Alternative Lengthening of Telomeres in 22 Human Cancer Cell Lines
John Foulke, Lysa-Anne Volpe, Michael Jackson, Luping Chen, Anupreet Bal, Chris White, Fang Tian
ATCC, Manassas, VA
Abstract: Immortalization of human cells and unlimited cell growth is often associated with the maintenance of telomere during cell proliferation. Although the telomere maintenance mechanism usually occurs through up-regulation of telomerase activity, 15% human cancers use a mechanism known as alternative lengthening of telomeres (ALT) which is independent of telomerase activity. ALT, which is dependent on homologous recombination, has been proposed as an important target for cancer therapy. In this study, a total of 22 human cancer cell lines were tested for ALT activity. These cell lines represent sarcoma and carcinoma derived from brain, bone, fat, skin, muscle and fibrous tissue. Telomeric C-Circle DNA, the first specific molecule discovered for ALT, was analyzed to indicate whether ALT activity is present. In addition, cancer gene mutations of these 22 lines were analyzed in parallel with ALT activity.
p53 Mutation Status and Functional Test in p53 Mutation Human Cancer Cell Line Panels
Afshin Sohrabi, Adam Brewer, John Foulke, Michael Jackson, Luping Chen, Anupreet Bal, Chris White, Kurt Langenbach, Fang Tian, ATCC, Manassas, VA
Abstract:
Introduction: Transcription factor p53 is a major tumor suppressor. It controls the cellular response to stress signals through the induction of cell-cycle arrest, apoptosis and senescence. In addition, p53 regulates key stages of metabolism, tumor metastasis and invasion. As such, p53 has been described as "the guardian of the genome". At least 50 percent of human tumors contain mutations or deletions of TP53 which is the gene code p53 protein. In another 50 percent, p53 itself is not mutated but the pathway is partially abrogated. Approximately 95% of p53 mutations lie in the core DNA-binding domain, and 40% of these involve several “hotspot” mutations which severely restrain p53 function. Although considerable energy has been focused on the p53 pathway and the p53 mutation in the past, the reactivation of mutant p53 is becoming to a new focus. Cell line panel containing wild type p53 cell lines and various p53 hot spot mutation cell lines could be an essential tool for studying molecular pathology of cancer and the novel drug discovery. Materials and Methods: Forty-seven common used human cancer cell lines derived from breast, lung, colon, pancreas as well as haematopoietic and lymphoid tissue were selected and sequenced. p53 hotspots mutations at codon 175, 220, 245, 248, 249, 255, 265, 273, 275, and 282 which lie in exons 5 - 8 of the p53 gene reference sequence were analyzed. Moreover, the selected cells lines were composed to p53 mutation panels. The p21 mRNA expression level and protein level in cells treated with or without etoposide were also evaluated. Results: In this study, we tested p53 hot spot mutations in about 47 selected ATCC distributed cell lines, and confirmed most of the published studies on p53 mutations. We have discovered that a few tumor cell lines that yielded different p53 genotypes than had previously been reported. Further, the p53 function between WT and mutant p53 cell lines were compared by determining the p53 downstream target p21 expression under DNA damage condition. Conclusions: To obtain valid and reproducible experiment data, it is essential for common used cell lines to be clearly characterized at the molecular level. Although numerous studies on p53 mutations have been published, the information in literature is a mixture of confirmed and controversial p53 status. Our study clearly showed the sequencing and functional testing of p53 mutation cell line panels, which provide reliable tools for p53 research in cancer biology and therapy filed. (Abstract has been published in The Journal of Molecular Diagnostics, Volume 14, Issue 6.)
Poster presented at the Parenteral Drug Association’s (PDA) Global Conference on Pharmaceutical Microbiology in Bethesda, Maryland on October 22-24, 2012.
Luncheon presentation by Fang Tian, PhD, Sr Scientist with ATCC at the annual Discovery on Target meeting, October, 2012.
ATCC poster presented at the
American Association of Cancer Research meeting in Chicago, Illinois on April 1-4, 2012:
Standardized and validated ATCC breast cancer panels: A tool for the rapid validation of biological hypotheses
Chengkang (CK) Zhang1, Michal Elias-Bachrach2, Sherry Challberg1, David Bettoun2
1 ATCC Cell Systems, Gaithersburg, MD;
2 HaRo Pharmaceutical, Merion Station, PA
Session ID: Tumor Biology 49
Session Date and Time: Wednesday April 4, 2012 8:00 AM - 12:00 PM
Location: Hall F, Poster Section 15
Abstract Number: 5268
Abstract: Advances in personalized medicine rely on knowledge of validated markers that predict response to treatment. The availability of such information could significantly impact preclinical drug optimization and reduce the cost of clinical trials. Conversely, improving drug efficacy based on clinical data requires performing studies in defined biological systems. These defined systems typically are clonal cell lines, which have been key in advancing our understanding of numerous disease states, including cancer. A number of recent comprehensive studies have demonstrated that individual cell lines are restricted along discreet lineages and therefore do not recapitulate tumor heterogeneity observed in patients However, the use of a combination of such cell lines reflected the clinical diversity observed in patient. (Neve et al., 2006; Kao J et al., 2006; Keller PJ et al., 2010). We postulated that the use of carefully characterized cell lines combined into biologically relevant panels might provide valuable insights into studies on target validation, lead optimization, mechanisms of action, and evaluation of efficacy in various patient subgroups. The lack of uniform culture conditions among various tumor cell lines hampers the design and conduct of experiments using such cell line panels and complicates the interpretation of the results obtained from them. We therefore undertook to combine panels of breast cancer cell lines that represented major clinical manifestations of the disease and culture them under standardized conditions. We were able to establish culture conditions that maintained the expression of mRNA and cell surface proteins for validated therapeutic and clinical markers. Using a small collection of clinically validated and investigational compounds we provide several examples about how carefully selected and characterized cell panels can be used to carry out pharmacology studies and address questions pertinent to tumor biology and anti-cancer drug development.
Authored by Liz Kerrigan and Raymond W. Nims and published in Regenerative Med. 6(2): 225-260, 2011.
Abstract: Authentication of human tissues, cell lines and primary cell cultures (including stem cell preparations) used as therapeutic modalities is often performed using phenotyping and technologies capable of assessing identity to the species level (e.g., isoenzyme analysis and/or karyotyping). This authentication paradigm alone cannot provide assurance that the correct human cell preparation is administered, so careful labeling and tracking of cells from the donor, during manufacture and as part of the final product are also employed. Precise, accurate identification of human cells to the individual donor level could, however, significantly reduce the risks of exposing human subjects to misidentified cells. The availability of a standardized method for achieving this will provide a way to improve the safety profile of human cell-based products by providing assurance that a given lot of cells originated from the intended donor and were not inadvertently mixed or replaced with cells from other donors. In support of this goal, an international team of scientists has prepared a consensus standard on authentication of human cells using short tandem repeat profiling. Associated with the standard itself will be the establishment and maintenance of a public database of short tandem repeat profiles for commonly used cell lines.
Authored by Xu, X. et al. and published in Nature Biotechnology 29(8): 735-742, 2011. PubMed: 21804562
Abstract: Chinese hamster ovary (CHO)–derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.
Innovation Showcase presented by ATCC at the ISSCR Annual Meeting, June 2011
ATCC has established an iPS Cell Repository to accession and globally distribute highly qualified, standardized cell lines that have been reprogrammed via expression of OSKM from a diverse range of tissues and disease states. Biorepositories accelerate research progress by managing the roadblocks often associated with the distribution of biological materials.
Noelle Strubczewski, Dezhong Yin, Yukari Tokuyama, Chengkang Zhang and Will Rust; ATCC, Manassas, VA.
Poster presented at the ISSCR Annual Meeting, June 2011
The ability to reprogram various types of somatic cells to generate human induced Pluripotent Stem Cells (iPSC) offers a new paradigm for modeling human tissue development and disease progression. These cells can also serve as a source for differentiated cells that can facilitate drug testing or enable cell therapy. However, the impact on these fields is largely determined by the breadth and genetic diversity of cell lines, and the accessibility of these lines to biomedical researchers around the world. The ATCC has established an integration-free reprogramming method using the Yamanaka factors delivered on episomal plasmids. We generate iPSC’s from primary human fibroblasts of healthy and diseased donors. Our primary fibroblasts are harvested from transplant and cadaveric organs with donor consent, or from the ATCC catalog, and are accompanied by donor medical history. Here we report the creation and characterization of a human iPSC from fibroblasts of a patient with a severe form of Osteogenesis imperfecta.
W. Ullmer, J. K. Cooper, R. Marlow, B. Buck, T. Irish, ATCC, Manassas, VA.
Poster presented at the ASM Annual Meeting, May 2011
Background
The continuing emergence of viruses causing significant respiratory illness in humans highlights the need for in vitro models of the human airway epithelium to study virus biology and pathogenesis. In this study, we investigate the ability of ATCC® Primary Cell Solutions™ Human Small Airway Epithelial Cells (PCS-301-010) to differentiate into pseudostratified epithelium, as well as their susceptibility to infection by a panel of respiratory viruses, including isolates of the 2009 H1N1 influenza pandemic.
Methods
Cells were differentiated at an air-liquid interface for 28 days, with differentiation being determined by the expression of markers for cilia formation and secretory protein production. After 28 days of differentiation, the cells were infected with viruses, including 5 isolates of 2009 H1N1 influenza A virus, influenza B virus (B/Lee/40), respiratory syncytial virus (Long), parainfluenzavirus 3 (C 243), rhinovirus 16 (11757), adenovirus type 5 (Adenoid 75), and coronavirus (229E). Infection was monitored for 4 days, and resultant viral yields were determined by plaque assay for influenza viruses and 50% tissue culture infective dose (TCID50) for all other viruses.
Results
Differentiation of the small airway epithelial cells was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of genes involved in formation of cilia and Clara cell secretory protein (CCSP) were determined to be up-regulated during the differentiation process. We observed positive immunofluorescent staining for all of the viruses tested. Viral yields varied, but typically ranged between 102.5 and 105.5 IU/mL by 96 hours post-infection.
Conclusions
This work demonstrates the ability of primary human small airway epithelial cells to be used as an in vitro model that more closely represents the human small airway epithelium than standard cell culture. This model system may be a useful tool for the investigation of viral infections of the respiratory epithelium.
By Matthew Boley, Kurt Langenbach and Brian Beck, BioServices Division, ATCC.
Poster presented at the ASCB Annual Meeting, December 2010
Abstract: The pore-forming epsilon toxin, produced by the ubiquitous anaerobe Clostridium perfringens, is a CDC/USDA overlap class B select agent and is of particular concern to the agricultural and biodefense communities. It is our hope that increased availability of human and animal epsilon-sensitive cell lines will facilitate research towards cellular receptor discovery and understanding trafficking mechanisms, subsequently strengthening the basis for toxin inhibition, treatment and countermeasure development. ATCC’s extensive cell biology collection was reviewed for promising epsilon-sensitive candidates, which were selected, then ranked according to target tissue, species origin and other criteria. Eighteen cell lines were chosen and screened for toxin susceptibility using the real-time, label-free, Roche xCELLigence™ System, which measures changes in impedance. Cell lines identified to be susceptible to the preliminary toxin challenge (100nM) were subsequently evaluated with a more expansive dosage range to determine EC50’s and time to cytotoxicity. This work was performed using the xCELLigence System and a parallel battery of complimentary tests including a classical cytotoxicity assay, observation of cell morphology as well as the use of fluorescent cellular staining dyes to verify observed xCELLigence System and cytotoxicity data and to investigate observed blebbing and apparent syncytia formation. Twelve cell lines showed increased sensitivity with two cell lines exhibiting 8 and 20-fold sensitivity below the most sensitive cell line published to date. The identification of authenticated human and animal cell lines with differential responses to epsilon toxin provides a multifarious platform to investigate systems biology of receptor and cell-trafficking mechanisms for the purpose of cell-based toxin inhibition, treatment and countermeasure development.
ATCC Technology Assessment of Roche xCELLigence™ System — an Electronic Impedance-Based Cell Sensing Unit
By Kurt J. Langenbach Ph.D., BioServices Division, ATCC, published online December 2010
Abstract: This article describes our systematic investigation into the utility of employing electronic impedance-based cell sensing measurement systems to evaluate changes in cell behavior. Our studies allowed us to evaluate multiple aspects of the system including sensitivity and flexibility. Results indicate that the xCELLigence System offers dynamic, real-time, label-free and non-invasive analysis of a variety of cellular events. In some scenarios, it offers considerable labor and reagent savings when compared to more classical approaches. In addition we found that the xCELLigence System offers an efficient way to optimize cell-based assays and provides data that is almost impossible to capture using typical endpoint assays.
This article describes our systematic investigation into the utility of employing electronic impedance-based cell sensing measurement systems to evaluate changes in cell behavior. Our studies allowed us to evaluate multiple aspects of the system including sensitivity and flexibility. Results indicate that the xCELLigence System offers dynamic, real-time, label-free and non-invasive analysis of a variety of cellular events. In some scenarios, it offers considerable labor and reagent savings when compared to more classical approaches. In addition we found that the xCELLigence System offers an efficient way to optimize cell-based assays and provides data that is almost impossible to capture using typical endpoint assays.
This BioTechniques webinar – presented on Monday, October 18, 2010 at 12pm EDT, 9am PDT – is sponsored by Roche and features research conducted by ATCC scientists.
Abstract: Cell analysis using endpoint assays commonly relies on labels, reporters, or dyes, and can be limited to measuring cell response at a single time point. Additionally, this approach can present technological challenges and involve labor-intensive protocols when used to investigate dynamic cell responses. Impedance-based cell monitoring allows for label-free, non-invasive detection of cellular response in real time. This technology enables more comprehensive cell response profiles and physiologically relevant data to assess cell behavior for a broad range of applications.
In this webinar, you will:
Learn about a wide range of research applications utilizing real-time label-free cell analysis
Discuss the advantages of using a single, non-invasive assay to continuously monitor changes in cell morphology and adhesion in response to multiple signaling pathways
Hear about the application of real-time label-free technology to develop and optimize cellular assays as well as improve cell characterization.
Ask the experts your questions live!
By Jeff Irelan, ACEA Biosciences, Inc. and Jonathan H. Morgan, ATCC
Abstract: G-coupled protein receptors (GPCRs), also known as 7-transmembrane proteins, constitute the single largest class of therapeutic targets for clinical and investigational drugs. New technology for assaying receptor function in a cellular context has greatly increased the identification of novel regulators and reduced attrition rates (defined as failure in preclinical testing/clinical trials) for candidate compounds. The xCELLigence™ System from Roche Applied Science assesses real-time endogenous GPCR function in disease-relevant cells without using exogenous labels. Impedance-based real-time kinetic recordings can detect all second messenger mediated GPCR responses during the course of an experiment. In the present study, we show that the xCELLigence System is a sensitive and robust assay for continuously measuring endogenous GPCR function. A panel of 43 ligands encompassing 24 therapeutically relevant receptor families was examined, producing functional GPCR profiles for the commonly used cell lines, HeLa, U-2 OS, SH-SY5Y and CHO-K1 (ATCC® CCL-2™, HTB-962™, CRL-2266™ and CCL-61™, in that order), as well as the disease-relevant human primary cells: HUVECs (PCS-100-010) and mixed renal epithelial cells (PCS-400-012).
Online article published August 08, 2010 at www.biotechniques.com. This article includes contributing quotes from Amanda Capes-Davies, a member of the ATCC Standards Development Organization Workgroup ASN-0002.
Abstract: After 50 years of skepticism, finger pointing and unenforced protocols, sentiments are growing for mandatory cell line authentication as a condition for funding and publication. The author investigates the current state of cell line contamination and finds how raising awareness could help cut off the supply of contaminated lines.
Authored by The ATCC Standards Development Organization Workgroup ASN-0002 and published online in the July 8, 2010 issue of In Vitro Cell. Dev. Biol.
Abstract: Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.
Authored by The ATCC Standards Development Organization Workgroup ASN-0002 and published in the May 7, 2010 issue of Nature Reviews Cancer.
Abstract: Cell lines are used extensively in research and drug development as models of normal and cancer tissues. However, a substantial proportion of cell lines are mislabelled or replaced by cells derived from a different individual, tissue or species. The scientific community has failed to tackle this problem and consequently thousands of misleading and potentially erroneous papers have been published using cell lines that are incorrectly identified. Recent efforts to develop a standard for the authentication of human cell lines using short tandem repeat profiling is an important step to eradicate this problem.
Webinar presented by Elizabeth Kerrigan, Director, Standards and Certification, ATCC and Jaspreet Sidhu, Ph.D., Vice President of Business Development and Pharmaceutical Microbiology, Molecular Epidemiology, Inc.
Abstract: In the pharmaceutical and personal care industries, products, processes and environments are quality control tested to prevent microbial contamination. Microbial strains with confirmed identity, viability and purity, produced by meticulous laboratory procedures that minimize subculturing, are important components of quality control testing programs. Responding to industry demands for rapid microbiological testing, instrumentation and associated databases have been developed that allow for the standardized testing of phenotypic and genotypic traits across a wide array of microorganisms. A polyphasic approach to identification provides strain confirmation and avoids the pitfalls of misidentification, painful recalls and regulatory repercussions. Case studies will be provided to illustrate key points. (Hosted by BrightTALK™)
Webinar presented by Yvonne Reid, PhD, Cell Biology Collection Scientist, ATCC on March 9, 2010.
Abstract: Animal cell lines are important in vitro systems and tools for scientists in diverse disciplines beyond basic cell biology. Cell line authentication and characterization are crucial in these fields, yet most research scientists under appreciate them. Over the years, numerous cell lines have been shown to be misidentified due, in part, to poor techniques, inadequate authentication protocols, and sharing of unauthenticated cell lines amongst researchers. Technological advances have given rise to improved capabilities. Cell line authentication and characterization now require a comprehensive strategy that employs several complementary technologies for systematic testing for morphology, microbial contaminations, cellular cross-contamination as well as functionality. The validity of conclusions drawn from research data is dependent on consistent and unequivocal verification of cell line identity and function. It is estimated that the financial loss incurred by poorly characterized or misidentified cell lines is in the millions of dollars. An overview of the current technologies used to authenticate and characterize animal cell lines was presented. (Hosted by Corning)
Webinar presented by Brian Douglass, Cell Biology Product Manager, ATCC.
Focus: Misidentification/cross-contamination, long-term subculturing and passage number, poor culture conditions and microbial contamination (mycoplasma).
You are invited to a series of free web-based technical seminars on cell culture. Co-sponsored by ATCC and The Society for In Vitro Biology (SIVB), the webinars are designed to provide novel tips, best practices and proven techniques to help with cell culture research needs.
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The Genomic Sequence of the Chinese Hamster Ovary (CHO)-K1 Cell Line
Authored by Xu, X. et al. and published in Nature Biotechnology 29(8): 735-742, 2011.
PubMed: 21804562 iPS Cell Repository for Human Tissue and Disease Models
View the video.