30 years with respect to the risks and benefits of immortalized tumorigenic cell lines , 26. ThiA characteristic of viruses is that viruses evolve, and due to mutations in their genetic material or recombination with other viruses, outbreaks of dangerous and potential lethal new viruses can occur. In these cases, fast development of vaccines is essential for global health. It would be of great importance if cell lines that are needed for the production of such vaccines could be selected upfront, at the start of a viral outbreak, based on scientific understanding instead of trial and error. In this study, we have made a start with the characterization of human tumor cell lines and their capacity to support viral replication. Five human cell lines, capable of growing in suspension and often used in research, but not currently qualified for vaccine production purposes, were selected, HL60, K562, KG1, THP-1, and U937 –32. ThesThe overall aim of this study is to generate data that will facilitate decision making on which substrate may be the best for the production of novel vaccine strains. As a first step for this, we investigated whether poliovirus, as a representative of the Picornaviridae family of viruses, can be propagated in the human suspension cell lines.2 humidified atmosphere. The Vero cell line was taken along as a positive control cell line and these cells were cultured in Virus Production Serum-Free Medium  supplemented with 2 mM glutamine (Life Technologies) at 37°C in a 5% CO2 humidified atmosphere. CHO suspension cells, which served as a negative control, were cultured in Ex-cell 302 medium (Sigma) in shaker flasks (Corning) gently rotating at 50–100 rpm at 37°C in a 5% CO2 humidified atmosphere.The human hematopoietic progenitor tumor cell lines HL60, U937, K562, KG1, and THP-1 were cultured in IMDM  (Invitrogen) or RPMI (THP-1) supplemented with 10% fetal bovine serum  and 10 U/mL penicillin-streptomycin (Gibco) at 37°C in a 5% CO6 cells/mL in 1 L of culture medium. Cultibag cultures were performed at 37°C, dissolved oxygen concentration of 50%, pH 7.2 at a constant rocking speed of 10 rpm, and an angle of 7°.To determine whether K562 cells were capable of growing in bioreactors, Cultibags (Sartorius Stedim) were inoculated with cells at a concentration of 0.3 × 106 cells were infected in T25 flasks with an MOI of 1. Cells and supernatants were harvested if full cytopathic effect (CPE) was observed or after 1 week of culture. After freeze-thawing three times to lyse the cells, samples were centrifuged at 3000 rpm to remove cellular debris and half of the supernatant was used to reinfect new hematopoietic cells. This was repeated for 4 times to allow the virus to adapt to the new cell lines in 5 passages. In the last infection round, 1 × 107 cells in a T175 flask were infected and samples were harvested at day 3 (VERO and U937) or day 6 to obtain small viral stocks that were used for subsequent experiments.To serially passage Sabin poliovirus type 1 on the hematopoietic cell lines, 1 × 10To determine replication kinetics, the susceptible tumor cell lines were infected with Sabin poliovirus type 1 from the parental virus or virus that was passaged for 5 times on the hematopoietic cell lines at MOI 1 or MOI 0.01, and samples of the supernatant and cellular lysates were harvested at different time points. After harvesting, the supernatant was centrifuged at 1000 rpm for 4 minutes to remove cells floating in the medium that did not release their progeny virus yet, and this fraction was added to the cellular fraction. Cells still attached to the flask were harvested by scraping the cells from the flask with a cell scraper (Becton Dickinson) in PBS. After centrifugation at 1000 rpm, PBS was removed and the cell pellet was resuspended in 1 mL of PBS. The cellular samples were freeze-thawed three times to release virus from the cells, and after centrifugation at 3000 rpm supernatant was transferred to a new tube. The virus titer in all the samples was determined by end-point titration on Vero cells.6 cells/mL, and samples of the culture medium were taken at days 3, 4, 5, and 6 after infection. Samples were filtered (0.22 μM filter) to remove viable cells and the virus titer and D-antigen levels in these samples were determined.K562 cells grown in Cultibags were infected at an MOI of 0.01 with passaged Sabin poliovirus type 1 when cells reached a concentration of 1.2–1.5 ×104 cells/100 μL in 96-well flat bottom plates in M199 medium containing 10% serum. Serial 10-fold dilutions were prepared from cellular lysates or supernatant from hematopoietic cells infected with Sabin poliovirus type 1 in serum-containing M199 medium, and 50 μL of these dilutions was added to 6 separate wells. After 7 days, all the wells were scored for the presence or absence of cytopathic effects (cpe), and the virus titer was determined using the Reed and Muench method, thereby calculating the 50% cell culture infective dose (CCID50)/mL. To calculate the plaque forming unit titer (pfu) from the CCDI50 value, the CCID50 was multiplied with 0.69 to generate the pfu titer/mL. To be able to compare the number of infectious Sabin type 1 polioviruses in the supernatant samples with the cellular lysate samples, the total amount of pfu in the samples was determined by multiplying the pfu/mL titer with the total amount of supernatant or cellular lysate that was harvested.To determine the virus titer of Sabin poliovirus type 1 that replicated in the different cell lines, the virus titer was determined by end-point titration. In short, adherent Vero cells were seeded at a concentration of 1 × 10To determine the presence of D-antigen in the virus samples, a sandwich ELISA was performed as described previously . BrieflyTo determine the expression of viral receptors on the different cell lines, FACS analyses were performed. PE-labeled antibodies directed against human CD155 (eBioScience), CD54 (BD Pharmingen), and CAR (Millipore) and FITC-labeled anti-human CD81 (BD Pharmingen) were used for flow cytometric analyses, with FITC- or PE-labeled IgG1 antibodies (BD Pharmingen) as controls. Antibody staining was performed in PBS supplemented with 0.1% BSA and 0.02% sodium-azide for 30 min at 4°C. After washing of the cells, the stained cells were analyzed on a Guava FACS (Merck Millipore) using Cell FlowJo software.7 CCID50/mL in all 5 passages. In CHO cells, Sabin poliovirus type 1 could only be detected in the first passage, and this is most likely due to the fact that cells were not washed after primary infection, suggesting that the virus titer is the result of virus that remained present in the culture medium, which was unable to infect the cells. In samples from passages 2–5 of Sabin poliovirus type 1 added to CHO cells, as expected no virus could be detected. A comparable pattern was observed after infection of THP-1 and HL60 cells with Sabin poliovirus type 1, suggesting that these two cell lines were refractory for the virus. The K562 and U937 cell lines were highly permissive for Sabin poliovirus type 1, resulting in high virus titers in all 5 viral passages. KG1 cells were also fully permissive for Sabin poliovirus type 1, but the amount of virus obtained after infection of KG1 cells was slightly lower compared to Sabin poliovirus type 1 that replicated in the other permissive cell lines. Microscopically it was observed that U937 cells infected with virus from passages 0–2 did not show clear cytopathic effects (cpe) after 6 days of culture, whereas cells infected with virus from passages 3, 4, and 5 were harvested after 3 days because full cpe was observed. A photographic overview of cells infected with virus from passage 4 is shown in To determine whether hematopoietic cell lines can support replication of Sabin poliovirus type 1, cells were infected with an MOI of 1 and cells together with supernatant were harvested at day 3  or day 6 after infection. After lysis and centrifugation of the cells, half of the supernatant was used for the reinfection of new cells. This procedure was repeated for 4 more times, and virus derived from passage 5 was used for additional experiments. In the samples derived from all 5 passages, the virus titer (CCID50/mL) was determined by a virus titration assay. As shown in 7 CCID50. In general, it was observed that passaged virus had a D-antigen level per virus that was slightly lower compared to virus replicated in Vero cells, varying between 1.2 and 2.3 DU/107 CCID50 depending on the cell line used.To be able to compare the D-antigen level per virus, the specific D-antigen level per infectious virus particle was calculated for the fifth viral passage in the permissive cell lines and is shown in To investigate whether the expression of CD155, the poliovirus receptor, on the surface of the different hematopoietic cell lines correlated with the capacity to propagate Sabin poliovirus type 1, human CD155 expression was determined using FACS analysis. In To determine the timing of viral replication and viral release in the supernatant and to compare original with passaged virus, samples of the culture medium of cells infected with an MOI of 1 were taken at different time points after infection, and the virus titer in these samples was determined. The MOI of 1 was chosen in order to determine the kinetics of a single replication round and not to look at the effects of repeated infection cycles. Sabin poliovirus type 1 from the original stock (passage 0) was compared with the passaged virus (passage 5). In Furthermore, it can be concluded from these data that, for all three hematopoietic cell lines tested, the virus titer of the passaged virus at day 2 in the supernatant is comparable to the virus titer obtained 2 days after replication of Sabin poliovirus type 1 in Vero cells. Also, the virus titer in the culture medium remained stable during the 7 days of the experiment. Altogether, these data indicate that, with respect to the viral kinetics, all three human hematopoietic cancer cell lines are efficiently producing poliovirus particles.For vaccine production purposes it is important that, after infection of cells with a low MOI, a high viral yield in the culture medium can be achieved. To investigate this, cells were infected with an MOI of 0.01 of passaged Sabin poliovirus type 1, and supernatant and cellular lysates were harvested separately at day 4 and day 7. The total viral titer in these samples was determined and is shown in 6 cells/mL in 1 L culture medium. After 4 days, in which the K562 cells grew to a concentration of 1.2–1.4 × 106 cells/mL, the cells were infected with the passaged Sabin poliovirus type 1 at an MOI of 0.01. At days 3, 4, 5, and 6, the cell viability was determined and samples of the culture medium were taken, in which the virus titer and D-antigen concentration were determined. As can be seen in 9 CCID50/mL in the culture medium already at day 3 after infection, which remained stable for at least the following 3 days. The D-antigen level at day 3 after infection  . Among tBecause of the experience that our group has with poliovirus production processes –45, it wFirst, it was determined whether the five selected cell lines were susceptible for poliovirus infection. K562, KG1, and U937 cell lines appeared to be capable of supporting replication of Sabin poliovirus type 1, whereas HL60 and THP-1 were not. All five cell lines did express CD155, the receptor for poliovirus entry. This means that receptor expression does not predict the capacity of a virus to replicate in a cell. Possible explanations for the lack of replication in the CD155 expressing HL60 and THP-1 cells could be that (i) CD155 expressed on these cell lines is nonfunctional, (ii) the nonsusceptible cell lines lack the expression of a coreceptor on the cellular membrane, or (iii) HL60 and THP-1 express an intracellular viral restriction factor. Since the tested cell lines have the capacity to differentiate towards several end-stages and it has been shown for other viruses that the differentiation stage of a cell can affect the susceptibility for a virus –35, it wIn the literature it has been described that human blood cell lines were susceptible for infection with poliovirus , and thaAnother study compared the replication capacity of Mahoney poliovirus on K562 and U937 cells to HeLa cells . CompareIn two more recent papers, the replication capacity of Sabin types 1, 2, and 3 or wild type polioviruses was studied on a panel of different suspension cell lines, deriving from humans, avians, or canines. In both studies the virus was not adapted to the new cell lines, but experiments were directly performed with virus produced on Vero cells. Vlecken et al. compared a number of adherent and suspension cell lines for the capacity to replicate Sabin poliovirus types 1, 2, and 3. Of the 5 cell lines tested , only the CAP cell line was capable of propagating Sabin type polioviruses, whereas all the other suspension cell lines were not capable of supporting viral replication . In the Since we already observed that also other Sabin poliovirus types are capable of replicating in the permissive three cell lines  and that all cell lines express other viral receptors, needed for efficient infection with rhinovirus, coxsackie, adenovirus, and hepatitis C virus, it would thus be interesting to determine the capacity of these viruses to replicate in these cell lines. Ultimately both the panel of (human) cell lines and viruses need to be expanded, in order to be able to predict upfront the suitability of cell lines for the production of specific viral vaccines, based on scientific understanding, thereby reducing timelines and costs in vaccine production processes.In conclusion, these data demonstrate that K562, KG1, and U937 cell lines are efficient in supporting the replication of Sabin poliovirus type 1. All five human hematopoietic cell lines expressed CD155, which thus did not explain susceptibility to viral replication, although we did not study the functionality of CD155. Furthermore, we have shown that in KG1 cells the passaged Sabin poliovirus type 1 replicated more rapidly than the control virus. Infection of K562, KG1, and U937 at an MOI of 0.01 resulted in a high viral titer in the culture medium after 4 days. Also, K562 cells grown and infected with adapted Sabin poliovirus type 1 in a disposable bioreactor system yielded high viral titers in the culture medium. Altogether, it is concluded that K562, KG1, and U937 cell lines can be used for the propagation of poliovirus and in follow-up studies the capacity of other (entero)viruses to replicate in these cell lines will be determined.


Conventional high-grade osteosarcoma is the most common primary bone cancer with relatively high incidence in young people. Recurrent and metastatic tumors are difficult to treat. We performed a kinase inhibitor screen in two osteosarcoma cell lines, which identified MEK1/2 inhibitors. These inhibitors were further validated in a panel of six osteosarcoma cell lines. Western blot analysis was performed to assess ERK activity and efficacy of MEK inhibition. A 3D culture system was used to validate results from 2D monolayer cultures. Gene expression analysis was performed to identify differentially expressed gene signatures in sensitive and resistant cell lines. Activation of the AKT signaling network was explored using Western blot and pharmacological inhibition. In the screen, Trametinib, AZD8330 and TAK-733 decreased cell viability by more than 50%. Validation in six osteosarcoma cell lines identified three cell lines as resistant and three as sensitive to the inhibitors. Western blot analysis of ERK activity revealed that sensitive lines had high constitutive ERK activity. Treatment with the three MEK inhibitors in a 3D culture system validated efficacy in inhibition of osteosarcoma viability. MEK1/2 inhibition represents a candidate treatment strategy for osteosarcomas displaying high MEK activity as determined by ERK phosphorylation status. Osteosarcoma is the most common primary malignant bone tumor occurring predominantly in children and adolescents, as well as in people older than 40 years of age. It is thought to arise from mesenchymal stem cells that are capable of producing osteoid , 2. At tScreening a kinase inhibitor library of pre-clinical or clinically approved drugs provides the possibility of identifying novel candidate treatments for osteosarcoma that can be translated to the clinic.Receptor tyrosine kinases (RTKs) are frequently hyperactive through mutation or overexpression in cancer causing aberrant activation of downstream signaling cascades, including Ras/Raf/MEK/ERK . This cain vivo  and performed a LIMMA analysis . We thenDose response curve fitting and statistical analyses were performed with GraphPad Prism 5.0 .


The introduction of animal origin free (AOF) media has significantly reduced the incidence of adventitious virus contamination in biological production systems. Nevertheless, contamination by the parvovirus Mouse Minute Virus (MMV) remains a continuing challenge. Although infrequent, infection of a fermenter can be catastrophic for a manufacturer, and can also have a potential impact on drug supply, patient safety and have regulatory implications.In this work, we evaluated engineering Chinese Hamster Ovary (CHO) cell lines to create a new host cell line that would be resistant to MMV infection by modifying the major receptors used by the virus to enter cells. The goal was to engineer a host cell line resistant to MMV infection, while maintaining productivity and product quality profiles. Our strategy is outlined below.Attachment to a cell surface receptor is a key first step in the infection cycle for viruses. While the exact functional receptor for MMV binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated . MoreoveMMV infectivity studies were conducted on the knock out clones identified in Figure CHO cell lines engineered to express lower surface sialic acid demonstrate increased resistance to MMV infection compared to the wild-type controls. While the complete absence of sialic acid on the SLC35A1 knock-out cell line led to almost complete resistance to MMV infection, the COSMC knock out clones were about 10x more resistant to MMV infection. The knock out of the COSMC gene exclusively truncates O-glycosylation, leaving the N-glycosylation pathway and the terminal sialic acids intact. The lower levels of cell surface sialic acid however, were able to confer the 10x resistance to MMV infection. Moreover, the Slc35A1 knock-out appears to result in complete abrogation of binding of the virus to the cell surface, even at time T = 0. The COSMC knock-out on the other hand appears to affect virus internalization and replication rather than initial binding of the virus to the cell surface. In contrast, the St3Gal4 -/- and the St3Gal4-/- + St3Gal6-/- knock-out clones appear to affect both cell surface binding as well as the internalization of the MMV virus, with the cumulative effect being similar to that of the COSMC knock-out.Our data demonstrate that viral resistance against MMV virus can be incorporated into CHO production cell lines, adding another level of "defense", against the devastating financial consequences of this virus infection. While the importance of cell surface sialic acid for virus binding and internalization is clearly evident, further studies on the downstream steps of the MMV life cycle may provide further targets for interrupting and eliminating the infection process, thus conferring a higher degree of resistance.


Motivation: Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets.Results: Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel.Availability and implementation: The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper.Contact:marcotte@icmb.utexas.edu or jon.young@utexas.eduSupplementary information:Supplementary data are available at Bioinformatics online. Non-small-cell lung cancer (NSCLC) remains a significant healthcare burden despite recent progress in drug discovery and development. Recent FDA-approved targeted therapies are only intended for appropriate subpopulations of patients. The drug Xalkori (crizotinib) is highly effective, but only for ∼4% of lung cancer patients . SimilarWhen identifying drug targets, one approach is to identify genes whose knockdown selectively leads to death of cancer cells but not matched normal cells. Such genes represent genetic vulnerabilities and potential drug targets. Several studies have been able to use whole genome RNAi to identify genetic vulnerabilities for cancer drug target discovery. A screen against all genes in two lung cancer cell lines identified proteasome members as candidate targets and discovered that small-molecule proteasome inhibition synergized with radiotherapy in a mouse xenograft model . Included among the cell lines are subtypes commonly observed in patients: adenocarcinoma, squamous-cell and large-cell carcinoma. As described previously  and confirmed to match the DNA fingerprint library maintained by ATCC and the Minna/Gazdar laboratory and confirmed to be free of mycoplasma by e-Myco kit (Boca Scientific).A larger pool of NSCLC cell lines encompassing the cell line panel described above was screened with the tankyrase inhibitors IWR-1-endo  and XAV 939 (Tocris) in an 8-point 4-fold dilution series (top dose = 100 µM) in 96-well plates. Cells were plated 24 h prior to the addition of drug, incubated for 4 days, and assayed using MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay) according to the manufacturer’s instructions (Promega). Cell number per well was determined empirically and ranged from 500 to 4000 per well, inversely proportional to doubling times . Dose response curves were generated and IC50s calculated using in-house software, DIVISA. All cells were grown in RPMI-1640 (Sigma) supplemented with 5% FBS and incubated at 37°C in a humidified atmosphere with 5% CORNAi screens of cancer cell lines from Project Achilles (http://metazoa.med.utoronto.ca). The full RNAi data were represented as a M where m is the number of genes, n is the number of cell lines, andThe gene sets examined for genetic vulnerabilities were protein complexes chosen from CORUM  curve was plotted from the test set consisting of the withheld genes, given that the corresponding training samples were statistically significant. This benchmarking procedure was repeated multiple times and a mean ROC curve was generated by vertical averaging.Independent of the 2-means clustering approach, a second method was employed to detect NSCLC genetic vulnerabilities without reliance on annotated gene sets. The entire RNAi knockdown dataset was represented as a matrix as described above, where each row is a gene knockdown and each column is a cell line. The Large Average Submatrix (LAS) biclustering algorithm was applied to this matrix to uncover biclusters in which the rows (genes) exhibit similar behavior across a set of columns (cell lines) . We alsoWe also considered alternative measures of gene set sensitivity. For every cell line within each gene set, the probability of observing the number of ‘hit’ genes (gene knockdowns producing non-viability) was computed according to a hypergeometric distribution. The resulting probabilities for each line were then multiplied together to obtain an overall score for the gene set. Statistical significance of the scores was found using a permutation test, as was done for the 2-means clustering.From a whole genome RNAi screen of NSCLC cell lines, we identified candidate drug targets in the form of genetic vulnerabilities specific to cell line subgroups. A couple of factors were considered when determining genetic vulnerabilities. First, given the heterogeneity of NSCLC, gene deletions that are almost universally toxic across the cell line panel would likely be toxic to other normal human cells as well. In addition, it is desirable to specifically target lung cancer cells but not normal cells, yet only one cell line in the panel was from a non-cancerous normal lineage. This suggests that in the interest of specificity, the number of cell lines constituting a genetic vulnerability should not be too large. On the other hand, a vulnerability consisting of a single cell line may be less likely to generalize to an appreciable number of patients. Therefore, the challenge stems from identifying groups of genes to which some, but not all, NSCLC cell lines are especially dependent. Ideally, these cell lines would represent a particular patient subpopulation. Another challenge was to avoid a combinatorially intractable problem of having to examine all possible combinations of cell lines against all possible combinations of genes. Novel applications of unsupervised learning algorithms were developed to overcome these challenges to prioritize potential NSCLC targets from RNAi sensitivities.The general workflow of this study is outlined in Supplementary Fig. S1). Fisher’s exact test also determined 33 of those 35 complexes to be highly statistically significant . We found that the standard k-means algorithm and the 1-D optimal k-means method, Ckmeans.1d.dp, yielded identical results although Ckmeans.1d.dp demonstrated marked runtime speedup. Finally, simulations of a synthetic dataset showed that the permutation test for statistical significance was not biased toward larger or smaller complexes .The selected gene sets we chose to examine were 2820 protein complexes. As calculated by the 2-means clustering approach, 35 had statistically significant scores at 10% FDR from a permutation test .In assessing the performance of our 2-means clustering by a leave-one-out strategy, only protein complexes above a certain size were considered. For complexes with at least five members, the 2-means clustering achieves a mean AUC of 0.62, with the average being computed over five iterations of withholding a random gene from each complex. When considering protein complexes containing at least eight members, a mean AUC of 0.66 is attained over eight iterations . Because no statistical significance was found, we did not carry out the benchmarking procedure as above.We applied our 2-means clustering method to data from Project Achilles on sensitivity of 19 NSCLC cell lines to shRNA knockdown of 5711 genes. Due to the lower coverage of genes compared to our whole genome knockdown dataset, the 2-means clustering is able to be applied to only a portion of many of the protein complexes. According to a permutation test, we were unable to find any statistically significant protein complexes at the same FDR 10% level previously used. The top scoring result is a ribosomal complex, followed by two proteasome complexes. A majority of the significant protein complexes found from our own RNAi dataset also maintain the general pattern of partitioning into sensitive and resistant cell lines in the Project Achilles experiment . Functional enrichment was not found for three of the top 10 results. In addition, searching each bicluster for enrichment of protein complexes yielded heavy enrichment for the spliceosome. For most of the biclusters, the functions of the enriched protein complexes match those found from the DAVID enrichment (Supplementary Table S3).LAS biclustering was employed as an independent and complementary approach to identifying candidate drug targets. The 2-means clustering approach relies on annotated gene sets, namely protein complexes, to address the challenge of selecting gene groups to interrogate for bimodal response to RNAi knockdown. On the other hand, biclustering offered an alternative strategy to tackle this challenge as it could find genetic vulnerabilities without regard to any prior annotation. The LAS algorithm found 22 statistically significant biclusters with Bonferroni-corrected th-ranked bicluster, was not discovered by the 2-means clustering as many of its genes either did not appear in our protein complex set or were only present individually in single complexes.Many of the protein complexes from 2-means clustering also participate in biological processes found in LAS biclustering . In partOne notable bicluster showed enrichment for the Wnt pathway . The H20Supplementary Fig. S3a), which precludes the use of a simple z-test comparing the complex members’ scores to the background distribution. Even if the data were normally distributed (as the Project Achilles data is), this method would not distinguish bimodal from half-toxic complexes , and in fact would prioritize universally toxic complexes.We evaluated several other methods to measure complex sensitivity. These approaches depended on annotated gene sets, as opposed to biclustering, which has no such constraints. Cell line viability results from our whole genome screen are not approximately normally distributed . With the large number of protein complexes being statistically significant, we felt that this method was less discriminative than the 2-means clustering approach in prioritizing complexes.We also considered using the hypergeometric distribution to assess the significance of multiple occurrences of sensitivity within a protein complex. From a permutation test, we found 544 statistically significant protein complexes at FDR 10%  targets the TRiC/CCT complex , which has been FDA-approved for multiple myeloma (In addition, translation and splicing emerged as strong genetic vulnerabilities from the 2-means analysis. Previously, translation has been proposed as a potential target in cancer . MoreoveSaccharomyces cerevisiae and Schizosaccharomyces pombe. A similar phenomenon may be occurring among our NSCLC cell line panel. Although the cell lines are not necessarily distantly related, they likely differ sufficiently due to different mutational compositions. Different yeast species flip protein complex essentiality as a result of adaptations to differing needs and environments, a phenomenon likely common to cancer cells as well. Moreover, particular NSCLC cell lines are largely resistant to loss of most, but not all, members of certain protein complexes. Those genes that are mostly essential in both sensitive and insensitive cell line subgroups could exhibit ‘moonlighting’ behavior by having multiple functions in both essential and nonessential processes.Some of the NSCLC genetic vulnerabilities that were found by our computational analysis include protein complexes that may appear to be entirely essential. It is perhaps surprising that certain cell lines are largely resistant to knockdown of many of these genes. One explanation may simply be a result of the strict thresholding of the RNAi data to produce binary readings of cell line viability, which could be affected by the <100% sensitivity of the assay. Another explanation may be provided by essential gene ‘moonlighting’ and ‘flipping’ of protein complex essentiality between distantly related species (The NSCLC genetic vulnerabilities uncovered by the computational analysis described here extends an earlier study (Novel candidate drug targets were found from computational analysis of a whole genome RNAi knockdown screen in NSCLC cell lines. The targets are protein complexes specific for particular lung cancer cell lines and function in splicing, translation and protein folding. Results of previous studies support further investigation of these protein complexes as avenues of therapeutic intervention in NSCLC. Moreover, the candidate targets provide an opportunity for drug repurposing, which could lead to reduced time in the drug development pipeline. Our results simultaneously establish lung cancer cell line subgroups and potentially novel druggable targets that are specific to each subgroup. This study contributes to a deeper understanding of therapeutically relevant events at the molecular scale in NSCLC.


However, the available annotations of cell lines are sparse, incomplete, and distributed in multiple repositories. Re-analyzing publicly available raw RNA-Seq data, we determined the human leukocyte antigen (HLA) type and abundance, identified expressed viruses and calculated gene expression of 1,082 cancer cell lines. Using the determined HLA types, public databases of cell line mutations, and existing HLA binding prediction algorithms, we predicted antigenic mutations in each cell line. We integrated the results into a comprehensive knowledgebase. Using the Django web framework, we provide an interactive user interface with advanced search capabilities to find and explore cell lines and an application programming interface to extract cell line information. The portal is available at The online version of this article (doi:10.1186/s13073-015-0240-5) contains supplementary material, which is available to authorized users. Within Django, data tables are described in Python models that are database agnostic, allowing one to run the system on, for example, SQLite or PostgreSQL. For performance reasons, the TCLP runs on PostgreSQL in conjunction with the webproxy NGINX and memcache for caching web requests.To store, integrate, display, and interrogate the data, we developed a platform based on Django, a python web framework . This provides the user direct access to underlying data models and bulk data retrieval. The user interface relies on and interacts with this API; advanced users can thus discover the available entry points or alternatively browse the API page at http://celllines.tron-mainz.de/api. Additional file In addition to the user interface, we provide an API based on the Django REST Framework  of murine type c retrovirus in the transcriptome of the bladder urothelial carcinoma cell line 253JBV, which might have confounding effects on experiments [et al. [The portal integrates mutation information for the analyzed cell lines from CCLE  and Klijet al. . For eacet al. .Using the determined HLA Class I and Class II types in conjunction with the mutations enabled us to define a catalog of HLA Class I and Class II neo-epitope candidates. Figure et al. [Such a list can be input for experiments searching for tumor HLA-ligands. As an example, Bassani-Sternberg et al.  recentlyThe TCLP allows searching for and listing gene expression values from a selected cell line. The table enables the user to filter via the gene name or to define a RPKM value range. The table dynamically changes its content to display only the data fulfilling the given criteria. The gene name is linked to the NCBI platform for additional gene information. All expression data of the current cell line can be downloaded via a download button at the top of the table or through the corresponding API.Cell lines are critical model systems but cell line annotations have been heterogeneous and sparse. Here, we collected and annotated existing public cell line information with ontologies. With internally available computational pipelines, we reprocessed public raw data, including RNA-Seq datasets of 1,082 cancer cell lines, to generate novel annotations including HLA type, HLA expression, HLA Class I and Class II neo-epitope candidates, gene expression, and expressed viruses. Integrating the multiple annotations in one platform with an interactive interface and advanced search capabilities, researchers can effectively identify cell lines for their experiments and targets for therapeutic development.http://celllines.tron-mainz.de.The TRON Cell Line Portal is freely accessible at


Currently used drugs are associated with side effects and were not able to separate GvHD from the GvL-effect because of general T-cell suppression. This review focuses on murine models for GvHD and currently available treatment options involving antibodies and applications for the therapeutic use of aptamers as well as strategies for targeting immune responses by allogenic antigens.Prevention of Graft- IntroductionMechanismMurine modelsAntibody treatment for GvHDAntibody treatment concerning T cellsAntibody treatment concerning regulatory T cellsAntibody treatment concerning B cellsAptamers as alternative to antibodiesConclusionversus-Host-Disease (GvHD) is still a life-threatening disease which can occur as consequence of HSCT. Van Putten et al. in 1967 showed that lethally irradiated mice infused with allogeneic bone marrow and spleen cells recovered from the radiation injury and bone marrow aplasia, but they died of a so-called ‘secondary disease’ [Allogeneic haematopoietic stem cell transplantation (HSCT) is an important therapy for many haematological or epithelial malignancies as well as shows promising results in non-malignant diseases . The empdisease’ . The symdisease’ . This didisease’ .Concurrently, experimentation by Billingham further detailed the occurrence of three essential elements in the development of GvHD, which was supported by their analysis . First, The MHC includes specific proteins known as human leucocyte antigens (HLA), which are expressed on the cell surfaces of all nucleated human body cells. Analysis showed that these HLA proteins are an essential factor in the activation of allogeneic T-cells ,9.versus-Leukaemia (GvL) effect with a link to GvHD was initially observed in murine models. Here, immune cells from the donor within the graft are able to eliminate leukaemia or tumour cells remaining within the host.In addition to the three postulated causes for the GvHD, an immunological mechanism described as Graft-versus-Host-Disease is initiated by mature CD4+ and/or CD8+ T-cells that accompany allogeneic HSCT [1-cells further activate CD8+ T-cells by the secretion of INF-γ and IL-2, which lead to an apoptosis of host cells [Graft-eic HSCT . To prevst cells .+ T cells, the direct cognate interactions with recipient tissues are not necessary [Alloimmune T-cell responses against multiple minor histocompatibility antigens (miHAs) show an immunodominance. The presence of specific immunodominant antigens is able to predict GvHD manifestations and is also able to describe the clinical and histological phenotype. The onset of GvHD caused by MHC-mismatched transplantation results from recipient APCs that survive chemotherapy and/or radiation therapy which is beneficial in immuno-suppression. Although the manifestation of GvHD can be initiated by dendritic cells (DC) and Langerhans cells, donor-derived APCs are also essential because of a cross-presentation of recipient antigens. Both recipient- and donor-derived APCs promote T-cell-mediated GvHD. Furthermore, priming of T cells in the spleen or lymph nodes and Peyer's patches also causes GvHD. Conversely, observations show that the models undergoing lethal irradiation prior to the transplantation have no T-cell priming in Peyer's patches requirement. The phenotype of GvHD has been defined because of the influence of either activating or suppressing T-cell co-stimulatory molecules. In MHC-mismatched GvHD mediated by CD4ecessary .+CD25+ regulatory T-cells in the donor or recipient can suppress the GvHD, as well as, the recipient natural killer T-cells [Different molecular mechanisms have been identified as inducers of GvHD, including the CD95–CD95 ligand, whose accumulation of CD95 ligand, observed through histological techniques, is involved in host tissue damage . It was  T-cells .The therapeutic options include a high-dose regimen of prednisone  and furtStrategies for GvHD prevention and treatment were often not able to distinguish between different T-cell populations and therefore, not between GvHD and the GvL-effect.+ T-cells and CD8+ T-cells. These often result in an inflammatory cytokine storm. This massive immune reaction is capable of inducing GvHD in the target tissues without the requirement for cognate T-cell interaction with MHC on the tissue [+ T-cells trigger the development of GvHD primarily by their cytolytic function, which requires the TCR to engage MHC on the target tissue [+ T-cells or CD8+ T-cells, or both T-cell populations, depending on the specific strain combination, may play a significant role in the disease. Developing murine models addressing these mechanisms have helped to understand and refine various other mechanisms of GvHD [In murine models, the onset of GvHD can be distinguished because of either MHC class I, class II, or both. Furthermore, isolated multiple miHAs are also responsible for GvHD development in these models. Although observations showed that the influence from multiple miHAs was limited relative to that induced by full MHC disparities . The GvHe tissue . CD8+ T-t tissue . The ind of GvHD . Murine wt mice have been shown to be more resistant than Balb/cwt mice, and in addition, F1 hybrids are usually more resistant than the parental strain. In general, the TBI dose and its intensity play a pivotal role in the manifestation of the inflammatory expression of GvHD. Although BMT models undergoing low TBI doses and a high donor T-cell dose will result in GvHD, which is dominated by a later onset of T-cell-dependent pathology [In some cases, recipient animals undergo radiation treatment in the murine models for GvHD. Considerable care has to be taken because inbred mice have shown differing reactions to radiation. Analysis as to the maximum tolerated total body irradiation (TBI) doses for a particular strain has to be determined before experimentation to avoid organ damage. C57BL/6athology . The cheathology .et al. [et al. have developed a GvHD-model in which C57BL/6 TTG mice received transplants. The host developed a severe GvHD within 45 days after transplantation. The engraftment was initially observed after 12 days associated with a GvHD development within the host animals [wt mice as possible donors and Balb/cwt as recipient mice [et al. allows the direct investigation of antibodies against human targets (CD4) [b was examined after transplantation. Human CD4+, HLA-DR+ and H2Kb+ cells representing either the donor or the host cells haematopoiesis were expressed from day 12 [Laub et al.  develope animals . The manent mice . The modts (CD4) . The recm day 12 .in vivo.The characterization of available inbred strains as well as specific knockout or transgenic animals have helped to develop specific murine models which can be used as well for the investigation of GvH responses 4 isotypes, for example, are less immunogenic than IgG1 isotypes because of the low potential for complement activation. Besides, the humanization of antibodies leads to chimeric isotypes that are less immunogenic than their originally murine counterparts. Accordingly, the investigation of alternative or improved therapeutic approaches or procedures, the treatment with antibodies or other biologicals without the need of conventional immuno-suppressive drugs are still warranted.For the treatment of GvHD, immuno-suppressive strategies against key elements of T-cell reactions were already investigated in several pre- and clinical studies. For the therapy of acute GvHD, most experiences are available for OKT3® or interleukin-2 receptor antibodies and for chronic GvHD with anti-CD20 antibodies ,23. Howeet al. observed also a linkage with signalling pathways and structures involved in T-cell activation as well as APC–T cell interaction [−CD8− thymocytes and its expression level is not changed by various stimulation procedures. Moreover, murine T cells are not observed to be activated via CD26. Therefore, for the analysis of CD26-mediated immune regulation leading to clinical applications, it was necessary to establish a GvHD-model (xenogeneic GvHD murine model) which is triggered by human T-cells. These models are generated by the transplantation of human T-cells into NOD-SCID (severe combined immuno-deficiency) mice. GvHD scores were determined by manifestation of rough hair, loss of weight and mortality after the alteration of the transplanted human T-cells into effector cells in the murine organs. The additional examination of the cytotoxic properties of human CD8+ T-cells after a CD26-mediated co-stimulation in vitro, demonstrated that the co-stimulation induced a secretion of inflammatory cytokines such as TNF-α, IFN-γ and soluble Fas Ligand, and also enhanced the expression of granzyme B. These results supported the observation that the cytotoxic function in human CD8+ T-cells is activated via the CD26-mediated co-stimulation and plays a pivotal role in the manifestation of GvHD. A possible CD26 blockade by application of a humanized anti-CD26 monoclonal antibody significantly reduced the development of GvHD. The effect of blocking CD26 was exerted by suppression of cytotoxic activity of human CD8+ T cells in vivo. The anti-CD26 antibody and the available drug, abatacept (CTLA4-Ig) showed similar results in vivo. In addition, an increased dosage of CTLA4-Ig showed a higher suppressive effect on GvHD but sustained suppression of engraftment of transplanted human T cells. The same dose of huCD26mAb showed no delay in engraftment. These data by Hatano et al. showed that CD26-mediated co-stimulatory activation in human CD8+ T-cells is involved in the pathogenesis of GvHD, and blocking the CD26-mediated co-stimulation resulted in prophylaxis and treatment of GvHD [The membrane-bound glycoprotein CD26 (110-kD) is multifunctional and shows dipeptidyl peptidase IV enzyme activity which is present on a wide variety of cells . CD26 iseraction . In addi of GvHD .et al. have investigated the role of CD28 and CTLA4 in the T-cell response to alloantigens in vivo by using an established GvHD-model in sub-lethally irradiated mice [in vivo [in vitro. On the other hand, proliferation was prevented in vivo. These results recognizing nearly complete modulation of CD28 in vivo affected by anti-CD28 mAb were documented in the research by Yu et al. For further understanding of why this difference in modulation occurs, intensive investigation has to be continued. One possibility could include the interaction with Fc receptors which cause an extensive mobilization of CD28 molecules into intracellular contact caps in vivo.Yu 1 mice.Blocking CTLA4-signals increased GvHD, but was clearly independent from the CD28 expression on the investigated donor T cells. The CTLA4 expression and function itself were not affected by the treatment with an anti-CD28 mAb. Interestingly, in this setting CTLA4 retained its ability to decrease T-cell responses and to protect from acute GvHD manifestation in the absence of CD28. This characteristic indicates a possible cross-linking of CTLA4 that inhibits signalling events triggered through the TCR. CTLA4 might also inhibit other co-stimulatory signals or through CD134. Therefore, these results support a possible GvHD prevention through the maintaining of these CTLA4-negative regulatory signals. Moreover, observations identified that treatment with anti-CD28 mAb causes an expansion of B cells in normal mice but no effect was detected in sub-lethally irradiated dm2B6Fet al. have documented that treatment with anti-CD154 mAb improves the manifestations of GvHD induced by CD28−/− T-cells [Saito  T-cells . The pos T-cells .et al. described the possible mechanism for the development of acute GvHD [Blazar ute GvHD . Their rute GvHD .et al. have shown that an epitope-specific ex vivo modulation of an allogeneic haematopoietic stem cell graft by an anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumour capacity of the graft (GvL) and the long-term suppression of GvHD [1 was tested in murine GvHD and tumour models. Here, the survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 hrs) pre-incubated graft even when tumour cells were co-transplanted or when recipient mice were treated by the antibody before transplantation. It was also possible to transfer the immune tolerance from GvHD-free recipient chimaeras into third party recipient mice without the need of re-application of MAX.16H5 IgG1 anti-human CD4 antibodies [Fricke  of GvHD . To disttibodies .et al. investigated several anti-Fas ligand inhibitory mAb also called FLIM [1 mice were lethally irradiated. Then, spleen and bone marrow cells taken from semi-allogeneic C57BL/6 mice were injected (died within 30 days). Cells from B6-gld/gld mice  survived for >80 days. A mAb (FLIM58) or Fas-Fc in these models lead to a reduction both in weight loss and mortality rate which was caused by GvHD. Interestingly, skin lesions, lymphoid hypoplasia and mononuclear cell infiltration into the liver were not improved. FLIM58 was more effective than a monoclonal antibody directed against FasL (Fas-Fc). In conclusion, FasL directed antibodies showed positive effects in the treatment of GvHD. The authors suggest that FasL might be one of the key mediators in lethal GvHD development [Miwa led FLIM . In thiselopment .et al. proposed a possible clarification for antibody mediated GvHD prevention [+ T-cells. Furthermore, CD83 which is also involved in B-cell maturation, peripheral B-cell function, homeostasis and DC maturation might act as a possible candidate for antibody mediated GvHD prevention.Studies from Wang evention . ResultsMoreover, in a mouse model using human T-cell peripheral blood mononuclear cells (PBMCs) transplanted into SCID mice, a following treatment with anti-CD83 antibodies prevented GvHD in a dose-dependent manner and the promising results also indicated the preservation of the GvL-effect .+CD25+FoxP3+ (Treg)) have been shown to decrease GvHD while preserving the GvL-effects [regex vivo using cytokines (IL-2/TGFb) and antibodies (anti-CD3/anti-CD28) was investigated. Another approach for the prevention of GvHD is the development of specific strategies to expand Tregin vivo.Regulatory T-cells  is a member of the TNF super family and its agonistic antibody (4C12) was reported to preferentially activate Tansplant . This ap1κ mAb that reacts with human CD74, the HLA class II-associated invariant chain [in vitro and in xenograft models, which has lead to the ongoing clinical evaluation of milatuzumab in relapsed or refractory B-cell malignancies. Murine studies have demonstrated that milatuzumab is capable of modulating human B-cell proliferation, migration, and adhesion molecule expression, which clearly shows the therapeutic potential of this mAb in autoimmune diseases. As an HLA class II invariant chain molecule, CD74 is widely expressed in both haematopoietic and non-haematopoietic APCs, which include B-cells, monocytes, macrophages, Langerhans cells, DCs, endothelial and certain epithelial cells. Since both recipient and donor APCs, including non-haematopoietic APCs, play critical roles in the initiation of GvHD, milatuzumab might have therapeutic potential for GvHD by altering recipient and/or donor APCs. It inhibits allogeneic T-cell proliferation in specific leucocyte reactions. In a developed human/mouse xenogeneic SCID mouse model in which GvHD is induced and mediated by transplantation of human CD4+ T-cells and DCs, milatuzumab effectively prevents the manifestations of acute GvHD. It is also able to suppress the serum levels of secreted human IFN-γ and IL-5, and also decreases the infiltration of human lymphocytes into GvHD target organs . The therapy with milatuzumab significantly promotes survival (90% versus 20%) and does not affect the number of cytomegalovirus-specific, IFN-γ–producing human CD8+ T-cells in allogeneic mixed leucocyte reactions [Milatuzumab (hLL1) is a humanized IgGnt chain . Previoueactions . The anteactions .in vivo studies.The prevention of GvHD with antibodies directed against specific targets mentioned in this review are also summarized in Table in vitro selection process of a combinatorial oligonucleotide pool against a target molecule of biological or therapeutic interest, by using the SELEX  technique. Exposure of target molecules to the oligonucleotide library is followed by elution of target-binders and amplification of those by PCR. Reiterative SELEX cycles are performed until the initial combinatorial pool containing of up to 1015 different sequences has been purified to a homogeneous fraction of target-binders. Aptamers are identified from this pool by cloning and DNA sequencing is further classified by structural conserved motifs in their previous random regions and binding affinity and selectivity [in vivo applications and as therapeutic agents are promising, as chemical modifications are routinely introduced providing stability to nuclease attack and largely prolonging aptamer half-life in the plasma.Aptamers are single-stranded DNA or RNA molecules, developed by an ectivity . ApplicaAptamers rival monoclonal antibodies in many applications, including diagnosis and therapy. Moreover, aptamer-target interactions reveal low dissociation constants, ranging from nanomolar to femtomolar, similar to those between monoclonal antibodies and antigens.in vitro and no organisms are required. There is no variation in the synthesis of different batches, they are thermostable, have low production costs and present low or no immunogenicity. Aptamers are flexible molecules and being capable to bind to hidden epitopes, which cannot be reached by antibodies [Unlike monoclonal antibodies which have high immunogenicity, high cost of production, lot-to-lot variation and are sensitive to elevated temperature, aptamers are produced tibodies . A varietibodies –40, incltibodies .Aptamers have also been developed for interfering with immune responses, for instance by binding and inactivation of interleukin-6 or TNF-α ,43. Auto+ T-lymphocyte function, provided evidence that T lymphocyte-mediated immune response can be successfully targeted by using the SELEX technology [Aptamers developed against the CD4 antigen inhibiting CD4chnology . Furtherchnology ,50. Aptachnology . CD28 onchnology –54. Recechnology . Having Moreover, an aptamer that targets OX40 on activated T-cells which stimulates cell proliferation and interferon-γ production was developed. The authors suggest that an engineered form of this aptamer able to block OX40-OX40L interaction may be a promise for the treatment of immune-mediated disease such as GvHD . The intInnate immunity pathways, such as for protection against viral infection, involves stimulation of the TLR receptor pathway or the RNA helicase RIG-I, leading to down-stream signalling and activation of transcription factors NF-κ B, IRF3 and IRF7 . NF-κ- sin vitro selection are protected against nuclease activity by the incorporation of 2′F- or 2′-NH2-pyrimidines [2-pyrimidines by 2′-O-methyl-nucleotides, as shown successfully for the FDA-approved anti-VEGF165 aptamer [Aptamers inhibiting cytotoxic T lymphocyte activation involved in immune disorders are promising tools for evaluation in clinical trials. Previous conducted phase I and II clinical trials have evaluated the use of aptamers in therapy . The proimidines . For the aptamer ,60.Rapid renal clearance and accumulation of aptamers in tissues following repeated applications require further attention. Pharmacokinetics of aptamers can be improved by coupling them to high molecular weight moieties such as polyethylene glycol or cholesterol, aiming to resolve the problem of tissue accumulation following repeated aptamer treatment and to prevent aptamers from forming basophilic granules in the cytoplasm of renal proximal tubular epithelial and reticuloendothelial cells and in the liver . Improveversus-Host-Disease is still considered as a life-threatening disease after allogeneic HSCT and classified into an acute and chronic form based on the time of onset [e.g. cyclosporine) with methotrexate. Other regimens for GvHD prophylaxis include antibodies directed against the anti-tumour necrosis factor and manipulation of the graft such as T-cell depletion. First-line therapy options include high-dose prednisone [Graft-of onset ,62. Boneof onset , peripheof onset  and umbiof onset  are the of onset –68. T ceof onset . Currentednisone . Only 50ednisone . Studiesednisone ,72.Further improvements and more powerful tools to analyse the existing murine GvHD-models will undoubtedly provide explanations for better comprehension of this complex immunological process in the future. Of particular importance is the identification of therapeutic options including novel antibodies and aptamers to prevent or cure GvHD.


NUP210 (encoding a main component of the nuclear pore complex) was the only gene in which the two modifications were not detected in another normal cell line. RNA-Seq analysis revealed that NUP210 was aberrantly overexpressed among the 26 lung adenocarcinoma cell lines, although the frequency of NUP210 overexpression was lower (19.3%) in 57 lung adenocarcinoma tissue samples studied and stored in another database. This study provides a basis to discover epigenetic biomarkers highly specific to a certain cancer, based on multi-omics data at the cell population level.Epigenetic mechanisms such as DNA methylation or histone modifications are essential for the regulation of gene expression and development of tissues. Alteration of epigenetic modifications can be used as an epigenetic biomarker for diagnosis and as promising targets for epigenetic therapy. A recent study explored cancer-cell specific epigenetic biomarkers by examining different types of epigenetic modifications simultaneously. However, it was based on microarrays and reported biomarkers that were also present in normal cells at a low frequency. Here, we first analyzed multi-omics data  obtained from 26 lung adenocarcinoma cell lines and a normal cell line. We identified six genes with both H3K27ac and H3K4me3 histone modifications in their promoter regions, which were not present in the normal cell line, but present in ≥85% (22 out of 26) and ≤96% (25 out of 26) of the lung adenocarcinoma cell lines. Of these genes,  The epigenetic mechanism is vital in regulating gene expression. Failure of the epigenetic modifications can result in inappropriate activation or inhibition of various signaling pathways and lead to diseases such as cancer –3. CanceEpigenetic alterations, including aberrant DNA methylation in the promoter and alterations in histone modifications, can be used as epigenetic biomarkers of cancer diagnosis and promising targets for epigenetic cancer therapy  that aimHowever, epigenetic alterations have physiological functions in both normal and cancer cells . Thus, tToward the development of epigenetic therapies with higher cancer cell specificity, a recent genome-wide study  exploredHowever, the recent genome-wide study that reported the dual modifications , was conNUP210 gene encoding a main component of the nuclear pore complex presented both H3K27ac and H3K4me3 histone modifications in its promoter regions among ≥85% (22 out of 26) and ≤96% (25 out of 26) of the lung adenocarcinoma cell lines. NUP210 was the only gene not showing the dual modifications in either SAEC or another normal cell line, Normal adult Human Lung Fibroblasts (NHLF). It was also one of the two genes showing aberrant overexpression among all 26 lung adenocarcinoma cell lines compared to the normal cell line SAEC, although the extent of overexpression was usually smaller in the other paired datasets. The results indicate that NUP210 gene is the most promising epigenetic biomarker for lung adenocarcinoma.We determined that https://github.com/taoliu/MACS/).We used ChIP-seq data of 26 lung adenocarcinoma cell lines and SAEC from the DBTSS database  11]. To. To11]. For each lung adenocarcinoma cell line, we extracted histone modifications that were not detected in SAEC. From the histone modifications, we calculated frequency of shared histone modifications among the 26 lung adenocarcinoma cell lines (as a population) by bedtools version 2.22.1 . These hhttp://genome-mirror.duhs.duke.edu/ENCODE/cellTypes.html). We examined whether the histone modifications for genes of interest in lung adenocarcinoma cell lines are also absent in NHLF cells.We examined ChIP-Seq data of NHLF, another only one normal lung cell line for which ChIP-Seq experiments have been conducted and their peaks were inferred and made publicly available by Bernstein–Broad Institute (We examined the expression levels of genes of interest in each of the 26 lung adenocarcinoma cell lines and SAEC by using RNA-Seq data stored in the DBTSS database. We examined which gene was aberrantly expressed in which lung adenocarcinoma cell line according to the following criteria : (i) genWe examined chromatin states of the promoters defined as up to ±1.5 kb from the most upstream TSSs  in the gWe examined the distribution of Single Nucleotide Polymorphisms (SNPs) in the promoters defined as up to ±1.5 kb from the most upstream TSSs  and geneWe focused on the following histone lysine methyltransferases, histone lysine demethylases, histone acetyltransferases, and histone deacetylases, which are associated with the six types of histone modifications. We compared their expression levels (in RPKM) in each of the 26 lung adenocarcinoma cell lines with those of SAEC. The fold-changes of expression levels were analyzed according to the following criteria: genes with ≥2- or ≤1/4-fold RPKM of SAEC if the genes were transcribed (>0 RPKM) in SAEC.https://hive.biochemistry.gwu.edu/tools/bioxpress/) , lung squamous cell carcinoma [Lung_Squ], rectum adenocarcinoma [Rectum_Ade], colon adenocarcinoma [Colon_Ade], prostate adenocarcinoma [Prostate_Ade], breast invasive carcinoma [Breast_Inv], thyroid carcinoma [Thyroid], kidney renal papillary cell carcinoma [Kidney_Pap], kidney renal clear cell carcinoma [Kidney_Cle], kidney chromophobe [Kidney_Chr], and liver hepatocellular carcinoma [Liver_Hep]. These consist of 57, 50, 6, 26, 50, 113, 59, 30, 72, 25, and 50 paired samples, respectively (https://www.ebi.ac.uk/gxa/baseline/experiments) and examined whether the aberrant gene expression in lung adenocarcinoma cell lines is also detected in a large dataset using the following criteria [We conducted validation in terms of aberrant gene expression. We examined RNA-Seq data of 538 pairs of tumor compared with those of normal tissue samples from the same cancer patients, which are available in the BioXpress database (xpress/)  collecteectively , all of ectively , in the criteria : (i) genTo identify genes presenting epigenetic modifications highly specific to lung adenocarcinoma, we analyzed ChIP-Seq data of six types of histone modifications  in 26 lung adenocarcinoma cell lines and SAEC  stored iFrom the histone modifications detected in at least one lung adenocarcinoma cell line, but not in SAEC, we calculated the frequency of shared histone modifications among the 26 lung adenocarcinoma cell lines (as a population). We extracted those that were detected in equal to or more than 85% (22 out of 26) of the cell lines in the promoter regions defined as up to ±1.5 kb from the most upstream TSSs  and condNUP210 and PKN1) is shown in Among them, as indicated in The read coverage was much higher in most of the 26 lung adenocarcinoma cell lines than that in SAEC. The read coverage of the other genes is shown in The analyses above depend on a single non-carcinoma cell line, SAEC. In order to validate that the histone modifications are highly specific to lung adenocarcinoma, we examined whether they were also absent in another normal cell line, NHLF. All four genes in NUP210 did not carry H3K27ac or H3K4me3 marks in its promoter region in NHLF (PKN1 (member of the protein kinase C superfamily) carried the dual histone modifications in the promoter region, differences were observed in the location of the H3K27ac histone modifications compared to those highly shared among the lung adenocarcinoma cell lines (PKN1 was located near the TSS in the lung adenocarcinoma cell lines (chr19:14544446–14544776 on hg19)  did not carry H3K27ac histone modification in its promoter region in NHLF  -A, whileon hg19) -B. In ad in NHLF . These rNUP210, PKN1, and PPP1R9A), we examined their functions and known relationships with cancer  in each of the lung adenocarcinoma cell lines and compared it with that in SAEC  PKN1, and (c) PPP1R9A). The number of lung adenocarcinoma cell lines aberrantly expressing NUP210, PKN1, and PPP1R9A was 26, 26, and 8, respectively , respectively.It is worth noting that NUP210, PKN1, and PPP1R9A promoter regions (±1.5 kb from TSS) that were inferred along each genome in the previous study [We also assessed chromatin states in us study . For eacus study .NUP210, PKN1, and PPP1R9A genes were estimated to be inactive in SAEC. In contrast, chromatin states of the promoter regions of the three genes were estimated to be “active promoter” in equal to or more than 85% of the 26 lung adenocarcinoma cell lines. These results support that the dual chromatin marks (H3K27ac and H3K4me3) highly specific to lung adenocarcinoma change the chromatin structure, which can lead to the aberrant expression of these genes.Promoter regions of the NUP210 and PKN1, which were aberrantly expressed in all of the 26 lung adenocarcinoma cell lines. The locations in each of the 26 lung adenocarcinoma cell lines are shown in NUP210 and PKN1. These results support that histone modifications are the primary cause of aberrant gene expression in lung adenocarcinoma.Association between gene expression and genetic polymorphisms has been of interest . In ordeIn order to explore potential causes of histone modifications highly specific to lung adenocarcinoma, we investigated the expression levels of “writers” and “erasers” of these modifications; histone lysine methyltransferases, histone lysine demethylases , 41, hisSMYD3, and a histone acetyltransferase, HAT1, was considerably increased among 21 and 11 lung adenocarcinoma cell lines, respectively. Similarly, no considerable decrease in the gene expression of histone lysine demethylase and histone deacetylase was observed (according to a criterion ≤1/4-fold RPKM of SAEC) in more than 22 cell lines. At most, the gene expression of a histone demethylase, KDM5D, and a histone deacetylase, HDAC11, was considerably decreased among 18 and 4 lung adenocarcinoma cell lines, respectively.No considerable increase in the gene expression of histone lysine methyltransferases and histone acetyltransferases was detected (according to a criterion ≥2-fold RPKM of SAEC) among more than 22 lung adenocarcinoma cell lines. At most, the gene expression of a histone lysine methyltransferase, SMYD3 and MLL2   , although the frequency of aberrant gene expression in the other 10 types of carcinoma was 0–12.0% (PKN1) and 0–12.0% (PPP1R9A), no lung adenocarcinoma tissue sample showed aberrant gene expression  and ≤ 96% (25 out of 26) of the lung adenocarcinoma cell lines. They are more highly specific to cancer cells than the previously reported ones [In previous studies, the sensitivity of DNA methylation biomarkers for gastric cancer was reported to range from 56% to 96%  and fromThe dataset was generated and analyzed in a previous study . Comparehttp://ihec-epigenomes.org/). In addition, we did not analyze DNA methylation because there was no available data of BS-Seq of the two normal cell lines Thus, we did not investigate the association between a certain histone modification and DNA methylation as previously described in a previous study using microarray data [A limitation of the present study is that we were able to use the data of only two normal cell lines (SAEC and NHLF). No other ChIP-Seq data (including input control) of a normal cell line from an adult lung tissue is available at the International Human Epigenome Consortium (IHEC) database  endoplasmic reticulum homeostasis [NUP210 is usually upregulated in tumors  [NUP210 among other lung adenocarcinoma cells and normal cells.Despite these limitations, to our knowledge, this is the first study revealing epigenetic markers highly specific to lung adenocarcinoma based on multi-omics data at the population level of cancer and normal cells . The most important finding was the dual modifications of H3K27ac and H3K4me3 in the promoter region of ll lines  Tables (eostasis  and a rerostate) . In ordeThe present study provides a basis to discover epigenetic biomarkers highly specific to a specific type of cancer for diagnosis and epigenetic therapy based on the multi-omics data at the cell population level.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(DOCX)Click here for additional data file.S1 FigThe number of six types of histone modifications not detected in SAEC, but observed in each of the 26 lung adenocarcinoma cell lines is shown. (a) H3K27ac (top left), (b) H3K4me3 (top right), (c) H3K4me1 (middle left), (d) H3K9me3 (middle right), (e) H3K36me3 (bottom left), and (f) H3K27me3 (bottom right).(TIFF)Click here for additional data file.S2 FigNFE2L3, (b) ETV4, (c) PRTG, and (d) TMEM86A. The blue arrows indicate the locations of the four genes and the red arrows indicate regions of the histone modifications. The Small Airway Epithelial Cell (SAEC) at the bottom is a normal control cell line and the others are the 26 lung adenocarcinoma cell lines. H3K4me3 read coverage is shown in a range of 0–300 in (a), 0–400 in (b), 0–500 in (c), and 0–300 in (d), respectively. The genomic positions in this figure are based on hg38.H3K4me3 histone modifications are located in the promoter regions of (a) (TIFF)Click here for additional data file.S3 FigPPP1R9A, (b) IGF2BP3, (c) HOXC4, and (d) FAM102B. The left panels represent H3K27ac and the right panels represent H3K4me3 modifications. The blue arrows indicate the locations of the four genes and the red arrows indicate regions of histone modifications. Small Airway Epithelial Cell line (SAEC) at the bottom is a normal control cell line and the others are the 26 lung adenocarcinoma cell lines. H3K27ac and H3K4me3 read coverage is shown in a range of 0–150 and 0–300 in (a), 0–300, and 0–500 in (b), 0–300 and 0–500 in (c), and 0–100 and 0–500 in (d), respectively. The genomic positions in this figure are based on hg38.H3K27ac and H3K4me3 histone modifications specific to the 26 lung adenocarcinoma cell lines are located in the promoter regions of (a) (TIFF)Click here for additional data file.S4 FigLocations of H3K4me3 peaks in a normal cell line, Normal adult Human Lung Fibroblasts (NHLF) are shown for four genes in (TIFF)Click here for additional data file.S5 FigIGF2BP3 (top), (b) HOXC4 (middle), and (c) FAM102B (bottom). Red rectangles indicate H3K27ac histone modifications and blue rectangles indicate H3K4me3 histone modifications that are highly specific to the lung adenocarcinoma cell lines. Gray rectangles are H3K27ac (upper) and H3K4me3 (lower) modifications in NHLF. The shade of gray indicates signal enrichment, which is calculated as the number of sequenced tags overlapping a 25 bp window centered at that position according to UCSC (the darker gray indicates higher numbers). Bottom lines in the figure indicate location of each gene. All H3K27ac and H3K4me3 read coverage in NHLF is shown in a range of 0–150. The genomic positions in this figure are based on hg19.Locations of H3K27ac and H3K4me3 peaks in a normal cell line, Normal adult Human Lung Fibroblasts (NHLF) are shown for the three genes in (TIFF)Click here for additional data file.S6 FigChromatin states inferred by ChromHMM for the genes listed in (TIFF)Click here for additional data file.S7 FigNUP210 (top), (b) PKN1 (bottom). The left panels represent the promoter regions and the right panels represent the gene bodies (exons and introns). The bar lines indicate the SNP locations. The blue arrows indicate the locations of each gene.Distributions of SNPs in each of the 26 lung adenocarcinoma cell lines are shown for two genes listed in (TIFF)Click here for additional data file.S8 FigNFE2L3, (b) ETV4, (c) PRTG, (d) TMEM86A, (e) IGF2BP3, (f) HOXC4, and (g) FAM102B. Navy bars indicate transcriptional aberrations compared with SAEC. The red bar at the right end shows expression levels in SAEC. RPKM values at the bottom in the table indicate averages among the 26 lung adenocarcinoma cell lines.The RPKM values of four genes in (TIFF)Click here for additional data file.S9 FigNFE2L3 (top left), (b) PRTG (top right), (c) TMEM86A (middle left), (d) PPP1R9A (middle right), (e) HOXC4 (bottom left), and (f) FAM102B (bottom right).The expression levels of three genes listed in (TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.


NBQ’s are unnatural alkaloids possessing a positive charge that could facilitate interaction with cell organelles. The anticancer activities of these compounds were evaluated through the National Cancer Institute (NCI) 60 cell line screening which represents diverse histologies. The screening was performed at 10 µM on all cell lines. Results from the NCI screening indicated cytotoxicity activity on six cell lines. In order to explore a possible mechanism of action, a detailed biological activity study of NBQ 95 and NBQ 38 was performed on A431 human epidermoid carcinoma cells to determine an apoptotic pathway involving, cell cycle changes, DNA fragmentation, mutations, mitochondrial membrane permeabilization and caspases activation. DNA fragmentation, cell cycle effects, mutagenesis, mitochondrial permeabilization and activation of caspases were determined by fluorimetry and differential imaging. Our data showed that A431 growth was inhibited with an average IC50 of 30 µM. In terms of the mechanism, these compounds interacted with DNA causing fragmentation and cell cycle arrest at sub G0/G1 stage. Mutagenesis was higher for NBQ 38 and moderate for NBQ 95 Mitochon-drial permeabilization was observed with NBQ 38 and slightly for NBQ 95. Both compounds caused activation of Caspases 3 and 7 suggesting an apoptotic cell death pathway through an intrinsic mechanism. This study reports evidence of the toxicity of these novel compounds with overlapping structural and mechanistic similarities to ellipticine, a known anti-tumor compound.This study reports the capacity of three nitro substituted benzazolo[3,2- Podophyllum quinolinium salts (BQS). The structural features in this series of compounds shown in The compounds presented in this study  belong to a family of unnatural alkaloids known as benzazoloquinolinium salts (NBQ) were synthesized as reported previously quinolinium salts i.e. NBQ 95 (NSC D-763304), NBQ 38 (NSC: D-763305) and NBQ 97 (NSC: D-763306) were screened through the National Cancer Institute’s Developmental Therapeutics Program  at 610 nm. Data was calculated and normalized by subtracting the background emission of the negative control. One way ANOVA with Tukey post test was performed to compare data and determine significance.The determination of Caspases 3 and 7 activation which is a hallmark of apoptosis in response to therapeutic drugs  was perfa]quinolinium salts represent unnatural alkaloids and have some structural similarity with Ellipticine an alkaloid isolated from Apocyanaceae family of plants, which has been reported as an antineoplastic agent, for which the mode of action is considered to be based on DNA intercalation and inhibition of topoisomerase II quinolinium salts (BQS) to inhibit cell viability after 48 hours of exposure were evaluated on the A431 epidermoid carcinoma cell line. Cells in culture treated with doses in the range of 10 uM to 150 uM showed a clear dose response with both compounds. The obtained IC50 values at 48 hours were 36 µM for NBQ38 and 28 µM for NBQ95, shown in The capacity and comparison of the two benzazolo quinolinium salts toward the NCI 60 cell panel as well as a general evaluation of their potential mode of action. Herein, we also report the biological capacity of the two most active compounds to induce DNA fragmentation and mutation, produce cell cycle arrest and cause cell death in an apoptotic fashion involving mitochondrial membrane depolarization and activation of Caspases 3 and 7.This study describes the cell viability inhibitory activity of three novel unnatural alkaloids nitro substituted benzazolo[3,2-The results from the NCI 60 cell line screening panel based on a one dose (10 µM) analysis are shown in The broad spectrum of tissue types that reported susceptibility to both NBQ 95 and NBQ 38 guided us into evaluating biological activities that could provide insights into the mode of action of these compounds. The 60 cell lines on the NCI screening panel have varied expression level of the epidermal growth factor receptor (EGFR) and p53 genes. While, A431 cells express abnormally high levels of the EGFR and contain no functional p53. As such, these two genes are the differentiating factors and their expression levels in A431 cells appear to represent similarity to many of the cell lines in the NCI panel in which our compound show activity. Therefore, the mechanistic study was carried with A431 cells. The structural similarities of NBQ 38 and NBQ 95 with ellipticine motivated us to consider and analyze the capacity of these agents to interact with DNA, cause cell cycle arrest and apoptosis induction including changes to mitochondrial membrane potential and caspases activation.50 concentrations of 36 µM and 28 µM for NBQ 38 and NBQ 95 respectively and although lower than those reported for ellipticine, in other cell lines (up to 10 µM) [0/G1 similarly to what has been reported for ellipticine [The cell viability inhibition analysis on A431 cells at doses ranging from 10 µM to 150 µM, reported ICo 10 µM)  we consio 10 µM) . The repipticine ,41. Thisipticine ,43. Anotipticine .DNA damage is known to provoke a downstream effect in which the depolarization of mitochondrial membrane and activation of effector caspases, are the most probable events suggesting an apoptotic pathway for cell death . This haAs reported in the literature ,48 anothIn conclusion, this study reports the effects of NBQ 38, NBQ 95, and NBQ 97, three novel ellipticine analogues, on the NCI 60 cell line panel with toxicities of 40% to 93% in cells from varies tissue types. Furthermore, the possible mechanism of action of NBQ 38 and NBQ 95 has been studied with A431 cells. These two drugs demonstrated cytotoxicity comparable to its structural analogue, ellipticine including overlapping mechanisms of action. The results show that these compounds interacted with DNA causing effects such as mutagenesis as well as cell cycle arrest followed by mitochondrial membrane depolarization and the activation of Caspases 3 and 7 and DNA fragmentation.N38N95