CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL.The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by  CYBA (16q24.3), CYBB (Xp11.4), NCF1 (7q11.23), NCF2 (1q25.3) and NCF4 (22q12.3) genes, respectively. The function of NADPH oxidase has been historically associated predominantly with phagocytes and their role in host defense. Phagocytic cells undergo a process called oxidative burst to generate large amounts of superoxide anion and other secondary ROS (reactive oxygen species) of microbicidal function. In line with this observation, genetic defects in any of the NADPH oxidase genes cause impaired functionality of phagocytes, immunodeficiency and manifest in chronic granulomatous disease characterized by recurrent and severe infections including pneumonia, infectious dermatitis or osteomyelitis (Online Mendelian Inheritance in Man database - OMIM): CYBA 233690, CYBB 306400, NCF1 233700, NCF2 233710, NCF4 613960) [The NADPH oxidase is a multi-protein enzyme consisting of two membrane bound subunits, the p22-phox and gp91-phox and three cytoplasmic subunits, the p47-phox, p67-phox and p40-phox [BA 16q24., CYBB  cell lines reported in our previous study [In light of the above and intrigued by the transcriptional downregulation of the us study , we inveCYBB downregulation in cHL cell lines led us to analyze these cell lines for deletions of genes encoding components of the NADPH oxidase complex. By mining SNP microarray data we identified deletions of CYBB, that is located on the X chromosome, in 2/7 (29%) cHL cell lines including a heterozygous deletion in the L540 cell line derived from a female cHL patient and the previously described homozygous deletion in KMH2 [CYBB in the heterozygous L540 cell line and further mutations in the other five cell lines (excluding KMH2) - out of which four are derived from male patients - we sequenced the entire coding sequence and exon-intron boundaries of the gene, but no mutations were identified. We extended the analysis to a copy number screen of the CYBB gene in 18 primary cHL cases and analyzed lymph node cryosections by combined immunophenotyping and interphase cytogenetics. Altogether we identified 8/18 (44%) cases with a signal constellation indicative for deletions of the CYBB gene with regard to the sex of the patients and the ploidy of the cases. These included six deletions restricted to the p arm of the X chromosome harbouring the CYBB locus with retained X centromere, and two deletions of the entire X chromosome. No cases with complete CYBB loss were identified.Our recent observation of  in KMH2 . To idenCYBA locus in 3/7 (43%) cHL cell lines including losses in HDLM2 and L540 and loss of heterozygosity (LOH) in the KMH2 cell line. LOH of the NCF2 locus was observed with a similar frequency, that is 3/7 (43%) cell lines, in L428, KMH2, UHO1, and of the NCF4 locus in one cell line, namely UHO1  and complete downregulation  for NCF1 (p<0.001) in the four cHL cell lines as compared to the B-cell controls. For the NCF4 gene lower expression was observed in 3/4 cHL cell lines  but not of the CYBA, CYBB, NCF2 and NCF4 genes. In line with this finding, we observed significantly lower expression of NCF1 on protein level in cHL cell lines compared to non-Hodgkin lymphoma cell lines (p<0.005)  . Thus, combining genomic data and expression analysis on mRNA as well as protein level provides a strong rationale for the hypothesis of NADPH oxidase impairment resulting in reduced ROS synthesis capacity in cHL.in situ hybridisation to the CYBB locus in primary biopsies showed recurrent deletions of the gene in HRS cells we analysed to what extent these changes corresponded to altered CYBB protein expression. By immunohistochemistry we investigated 14 of the 18 cases studied by interphase cytogenetics for expression of the CYBB protein. Remarkably, in all of these 14 cases we observed complete loss of CYBB protein expression in all HRS cells irrespective of the presence or absence of a genomic deletion. In contrast, non-neoplastic lymphatic cells and macrophages stained positive for the CYBB protein (As  protein . This su+ positive cell lines including one T-cell lymphoma and one nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) cell line and observed a direct increase of superoxide anion production. As this demonstrated the usefulness of this approach, we analyzed six cHL cell lines that are characteristic for CD30 overexpression and were previously reported to have an active CD30 signalling pathway [- non-Hodgkin lymphoma cell lines as negative controls  and weak 2.9-fold increase in the CD30- control cell lines (negative control). The cHL cell lines in turn, despite being CD30 positive, showed only a background activation of 1.9-fold suggesting an impaired functionality of the NADPH oxidase. We interpret the weak increase of superoxide anion production in the cHL cell lines and the CD30- control cell lines as an unspecific reaction of the anti-CD30 antibody Ki-1-positive tumor cell culture supernatant used for stimulation.In line with the findings on and UHO1 . Howevercroarray . Taken tIt has been reported that CD30 signalling causes ROS production by the mitochondrial pathway, whereas inhibitors of the  NADPH oxidase complex did not affect the ROS levels measured in this study . However-. We therefore measured CD30 expression of LM1, DG-75, DAUDI, and L428 cell lines using an APC-labeled monoclonal antibody and compared the fluorescence intensities to a control antibody that was matched for isotype, concentration, and fluorochrome label (data not shown). While LM1 and DG-75 cell lines indeed showed a minimally higher CD30 labelling compared to Daudi, this difference did not explain the observed increase in ROS production of LM1 and DG-75 relative to Daudi cells. ROS formation in LM1 and DG-75 is therefore likely triggered by a CD30-independent mechanism caused by unspecific binding of the antibody. Noteworthy, none of the six cHL cell lines showed a similar increase above background level.Noteworthy, two of the control cell lines in our experiment, namely LM1 and DG-75, showed an increase of superoxide anion production above the background level despite being reported to be CD30+ DEV cell line used in this experiment is derived from a case of NLPHL [Interestingly, the CD30of NLPHL , a rare of NLPHL . TherefoNCF1 gene reported here.In line with this hypothesis it was demonstrated that ROS signalling is necessary for normal B-cell differentiation . Besides+ cell lines and the strong and constitutive CD30 expression in primary HRS cells of cHL, one may speculate that the inactivation or downregulation of NADPH oxidase represents a strategy of the HRS cells to escape from an overwhelming and toxic ROS production, that could otherwise impair HRS cell survival.In light of the induction of ROS by CD30 signaling in several CD30In summary, in this study we show multiple alterations targeting the NADPH oxidase genes and impaired functionality of the enzyme in vivo. Moreover, we suggest that the loss of ROS signaling during B-cell lineage development may potentially contribute to the loss of B-cell phenotype of HRS cells.2 with the exception of Granta 519 which was cultured in DMEM medium. DNA and / or cells from seven cHL cell lines, i.e. L428, HDLM2, KMH2, L1236, SUPHD1, UHO1, L540 were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)  or were kindly provided by Dr. Andreas Bräuninger  . Cell lDNA from cHL cell lines L428, HDLM2, KMH2, L1236, SUPHD1, UHO1 and L540 was hybridized to genome-wide human SNP array 6.0  as described before [CYBB gene. Primer sequences for the mutation screening were designed using the Primer3 v. 0.4.0 software (http://frodo.wi.mit.edu/primer3/) and are available on request. DNA genomic sequences were downloaded from the UCSC Genome Browser (www.genome.ucsc.edu). PCR products encompassing each exon together with the 5’ and 3’ splicing sites were Sanger sequenced using both the forward and reverse primer by standard procedures. The fluorograms were analyzed using the Chromas Lite 2.01 software. The cHL cell lines L428, HDLM2, L1236, SUPHD1, UHO1 and L540 were analyzed for mutations in the SpectrumGreen  spanning the CYBB locus together with the centromeric CEPX SpectrumOrange (Abbott/Vysis) probe was used as described before [+ cells /case were evaluated independently by two observers. For FICTION (Fluorescence Immunophenotyping and interphase Cytogenetic as a Tool for Investigation Of Neoplasia) the Bacterial Artificial Chromosome (BAC) probe RP11-299O2 labeled in d before ,38. CryoCYBB probe was lower than the signal number of the CEPX probe and lower than the expected number of CEPX signals in at least 30% HRS cell nuclei / case; (ii) if the signal number of the CYBB probe was lower than the expected number of CEPX signals in at least 30% HRS cell nuclei / case. In the first case a deletion of the X p arm harbouring the CYBB locus was scored and in the second a deletion of whole chromosome X.The threshold for the detection of a deletion was arbitrarily set to 30%. In detail, a deletion was scored in two cases: (i) if the signal number of the The expected number of CEPX signals was estimated based on the ploidy of the case and the sex of the patient. Ploidy levels of the cases were estimated by taking median signal numbers for the chromosome enumeration probes CEP6  CEP10  equipped with appropriate filter sets  and documented using an ISIS imaging system . CYBA, CYBB, NCF1, NCF2 and NCF4 genes was extracted and visualised using the GeneCluster 2.0 software. Relative expression of the analyzed genes across the samples was compared using t-test. The gene expression dataset is available at http://ICG.cpmc.columbia.edu/faculty.htm and http://www.ncbi.nlm.nih.gov/geo .Published gene expression profiles from Affymetrix U95 array of L428, HDLM2, KMH2 and L1236 cHL cell lines and normal B-cell controls   and U133The cHL cell lines L428, HDLM2, KMH2, L1236, UHO1 and L540 and control cell lines LM1, Karpas 299, DEV, DG-75, Ca-46, Karpas 422, Daudi and Granta 519 were analyzed for NCF1 protein expression. Primary anti-NCF1 / p47-phox (ab795)  antibody detected by the secondary anti-Goat IgG (H+L) antibody conjugated with alkaline phosphatase  was used.Chemicals were purchased from Sigma-Aldrich  unless otherwise stated. Cells were harvested, washed twice with PBS (phosphate-buffered sodium) and lysed for 15 min at 4°C in lysis buffer , protease inhibitor cocktail  and sonicated for 5 s on ice. The homogenate cells were centrifuged at 1000 x g for 10 min at 4°C. Protein concentration of cell lysates was determined by the bicinchoninic acid method using the BCA Protein Assay Reagents . Proteins were fractionated by 12.5% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked for 30 min at room temperature in Tris-buffered saline containing 0.1% (v/v) Tween-20, 5% milk powder and washed twice in Tris-buffered saline containing 0.1% (v/v) Tween-20. After incubation at room temperature with primary antibodies, membranes were washed with Tris-buffered saline containing 0.1% (v/v) Tween-20 and incubated with a 1:5,000 dilution of secondary anti-mouse horseradish peroxidase conjugated antibodies for 2 h at room temperature. Membranes were washed and developed using ECL detection reagent . Developed membranes were exposed to x-ray film . Antibodies against actin (C-11) SC-1615 (Santa Cruz) were used to verify equal loading of the lanes. Western blot quantification was done by densitometric analysis of the scanned films using Molecular Dynamics Personal Densitometer  and the Image Quant 5.2 software (Molecular Dynamics). Relative protein quantity in relation to actin was measured and calculated for each cell line as arbitrary units (AU). For each of the analyzed proteins 6 independent Western blots were performed and a mean value of the quantifications was calculated.Immunohistochemical staining of CYBB protein was performed using a mouse monoclonal antibody as follows. FFPE tissue section (1-2 µm) were processed for antigen retrieval by boiling in citrate puffer of pH6 in a pressure cooker for 3 minutes. Incubation by the primary antibody against CYBB  (1:100 dilution) was performed for 1 h at room temperature. Immunoperoxidase staining was developed using a diaminobenzidine chromogen kit . Counterstaining was done with Hemalaun.The sections were evaluated with a Olympus BX43 microscope equipped with a CCD camera DP 72 (Olympus) and documented with CellSens Entry (Olympus) software.The cHL cell lines L428, HDLM2, KMH2, L1236, UHO1 and L540 and control cell lines Karpas 299, DEV, LM1, DG-75, Ca-46, Karpas 422, Daudi and Granta 519 were analyzed for superoxide anion production. To stimulate superoxide anions synthesis we used anti-CD30 antibody from Ki-1-positive tumor cell culture supernatant that was kindly provided by Dr. H.P. Hansen . The supernatant was purified using protein G Sepharose  and diluted on the protein G matrix  with Glycin / HCl buffer pH 2.7. The antibody was stored in phosphate-buffered solution, pH 7.2. Prior to stimulation, cells were harvested and diluted to a concentration of 3.10E5 cells/150 µl in fresh RPMI 1640 medium or DMEM. Cells were incubated simultaneously with anti-CD30 antibody and 0.03 mM DHE (dihydroethidium)  for 30 min. at 37°C in the dark. 2·-) was determined using the oxidation-sensitive fluorescent probe DHE resulting in a color shift of the dyes as described before [Intracellular level of superoxide anion . The % RFUs of the cell lines incubated with the anti-CD30 antibody were calculated relatively to untreated cells and presented as fold change of superoxide anion production.+ lymphoma cell lines as positive control; 6 CD30- lymphoma cell lines as negative controls, and 6 CD30+ cHL cell lines  the authors are free to use archival material sent to the Department of Pathology, Lymph Node Registry of the University Kiel, after the diagnostic process is finished. If the specimen are used anonymized, clinical data, which were available for the diagnostic process can be used too. Obtaining new clinical data about the patients need a patient informed consent. However, the latter is not the case in our study. We only used anonymized specimen and gender and age as the only clinical variables.Table S1Characteristic of cell lines used in the study.(DOC)Click here for additional data file.


Problem-based learning (PBL) has become the most significant innovation in medical education of the past 40 years. In contrast to exam-centered, lecture-based conventional curricula, PBL is a comprehensive curricular strategy that fosters student-centred learning and the skills desired in physicians. The rapid spread of PBL has produced many variants. One of the most common is 'hybrid PBL' where conventional teaching methods are implemented alongside PBL. This paper contends that the mixing of these two opposing educational philosophies can undermine PBL and nullify its positive benefits. Schools using hybrid PBL and lacking medical education expertise may end up with a dysfunctional curriculum worse off than the traditional approach.For hybrid PBL schools with a dysfunctional curriculum, standard PBL is a cost-feasible option that confers the benefits of the PBL approach. This paper describes the signs of a dysfunctional PBL curriculum to aid hybrid PBL schools in recognising curricular breakdown. Next it discusses alternative curricular strategies and costs associated with PBL. It then details the four critical factors for successful conversion to standard PBL: dealing with staff resistance, understanding the role of lectures, adequate time for preparation and support from the administrative leadership.Hybrid PBL curricula without oversight by staff with medical education expertise can degenerate into dysfunctional curricula inferior even to the traditional approach from which PBL emerged. Such schools should inspect their curriculum periodically for signs of dysfunction to enable timely corrective action. A decision to convert fully to standard PBL is cost feasible but will require time, expertise and commitment which is only sustainable with supportive leadership. Problem-based learning (PBL) is the single most important innovation in medical education of the past 40 years. It was As the McMaster model became known, staff from Maastricht (Netherlands) and Newcastle  spent time at McMaster in the 1970s before returning to implement PBL at their new medical schools. In 1979Among the leading debates on what constitutes PBL has been the implementation approach (method or philosophy) and the basic type (pure or hybrid).While some schools revamped their curriculum to incorporate the PBL approach, others make that claim by simply adding PBL tutorials onto an otherwise unchanged conventional curriculum. However, PBL’s pioneers understood it as “a whole curriculum, not a teaching method that can be used alongside other methods”. In settThe term ‘hybrid’ PBL is thought to originate from Harvard’s New Pathway curriculum in which “the scope, frequency and format of lectures and laboratory sessions could be effectively altered to dovetail with active problem-based discussions”. A 2003 Kwan and Tam divided In a standard PBL curriculum, lectures and other inputs serve as instructional scaffolding to support learning by the PBL approach. In contrast, lectures in a conventional curriculum are intended for content experts to directly transmit knowledge to the students. While it is common to add more lectures to a lecture-based curriculum, excessive didactic sessions in a PBL curriculum conflicts with its student-centered, independent learning philosophy. Despite the prevalence of hybrid PBL, there is scant literature on how to diagnose and manage a dysfunctioning hybrid PBL curriculum. In this article, I describe the signs of a dysfunctional PBL curriculum to aid hybrid PBL schools assess the state of their curriculum. This is followed by a review of the issues surrounding adoption of PBL in the light of resource requirements and alternative curricular strategies. I then discuss the critical issues and steps for steering a failing curriculum back towards standard PBL.Schools with hybrid PBL curricula that lack curriculum development expertise should undertake regular inspection of their curricula for one or more of the following signs of dysfunction:PBL sessions may exist alongside lectures such that over the course of a preclinical year, students tackle weekly multidisciplinary problems and yet attend hundreds of discipline-based lectures which often cover the same ground. If the week’s lecture contents effectively ‘solve’ the trigger problem, the active inquiry processes fostered by PBL will be subverted. This coWhere summative assessments are frequent and fact-based, as they usually are in lecture-based curricula, students are driven to eschew deep learning approaches and favor cramming to maximize their grades, a scenario which PBL was designed to avert. Where the lecture load is heavy and staff are required to contribute assessment questions from the lectures they delivered, the study of lecture contents alone is sufficient to pass the assessments. Since the contribution of PBL to final grades may just be a small percentage awarded for attendance at PBL tutorials, students will not be motivated to put effort into PBL aside from attending the sessions. Such a hybrid curriculum is in essence a conventional lecture-based curriculum except that students have the extra burden of going through the motions of attending PBL sessions.Schools with a dysfunctional PBL curriculum often lack medical education expertise. Staff are usually unaware of PBL’s curricular philosophy and typically view PBL as one of many teaching methods. Hence little or no resources is allocated for its maintenance or monitoring. In such schools, PBL problems tend to be reused without review, thus perpetuating any errors or content overlap with other didactic sessions. If student numbers increase without recruitment of more tutors, the size of PBL groups would inevitably increase beyond the typical recommended maximum of 8 to 10 students. This hiThe implementation of PBL is often opposed by staff favoring the conventional curriculum. Curriculum review meetings are often used by PBL opponents to add more discipline-specific teaching sessions. They may request new lectures by citing disappointing assessment results from topics learnt through PBL. However, poor results from lecture-taught topics never lead to the removal of those lectures or their conversion into PBL problems. Such undermining of PBL may go unchallenged at preclinical curriculum reviews when basic science staff are invited to attend as a representative of their discipline, with no one assigned to represent PBL.Schools that do not accept PBL as an overall curriculum strategy often perpetuate the situation through their staff development programmes. Many tutor training workshops merely teach the tutorial process without presenting PBL as a whole-curriculum philosophy. Tutors trained in this manner may regard PBL groups as no different from the small groups they tutored in conventional curricula. Hence these tutors may operate by frequently intervening with their own didactic questions and then supplying the answers, thus disrupting the PBL process. Students will also be unmotivated to put effort in analyzing a problem if the tutors see themselves as information providers whose job it is to disseminate the problem’s learning objectives.Where case-writing workshops do not emphasize the difference between PBL and other teaching methods, case writers may perceive PBL problems as performing the same role as standalone clinical cases used during clerkships. Hence they may write scenarios which are not open-ended problems that stimulate inquiry, but “a simple problem with a well-defined solution, which results in a scavenger hunt for information from resources that the professor has provided”. IgnoranKwan and Tam cited a quote that “Poor teaching is bad, but poor PBL is even worse”. In a leSchools with a dysfunctional PBL curriculum have the option of returning to the traditional curriculum, adopting standard PBL or some other curricular model. PBL is often regarded as being resource intensive. While cost is a constraint, an ineffective curriculum is equally unacceptable. In contrast to the traditional curriculum, PBL's curricular approach meets the recommendations of accrediting bodies such as the Liaison Committee on Medical Education (independent study and active learning) and the In CBL, students in small groups use knowledge from advance reading to solve a clinical problem under the direction of a facilitator familiar with the subject matter. CBL canAlthough PBL is described as a resource intensive curriculum model, there are few studies that compare costing for different models. Most ofA dysfunctioning hybrid PBL curriculum is inferior even to the conventional curriculum from which PBL arose. Schools that want to resolve this problem and derive the benefits of PBL should convert to standard PBL. Successful conversion will depend on the able handling of 4 critical factors: the nature of resistance to PBL, use of lectures in standard PBL, importance of adequate preparation and the necessity for supportive leadership.A large part of resistance towards PBL is natural resistance against change, as would be expected from staff who have spent an entire career in lecture-based curricula. For them, changing to PBL would mean giving up years of accumulated lecture materials to become a novice small group tutor. Also, many think little of PBL because they had never understood it as a comprehensive curriculum philosophy, but as just one of many methods to run small group tutorials. Still others have been in schools where aggressive PBL proponents effected a curriculum change to PBL without spending sufficient time selling it to them. Consequently, the mere mention of PBL can evoke responses ranging from polite disagreement to vehement loathing.At a more fundamental level, Margetson has expounded on hostility to PBL arising from one’s conception of how knowledge is acquired. Some PBfirst in the learning process”[Hamdy has succinctly stated that “Having lectures or resource sessions given by faculty to support students’ learning is not against the PBL religion”. Barrowsprocess” because process” and highprocess”- a task process”.Lectures in a PBL curriculum should be consistent with the PBL philosophy of active, deep and self directed learning. Lectures can foster self directed learning when students know the contents will help them to fully engage with their PBL problem. A lecture can also be a part of student-directed learning if students request for it because they decided it is critical to their learning at that time. An intePBL was implemented at Maastricht and Newcastle only after some of their staff spent months to years learning it at McMaster. Walton The effective establishment and maintenance of a PBL curriculum requires a raft of supporting policies such as funding staff visits to prominent PBL centres, recognition and reward for staff involvement in PBL, provision of equipped PBL rooms and a student assessment system compatible with PBL. Hence conversion to standard PBL requires the endorsement, and at times the enforcement of a PBL advocate who holds administrative authority in the school and/or the university. This is particularly necessary when staff are required to make difficult changes such as reduction or removal of lectures. The staff in a hybrid PBL school are often divided by curricular philosophy such that the balance of opinion may change with the departure of PBL enthusiasts or arrival of traditionalists. Des Marchais has warned that “Educational reform demands strong and innovative leadership, because the pressure to revert to tradition is always present and may erode the system even as it is put in place”. Hence sAnother important leadership role for supporting the conversion of hybrid curricula to standard PBL is that which can be played by PBL organizations. Schools intending to adopt PBL but lacking curriculum development expertise will need help in practicalities such as the frequency of tutorial sessions, the number of students per tutorial group and the hours of self directed learning per PBL problem. There are no strict rules governing these issues but information on regional best practices will be a helpful starting point. They would also want to know the established PBL schools that are prepared to host staff from other schools who wants to learn from them. A regional medical education or PBL association would be well placed as the central channel for such information.PBL has revolutionized medical education because it fosters the skills required by practising physicians. Neville’s review of the 40 years of PBL implementation predicts the PBL curriculum of the future to be a hybrid in which “students are prepared didactically with fundamental concepts on which they elaborate in small-group tutorials”. This isNot applicable.TBL: Team-based learning.The authors declare that they have no competing interests.WL conceived and drafted the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/12/89/prepub


Hybridoma technology was established in the 2nd half of the 20th century and in the view of current protein production it might seem old-fashioned. Despite, it is commonly used to produce monoclonal antibodies (mAbs) for R & D, clinical diagnostics or medical applications and the demand for mAbs produced by hybridomas is still high. However, compared to CHO, only a few serum-free hybridoma media are available and even less suppliers for chemically defined products are on the market. In this work, a new chemically defined medium and feed were developed to bring hybridoma processes to the next level and to target the existing gap in the market.HybriMACS CD medium was developed using various research and production hybridoma cell lines from Bielefeld University  and industrial partners. HybriMACS CD medium was supplemented with 8 mM L-Glutamine for routine cultivation, batch and perfusion processes. For optimal performance of MF20 hybridoma cells (DSHB at the University of Iowa) the HybriMACS CD medium was supplemented with insulin (4 mg/L) or IGF (0.04 mg/L).2 and a relative humidity of 80%. For bioreactor cultivations closed-loop controlled 2 L benchtop systems were used with parameters set to 37 °C, 40% DO and pH 7.1 +/- 0.05. Automated viable cell counting was performed using a Cedex (Innovatis). Monoclonal antibody (mAb) concentrations were determined with Protein A HPLC or ELISA (MF20 cell line).All cultivations were carried out using standard conditions. Briefly, precultures and batch cultivations were performed in 125 mL and 250 mL Erlenmeyer flasks. Incubator conditions were set to 37 °C, 5% COHybridoma cell growth in HybriMACS CD medium was compared to 12 competitor products in the time course of several passages and a final batch cultivation. For a mouse-mouse hybridoma cell line, maximum viable cell density (vcd) in HybriMACS CD was highest and for a rat-mouse hybridoma cell line second-highest compared to growth in the 12 competitor media, as shown in Figure Easy adaption from serum-containing medium was verified by direct thawing of five different hybridoma cell lines in HybriMACS CD Figure . Further6 cells/mL in uncontrolled and controlled batch processes using HybriMACS CD. For uncontrolled fed batch cultivations 1.0 × 107 cells/mL were observed as maximum viable cell density, while controlled fed batch processes reached values above 1.5 × 107 cells/mL. The final antibody titer was increased at least by a factor of 5 in uncontrolled fed batches and up to 10 times in controlled fed batch cultivations using HybriMACS Feed Supplement. Exemplary results of controlled as well as uncontrolled batch and fed batch cultivations are shown in Table The majority of tested cell lines reached a maximum cell density above 2.5 to 5.0 × 10HybriMACS CD is a chemically-defined, protein-free medium composition with no need for growth hormone supplementation. The specially designed formulation supports direct adaption of serum-dependent hybridoma cells, even when starting from a serum-containing cell bank. In addition, the developed medium formulation enables stable long-term growth of hybridoma cell lines, supporting an unrestricted utilization in diverse processes. HybriMACS CD is suitable for bioreactor batch and perfusion processes reaching high cell densities and commonly accepted amounts of antibody. A specially tailored HybriMACS Feed Supplement increased final antibody titer at least by a factor of 5 to 10 for all tested hybridoma cell lines. This improvement can be further increased by customization of the generic feed regime, while maintaining suitable glucose and glutamine concentrations.


It has been reported that retinoid  inhibits growth in HTLV-1-positive T-cell lines and fresh cells from patients with adult T-cell leukemia (ATL) in vitro. In this study, we focused in role of cellular senescence in the treatment with retinoid.1. Cellular senescence was observed by senescence-associated β-galacsidase  staining.2. Gene expression of cyclin dependent kinase, Tax was performed by RT-PCR.3. To determine whether or not telomere attrition was occurred, TRAP assay was performed.1. SA β-Gal positive cells were observed in spontaneous culture without retinoid on HTLV-I (+) T-cell lines  or primary cells from acute ATL patients, but not on HTLV-I (-) T cell lines (Jurkat and MOLT-4). By treatment with retinoid, those number of SA β-Gal positive cells was increased significantly on HTLV-I (+) T-cell lines, but not on HTLV-I (-) T-cell lines (MOLT-4 and Jurkat).2. Expression of CDK inhibitor, p16INK4a , was enhanced in all of HTLV-I (+) T-cell lines. 3. In TRAP assay, no inhibition of telomere activity was observed in retinoid treated HTLV-I (+) T-cell lines, indicative of premature cellular senescence.It has been reported that oncogene induced stress (OIS) induced cellular senescence. Tax gene may act as an oncogene in ATL cells, and retinoid facilitated the cellular senescence resulting in cell death. Taken together, retinoid therapy may be a reasonable therapy with cellular senescence induction in addition of OIS.


We reviewed the phenotypic and molecular characteristics of MCF10DCIS.com and the SUM cell lines based on numerous studies performed over the years. The major signaling pathways that give rise to the phenotype of these cells may serve as a good resource of information when researchers in drug discovery and development use these cells to identify novel targets and biomarkers. Major signaling pathways and mutations affecting the coding sequence are also described providing important information when using these cells as a model in a variety of studies. However, for the last several years the relevance of cell lines in clinical cancer research has been criticized due to the properties of cell lines that differ when compared with primary tumor cells. New technologies such as next generation sequencing have enabled molecular characterization and identification of signaling pathways in specific cell lines. This characterization has facilitated the identification of cancer cell lines that are more clinically relevant for biological experiments as well as drug research and development. In addition, well characterized cells enable the identification of potential biomarkers for the development of companion diagnostics. Although there are still limitations with the relevance to the clinic, well characterized cancer cell lines will continue to be an important source for drug R&D and studying cancer biology. Human tumor-derived cell lines grown In this paper, we review relevant information available in numerous studies that encompass the characterization of the different breast cancer cell lines available at Asterand US. This review will be a valuable resource for researchers in academia and industry for the use of the relevant cell type in their research. The MCF10DCIS cell line was licensed to Asterand US by Wayne State University and the SUM cell lines were licensed to Asterand US by the University of Michigan. in situ. Solid or comedo growth patterns are high grade ductal carcinoma in situ [in vitro cultures including MCF10DCIS to mimic the three grades of breast cancer along the metastatic cascade namely, nonmalignant, noninvasive carcinoma, and invasive carcinoma in vivo. MCF10DCIS.com is a clonal breast cancer cell line derived from a xenograft originating from premalignant MCF10AT cells that were injected into severe combined immune-deficient mice. The morphology of the MCF10DCIS cell line is shown in  in situ  used sixCD44 is a cell-surface glycoprotein involved in cell-cell and cell extracellular matrix interactions including the migration and invasion of cancer cells and has been used as a key cancer stem cell-surface marker in various malignancies including breast cancer . So et aCourtney and coworkers  describein vitro, in both monolayers and spheroids [ MCF10DCIS.com cells were found to undergo spontaneous apoptosis pheroids . Shekharpheroids  evaluatein situ and subsequently in invasive ductal carcinoma (IDC) [Malignant precursor cells with metastatic potential may already develop at early stages of tumorigenesis . In addima (IDC) . Stromalma (IDC) . SDF-1 hma (IDC) . Its effma (IDC) . Tumors ma (IDC) . This st In addition, elevated levels of MMP-2, MMP-3, MMP-9, and MMP-11 were observed in the stroma and epithelia of solid DCIS lesions prior to conversion to comedo-DCIS. The MMPs are a large family of proteases which include the stromelysins, collagenases, gelatinases, elastases, and the membrane-type MMPs. Overexpression of MMPs associated with metastasis has been reported in several cancers including breast . The rolβ-galactoside-binding protein that is expressed by various types of human cells and plays an important role in cancer cell growth, transformation, apoptosis, angiogenesis, adhesion, invasion, and metastasis [Galectin-3 is a mammalian tastasis . The imptastasis . Amm andtastasis  highlighShevde and coworkers  found thThere are 11 SUM cell lines, ten of which are provided by Asterand US as shown in The isolation and culture of these cell lines designated SUM44PE, SUM52PE, and SUM102PT have been described in detail in several publications –33. Foroα, β-catenin, and angiogenic features [TP53, and other genes expression status. Laboratory investigations of SUM149 and SUM190 demonstrated radio resistance of these cell lines. Woodward et al. [κB pathway. Inflammatory breast cancer (IBC) is among the most invasive, metastatic and lethal variant of human breast cancer . IBC's hfeatures . There hfeatures . Recentlfeatures . Moleculfeatures , 40. SUMfeatures . Forozanfeatures  characted et al.  studied d et al.  investigd et al.  showed td et al.  indicateneu). SUM1315MO2 and SUM 149PT both of basal origin were found to have BRCA1 mutations and BRCA1 allelic loss [BRCA1 and BRCA2 are human genes that belong to a class of genes known as tumor suppressors. The names BRCA1 and BRCA2 stand for breast cancer susceptibility gene 1 and breast cancer susceptibility gene 2, respectively. BRCA1 has a central role in several pathways coordinating various cellular processes in response to DNA damage, including DNA repair and preservation of genomic integrity . BRCA1 alic loss . In the lic loss  identifilic loss . The 185The CHEK2 gene encodes a serine/threonine kinase. CHEK2 has been described as a tumor suppressor with proapoptotic, cell cycle checkpoint, and mitotic functions . ATM andInherited mutations in the CHEK2 gene have been identified in some cases of breast cancer. For example, a deletionmutation at nucleotide position 1100 is associated with an increased risk of breast cancer, particularly in the European population. Association studies estimated the risk of breast cancer carriers of 1100delC to be increased by 2.7-fold in women of Northern and Eastern European descent , 52. Thep53 is a nuclear transcription factor and transactivates numerous target genes involved in the induction of cell cycle arrest and/or apoptosis . MutatioHDM2, a negative regulator of the p53 pathway, is amplified and the protein is overexpressed in the luminal subtype SUM52PE cells . HDM2 waAnother important partner of the p53 pathway is p14ARF with a role of keeping HDM2 localized in the nucleolus and preventing it from degrading p53 . The ind1. The expression of this gene is tightly controlled by p53, through which this protein mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli. p21 was barely detectable in 4 SUM cell lines  and showed normal protein expression in the 4 SUM cell lines SUM 229PE, 185PE, 190PT, and 44PE. The remaining 2 cell lines were not analyzed [p21 is a potent cyclin-dependent kinase inhibitor and inhibits the activity of cyclin-CDK2 or -CDK1 complexes, and thus functions as a regulator of cell cycle progression in Ganalyzed .1 to S and G2 to M phase transitions [CDKN2A, p16Ink4A) also known as tumor suppressor 1 (MTS-1), is a tumor suppressor protein, encoded by the CDKN2A gene and plays an important role in regulating the cell cycle. Mutations in p16 increase the risk of developing a variety of cancers [The Retinoblastoma protein (RB1) is a tumor suppressor that is a crucial regulator of appropriate cell cycle progression, including Gnsitions . In cellnsitions . RB1 dernsitions . RB1 is  cancers . Loss of cancers . Sørlie  cancers  performeCCND1) is observed in as many as 50% of breast cancers, wherein it is believed to drive aberrant phosphorylation or inactivation of RB protein [The p16 gene was deleted in basal cell subtypes SUM102PT, SUM1315MO2, SUM149PT, and SUM229PE. The remaining three cell lines did not have detectable expression of p16 transcript or protein. No p16 deletions were found in the luminal cell subtypes. The p16 gene was found to be methylated in luminal subtype SUM 44PE and the transcript and protein levels were barely detectable . Despite protein . In brea protein . Hollest protein  suggesteMutations that activated PIK3CA and RAS pathways in SUM cell lines have been identified  as shownα-catenin, β-catenin, γ-catenin, p120-catenin, and the transmembrane protein E-cadherin. Extracellular E-cadherin forms homodimers with E-cadherin proteins on adjacent epithelial cells in a calcium-dependent manner. Decreases in this adhesion ability of the cell have been linked to metastasis and tumor progression [ The E-cadherin/catenin protein complex maintains the integrity of epithelial tissues through cell-cell adhesion , 72. Thegression .α-, β-, and γ-catenin protein expressions were determined by immunohistochemistry. E-cadherin gene status was analyzed for gene mutations and promoter methylation status. All the spindle shaped, basal morphology SUM cell lines showed promoter hypermethylation indicating inactivation of E-cadherin. SUM1315MO2 had barely detectable γ-catenin protein expression and no E-cadherin expression. SUM159PT also did not show E-cadherin protein expression. SUM44PE with luminal subtype and rounded morphology had a protein truncating mutation in the E-cadherin gene with no detectable E-cadherin or α-catenin and barely detectable γ-catenin protein expression. Hollestelle and colleagues  used humEpithelial cell adhesion molecule (EpCAM) also known as CD326 is a type I transmembrane 39–42 kDa glycoprotein that functions as a homophilic, epithelial-specific intercellular cell-adhesion molecule. The 314 amino acid-long EpCAM protein comprises a large extracellular domain with an epidermal growth-factor- (EGF-) like domain and a putative thyroglobulin (TY) domain, a single transmembrane region, and a short (26 amino acids) cytoplasm tail . Recent Interestingly, some SUM cell lines express markers of some of cancer stem cell (CSC) populations namely, ALDH, CD44+, and CD24− , 84. ALDThe phenomenon of heterogeneity has also been well documented in breast cancer . This hain vitro and showed an increased number of stem cells in both cell lines. The authors used the Markov mathematical model in which cells transition stochastically between states to predict the conversions that were most likely to have occurred to achieve this outcome. Subpopulations of SUM149PT and SUM159PT cells, selected for a given phenotypic state both luminal and basal cells, were shown to have the ability to give rise to stem cells . SUM149P Mutations affecting the coding sequence in these cell lines are reported in the COSMIC database and other resources are shown in The PI3K  pathway is one of the most important pathways in cancer metabolism and growth. Mutations in the PI3K  pathway are frequent in breast cancer and result in resistance to HER2-targeted agents hormonal agents . The PI3Hormone receptor-positive breast cancer therapies have been the main drug targets for several decades. HER2 became an accepted therapeutic target in standard breast cancer practice. Trastuzumab, a monoclonal antibody that interferes with the HER2/neu receptor, has been developed by Genentech. In addition to the treatment with trastuzumab other approaches have been developed , 101. OnRecently, HER3 and its physiologic ligand heregulin (HRG) have been implicated in the development of resistance to antiestrogen therapies . PresentBRCA1 mutations have the triple-negative phenotype clustered within the basal group [BRCA1 or BRCA2 mutations with advanced breast cancer that were treated with PARP inhibitor olaparib [Triple-negative breast cancer (TNBC) is an aggressive disease with about 70% of breast tumors falling within the basal-like group of breast cancers . A largeal group . In 2009al group  showed tolaparib . Oral ololaparib . The Hooolaparib  ongoing olaparib  studyingRecently the cancer genome network (TCGA) reported a comprehensive molecular analysis of breast cancer tumors with a catalogue of likely genomic drivers of the most common breast cancer subtypes . They caThis is a summary of several studies performed over the years that reveal the signaling pathways governing the MCF10DCIS.com and SUM cell lines. Breast tumors can be classified by their intrinsic subtypes. These subtypes have been associated with differences in survival with the basal-like and HER2-positive subtypes having the shortest survival times . There aAs we complete this paper, there are 538 clinical trials focused on breast cancer currently in progress. The breast cancer clinical trials are categorized either by type of breast cancer such as inflammatory or triple negative breast cancer or by response and resistance to treatments . Most of


The aim of this study was to evaluate and compare the cytotoxicity induced by two resin-based sealers, 2Seal and AH Plus, in two osteoblast-like cell lines, MG-63 and Saos-2. Using sterile discs of both sealers in complete media, 24- and 72-h extracts were prepared. The extracts were exchanged with Saos-2 or MG-63 cell culture media at 75% confluence, and after 24 h incubation, cell viability tests were performed for each extract and cell line using MTT and trypan blue dye exclusion assays. Corresponding incubated media were used as negative control groups. For both extracts and sealers, cytotoxicity was observed in both cell lines. For Saos-2, there was no statistical difference in toxicity between the sealers for either extract (p > 0.05). For MG-63, the 2Seal 24-h extract and the AH Plus 72-h extract had greater cytotoxicity than the other extracts (p < 0.05(. Both AH Plus and 2Seal demonstrated significant cytotoxicity in these two cell lines. In contrast to 2Seal, the cytotoxicity of AH Plus in the MG-63 cell line increased with extraction time from 24 to 72 h. The AH Plus and 2Seal 24-h extracts showed different levels of cytotoxicity in the MG-63 cell line, while in the Saos-2 cell line there were no detectable differences. This may reflect higher sensitivity of the MG-63 cell line compared to Saos-2 toward cytotoxicity induced by these two sealers, or different kinetics of toxicant release from the sealers. Substances used for root canal sealing along with endodontics treatment procedures have improved considerably during the last two centuries . DespiteNumerous cell lines including those obtained from human periodontal fibroblasts have been used for dental materials cytotoxicity assays, 9. Alsoin vitro biocompatibilitiy results would depend on the selected method of cytotoxicity assay  with RPMI-1640  supplemented with 10% FBS  and 1% PenStrep® . After 24 hours, when cells reached 70-80% confluence, the media were changed by the corresponding sealers extracts and after another 24 hours the viability of the cells were measured. Saos-2 and MG-63 cell lines  were seeded in 24 well plates  in 24 wells cell culture plates. The supernatants were collected after 24 or 72 hrs and replaced with either MG-63 or Saos-2 culture media. Complete media incubated in empty wells was used as negative control.According to the manufacturers’ instruction, components of either AH Plus  or 2Seal  were mixed and cylindrical discs (3 mm diameter and 3 mm thickness) were made in a laminar flow safety cabinet using a stainless steel mould. Then discs were aseptically incubated at 37Sealers cytotoxicity assaysOC. The purple/blue formazan precipitate was dissolved in 800 μl of acidic isopropyl alcohol (0.04 N) and the colored solution absorbance wasread at 570 nm and 630 nm (as reference wavelength) using a UV-Vis spectrophotometer. For TB assay, numbers of viable cells after 24 hours exposure to the extracts were counted for each triplicate and presented as percentage of the control.All experiments were performed in triplicates. The viability of the cells was measured after Saos-2 or MG-63 cell lines exposure to the sealers extracts using MTT assay  and TrypStatistical AnalysisThe results have been presented as Mean ± Standard Error. Different groups’ means were compared by t-Student and one-way analysis of variance (ANOVA) and Tukey's Post Hoc tests. The statistical significance was set at p< 0.05.The results of MTT assays for both 24 and 72 hrs extracts show significant cytotoxic effects in MG-63 and Saos-2 cell lines induced by 2Seal and AH Plus  and 2. TEvaluation of cytotoxity induced by root canal filling materials using human cell lines has been widely used in recent decades to predict and compare the new materials biocompatibilities , 6. In tDespite of worldwide AH Plus’ common use, it causes cytotoxic effects on different cell lines, like other root canal filling materials  , 24, 25.It has been shown that with different sealer materials, extraction time might have great effects on the final observed cytotoxicities. For instance with 24 hrs extraction time, Endion has showed higher toxicity on Saos-2 cell line compared to AH Plus, while with 72 hrs extraction time, AH Plus toxicity exceeded . AH PlusAlternatively some reactive species might cross react with the serum proteins in the media with consequent decrease in toxicity during extraction time. Since AH Plus setting process is the result of a polymerization system called “Linear Epoxide-Amine Addition”, leaching non reacted monomers from this polymer matrix, might be another justification for its constant increasing  cytotoxicity with longer extraction time , 27, 28.On the other hand, there is little information concerning 2Seal cytotoxicity on different cell lines especially Saos-2 and MG-63 cell lines. The information provided by the manufacturer indicates that 2Seal (like AH Plus) is a polymer made from interaction between a bisphenol derivative with a diamine derivative. It is an epoxy resin sealer and leftover of both these two copolymers are toxic and might be responsible for 2Seal cytotoxicity. In an in vivo study conducted with 3 different sealers on rat molars, 2Seal showed the best biocompatibility in terms of producing less periapical inflammation compared to the other two sealers: RSA Roekoseal and Aptal Harz . In another study, 2Seal had showed histological effects similar to AH Plus on canine molars periapical tissues . In the


A number of immunoglobulin (Ig) secreting human hybrid cell lines were created using one-on-one somatic cell hybridization of a rare human tumor infiltrating B lymphocyte and a cell of a novel human cell line (WTM), developed in house and described earlier . These hThree different hybrid cell lines each representing the highest, medium and lowest ranges of Ig productions, were subject to culture temperature drops from 37°C to 36°C, 35°C or 34°C for up to 168 hours with 24-hour data point intervals. In case of prolonged mild hypothermia, the cell line with Ig production most susceptible to temperature drops was maintained at various temperatures below 37°C  for at least 5 passages with each passage lasting 120 hours and the data taken at a 24-hour interval. At each data point for each of the hybrid cell lines at a given temperature interval, the sample was collected to determine cell concentration, cell size and Ig production.Whilst there was no observable effect of any of the short-term temperature drops on the cell growth or the cell size in any of the three hybrid cell lines, the level of Ig concentration consistently increased in all of them, with gains ranging from 67% and 320% and with Ig productivity peaking between 48 and 72 hours after the exposure to lower temperatures Figure .In contrast to short-temperature drop conditions, a prolonged exposure to mild hypothermic conditions (longer than 1 passage) led to a progressive decrease in cell size over 5 passages. This decrease in the cell size was accompanied by gradual 10-30% gains of Ig production with each passage after the initial 100 to 150% increase in Ig concentration immediately upon transfer to lower temperature (Table In conclusion, whilst lowering temperature in the culture resulted in overall increase in Ig concentration, our results suggest that there might be different mechanisms responsible for the increase in Ig productivity in response to short temperature drop and prolonged hypothermia.


Models predicted IC50 values in a 8-fold cross-validation and an independent blind test with coefficient of determination R2 of 0.72 and 0.64 respectively. Furthermore, models were able to predict with comparable accuracy (R2 of 0.61) IC50s of cell lines from a tissue not used in the training stage. Our in silico models can be used to optimise the experimental design of drug-cell screenings by estimating a large proportion of missing IC50 values rather than experimentally measuring them. The implications of our results go beyond virtual drug screening design: potentially thousands of drugs could be probed in silico to systematically test their potential efficacy as anti-tumour agents based on their structure, thus providing a computational framework to identify new drug repositioning opportunities as well as ultimately be useful for personalized medicine by linking the genomic traits of patients to drug sensitivity.Predicting the response of a specific cancer to a therapy is a major goal in modern oncology that should ultimately lead to a personalised treatment. High-throughput screenings of potentially active compounds against a panel of genomically heterogeneous cancer cell lines have unveiled multiple relationships between genomic alterations and drug responses. Various computational approaches have been proposed to predict sensitivity based on genomic features, while others have used the chemical properties of the drugs to ascertain their effect. In an effort to integrate these complementary approaches, we developed machine learning models to predict the response of cancer cell lines to drug treatment, quantified through IC In the context of cancer, libraries of chemical entities have been tested in this way against panels of cell lines grown in different conditions and with heterogeneous genomic backgrounds EWS-FLI1 translocation in Ewing’s sarcoma and sensitivity to PARP inhibitors, have been identified and further experimentally validated. Results of these studies have been made publicly available, providing unique resources that support the discovery of new predictive biomarkers for personalised cancer therapy.In these studies, using systematic statistical inference and regression methods, determinant such as oncogenic lesions, high or low levels of basal gene expression and other genotypic traits have been associated to profiles of increased sensitivity/resistance to specific compounds. For instance, by applying a multivariate analysis of variance 50, the half maximal (50%) inhibitory concentration of a substance with respect to cell viability) is often not complete. Although many steps are automated, filling experimentally each gap could be expensive and laborious 50s and estimate them for novel cell lines would be of great value for drug screening design.Increasing further the size of the considered cell-line/compound panels would be very beneficial, as it provides the basis to improve the accuracy and predictive power of the inferred associations. However, this requires larger infrastructures and the cost grows with the screening size. In addition, due to various technical and logistical reasons in a high-throughput screen in silico identification of potentially effective drugs for treating a specific cancer could be used for drug repositioning Furthermore, a robust prediction tool for in silico methods to accurately predict the effectiveness of drugs based on the molecular making of tumours  would be a major milestone towards personalized therapies for cancer patients based on molecular biomarkers Ultimately, 50. We focused on the most comprehensive cancer drug screening dataset available to date, from the “Genomics of Drug Sensitivity in Cancer” (GDSC) project 50 profile across the panel of cell lines based on the genomic background of each cell, as characterised by microsatellite instability status (1 =  unstable or 0 =  stable), somatic coding variants in the coding sequence of 77 cancer genes (1 =  any change in protein sequence and 0 =  wild type) and copy number alterations denoting gene amplification and deletion of those cancer genes . However, the predictive power of these initial models was limited, especially for those drugs without a well-known oncogene-to-drug response dependency.We therefore investigated whether it is possible to build machine learning models  that can predict drug sensitivity using cell line screening experimental data, where cell lines are treated with variable concentration of a given drug and the resulting dose-response curve summarized by an ICWe reasoned that cancer cell sensitivity to drug molecules is driven by features from both cells and drugs. Whereas cell features are ultimately connected to the inner workings of the cell, drug features include physicochemical properties that are correlated with the ability of the molecule to cross the cell membrane (e.g. lipophilicity) or its selectivity to intracellular targets .Indeed, extensive work has been done on Quantitative Structure-Activity Relationship (QSAR) approaches to predicting whole-cell activity of molecules based of their chemical properties We therefore extended our machine learning models to include as input chemical features from the drugs, besides the molecular characterization of the cell lines see . This inhttp://padel.nus.edu.sg/software/padeldescriptor/).Chemical descriptors were generated with PaDEL software 50 values .For building our model, we used GDSC screening data from 608 genomically characterised cell lines and 111 drugs for which chemical information were available (see 50) values on the test sets with an averaged Pearson correlation coefficient (Rp), coefficient of determination (2R) and root mean square error (RMSE) . Furthermore, we conducted a blind test using 13,565 new experimental IC50 values only received after training our models in order to verify our cross-validation results . The results on the blind test were almost as good as in the cross-validation, obtaining an Rp of 0.79, 2R of 0.64 and an RMSE of 0.97   of 0.85,r (RMSE) . Alterna Fig S1, . The acche total .50 values predicted for the test set using our model recapitulate associations manifested in the experimental data, for instance, whether a given mutation is causing sensitivity or resistance against a drug 50 values, we correctly captured 79% (168/213) of the significant observations with the same t-test tendency (positive or negative effect on drug sensitivity) identified with the experimental IC50s. When only considering significant associations from our model , we correctly predicted 28% (59/213) of all experimentally identified associations. Where we failed to detect an association the ANOVA effect size is often small, or the experimental correlation is associated with a mutation either not or infrequently represented within the subset of cell lines with predicted IC50 values. Notably, as example of the utility of this approach, using only predicted IC50 values we identified known drug-to-oncogene associations such as sensitivity of BRAF-mutated cells lines to MEK1/2-inhibitors  to identify drug-to-oncogene associations, we investigated how well the IChibitors  [20]. Thp of 0.82, 2R of 0.68 and an RMSE of 0.89  demonstrating the accuracy of our model to predict IC50 values for completely new cell lines. In an additional simulation, we left out all cancer cell lines from a specific tissue, e.g. we removed all lung cancer cell lines (106 out of 608 cell lines) and still obtained an Rp of 0.79, 2R of 0.61 and RMSE of 0.99 .In addition, we assessed the predictive power of our model for unknown cell lines. Therefore, we applied a more stringent 8-fold cross-validation, where a cell line was either included in the train or test set. These models achieved an Rin silico multi-drug models to impute missing IC50 values with non-parametric machine learning algorithms such as neural networks and random forests. As output for our method, we chose to explore IC50 values as generated by Garnett et al. Our results show that by using genomic features from the cell lines and chemical information from drugs, it is possible to build 2. Thanks to the use of chemical descriptors, multi-drug models are trained with a volume of data that is two orders of magnitude bigger than the data to train each single-drug model. This larger dataset weights the difficulty in training heterogeneous response values across drugs.The Pearson correlation  and coefin silico identification of genomic events associated with altered drug sensitivity, which is only possible when genomic properties are considered.In several instances, the use of multi-drug models permitted the Although our models did not capture all known gene to drug associations, we anticipate that as larger drug sensitivity and genomic datasets become available in coming years the predictive power of these models will increase. We believe that the predictive power of our models is due to the large number of cell lines and broad range of drugs in the GDSC panel that samples intensively the chemical space of common cancer drugs (chemotherapeutic and kinase inhibitors). It remains to be determined how these models will predict completely unknown families of therapeutic agents.in silico resource for predicting treatment outcomes and elucidating compound mode of action The predictive ability of our methods for individual values is still limited and could be further improved by extending the set of input features with additional layers of molecular characterization of the cell lines, such as basal transcriptional profiles and phosphoproteomic data. These data types have been used to predict drug responses in various contexts in silico predicted impinged pathway Our method uses purely experimental data, but additional predictive power can be expected from including knowledge of the underlying network Incorporation of these additional features will require a scheme to prioritize the input features based on their impact on the final trained model. Associations between features and outcomes could be explicitly unveiled by integrating in our models feature selections criteria and dimensionality reduction techniques.In terms of predictive models, we have used standard machine learning methods , given their flexibility and robustness as predictive models. A fertile ground for further research is investigating the application of other modeling techniques, including linear regression methods .50 on cell lines not screened yet, predictions from these models can be used to decide whether it is worthwhile expanding the panel of cell lines, or rather focus on a few selected ones.Our results also show that one can estimate the accuracy of prediction for different degrees of sparseness in the data, which may have utility when designing experiments where coverage has to be balanced with accuracy. Furthermore, because models are able to predict ICin silico, based on their chemical features and similarity. These predictions can help to evaluate the potential activity of new drugs, e.g. from large chemical libraries, to be screened. Furthermore, predictions on clinically approved drugs is expected to reveal candidates for drug repurposing and potentially identify specific disease sub-types that would be most responsive The implications of our results go beyond their utility to optimise the experimental design of drug screenings. Once a model is built, it could be used to systematically test the potential effect of novel drugs We used the data from the Genomics of Drug Sensitivity in Cancer project 50 value (drug concentration in μM units required to eradicate 50% of the cancer cells). Currently, the dataset contains 38,930 IC50 values out of these 67,488 (58%), with missing values mostly due to logistic reasons such as co-ordinating measurements from various screening centres. The log IC50 ranges from –7.40  to 6.91 . Note that extremely large and small values are extrapolations in the IC50 that have no clinical relevance. We use these ranges in this study as those are the ones used in the paper Garnett et al. The resulting matrix of 608 cell lines by 111 drugs will have 67,488 possible drug response curves, each summarised by its IC50 values, which were obtained after training took place, or put it differently, a batch of new experimental data was generated to independently validate our models. To sum up, 58% of the IC50 values are in the original dataset , an additional 18% are used for the blind test (independent test).We generated test sets during the cross-validation for estimating the expected error . However, even cross-validation can overestimate the prospective performance of machine learning methods. Therefore, we conducted a truly blind test in order to demonstrate the prospective capabilities of our cross-validated models to impute missing IC50 values in the 608 cell lines by 111 drugs matrix . Our blind test contains 13,565 newly generated ICThere are two different input data streams in our method: the genomic background for each cancer cell line, and the chemical properties of a drug. For the first input data stream, cancer cell lines are characterised by the mutational status of 77 oncogenes, where each of them is further described by copy number variation (any high grade amplification or homozygous deletion of a cancer gene) and sequence variation . Additionally, there is one binary feature for the microsatellite stability status of each cell line. The cell line features were encoded as followed:Microsatellite instability status Sequence variation Copy number variation All mutations considered, we have 77 possible copy number variations plus 77 possible sequence variations and one microsatellite stability value, which sums up to 155 possible cell line features. However, a few mutational features are missing for some cell lines, and we conservatively removed a feature in case it was missing for any cell line. This led to a final set of 138 genomic features characterising each cancer cell line.http://padel.nus.edu.sg/software/padeldescriptor/) The second input data stream incorporates 1D and 2D chemical properties of each drug. We generated these chemical features using the PaDEL software , other six were destined for training the model and the remaining piece was used for cross-training. Cross-validation is a process used to avoid under- and overfitting We used an 8-fold cross-validation for building our models. Therefore, we separated the original dataset into eight equally sized sets of ICFurthermore, we used a more stringent version of the above described 8-fold cross-validation. We ensured that test, train and cross-train set are not sharing any cell line, which might occur in the non-stringent version (described above). For instance, assume cell line C1 is treated with the drugs D1, D2 and D3; For the non-stringent cross-validation, the combination C1–D1, C1–D2 and C1–D3 might be distributed over test, train and cross-train set; for the stringent cross-validation, every combination with C1 is exclusively occurring in one of those three sets.http://www.heatonresearch.com/encog) 50) value.For the neural networks, we used the Java implementation from Encog 3.0.1  also into a range from 0 to 1, which was done with the following logistic-like function:As perceptron activation function for enabling the network to predict non-linear behaviour, we used the sigmoid function, which returns values in an interval from 0 to 1. Therefore, we had to normalise the IC: Observed/expected IC50 value, which has to be a positive number greater than zero.We trained the network with the resilient error backpropagation implementation from Encog with default parameters We also carried out random forest http://www.cancerrxgene.org/. The training set is based on release v1.0 from June 2012. Newly generated IC50 values of the blind test are published in release v1.1 from July 2012, which are not part of Release v1.0.The dataset is fully accessible of the Genomics of Drug Sensitivity in Cancer project http://www.heatonresearch.com/encog). For the random forest model, the R package randomForest (version 4.6–6) http://cran.r-project.org/web/packages/randomForest/index.html).The Encog Machine Learning Framework (version 3.0.1) Figure S1Blind test of multi-drug model. The training dataset holds 38,930 IC50 values, that is ∼58% of all possible drug-to-cell line combinations. For the blind test, 13,565 novel IC50 values were generated, an∼18% additional data points which were not included in the training dataset. For obtaining the predicted log(IC50) values, we averaged the output of each model . The prediction on the blind test was slightly worse than that estimated by cross-validation (RMSE) was increased from 0.83 to 0.97, coefficient of determination (R2) declined from 0.72 to 0.64 and the Pearson correlation coefficient (pR) was decreased from 0.85 to 0.79. This small performance decrease is due to the fact that blind test data points are not selected at random: these tend to come from drug-cell combinations that are not optimally represented in the training set .lidation : root me(TIFF)Click here for additional data file.Figure S250) values leaving out cell lines.Correlation between predicted to experimental observed logClick here for additional data file.Figure S350) values leaving out all lung cell lines.Correlation between predicted to experimental observed log, which we were able to predict with minor performance reduction compared to including all cell lines (RMSE) increased from 0.83 to 0.99, coefficient of determination (R2) declined from 0.72 to 0.61 and the Pearson correlation coefficient (pR) decreased from 0.85 to 0.79.ll lines : root me(TIFF)Click here for additional data file.Table S1Drug list.(CSV)Click here for additional data file.Text S1Performance measurements.(DOC)Click here for additional data file.Text S2Comparison of imputation methods and machine learning approach.(DOC)Click here for additional data file.Text S3Random Forest.(DOC)Click here for additional data file.


KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca2+-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3–7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer. The α-subunit can form macromolecular complexes with four different types of auxillary β-subunits and local Ca2+ influx channels 2+, membrane voltage, pH, shear stress, carbon monoxide, phosphorylation states, as well as G-proteins and steroid hormones, it is generally difficult to predict the role of BK channels in a given tissue 2), PI3K and PTEN can regulate BK channel activity BK (KCNMA1) potassium channels are a diverse class of ion channels expressed in many different cell types KCNMA1 in 16% of late-stage prostate cancers, identifying KCNMA1 as one of the most common amplifications in prostate cancer KCNMA1 by siRNA and specific BK channel blockade by iberiotoxin inhibited cell proliferation of the prostate cancer cell line PC3, which carries an amplification of KCNMA1. This study suggested a specific role of KCNMA1 in the transition from hormone-sensitive to hormone-insensitive and castration-refractory prostate cancer In many diseases, defective regulation/or expression of BK channels have repeatedly been associated with altered cell cycle progression KCNMA1 amplification in cancers beyond prostate cancer, and to explore its functional and prognostic consequences.The aim of this study was to investigate the prevalence of KCNMA1 gene sequence and CEP 10 , were utilized as described previously . The following criteria were applied for amplified FISH-amplified results: KCNMA1-probe/centromer 10-signal ratio of at least 2, and at least 5 signals of KCNMA1-probe.The bacterial artificial chromosome (BAC) RP11-428p16, which contains major parts of the Formalin-fixed and paraffin-embedded clinical specimens from the archive of the Institute of Pathology, University Hospital Basel, Switzerland, were used for the construction of all TMAs 2. Trypsin-EDTA (Amimed) was used as a transferring reagent. Cell lines MFM223 T47D Transfection was performed following the small-interferent RNA (siRNA) sequences transfection protocol for Lipofectamine 2000 . 4 hours after transfection cell culture medium was replaced and supplemented with 10% FCS, to trigger normal cell growth. Nonsense RNAi (nsRNA) was used as a negative control for KCNMA1 siRNA. Relative levels of KCNMA1 expression were determined in replicate cultures by real-time PCR. For proliferation assays replicate cultures were grown in parallel, to allow repeated cell harvesting and counting. Cell counting was performed, using a ‘CASY’ cell counter  and standard counting procedures were followed to determine cell quantity. The efficiency of the RNAi on KCNMA1 knock-down was controlled in replicate cultures that were grown in parallel.Trypsinized cells were harvested and washed in PBS twice. Aliquots of 100 µL were centrifuged for 2 minutes and dried with a ventilator. Frozen tissue samples and cytospins were incubated with an anti-KCNMA1 polyclonal antibody and prepared as previously described Standard indirect immunoperoxidase procedures were used for the detection of Ki67 , estrogen receptor-α  and androgen receptor  in paraffin-embedded PCA tissue sections by standard protocols, as previously described TggCCTCCTCCATggTgA, KCNMA1-rev: TTCTgggCCTCCTTCgTCT, GAPDH-for: gAAggTgAAggTCggAgTC, GAPDH-rev: gAAgATggTgATgggATTTC, KCNMA1-FL: AgCgTCCgCCAgAgCAAgAT-FL, KCNMA1-LC: LC640-ATgAAgAggCCCCCgAAgAAAgT-PH, GAPDH-FL: AggggTCATTgATggCAACAATATCCA-FL, GAPDH-LC: LC640-TTTACCAgAgTTAAAAgCAgCCCTggTg-PH. PCR conditions were: Activation 10 min 95°C, annealing 5 s at 54°C, elongation 15 s at 72°C, 40 cycles. Relative levels of expression  were determined according to Total RNA from cell lines was extracted using RNeasy Mini Kit  according to the instructions of the manufacturer. RNA concentrations were determined with Nanodrop spectrophotometer . Real-time PCR was performed using the LightCycler system and LightCycler FastStart DNA Master Hybridization Probes kit . Primers were obtained from TIB MolBiol : KCNMA1-for: 2PO4 0.4, K2HPO4 1.6, D-glucose 5, MgCl2 1, Ca-gluconate 1.3, pH 7.4). Experiments were performed in the fast whole cell configuration as described previously 2PO4 1.2, Na2HPO4 4.8, EGTA 1, Ca-gluconate 0.758, MgCl2 1.034, D-glucose 5, ATP 3. The pH was set to 7.2 and Ca2+ activity was 0.1 µM. The access conductance was measured continuously, remaining between 60–100 nS. Membrane conductance Gm was calculated from the measured current (I) and Vc values according to Ohm's law.MCF-7 and MFM-223 cells grown on glass cover slips were mounted on the stage of an inverted microscope , kept at 37°C and continuously perfused with Ringer solution of the following composition (mmol/l): NaCl 145, KHβ-estradiol  was dissolved in 100% ethanol to build a stock solution of 0.5 µM, which was further diluted to a final concentration of 10 nM, using the appropriate cell culture medium. Paxilline  was dissolved in 100% DMSO to build a stock solution of 50 mM, and then further diluted in phenol red-free medium supplemented with 5% dextran-coated charcoal-stripped FCS 17KCNMA1 in 16% of advanced-stage human prostate cancers KCNMA1 amplification in the whole range of human cancer and analyzed a large multi-tumour TMA, containing 2445 tissue spots from 118 different tumour types by FISH, using a KCNMA1-specific BAC probe (KCNMA1 was restricted to 10 of 603 (1.7%) gynecological tumours known to be influenced by estrogens (KCNMA1 was found in 23 of 1200 breast cancers (1.9%) and was significantly associated with high tumour grade (p<0.001), high tumour cell proliferation (p<0.01) and poor tumour-specific survival (Log-Rank p = 0.031).Previously, we reported amplification of AC probe Table 1.KCNMA1 and BK expression in these specimens, suggesting that KCNMA1 can also be up regulated by mechanisms other than genomic amplification (KCNMA1-amplified breast cancer (MFM223) and prostate cancer  cell lines were strongly positive for BK channels, while non-amplified breast cancer cell lines T47D and MCF7 were only weakly positive or negative as evaluated by immunofluorescence  using two tissue cores per specimen. The specimens were also analyzed by IHC for expression of ER-α, AR and Ki67 (Mib1). KCNMA1 amplification was significantly associated with high tumour cell proliferation  (KCNMA1 amplified tumours but in only one of nine (11%) KCNMA1 amplified tumours (p<0.05). Immunohistochemical AR positivity was not associated with proliferation (data not shown). ER-α was not associated with KCNMA1-amplification status of Ki67 (data not shown).To explore the association between amplification of p<0.002) Figure 4KCNMA1 amplified breast (MFM223) and prostate (PC3) cell lines revealed highest expression of BK-mRNA, with transcript levels in MFM223 being over 100-times higher than in MCF7  hormone sensitive versus (b) hormone insensitive organs. The average rank of KCNMA1-mRNA expression was significantly higher in class (a) than in class (b)  , 35% (MCF7), and 13% (MFM223) Figure 6+ currents were present in both MCF7 and MFM223 cell lines. Furthermore, we found that 17β-estradiol stimulated whole-cell K+ outward currents in both cell lines resulting in a hyperpolarisation of their membrane potential  concentration (10 µM). This finding suggests a different sex hormone-sensitivity towards KCNMA1 stimulation in these cell lines (+ outward currents activated by 17β-estradiol or NS1619 were inhibited by the BK channel blocker paxilline (5 µM). Taken together, these results indicate that breast cancer cells exhibit functional BK currents. Fast proliferating MFM223 cells have larger KCNMA1 currents with pharmacological properties different to MCF7 cells, possibly due to differential expression of ß-subunits (KCNMB1-4).Electrophysiological analysis revealed that BK KKCNMA1 to suppress KCNMA1-currents in both MCF7 and MFM223 cells. While 17β-estradiol clearly activated whole-cell K+ outward currents in non-treated MCF7 (10 nM) and MFM223 (10 µM) cells and in cells transfected with scrambled RNA, K+ current activation by 17β-estradiol was strongly reduced in both cell lines after siRNA-knockdown of KCNMA1  is located at 10q22.3 and was recently found to be amplified in a proportion of castration-refractory human prostate cancers, suggesting an involvement in the progression of the disease KCNMA1 amplification beyond prostate cancer. Our findings provide the first evidence that breast, ovarian and endometrial cancers carry also KCNMA1 amplification. Highest prevalence was found in invasive ductal breast cancer and serous carcinoma of the ovary and endometrium (3–7%). Our results demonstrate that KCNMA1 amplification is associated with high tumour stage, high tumour grade, high tumour cell proliferation, and poor prognosis.KKCNMA1 was amplified in MFM223, a rapidly growing metastatic human breast cancer cell line, and associated with strongly up-regulated mRNA- and protein expression. KCNMA1-mRNA expression was by far highest in MFM223 among all 26 analyzed cancer cell lines using quantitative real-time PCR, and more than 100-fold higher than in the non-amplified breast cancer cell line MCF7. Thus, it appears paradoxical that there was no inhibition of proliferation by siRNA or paxilline in MFM223 as opposed to the breast cancer cell lines MCF7 and T47D. One could hypothesize that the massive overexpression renders the MFM223 cells resistant to inhibition. Moreover this cell line essentially grows estrogen independent and demonstrates pharmacological properties different to other BK-expressing cancer cells, which is possibly due to differential expression of ß-subunits. In contrast to MCF7 and T47D, MFM223 has a low ER and PR expression but a very strong AR expression, Although expression and function of KCNMA1 is related to cell cycle control and correlates with proliferation and migration, it is not a simple task to unravel the underlying mechanisms β-estradiol, while low concentrations (10 nM) were sufficient to activate BK currents in MCF7 cells. A direct activator of KCNMA1, NS1619 (10 µM), significantly activated BK currents in MFK223 but not in MCF7 cells. These results suggest that, apart from differences in the level of KCNMA1 expression, there are probably differential expression levels of KCNMB1-4 subunits in both cell lines, which may lead to differential sensitivity towards physiological agonists (17β-estradiol) and pharmacological compounds (NS1619). Alternative pre-mRNA splicing is another mechanism that could explain different function of BK channels in different tissues, tumour types and cellular conditions Along this line, in the non-estrogen sensitive MFM223 cells, BK currents were activated only by non-physiologically high concentrations of 17KCNMA1 amplification to carcinomas from sex hormone-regulated organs is highly intriguing and also suggests an interaction of KCNMA1 within the hormonal context of these tumours. This is supported by our data, showing that 17β-estradiol induces cell proliferation in MCF7 and functional activation of BK channel in MCF7 and MFM223, which is abrogated by siRNA or by paxilline. This is also in line with a previous study where the BK blocker iberiotoxin lead to reduced proliferation in the breast cancer cell line MDA-MB-231 The restriction of KCNMA1 amplification and AR expression. Almost all  KCNMA1 amplified, but only 11 of 25 (44%) non-amplified breast cancers were AR-negative. The role of AR in breast cancer has not yet been fully elucidated, but it has been postulated that AR has an anti-proliferative and favourable prognostic effect in ER-positive breast cancer, whereas it has a growth-stimulating and unfavourable prognostic effect in ER-negative breast cancer We found an inverse association between β-estradiol but also testosterone can relax smooth muscle by potentiating BK channels KCNMA1 amplification occur in hormone-regulated cancers. Further studies are needed to elucidate the complex interplay between sex hormones and KCNMA1 in breast- and prostate cancer that is likely to be influenced by the type of regulatory ß-subunits of the BK channel, alternative splicing, the background of ER and/or AR status, and anti-hormone therapy.It has previously been demonstrated that not only 17KCNMA1 gene copy number. Thus, the presence of the KCNMA1 amplification in tumour specimens and MFM223 might rather highlight KCNMA1 as a gene with oncogenic relevance, while mechanisms other than amplification seem to be responsible for KCNMA1 overexpression in most of these tumours.Unfortunately, there is currently no reliable antibody for formalin-fixed and paraffin-embedded material. Therefore, we could not test the prevalence and significance of KCNMA1 protein expression in a large number of clinical cancer specimens. However, immunofluorescence revealed moderate to strong KCNMA1 expression in eight out of nine frozen breast cancer specimens irrespectively of KCNMA1 amplification occurs in carcinomas of the breast, ovary and endometrium. The prevalence of the amplification across different human cancer types points towards a broader general oncogenic potential of KCNMA1 than previously assumed.In conclusion, we report for the first time that