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  • 1
    Language: English
    In: European Journal of Cancer, July 2013, Vol.49(11), pp.2587-2595
    Description: 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). 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 ( Cr). 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. 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.
    Keywords: Frameshift Antigens ; Microsatellite Instability ; Haematological Malignancies ; Medicine
    ISSN: 0959-8049
    E-ISSN: 1879-0852
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  • 2
    Language: English
    In: International Journal of Cancer, 01 March 2013, Vol.132(5), pp.1232-1234
    Keywords: Medicine;
    ISSN: 0020-7136
    E-ISSN: 1097-0215
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  • 3
    In: International Journal of Cancer, 01 March 2017, Vol.140(5), pp.1209-1214
    Description: 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. What's new? Tumor cell lines are essential research tools, but a significant percentage is contaminated with other cell lines and/or mycoplasma. In our study of leukemia and lymphoma cell lines, the authors found that the incidence of mycoplasma‐contamination has decreased significantly over the past 25 years. However, cross‐contamination of cell lines still remains at an unacceptably high level, particularly among cell lines circulating unchecked between different laboratories. Researchers are urged to use authenticated cell lines, to ensure the accuracy of clinical and experimental results.
    Keywords: Authentification ; Cell Lines ; Leukemia ; Lymphoma ; Mycoplasma
    ISSN: 0020-7136
    E-ISSN: 1097-0215
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  • 4
    Language: English
    In: International Journal of Cancer, 15 April 2013, Vol.132(8), pp.1954-1958
    Description: 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. What's new? The KS Y1 and SLK cell lines are widely used for the study of Kaposi's sarcoma. KS Y1 cells, however, were discovered by ATCC to have a similar short tandem repeat (STR) profile as T24 urinary bladder cancer cells. Investigation of SLK cells here reveals that these cells also are contaminated, having an STR profile indistinguishable from that of Caki‐1 renal cell carcinoma cells. The results indicate that SLK cells should not be used as model systems for Kaposi's sarcoma.
    Keywords: Kaposi'S Sarcoma ; Kshv ; Hhv‐8 ; Cross‐Contamination ; Quality Control
    ISSN: 0020-7136
    E-ISSN: 1097-0215
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  • 5
    Language: English
    In: PLoS ONE, 01 January 2014, Vol.9(9), p.e108758
    Description: 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.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 6
    Language: English
    In: International Journal of Cancer, 15 January 2013, Vol.132(2), pp.308-314
    Description: 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 for STR profiling and ∼1 × 10 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.
    Keywords: Multiplex Cell Authentication Mca ; Snp ; Str Profiling ; Luminex ; Cell Line ; Cross‐Contamination ; Mmr Deficiency
    ISSN: 0020-7136
    E-ISSN: 1097-0215
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  • 7
    Language: English
    In: International Journal of Cancer, 01 January 2010, Vol.126(1), pp.303-304
    Keywords: Databases, Genetic ; Microsatellite Repeats ; Neoplasms -- Pathology;
    ISSN: 0020-7136
    E-ISSN: 1097-0215
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  • 8
    Language: English
    In: Radiation and Environmental Biophysics, 2011, Vol.50(3), pp.345-352
    Description: An improved assessment of the biological effects and related risks of low doses of ionizing radiation is currently an important issue in radiation biology. Irradiations using microbeams are particularly well suited for precise and localized dose depositions, whereas recombinant cell lines with fluorescent proteins allow the live observation of radiation-induced foci. Living cells of the fibrosarcoma cell line HT-1080 stably expressing 53BP1 or full-length reconstituted MDC1 fused to Green Fluorescent Protein (GFP) were irradiated with protons and α-particles of linear energy transfers (LETs) of 15 and 75 keV/μm, respectively. Using a microbeam, the irradiations were carried out in line patterns, which facilitated the discrimination between undefined background and radiation-induced foci. As expected, foci formation and respective kinetics from α-particle irradiations with a high LET of 75 keV/μm could be detected in a reliable manner by both fusion proteins, as reported previously. Colocalization of γ-H2AX foci confirmed the DSB nature of the detected foci. As a novel result, the application of protons with low LET of 15 keV/μm generated 53BP1- and MDC1-mediated foci of almost equal size and slightly different kinetics. This new data expands the capability of 53BP1 and wild-type MDC1 on visible foci formation in living cells after irradiation with low-LET particles. Furthermore, the kinetics in HT-1080 cells for α-particle irradiation show a delay of about 20 s for 53BP1 foci detection compared to wild-type MDC1, confirming the hierarchical assembly of both proteins. Preliminary data for proton irradiations are shown and also these indicate a delay for 53BP1 versus MDC1.
    Keywords: Radiation (Physics) ; Sarcoma ; Fluorescence ; Universities And Colleges ; Proteins;
    ISSN: 0301-634X
    E-ISSN: 1432-2099
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  • 9
    Language: English
    In: PLoS ONE, Sept 30, 2014, Vol.9(9)
    Description: 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.
    Keywords: Tumor Proteins ; Apoptosis ; Phosphotransferases ; Neuroblastoma
    ISSN: 1932-6203
    Source: Cengage Learning, Inc.
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  • 10
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2013, Vol.946, pp.27-38
    Description: 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.
    Keywords: DNA Contamination ; Microsatellite Repeats ; DNA Fingerprinting -- Methods ; DNA, Mitochondrial -- Genetics
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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