37 research outputs found

    Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in Human and Other Primate Cell Lines

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    The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections

    Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

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    A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant

    Screening human cell lines for viral infections applying RNA-Seq data analysis.

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    Monitoring viral infections of cell cultures is largely neglected although the viruses may have an impact on the physiology of cells and may constitute a biohazard regarding laboratory safety and safety of bioactive agents produced by cell cultures. PCR, immunological assays, and enzyme activity tests represent common methods to detect virus infections. We have screened more than 300 Cancer Cell Line Encyclopedia RNA sequencing and 60 whole exome sequencing human cell lines data sets for specific viral sequences and general viral nucleotide and protein sequence assessment applying the Taxonomer bioinformatics tool developed by IDbyDNA. The results were compared with our previous findings from virus specific PCR analyses. Both, the results obtained from the direct alignment method and the Taxonomer alignment method revealed a complete concordance with the PCR results: twenty cell lines were found to be infected with five virus species. Taxonomer further uncovered a bovine polyomavirus infection in the breast cancer cell line SK-BR-3 most likely introduced by contaminated fetal bovine serum. RNA-Seq data sets were more sensitive for virus detection although a significant proportion of cell lines revealed low numbers of virus specific alignments attributable to low level nucleotide contamination during RNA preparation or sequencing procedure. Low quality reads leading to Taxonomer false positive results can be eliminated by trimming the sequence data before analysis. One further important result is that no viruses were detected that had never been shown to occur in cell cultures. The results prove that the currently applied testing of cell cultures is adequate for the detection of contamination and for the risk assessment of cell cultures. The results emphasize that next generation sequencing is an efficient tool to determine the viral infection status of human cells

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

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    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

    Prevalence and characterization of murine leukemia virus contamination in human cell lines.

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    Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%

    Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma.

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    Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors

    PCR-Assays for the sensitive and reliable detection of MLV sequences in cell line DNA.

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    <p>The upper two panels show the ethidium bromide stained gels of the MLV-specific PCR assays performed with the outer (first panel) and inner primers (second panel). The sizes of the MLV-specific bands are 604–642 bp and 150–174 bp, respectively, depending on the MLV genotype. Some cell lines produce an unspecific band with the outer primers (also seen in some MLV-negative cell lines). The inner primers usually also produce several unspecific bands seen only in MLV-positive cell lines. The cell lines KYSE-30 and NAMALWA are MLV-negative in the assay, whereas the cell lines from the same series or subclones are MLV-positive, respectively. The third panel demonstrates the integrity of the DNAs used for the analyses by amplification of the human ABL gene. The lower panel shows contamination of the genomic DNA with mouse DNA by amplification of mouse specific IAP coding sequences. The PCR produces a double band of approximately 300 bp. The cell line NCEB-1 harbors several mouse-derived chromosomes.</p

    Neighbor joining tree of full genomic MLV sequences.

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    <p>The chart demonstrates the relationship of the complete MLV genomes. The alignment and the tree construction were performed with the MEGA6 software. The investigated sequences produce the same major groups of MLV subtypes as found for the partial sequences. The scale bar represents the number of nucleotide substitutions per site. * Sequences obtained from the NCBI sequence database.</p
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