Publications by authors named "Wilhelm G Dirks"

45 Publications

First report on establishment and characterization of a carcinosarcoma tumour cell line model of the bladder.

Sci Rep 2021 Mar 16;11(1):6030. Epub 2021 Mar 16.

Surgical Department, University Hospital Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167, Mannheim, Germany.

Carcinosarcoma of the urinary bladder is a very rare and aggressive subtype of bladder cancer with poor prognosis. Characteristically carcinosarcomas exhibit biphasic nature with both epithelial and mesenchymal differentiation. Limited information is available regarding its clinical features and appropriate treatments due to its rarity. Development of tumour models can further our understanding of bladder carcinosarcoma. We report establishment and characterization of the first-ever bladder carcinosarcoma cell line MaS-3. It is established by the outgrow method from 86 year-old caucasian male who underwent a radical pelvic resection after neoadjuvant radiotherapy. MaS-3 showed carcinosarcoma profile with high conformity with to the original tumour in terms of immunocytochemistry. Proteome analysis also aligned the MaS-3 cell line with the carcinosarcoma specimen rather than corresponding non-malignant tissue. Chemotherapy sensitivity testing revealed a great sensitivity of MaS-3 growth to 5-Fluorouracil, Gemcitabine and Cisplatin, with almost no impact of Irinotecan. Additionally, the suitability of MaS-3 for 3D in vitro experiments was also demonstrated. The newly established cell line MaS-3 shows typical characteristics of the tumour and may thus be a useful in vitro model system for studying the tumour biology and developing future of treatments of this rare but very aggressive entity.
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http://dx.doi.org/10.1038/s41598-021-85400-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971026PMC
March 2021

Modulators of histone demethylase JMJD1C selectively target leukemic stem cells.

FEBS Open Bio 2021 Jan 16;11(1):265-277. Epub 2020 Dec 16.

Laboratory for Stem Cell and Regenerative Medicine & Clinical Research Center, The Affiliated Hospital of Weifang Medical University, China.

Leukemic stem cells (LSCs) comprise a very rare cell population that results in the development of acute myeloid leukemia. The selective targeting of drivers in LSCs with small molecule inhibitors holds promise for treatment of acute myeloid leukemia. Recently, we reported the identification of inhibitors of the histone lysine demethylase JMJD1C that preferentially kill MLL rearranged acute leukemia cells. Here, we report the identification of jumonji domain modulator #7 (JDM-7). Surface plasmon resonance analysis showed that JDM-7 binds to JMJD1C and its family homolog JMJD1B. JDM-7 did not significantly suppress cell proliferation in liquid cell culture at higher doses, although it led to a significant decrease in semi-solid colony formation experiments at lower concentrations. Moreover, low doses of JDM-7 did not suppress the proliferation of erythroid progenitor cells. We identified that JDM-7 downregulates the LSC self-renewal gene HOXA9 in leukemia cells. We further found that the structure of JDM-7 is similar to that of tadalafil, a drug approved by the US Food and Drug Administration. Molecular docking and surface plasmon resonance analysis showed that tadalafil binds to JMJD1C. Moreover, similar to JDM-7, tadalafil suppressed colony formation of leukemia cells in semi-solid cell culture at a concentration that did not affect primary umbilical cord blood cells. In summary, we have identified JDM-7 and tadalafil as potential JMJD1C modulators that selectively inhibit the growth of LSCs.
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http://dx.doi.org/10.1002/2211-5463.13054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780120PMC
January 2021

DNMT3A R882H mutation in acute myeloid leukemia cell line SET-2.

Leuk Res 2020 01 9;88:106270. Epub 2019 Nov 9.

Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

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http://dx.doi.org/10.1016/j.leukres.2019.106270DOI Listing
January 2020

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells.

PLoS Genet 2019 10 4;15(10):e1008355. Epub 2019 Oct 4.

Cancer Research Center of Marseille; CNRS; Inserm; Institut Paoli-Calmettes; Aix-Marseille Université, Marseille, France.

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.
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http://dx.doi.org/10.1371/journal.pgen.1008355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795472PMC
October 2019

Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines.

Cells 2019 10 2;8(10). Epub 2019 Oct 2.

Industry Biotechnology Centre and School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.

The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.
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http://dx.doi.org/10.3390/cells8101194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830094PMC
October 2019

Small molecular modulators of JMJD1C preferentially inhibit growth of leukemia cells.

Int J Cancer 2020 01 25;146(2):400-412. Epub 2019 Jul 25.

Laboratory for Stem Cell and Regenerative Medicine, The Affiliated Hospital of Weifang Medical University, Weifang, Shandong, China.

Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells.
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http://dx.doi.org/10.1002/ijc.32552DOI Listing
January 2020

Cell Lines as Biological Models: Practical Steps for More Reliable Research.

Chem Res Toxicol 2019 09 15;32(9):1733-1736. Epub 2019 Jun 15.

American Type Culture Collection (ATCC) , Manassas , Virginia 20110 , United States.

Research in toxicology relies on models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control testing. This article sets out recommendations to improve the reliability of cell-based research.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748863PMC
September 2019

The LL-100 panel: 100 cell lines for blood cancer studies.

Sci Rep 2019 06 3;9(1):8218. Epub 2019 Jun 3.

Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Lines, Braunschweig, Germany.

For many years, immortalized cell lines have been used as model systems for cancer research. Cell line panels were established for basic research and drug development, but did not cover the full spectrum of leukemia and lymphoma. Therefore, we now developed a novel panel (LL-100), 100 cell lines covering 22 entities of human leukemia and lymphoma including T-cell, B-cell and myeloid malignancies. Importantly, all cell lines are unequivocally authenticated and assigned to the correct tissue. Cell line samples were proven to be free of mycoplasma and non-inherent virus contamination. Whole exome sequencing and RNA-sequencing of the 100 cell lines were conducted with a uniform methodology to complement existing data on these publicly available cell lines. We show that such comprehensive sequencing data can be used to find lymphoma-subtype-characteristic copy number aberrations, mRNA isoforms, transcription factor activities and expression patterns of NKL homeobox genes. These exemplary studies confirm that the novel LL-100 panel will be useful for understanding the function of oncogenes and tumor suppressor genes and to develop targeted therapies.
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http://dx.doi.org/10.1038/s41598-019-44491-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547646PMC
June 2019

Epstein-Barr virus (EBV) activates NKL homeobox gene HLX in DLBCL.

PLoS One 2019 29;14(5):e0216898. Epub 2019 May 29.

Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

NKL homeobox genes encode developmental transcription factors regulating basic processes in cell differentiation. According to their physiological expression pattern in early hematopoiesis and lymphopoiesis, particular members of this homeobox gene subclass constitute an NKL-code. B-cell specific NKL-code genes generate a regulatory network and their deregulation is implicated in B-cell lymphomagenesis. Epstein-Barr virus (EBV) infects B-cells and influences the activity of signalling pathways including JAK/STAT and several genes encoding developmental regulators. Therefore, EBV-infection impacts the pathogenesis and the outcome of B-cell malignancies including Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL). Here, we isolated EBV-positive and EBV-negative subclones from the DLBCL derived cell line DOHH-2. These subclones served as models to investigate the role of EBV in deregulation of the B-cell specific NKL-code members HHEX, HLX, MSX1 and NKX6-3. We showed that the EBV-encoded factors LMP1 and LMP2A activated the expression of HLX via STAT3. HLX in turn repressed NKX6-3, SPIB and IL4R which normally mediate plasma cell differentiation. In addition, HLX repressed the pro-apoptotic factor BCL2L11/BIM and hence supported cell survival. Thus, EBV aberrantly activated HLX in DLBCL, thereby disturbing both B-cell differentiation and apoptosis. The results of our study appreciate the pathogenic role of EBV in NKL homeobox gene deregulation and B-cell malignancies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216898PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541347PMC
January 2020

RBFOX2 and alternative splicing in B-cell lymphoma.

Blood Cancer J 2018 08 10;8(8):77. Epub 2018 Aug 10.

Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

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http://dx.doi.org/10.1038/s41408-018-0114-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086906PMC
August 2018

Authentication of M14 melanoma cell line proves misidentification of MDA-MB-435 breast cancer cell line.

Int J Cancer 2018 02 10;142(3):561-572. Epub 2017 Oct 10.

International Cell Line Authentication Committee (ICLAC).

A variety of analytical approaches have indicated that melanoma cell line UCLA-SO-M14 (M14) and breast carcinoma cell line MDA-MB-435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross-contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA-MB-435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA-MB-435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA-MB-435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA-MB-435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.
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http://dx.doi.org/10.1002/ijc.31067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762610PMC
February 2018

Fetal Bovine Serum (FBS): Past - Present - Future.

ALTEX 2018 9;35(1):99-118. Epub 2017 Aug 9.

Division of Physiology, Medical University Innsbruck, Innsbruck, Austria.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.
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http://dx.doi.org/10.14573/altex.1705101DOI Listing
August 2018

KDM3B shows tumor-suppressive activity and transcriptionally regulates HOXA1 through retinoic acid response elements in acute myeloid leukemia.

Leuk Lymphoma 2018 01 25;59(1):204-213. Epub 2017 May 25.

a Laboratory for Stem Cell and Regenerative Medicine , The Affiliated Hospital of Weifang Medical University , Weifang , Shandong , China.

KDM3B reportedly shows both tumor-suppressive and tumor-promoting activities in leukemia. The function of KDM3B is likely cell-type dependent and its seeming functional discordance may reflect its phenotypic dependence on downstream targets. Here, we first showed the underexpression of KDM3B in acute myeloid leukemia (AML) patients and AML cell lines with MLL-AF6/9 or PML-RARA translocations. Overexpression of KDM3B repressed colony formation of AML cell line with 5q deletion. We then performed global microarray profiling to identify potential downstream targets of KDM3B, notably HOXA1, which was verified by real time PCR and Western blotting. We further showed KDM3B binding at retinoic acid response elements (RARE) but not at the promoter region of HOXA1 gene. KDM3B knockdown resulted in increased mono-methylation but decreased di-methylation of H3K9 at RARE while eschewing the promoter region of HOXA1. Collectively, we found that KDM3B exhibits potential tumor-suppressive activity and transcriptionally modulates HOXA1 expression via RARE in AML.
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http://dx.doi.org/10.1080/10428194.2017.1324156DOI Listing
January 2018

False and mycoplasma-contaminated leukemia-lymphoma cell lines: time for a reappraisal.

Int J Cancer 2017 Mar;140(5):1209-1214

Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

Leukemia-lymphoma cell lines are important research tools in a variety of fields. To represent adequate model systems it is of utmost importance that cell lines faithfully model the primary tumor material and are not cross-contaminated with unrelated cell material (or contaminated with mycoplasma). As it has been previously reported that cross-contaminated cell lines represent a significant problem, it is of interest to know whether any improvement in the prevalence of such "false cell lines" had occurred since we called the alert in 1999. A retrospective review of our data archives covered 848 cell lines received from 1990 to 2014 from 290 laboratories in 23 countries spanning the spectrum of leukemia-lymphoma entities. Two variables were considered: authenticity and freedom from mycoplasma infection. Regarding provenance, we separately considered primary sources (original investigators having established the cell lines or reference repositories) and secondary sources. The percentages of mycoplasma-contaminated cell lines decreased significantly over the 25-year timespan. Among primary sourced material: mycoplasma-contamination fell from 23% to 0%; among secondary sourced: from 48% to 21%. The corresponding figures for cross-contamination declined from 15% to 6%, while among material obtained from secondary sources prevalence remained remarkably high, throughout the time periods at 14-18%. Taken together, our data indicate that using non-authenticated cell lines from secondary sources carries a risk of about 1:6 for obtaining a false cell line. The use of authentic leukemia-lymphoma cell lines holds important translational value for their model character and the reproducibility of the laboratory data in the clinical arena.
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http://dx.doi.org/10.1002/ijc.30530DOI Listing
March 2017

Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines.

PLoS One 2015 23;10(11):e0139663. Epub 2015 Nov 23.

Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2-10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings highlight biallelic deletions as a major class of chromosomal lesion in PMBL cell lines, while endorsing the latter as preclinical models for hunting and testing new biomarkers and actionable targets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139663PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657880PMC
June 2016

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis.

Cell Cycle 2015 ;14(11):1704-15

a Institute of Clinical Chemistry and Laboratory Diagnostics; University Düsseldorf; Medical Faculty , Düsseldorf , Germany.

To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.
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http://dx.doi.org/10.1080/15384101.2014.1000182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614858PMC
March 2016

BCL6--regulated by AhR/ARNT and wild-type MEF2B--drives expression of germinal center markers MYBL1 and LMO2.

Haematologica 2015 Jun 13;100(6):801-9. Epub 2015 Mar 13.

Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig

Genetic heterogeneity is widespread in tumors, but poorly documented in cell lines. According to immunoglobulin hypermutation analysis, the diffuse large B-cell lymphoma cell line U-2932 comprises two subpopulations faithfully representing original tumor subclones. We set out to identify molecular causes underlying subclone-specific expression affecting 221 genes including surface markers and the germinal center oncogenes BCL6 and MYC. Genomic copy number variations explained 58/221 genes differentially expressed in the two U-2932 clones. Subclone-specific expression of the aryl-hydrocarbon receptor (AhR) and the resulting activity of the AhR/ARNT complex underlaid differential regulation of 11 genes including MEF2B. Knock-down and inhibitor experiments confirmed that AhR/ARNT regulates MEF2B, a key transcription factor for BCL6. AhR, MEF2B and BCL6 levels correlated not only in the U-2932 subclones but in the majority of 23 cell lines tested, indicting overexpression of AhR as a novel mechanism behind BCL6 diffuse large B-cell lymphoma. Enforced modulation of BCL6 affected 48/221 signature genes. Although BCL6 is known as a transcriptional repressor, 28 genes were up-regulated, including LMO2 and MYBL1 which, like BCL6, signify germinal center diffuse large B-cell lymphoma. Supporting the notion that BCL6 can induce gene expression, BCL6 and the majority of potential targets were co-regulated in a series of B-cell lines. In conclusion, genomic copy number aberrations, activation of AhR/ARNT, and overexpression of BCL6 are collectively responsible for differential expression of more than 100 genes in subclones of the U-2932 cell line. It is particularly interesting that BCL6 - regulated by AhR/ARNT and wild-type MEF2B - may drive expression of germinal center markers in diffuse large B-cell lymphoma.
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http://dx.doi.org/10.3324/haematol.2014.120048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450626PMC
June 2015

Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen.

Sci Rep 2015 Feb 3;5:8202. Epub 2015 Feb 3.

Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Paul Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany.

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.
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http://dx.doi.org/10.1038/srep08202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314641PMC
February 2015

Association between acquired resistance to PLX4032 (vemurafenib) and ATP-binding cassette transporter expression.

BMC Res Notes 2014 Oct 10;7:710. Epub 2014 Oct 10.

Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Paul Ehrlich-Str, 40, Frankfurt am Main 60596, Germany.

Background: Various kinase inhibitors are known to be ATP-binding cassette (ABC) transporter substrates and resistance acquisition to kinase inhibitors has been associated to increased ABC transporter expression. Here, we investigated the role of the ABC transporters ABCB1, ABCC1, and ABCG2 during melanoma cell resistance acquisition to the V600-mutant BRAF inhibitors PLX4032 (vemurafenib) and PLX4720. PLX4032 had previously been shown to interfere with ABCB1 and ABCG2. PLX4720 had been demonstrated to interact with ABCB1 but to a lower extent than PLX4032.

Findings: PLX4032 and PLX4720 affected ABCC1- and ABCG2-mediated drug transport in a similar fashion. In a panel of 16 V600E BRAF-mutated melanoma cell lines consisting of four parental cell lines and their sub-lines with acquired resistance to PLX4032, PLX4720, vincristine (cytotoxic ABCB1 and ABCC1 substrate), or mitoxantrone (cytotoxic ABCG2 substrate), we detected enhanced ABC transporter expression in 4/4 cytotoxic ABC transporter substrate-resistant, 3/4 PLX4720-resistant, and 1/4 PLX4032-resistant melanoma cell lines.

Conclusion: PLX4032 has the potential to induce ABC transporter expression but this potential is lower than that of PLX4720 or cytotoxic ABC transporter substrates. Since ABC transporters confer multi-drug resistance, this is of relevance for the design of next-line therapies.
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http://dx.doi.org/10.1186/1756-0500-7-710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197243PMC
October 2014

Aurora kinases as targets in drug-resistant neuroblastoma cells.

PLoS One 2014 30;9(9):e108758. Epub 2014 Sep 30.

Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Frankfurt am Main, Germany.

Aurora kinase inhibitors displayed activity in pre-clinical neuroblastoma models. Here, we studied the effects of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) and the aurora kinase inhibitor alisertib (MLN8237) that shows some specificity for aurora kinase A over aurora kinase B in a panel of neuroblastoma cell lines with acquired drug resistance. Both compounds displayed anti-neuroblastoma activity in the nanomolar range. The anti-neuroblastoma mechanism included inhibition of aurora kinase signalling as indicated by decreased phosphorylation of the aurora kinase substrate histone H3, cell cycle inhibition in G2/M phase, and induction of apoptosis. The activity of alisertib but not of tozasertib was affected by ABCB1 expression. Aurora kinase inhibitors induced a p53 response and their activity was enhanced in combination with the MDM2 inhibitor and p53 activator nutlin-3 in p53 wild-type cells. In conclusion, aurora kinases are potential drug targets in therapy-refractory neuroblastoma, in particular for the vast majority of p53 wild-type cases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182628PMC
June 2015

Localization of MLH3 at the centrosomes.

Int J Mol Sci 2014 Aug 11;15(8):13932-7. Epub 2014 Aug 11.

Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Lines, Braunschweig 38124, Germany.

Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLβ, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers.
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http://dx.doi.org/10.3390/ijms150813932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159832PMC
August 2014

Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.

J Cell Biochem 2013 Oct;114(10):2405-14

Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.
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http://dx.doi.org/10.1002/jcb.24591DOI Listing
October 2013

Frameshift-derived neoantigens constitute immunotherapeutic targets for patients with microsatellite-instable haematological malignancies: frameshift peptides for treating MSI+ blood cancers.

Eur J Cancer 2013 Jul 2;49(11):2587-95. Epub 2013 Apr 2.

Division of Molecular Oncology and Immunotherapy, Department of General Surgery, University of Rostock, Rostock, Germany. Electronic address:

Purpose: Microsatellite instability (MSI) resulting from loss of functional DNA mismatch repair was recently found in various haematological disorders. In coding sequences, MSI leads to frameshift mutations (FSMs) and the production of C-terminally altered proteins which are foreign to the immune system. Here, we wondered whether these frame-shifted peptide (FSP) sequences represent tumour-specific antigens also for MSI(+) leukaemia and lymphomas (L/L).

Material And Methods: A total of 33 coding region microsatellites were examined in MSI(+) L/L cell lines for the presence of FSMs. Thereafter, recognition of MSI(+) cells by established FSP-specific CD8(+) T cell lines was quantified using interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assays. In each experiment, MSI(+) L/L cell lines and T2 targets exogenously loaded with the cognate peptide (=internal control) were employed. Supplementary, lytic activity towards tumour cells was analysed by standard chromium release assay ((51)Cr).

Results: Mutational profiling of 33 coding microsatellite loci in nine MSI(+) L/L cell lines revealed instability in at least nine microsatellites. In each cell line, a distinct mutational profile was observed. Only three of the 33 loci were stable. FSP-specific and human leukocyte antigen-A2 (HLA-A2)-restricted T cells specifically recognised MSI(+) L/L cells endogenously expressing TGFβRII(-1), Caspase 5 (-1) and MSH3 (-1) in ELISpot assays. Moreover, specific killing of Caspase 5 (-1) and MSH3 (-1) expressing L/L cell lines was achieved in functional cytotoxicity assays.

Conclusion: Data presented here expand the importance of FSPs as shared and general tumour-specific antigens. Consequently, they open new avenues for specific immunotherapies not only for solid but also for MSI(+) haematological malignancies.
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http://dx.doi.org/10.1016/j.ejca.2013.02.035DOI Listing
July 2013

STR DNA typing of human cell lines: detection of intra- and interspecies cross-contamination.

Methods Mol Biol 2013 ;946:27-38

Department of Human and Animal Cell Lines, DSMZ, German Collection of Microorganisms and Cell Cultures and German Biological Resource Center, Braunschweig, Germany.

Inter- and intraspecies cross-contaminations (CCs) of human and animal cells represent a chronic problem in cell cultures leading to false data. Microsatellite loci in the human genome harboring short tandem repeat (STR) DNA markers allow individualization of cell lines at the DNA level. Thus, fluorescence polymerase chain reaction amplification of STR loci D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO, and Amelogenin for gender determination is the gold standard for authentication of human cell lines and represents an international reference technique. The major cell banks of the USA, Germany, and Japan (ATCC, DSMZ, JCRB, and RIKEN, respectively) have built compatible STR databases to ensure the availability of STR reference profiles. Upon determination of an STR profile of a human cell line, the suspected identity can be proven by online verification of customer-made STR data sets on the homepage of the DSMZ institute. Furthermore, an additional tetraplex PCR has been established to detect mitochondrial DNA sequences of rodent cells within a human cell culture population. Since authentic cell lines are the main prerequisite for rational research and biotechnology, the next sections describe a rapid and reliable method available to students, technicians, and scientists for certifying identity and purity of human cell lines of interest.
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http://dx.doi.org/10.1007/978-1-62703-128-8_3DOI Listing
May 2013

Match criteria for human cell line authentication: where do we draw the line?

Int J Cancer 2013 Jun 28;132(11):2510-9. Epub 2012 Nov 28.

CellBank Australia, Children's Medical Research Institute, Westmead, New South Wales, Australia.

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.
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http://dx.doi.org/10.1002/ijc.27931DOI Listing
June 2013

Kaposi's sarcoma-derived cell line SLK is not of endothelial origin, but is a contaminant from a known renal carcinoma cell line.

Int J Cancer 2013 Apr 12;132(8):1954-8. Epub 2012 Oct 12.

Division of Molecular and Experimental Surgery, Clinical Center Erlangen, Erlangen, Germany.

Kaposi's sarcoma (KS) is an endothelial cell-derived tumor. Investigations of the molecular mechanisms of KS pathogenesis and the identification of drugs for treatment of KS depend critically on valid cell-culture models. Two major immortalized cell lines are available for KS research. Recently, the KS cell line KS Y-1 has been shown to be cross-contaminated with the T24 urinary bladder cancer cell line (ATCC HTB-4). Here, we show by short tandem repeat profiling that the second KS cell line, SLK, is indistinguishable from the clear-cell renal-cell carcinoma cell line Caki-1. Immunocytochemical detection of cytokeratin expression confirmed the epithelial-cell origin of SLK cells. Our findings indicate that SLK cells are not of endothelial origin and should not be used in future studies as a model for KS-derived endothelial tumor cells. We suggest that in the future, more attention needs to be paid to the authenticity of cells in lines derived from human tissues.
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http://dx.doi.org/10.1002/ijc.27849DOI Listing
April 2013

Where have all the cell lines gone?

Int J Cancer 2013 Mar 16;132(5):1232-4. Epub 2012 Aug 16.

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http://dx.doi.org/10.1002/ijc.27752DOI Listing
March 2013

High-throughput SNP-based authentication of human cell lines.

Int J Cancer 2013 Jan 28;132(2):308-14. Epub 2012 Jun 28.

Department of Genome Modifications and Carcinogenesis, F020, Research Program Infection and Cancer, German Cancer Research Center, DKFZ, Heidelberg, Germany.

Use of false cell lines remains a major problem in biological research. Short tandem repeat (STR) profiling represents the gold standard technique for cell line authentication. However, mismatch repair (MMR)-deficient cell lines are characterized by microsatellite instability, which could force allelic drifts in combination with a selective outgrowth of otherwise persisting side lines, and, thus, are likely to be misclassified by STR profiling. On the basis of the high-throughput Luminex platform, we developed a 24-plex single nucleotide polymorphism profiling assay, called multiplex cell authentication (MCA), for determining authentication of human cell lines. MCA was evaluated by analyzing a collection of 436 human cell lines from the German Collection of Microorganisms and Cell Cultures, previously characterized by eight-loci STR profiling. Both assays showed a very high degree of concordance and similar average matching probabilities (~1 × 10(-8) for STR profiling and ~1 × 10(-9) for MCA). MCA enabled the detection of less than 3% of contaminating human cells. By analyzing MMR-deficient cell lines, evidence was obtained for a higher robustness of the MCA compared to STR profiling. In conclusion, MCA could complement routine cell line authentication and replace the standard authentication STR technique in case of MSI cell lines.
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http://dx.doi.org/10.1002/ijc.27675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492511PMC
January 2013