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.
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.
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.
What's new?
Histone demethylase JMJD1C represents one of the most promising therapeutic targets for a subtype of acute leukemia with inferior prognosis, MLLr AL. Here, the authors focused on the catalytic Jumonji domain of JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. They identified structurally‐akin JMJD1C inhibitors with the ability to selectively kill MLLr AL cells, regulate MLLr AL cell metabolism, and collaborate with all‐trans retinoic acid to repress MLLr AL. The findings helped identify the backbone of an inhibitor class that holds promise for the treatment of MLLr AL.
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.
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.
Fetal Bovine Serum (FBS): Past - Present - Future van der Valk, Jan; Bieback, Karen; Buta, Christiane ...
ALTEX, alternatives to animal experimentation,
2018, Letnik:
35, Številka:
1
Journal Article
Recenzirano
Odprti dostop
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.
Immortalized or continuous cell lines are invaluable tools in basic and preclinical research. However, the widespread use of misidentified cell lines is a serious threat to scientific ...reproducibility. Based on the experiences of mandatory cell line authentication at the International Journal of Cancer (IJC), we provide an overview of the issues pertinent to misidentified cell lines and discuss available solutions. We also summarize the lessons learned, revealing that at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified. About 4% of the considered manuscripts are rejected for severe cell line problems, and most are subsequently published in other journals. In order to diminish such malpractice and its consequences for the scientific record, we postulate that strict multi‐layered quality control is essential. Besides journals and publishers, we encourage scientists, research institutions, and funders to take action on the matter and revise their respective policies. Hence, we provide concrete recommendations on introducing regular authentication schemes and staff training, and discuss future steps for enhancing good cell culture practices.
This commentary discusses multi‐layered quality control of immortalized cell lines as an essential prerequisite for reliability of the scientific record and good cell culture practices.
Research on prostate cancer is mostly performed using cell lines derived from metastatic disease, not reflecting stages of tumor initiation or early progression. Establishment of cancer cell lines ...derived from the primary tumor site has not been described so far. By definition, cancer cells are able to be cultured indefinitely, whereas normal epithelial cells undergo senescence in vitro. Epithelial cells can be immortalized, accomplished by using viral integration of immortalization factors. Viral approaches, however, might be impaired by regulatory and safety issues as well as random integration into regulatory genetic elements, modifying precise gene expression. We intend to use surgical specimen of prostate cancer patients to (i) prove for establishment of cancer cell lines, and (ii) perform non-viral, Sleeping Beauty (SB) transposase-based immortalization of prostate epithelial cells.
Radical prostatectomy samples of prostate cancer patients (n = 4) were dissociated and cultured in vitro. Cells were cultivated either without or after non-viral, Sleeping-Beauty transposase-based stable transfection with immortalization factors SV40LT and hTERT. Established cell lines were analyzed in vitro and in vivo for characteristics of prostate (cancer) cells.
Initial cell cultures without genetic manipulation underwent senescence within ≤ 15 passages, demonstrating inability to successfully derive primary prostate cancer cell lines. By using SB transposase-based integration of immortalization factors, we were able to establish primary prostate cell lines. Three out of four cell lines displayed epithelial characteristics, however without expression of prostate (cancer) characteristics, e.g., androgen receptor. In vivo, one cell line exhibited tumorigenic potential, yet characteristics of prostate adenocarcinoma were absent.
Whereas no primary prostate cancer cell line could be established, we provide for the first-time immortalization of primary prostate cells using the SB transposase system, thereby preventing regulatory and molecular issues based on viral immortalization approaches. Although, none of the newly derived cell lines demonstrated prostate cancer characteristics, tumor formation was observed in one cell line. Given the non-prostate adenocarcinoma properties of the tumor, cells have presumably undergone oncogenic transformation rather than prostate cancer differentiation. Still, these cell lines might be used as a tool for research on prostate cancer initiation and early cancer progression.
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.
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.
What's new?
Short tandem repeat (STR) profiling is the recommended approach for authentication testing of human cell lines. To improve its effectiveness, however, a method by which STR can account for genetic drift arising from the passage of malignant cells is needed. Here, algorithms based on a set of match criteria, eight core STR loci, and amelogenin analysis were found to successfully discriminate between related and unrelated samples. The match criteria used here would bring greater rigor to the interpretation of STR profiling results.
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