RESUMEN
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.
Asunto(s)
Línea Celular Tumoral/microbiología , Leucemia/patología , Linfoma/patología , Mycoplasma/aislamiento & purificación , Dermatoglifia del ADN , Humanos , Hibridación in Situ , Cariotipificación , Estudios RetrospectivosRESUMEN
Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a mature, CD30-positive T-cell lymphoma lacking expression of the anaplastic lymphoma kinase (ALK). In contrast to ALK-positive ALCL, BIA-ALCL cells express cyclin D2 (CCND2) which controls cyclin dependent kinases 4 and 6 (CDK4/6). DNA methylation and expression analyses performed with cell lines and primary cells suggest that the expression of CCND2 in BIA-ALCL cell lines conforms to the physiological status of differentiated T-cells, and that it is not the consequence of genomic alterations as observed in other hematopoietic tumors. Using cell line model systems we show that treatment with the CDK4/6 inhibitor palbociclib effects dephosphorylation of the retinoblastoma protein (RB) and causes cell cycle arrest in G1 in BIA-ALCL. Moreover, we show that the PI3K/AKT inhibitor BEZ-235 induces dephosphorylation of the mTORC1 target S6 and of GSK3ß, indicators for translational inhibition and proteasomal degradation. Consequently, CCND2 protein levels declined after stimulation with BEZ-235, RB was dephosphorylated and the cell cycle was arrested in G1. Taken together, our data imply potential application of CDK4/6 inhibitors and PI3K/AKT inhibitors for the therapy of BIA-ALCL.
RESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular/microbiología , Desinfección/métodos , Macrólidos/administración & dosificación , Mycoplasma/efectos de los fármacos , Animales , Antibacterianos/administración & dosificación , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
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.
Asunto(s)
Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma de Efusión Primaria/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Imidazoles/farmacología , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Linfoma Anaplásico de Células Grandes/genética , Linfoma de Efusión Primaria/tratamiento farmacológico , Linfoma de Efusión Primaria/genética , Compuestos Macrocíclicos/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Quinolinas/farmacologíaRESUMEN
The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.
Asunto(s)
COVID-19/virología , Receptores Virales , SARS-CoV-2/fisiología , Internalización del Virus , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Línea Celular Tumoral , Humanos , Receptores Virales/genética , Receptores Virales/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
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.
Asunto(s)
Primates/virología , Virus/genética , Virus/aislamiento & purificación , Animales , Southern Blotting , Línea Celular , ADN Circular/análisis , VIH-1/genética , VIH-1/aislamiento & purificación , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/aislamiento & purificación , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Virus Linfotrópico T Tipo 2 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/aislamiento & purificación , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Retrovirus de los Simios/genética , Retrovirus de los Simios/aislamiento & purificación , Saimiri/virología , Proteínas Virales/análisisRESUMEN
EZH2 gain of function mutations (EZH2GOFmu) has been implicated in the pathogenesis of B-non-Hodgkin lymphoma. The EZH2-specific inhibitor GSK126 inhibits trimethylation of histone H3K27 and induces target gene expression. However, in 3/4 EZH2GOFmu B-NHL lymphoma cell lines, GSK126 (400 nM) did not induce growth arrest. Only at high doses (10 µM), the inhibitor was effective as antiproliferative agent, comparably in EZH2GOFmu, wild-type, and EZH2-negative cell lines, suggesting that at high concentrations, the antiproliferative effects of GSK126 are off-target effects. In sum, we could not confirm that B-NHL cell lines with EZH2GOFmu show a higher sensitivity to GSK126 than EZH2 wild-type cell lines do. Only 1/4 EZH2GOFmu B-NHL cell lines tested (PFEIFFER) were sensitive to GSK126 (400 nM) inducing growth arrest. If these results can be translated to patients, they raise the question of whether the presence of EZH2 activating mutations alone allows selection for targeted therapy with EZH2 inhibitors.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2 , Complejo Represivo Polycomb 2 , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Humanos , Metilación , Mutación , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismoRESUMEN
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.
Asunto(s)
Línea Celular/virología , Animales , Bovinos , Biología Computacional , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Control de Calidad , Análisis de Secuencia de ARNRESUMEN
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.
Asunto(s)
Infecciones por Virus de Epstein-Barr/metabolismo , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Proteínas de Homeodominio/biosíntesis , Linfoma de Células B Grandes Difuso/metabolismo , Proteínas de Neoplasias/biosíntesis , Factores de Transcripción/biosíntesis , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Supervivencia Celular , Infecciones por Virus de Epstein-Barr/patología , Humanos , Linfoma de Células B Grandes Difuso/patología , Proteínas de la Matriz Viral/metabolismoRESUMEN
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.
Asunto(s)
Leucemia/patología , Linfoma/patología , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN/genética , Exoma/genética , Exones/genética , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia/genética , Linfoma/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN/genética , ARN/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
Tumor cell lines are widely used as oncologic models and resources, forming, along with primary patient material and animal models, one of three major subjects for cancer investigation. With the advent of the Human Genome Project (HGP) and the ensuing provision of sequencing data and mapped clones, human cancer cell lines, notably those derived from leukemia-lymphoma (LL) have become increasingly productive tools for cancer gene ascertainment and characterization. Hence, the roles of putative novel cancer genes may be investigated using diverse panels of LL cell lines, both individually by PCR-based methods, and globally by transcriptional chip-profiling. Similar studies have also enabled the faithfulness with which cancer cell lines model their supposed in vivo counterparts to be quantified at last. Several recent transcriptional profiling studies indicate that of all tumor types well characterized human LL cell lines most accurately model the gene expression patterns of their corresponding primary tumors. Analysis using genomic arrays tells a similar story for the stability of chromosome rearrangements in LL cell lines. Well characterized LL cell lines also provide ideal tools for investigating the druggability of individual gene products, e.g. by measuring their transcript levels using q(uantitative)-PCR methods in cells subjected to treatments with small interfering (si)-RNAs. We provide a list of authentic, well characterized examples for prospective investigators, since many circulating cell lines have been cross-contaminated and describe DNA profiling methods which, together with classic and molecular cytogenetic analyses, inform authentication. We also review the problem of mycoplasma contamination and means for its eradication.
Asunto(s)
Línea Celular Tumoral , Leucemia/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Animales , Evaluación Preclínica de Medicamentos , Marcación de Gen , Humanos , Leucemia/genética , Leucemia/patología , Linfoma/genética , Linfoma/patología , ARN Interferente Pequeño/farmacología , Translocación GenéticaRESUMEN
Mycoplasma hyopneumoniae, an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular ß-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target ß-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.
Asunto(s)
Actinas/metabolismo , Adhesinas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Interacciones Huésped-Patógeno/fisiología , Mycoplasma hyopneumoniae/metabolismo , Mycoplasma hyopneumoniae/patogenicidad , Unión Proteica , Actinas/efectos de los fármacos , Animales , Anticuerpos Monoclonales/farmacología , Avidina/metabolismo , Biotinilación , Línea Celular , Cilios/metabolismo , Células Epiteliales/microbiología , Pulmón , Proteínas de la Membrana/metabolismo , Neumonía Porcina por Mycoplasma , PorcinosRESUMEN
Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members.
Asunto(s)
Apoptosis/genética , Línea Celular Tumoral/patología , Linfoma de Células B Grandes Difuso/patología , Compuestos de Anilina/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Linfoma de Células B Grandes Difuso/genética , Masculino , Persona de Mediana Edad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Sulfonamidas/administración & dosificaciónRESUMEN
Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin (IG) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5+, two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies.
Asunto(s)
Biomarcadores de Tumor/genética , Exoma/genética , Regulación de la Expresión Génica , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Linfoma de Células B/genética , Mutación , Antígenos CD5 , Línea Celular Tumoral , Células Clonales , Reordenamiento Génico , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologíaRESUMEN
Mycoplasma contamination of cell lines is one of the major problems in cell culture technology. The specific, sensitive, and reliable detection of mycoplasma contamination is an important part of mycoplasma control and should be an established method in every cell culture laboratory. New cell lines as well as cell lines in continuous culture must be tested in regular intervals. The polymerase chain reaction (PCR) methodology offers a fast and sensitive technique to monitor all cultures in a laboratory. The technique can also be used to determine the contaminating mycoplasma species. The described assay can be performed within 3 h, including sample preparation, DNA extraction, performing the PCR reaction, and analysis of the PCR products. Special precautions necessary to avoid false-negative results resulting from inhibitors of the Taq polymerase present in the crude samples and the interpretation of the results are also described.
Asunto(s)
Mycoplasma/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN , Mycoplasma/clasificación , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Mycoplasma contaminations have a multitude of effects on cultured cell lines that can potentially influence the results of experiments and pollute bioactive substances used in human medicine. The elimination of mycoplasma contaminations in cell cultures has become a practical alternative to discarding and re-establishing important or irreplaceable cell lines. Different quinolones, tetracyclins, and macrolides shown to have strong antimycoplasma properties are employed for the decontamination. We provide detailed descriptions to assure eradication of mycoplasma, to prevent formation of resistant mycoplasma strains, and to cure heavily contaminated and damaged cells. To date, no consistent and permanent alterations that affect the eukaryotic cells during or after the treatment have been detected.
Asunto(s)
Línea Celular/microbiología , Mycoplasma/aislamiento & purificación , Antibacterianos/farmacología , Mycoplasma/efectos de los fármacosRESUMEN
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%.
Asunto(s)
Virus de la Leucemia Murina/aislamiento & purificación , Línea Celular , ADN Viral/genética , Genoma Viral/genética , Genotipo , Humanos , Virus de la Leucemia Murina/genética , Reacción en Cadena de la PolimerasaAsunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación Missense , Sustitución de Aminoácidos/genética , Arginina/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , ADN Metiltransferasa 3A , Análisis Mutacional de ADN , Histidina/genética , HumanosRESUMEN
We have identified new mutations in the p53 gene in 3/11 growth factor-independent and in 2/8 growth factor-dependent human acute myeloid leukemia (AML)-derived cell lines by single-strand conformational polymorphism (SSCP) and sequencing analysis. MEG-01 had a triplet deletion at codon 304; F-36P, NB-4 and MV4-11 showed point mutations at codon 344. F-36P had a second point mutation at codon 270 and NB-4 additionally at codon 319. M-MOK had a nucleotide substitution at codon 191. The frequency of p53 mutations in the cytokine-independent cell lines was comparable to that in the cytokine-dependent lines. These results suggest that loss of Wild type (wt) p53 is not the decisive event causing tumor cells to proliferate in vitro without externally added growth factors.
Asunto(s)
Genes p53 , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-3/farmacología , Leucemia Mieloide/genética , Enfermedad Aguda , Western Blotting , División Celular/efectos de los fármacos , Codón/genética , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Humanos , Leucemia Mieloide/patología , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/efectos de los fármacos , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Human leukemia-lymphoma (LL) cell lines represent important tools for experimental research. Among the various problems associated with cell lines, the two most common concern contaminations: (1) cross-contamination with unrelated cells and (2) contamination with microorganisms, in particular mycoplasma. The bad news is that about one-third of the cell lines are either cross-contaminated or mycoplasma-infected or both. The good news is that there are means to recognize and overcome these problems. In cases where, during attempts to establish new LL cell lines, primary LL cultures are cross-contaminated with continuous cell lines, intended new cell lines simply cannot be established ("early" cross-contamination). In cases of "late" cross-contamination of existing LL cell lines where the intrusive cells have a growth advantage, the original ("uncontaminated") cell lines may still be available elsewhere. DNA fingerprinting and cytogenetic analysis appear to be the most suitable approaches to detect cross-contaminations and to authenticate LL cell lines. A different but related aspect of "false" LL cell lines is the frequent misclassification of cell lines whereby the actual cell type of the cell line does not correspond to the purported model character of the cell line. Mycoplasma infection can have a multitude of effects on the eukaryotic cells which, due to the variety of infecting mycoplasma species and many other contributing parameters, cannot be predicted, rendering resulting data questionable at best. Practical procedures for the detection and elimination of mycoplasma contamination have been developed. Diagnostic and preventive strategies in order to hem the alarming increase in "false" and mycoplasma-positive LL cell lines are recommended.