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Cover:Biology is complex, and under-
standing fundamental biologicalprocesses requires precision tools
to tease out small changes thatcan have dramatic effects. Asscreening activities move fromthe pharmaceutical setting to
academia, basic research scien-tists are gaining a new portfolio ofhighly sensitive, reliable reagents
and assays. Precision tools forprecision research. Cover copy-
right Promega Corporation.
Editor . .Michele Arduengo, Ph.D., ELS
Art Director . . . . . . . . . . .Karen Gaustad
Figure Production . . . . .Sara Klink, M.S.
Copyright 2008 Promega Corporation.All rights reserved. No part of this doc-ument may be reproduced, stored in a
retrieval system or transmitted in anyform or by any means, electronic,
mechanical, photocopy, recorded orotherwise, without prior written permis-
sion of Promega Corporation.2800 Woods Hollow Road
Madison, WI 53711-5399 USA
To receive Cell Notes, subscribe online or contact us by e-mail:
[email protected]. For information on contributing,
contact the Editor.
Tel: 608-274-4330 (ext. 2566)Fax: 608-277-2601
E-mail:[email protected]
Cell Notes is published by:Promega Corporation
2800 Woods Hollow RoadMadison, WI 53711-5399 USA
Tel: 608-274-4330
CELL NOTES ISSUE 22
Printed in USA 10/0816336-CN-CS
CN022
■ HTSHigh-Throughput Screening and Drug Discovery at St. Jude Children’s
Research Hospital 3
■ SCREENINGScreening for New Chemical Entities in Academic Settings 5In Vivo Imaging: Non-invasive Bioluminescent Imaging Improves
Understanding of Tumor Biology 6
■ GSH ASSAYHomogeneous Luminescence-Based Assay for Quantifying the
Glutathione Content in Mammalian Cells 7
■ ENZYME ASSAYSLuminogenic Enzyme Substrates: The Basis for a New Paradigm
in Assay Design 10
■ CELL CULTUREDoing Good Science: Authenticating Cell Line Identity 15
■ CELL SIGNALINGStudying MAP Kinase Signaling with a Small-Molecule Inhibitor, U0126 18
■ MISCELLANEOUSErratum 9Meetings 20Contact Us 20
IN THIS ISSUE
www.promega.com
Cell Notes Editorial Board: Simon T.M. Allard, Ph.D., Robert Deyes, M.S., Frank Fan, Ph.D., Pam Guthmiller, B.S., Eric Vincent, Ph.D., and John Watson, Ph.D.
Cell Notes is provided free to cell biologists in training and at the professional level. Wewelcome article submissions from external authors. Contact the editor([email protected]) for more information about article submission andreview.
DRUG SCREENING LATIN AMERICA CONFERENCE
EDITOR’S DESK
CELL NOTES ISSUE 22 2008 2 www.promega.com
Early during my graduate training, my adviser rushed into the lab in an excited dither about a new publications database availablefrom the National Library of Medicine. It was called Medline. My advisor gathered those of us who were present around the lab’ssingle computer.
“Here let me show you how it works.” We stood staring over his shoulder. “You can find all the papers someone has published justby typing in that person’s last name.” And, he proceeded to type his last name into the search box. After what seemed like hours,but was probably less than a minute, a list of publications appeared.
“Let's see, no, that's one of my wife’s papers. And that one. And that one. Wait a second. None of my papers are coming up.”
Repressing guffaws of roll-in-the-aisle laughter, my colleagues and I slowly backed away from the computer and returned to our labbenches while our adviser scratched his head.
Since that time the PubMed database has expanded hugely, and it has been joined by other literature databases such as StanfordUniversity’s HireWire® Press. You can find practically anything now, including papers written by my graduate advisor. Today entirepapers are now available online as PDF files; open access journals are revolutionizing the process of peer-reviewed publication, andalmost no one spends hours in the library searching through stacks of hard-copy journals for an elusive article.
Technology certainly changes things, and this issue of Cell Notes focuses on some of the changes that high-throughput technologiesare bringing to the basic research lab. Increasingly, drug discovery and other screening activities are moving into the academicresearch laboratory; we discuss some of these changes in the article on page 5. Our feature article about the HTS Core Facility at St. Jude Children’s Research Hospital shows how screening facilities are driving both basic and translational research outside thepharmaceutical industry (page 3).
For HTS to make a meaningful contribution to the life sciences, scientists need more than just robotics; they need easilyminiaturized, highly sensitive and reliable assays and reagents. Our articles about the GSH-Glo™ Assay (page 7), bioluminescentenzyme assays (page 10), and U0126 (page 18) describe some of those reagents. Not only do researchers’ assay reagents need tobe reliable, but they need assurance that the cell lines they use for their models are what they are advertised to be. Our article onpage 15 about cell line authentication highlights this important issue.
Things are changing, and at a tremendous pace. This issue of Cell Notes explores some of the changes that automation and high-throughput technologies are bringing to basic research.
Enjoy.
Michele Arduengo, Editor
26–27 February 2009Rio de Janeiro, Brazil
Join business and research leaders from developed and emerging markets to review the status ofdrug discovery activities in Latin America and discuss future trends and prospects.
Keynote Speakers: Hakim Djaballah Director, Memorial Sloan-Kettering Cancer Center;Ralph Garippa Research Leader, Roche Discovery Technologies; John Watson DirectorPharmacology and Biotechnology, Promega Corporation; Chiwai Wong Principal Investigator,Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences
Visit: www.selectbiosciences.com/conferences/DDLA2009 for more information and to register.
photo credit: James G. Howes
www.promega.com 3 CELL NOTES ISSUE 22 2008
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Introduction
In the past decade high-throughput screening (HTS) hasbeen used predominantly in the pharmaceutical andbiotechnology industries for drug discovery. That landscapehas changed greatly in recent years. Governmentinstitutions, universities and hospitals have established avariety of screening centers with somewhat mixed objectivesincluding identifying probes that can be accessed by thepublic, identifying drug leads for potential licensingopportunities and providing contract services forpharmaceutical companies (1–3). Furthermore, HTS alsohas expanded to emerging markets where unique collectionsof compounds or extracts, such as traditional Chinese herbalmedicines, are used (4).
In this article we highlight the HTS Core Facility at St. JudeChildren’s Research Hospital in Memphis, Tennessee, USA.St. Jude Children’s Research Hospital was founded in 1962by the late entertainer Danny Thomas to “advance cures,and means of prevention, for pediatric catastrophic diseasesthrough research and treatment” (5). At St. Jude, no child isdenied treatment on the basis of race or religion, and nofamily ever receives a bill from St. Jude for their child’streatment. St. Jude handles all costs not covered byinsurance and total treatment cost for patients with noinsurance.
The hospital’s vision to be the world leader in advancing thetreatment and prevention of catastrophic diseases inchildren is developed through excellence in patient care andthrough strong programs in basic, translational and clinicalmedicine. The HTS Core Facility is part of the Department ofChemical Biology and Therapeutics, which works, togetherwith other St. Jude research faculty, to discover and developnew chemical entities that increase understanding of thepathophysiology of catastrophic pediatric diseases or thatfunction as therapeutic leads for the treatment of suchdiseases.
Supporting Translational Research: The Roles and Goals ofthe HTS Core Facility
The HTS Core Facility at St. Jude was established in 2006.The facility at St. Jude uses both commercially availablechemical libraries and St. Jude’s own repository of chemicalcompounds designed by chemists in the Department ofChemical Biology and Therapeutics for probe developmentand drug discovery. St. Jude has the capacity to house 1 million chemical compounds on site.
The staff of the HTS Core Facility collaborate with researchfaculty at St. Jude to develop assays, validate them andminiaturize the assays so that they are amenable toautomation. After a screening assay is successfully
HIGH-THROUGHPUT SCREENING AND DRUG DISCOVERY AT ST. JUDECHILDREN’S RESEARCH HOSPITALTAOSHENG CHEN, PH.D., AND R. KIP GUY, PH.D., ST. JUDE CHILDREN’S RESEARCH HOSPITAL, MEMPHIS, TN
We highlight the role of the HTS Core Facility at St. Jude Children’s Research Hospital in Memphis, TN, USA. This facility has a significant focus on discovering and developing new chemical entities based on an extensive, on-site chemical library.
The campus of St. Jude Children’s Research Hospital in Memphis, Tennessee, USA (photo credit: St. Jude Biomedical Communications).
developed, the facility conducts the screening experimentsusing the assay and executes the appropriate secondary andADME/Tox screening to assist in determining mechanism ofaction, physical properties and off-target toxicities of anycompound “hits” from the primary screen.
In addition to conducting screening projects in collaborationwith St. Jude research faculty, the HTS staff manage thecompound store and evaluate and develop novel drugdiscovery technologies.
Creating A State-of-the-Art HTS Facility
The HTS Core Facility at St. Jude employs state-of-the-artautomation and assay technologies. The facility is located inthe Integrated Research Center. The facility can performbiochemical and cell-based (BSL2+ compliant) assays usingan on-site library of 525,000 compounds and full siRNA setsfor mouse and human genomes that are stored in a REMP-designed automated compound store with the capacity tostore 1 million compounds or other materials (e.g., RNAimolecules).
The facility has multiple integrated screening robots thatcontain various liquid-handlers and high-throughput platereaders for a variety of assay outputs including Alphascreen®,fluorescence intensity, fluorescence polarization, time-resolvedfluorescence, luminescence, and absorbance. The facility alsohas an automated microscope and flow cytometer for cell-based high-content screening.
The robots include Staubli robotic arms for moving multiwellplates from station to station; Liconic incubators, each with acapacity of up to 242 plates, which can be swapped amongdifferent docking stations during the run process; andscheduling software that controls the screening systemoperation and handles the deposition of all screening-relevant data.
Progress in HTS Research
Since its inception in 2006, the HTS facility has initiatedmultiple HTS projects. The work from the screening centerand the Department of Chemical Biology and Therapeutics atSt. Jude is already beginning to appear in the literature (6–9).The HTS center also is undertaking new technology initiativesincluding screens involving RNAi.
Literature Cited
1. Arduengo, P.M. (2005) Cell Notes 13, 22–4.
2. Arduengo, P.M. (2006) Cell Notes 16, 30–2.
3. Grooms, K. (2008) Promega Notes 99, 22–24.
4. Kang, Z. et al. (2008) Cell Notes 20, 9–11.
5. Scientific Report 2007. St. Jude Children’s Research Hospital.http://www.stjude.org/ (accessed July 24, 2008)
6. Arnold, A. et al. (2006) Sci. STKE. 341, 13.
7. Mills, N.L., Shelat, A.A. and Guy, R.K. (2007) J. Biomol. Scr. 12,946–55.
8. Agler, M. et al. (2007) J. Biomol. Scr. 12, 1029–41.
9. Chen, T. (2008) Curr. Opin. Chem. Biol. Jul. 26 (epub ahead ofprint) http://dx.doi.org/10.1016/j.cbpa.2008.07.001
Alphascreen is a registered trademark of Perkin Elmer, Inc.
The Chemical Library Storage and Retrieval Facility in the HTS Core Facility. St. Jude Children’s Research Hospital can store up to 1 million chemicalcompounds or small molecules (photo credit: St. Jude BiomedicalCommunications).
CELL NOTES ISSUE 22 2008 4 www.promega.com
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Introduction
Large pharmaceutical companies with significant financialleverage were the pillars of drug development in the mid- tolate 1980s. Two predominant factors influenced the rate ofsuccess in this field: resources and access to financial capital.Large organizations such as Pfizer, Wyeth and Bristol MeyersSquibb had both elements. In the mid-1990s, numerous NewChemical Entities (NCEs) were discovered and marketed.Financial success was synonymous with drug development.Indeed, the mid-1990s served as the “golden years” forpharmaceutical companies. Some companies such as Pfizersaw their stock value balloon within a short period of time.
However, in 2008, the picture is anything but rosy forpharmaceuticals. Most large traditional pharmaceuticalcompanies (excluding biotech companies) have seen theirstock decrease markedly from the “golden years.” Whatcaused this change and who are the new beneficiaries ofdrug development programs? The concept that onlypharmaceutical companies develop and test new drugs ischanging. The paradigm has been shifting to academia andother nonpharmaceutical entities. With extremely low drugdevelopment success rates, pharmaceutical companies havebeen outsourcing research to foreign countries with scientificexpertise and to academic research organizations.
From 1991–2000, 23% of the drugs that survived Phase I, IIand III trials were not approved by the FDA (1). Nearly onequarter of the drugs that were tested in expensive large,double-blind human clinical trials failed to produce anyrevenue for the company that invested in the development ofthat drug. This does not include those drug candidates thatfailed in Phase I or II trials. Only about 11% of all drugcandidates that make it to the clinical trial phases obtain FDAapproval (1). With these low success rates, pharmaceuticalcompanies have reevaluated their business models anddevelopment needs. As a result, drug discovery anddevelopment are taking place in other settings.
Moving Screening and NCE Development into Academia
Academic institutions provide a twofold advantage. First, labsspecializing in a particular area of research have a profoundunderstanding of mechanisms and overall knowledge toadvance a compound through multiple stages. The expertisemay be pivotal to the success of any particular compound.Second, academic research organizations employ numerouspostdoctoral fellows and other individuals with a high degreeof expertise who are generally not compensated at the samelevel as pharmaceutical researchers in large organizations.
Hence, this approach meets the scientific and businessneeds to propel compounds through the pipeline and reducedevelopment costs.
Development costs have been quoted as high as 1.7 billiondollars for an NCE when discovered solely by apharmaceutical company, almost double the widely accepted$897 million estimate published by the Tufts Center for theStudy of Drug Development (2). Increasing failure rates inclinical trials are thought to be driving increased drugdevelopment costs (2).
New York Area Bioluminescent Screening Symposium
Promega hosted a bioluminescent-screening symposium onMay 2, 2008, for researchers in academia within the NewYork City, USA, area. Approximately 50 researchers fromColumbia University, Rockefeller University, Albert EinsteinSchool of Medicine and several other prestigious institutionsattended. Attendees got a firsthand view of the type, qualityand quantity of research taking place within the NYC area.The goal of this symposium was to educate researchers onthe diversity of research and the techniques used to fullyachieve their research potential. Additionally, this served as aforum for Promega to display the tools and techniques thatare available to aid in the research and streamline theworkflow process.
In addition to academic research organizations, small start-up companies with access to capital such as IngeniousTargeting Laboratories (ITL) were present at the forum. ITL iscurrently engaged in numerous pharmaceutical developmentprograms focused on developing novel treatments forcardiovascular diseases, immunological disorders and skindiseases. Hence, the goal of this seminar was not just totarget academic research organizations but all involved with
SCREENING FOR NEW CHEMICAL ENTITIES IN ACADEMIC SETTINGSPOONAM JASSAL, PROMEGA CORPORATION
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the discovery process for novel drugs and therapies. This all-encompassing forum provided insight not only to research butalso served as a platform to share and exchange ideas andresearch techniques.
Summary
Large pharmaceutical companies have begun to outsourcenumerous projects in the hope of expediting the discovery offuture NCEs. Significant potential exists in the exchangebetween corporate and academic laboratories for learning,developing and sharing ideas, technology and strategies foranswering fundamental biological questions, conducting
translational research, and developing useful therapeutics.
References
1. Lowe, D. (2004) In the pipeline. http://pipeline.corante.com/archives/2004/09/20/drug_development_the_current_odds.php(accessed August 26, 2008)
2. Mullin, R. (2003) Drug development costs about $1.7 billion.Chem Eng. News 81, 8.
NON-INVASIVE BIOLUMINESCENTIMAGING IMPROVESUNDERSTANDING OF TUMORBIOLOGYDolores Hambardzumyan and Eric Holland, Department ofCancer Biology and Genetics, Sloan Kettering Institute
Gliomas and medulloblastomas are the most commonprimary brain tumors in adults and children, respectively.Although the standard of care for gliomas may have evolvedslightly over the last 50 years, the clinical outcome of thisdisease remains unchanged. Therefore, further research toimprove the treatment modalities is urgently needed. Animportant step forward is the development and validation ofmouse models that accurately recapitulate the complexity ofhuman tumors.
This effort has been greatly facilitated by the development ofpreclinical imaging technologies such as bioluminescentimaging (BLI). A reporter mouse line can be engineered toexpress luciferase from a promoter that responds to a specificbiological process. Luciferase activity can be measured in vivoand correlated with the strength of the pathway driving thetransgene construct. For example: we know that the E2F1promoter is regulated by Rb in cell cycle progression andappears to mediate tumor-selective transgene expression intumor cells. Therefore, the human E2F1 promoter was used todrive the firefly luciferase gene in a transgenic mouse model.Another reporter line was generated based on the fact thatSHH is activated in a subset of human medulloblastoma. Inthis reporter construct, luciferase is driven by a promoterresponsive to Gli, which is a downstream element in the SHHpathway.
Such reporter lines can be used as tools for discovery,revealing pathways previously not recognized as being activein specific tumor types. For example, using the Gli-luciferase
reporter mouse, we have demonstrated that the SHHpathway is active in PDGF-driven gliomas. We routinely useluciferase reporter lines for more accurate and detailedmeasurements of biological processes in preclinical trials byradiation- or chemotherapy. The imaging results from BLImust be validated with histological analysis of tumors. Braintumors, especially gliomas, are highly heterogeneous, andthere is high variability in response to different treatmentmodalities. Non-invasive imaging with BLI provides asignificant advantage and allows each one to serve as its owncontrol. BLI is less expensive than other small animal imagingtechnologies such as MRI and PET imaging. Finally, thetumors that have been generated in reporter lines also can beused to generate cell culture for in vitro studies; cells isolatedfrom those tumors can be cultured, and BLI can be used tomonitor the activity of different pathways.
SYMPOSIUM SIDEBAR: IN VIVO IMAGING
The Gli-Luc reporter mouse can be used for monitoring PDGF-inducedgliomas in mice in vivo. Black arrow points to the tumor.
Introduction
During the last decades it has become apparent thatphysiological levels of reactive oxygen species (ROS) play animportant role in various diseases, such as chronicinflammation and cancer (1). One factor that can modulateROS level is the nonprotein thiol glutathione (GSH) that isproduced by mammalian cells (2). This antioxidant caninteract with diverse ROS species and is consequentlyoxidized itself, thereby generally reducing cellular redoxstress. Additionally, biosynthesis of GSH can be up- ordownregulated by the cells when triggered by a huge numberof physiological and xenobiotic compounds (Figure 1). Animbalance of ROS can lead to protection or physiological
stress of the cells, which can in the latter case inducemanifold cellular defense mechanisms frequently leading tocell death, partially by apoptosis (3). Furthermore if GSHlevel is decreased, the capacity of cells for detoxification byphase II metabolism is reduced (Figure 1). Hence it isimportant to evaluate the mechanisms of action of differentnatural agents during preclinical drug development byinvestigating alterations in GSH levels.
Reagents, Materials and Instrumentation
In the experiments described here, we used the GSH-Glo™Glutathione Assay Kit (Cat.# V6911, V6912), buthionine-(S,R)-sulfoxime (BSO; Sigma), N-acetylcysteine (NAC;
HOMOGENEOUS LUMINESCENCE-BASED ASSAY FOR QUANTIFYING THEGLUTATHIONE CONTENT IN MAMMALIAN CELLSCHRISTIANE SCHERER, SILVIA CRISTOFANON, MARIO DICATO, MARC DIEDERICH*,LABORATOIRE DE BIOLOGIEMOLÉCULAIRE ET CELLULAIRE DU CANCER, HÔPITAL KIRCHBERG, 9, RUE EDWARD STEICHEN, L-2540 LUXEMBURG,LUXEMBURG, *CORRESPONDING AUTHOR. TEL: +352 2468 4040; FAX: +352 2468 4060; E-MAIL ADDRESS; [email protected]
Promega has developed a new luminescence-based assay to rapidly quantify the total intra- and extracellular glutathione (GSH)content of mammalian cells and tissues. This method is able to rapidly assess the basic level of this antioxidant, nonprotein thiol indifferent eukaryotic cell lines. In addition, the antioxidant or prooxidant activity of diverse xenobiotics affecting cellular glutathione levels can be investigated with little effort. Hence we describe experiments with the human leukemic cell line U937, where theintracellular glutathione biosynthesis/level is induced or reduced after treatments with N-acetylcysteine (NAC) or buthionine-(S,R)-sulfoxime (BSO), respectively.
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Figure 1. Schematic of the physiological roles of glutathione (GSH).
www.promega.com 7 CELL NOTES ISSUE 22 2008
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Sigma), Orion Microplate Luminometer (Berthold), and 96-well luminometer plates (Greiner Bio-one).
Cell Culture
U937 cells (human histiocytic lymphoma, DSMZ) werecultured in RPMI 1640 medium (Bio-Whittaker) containing10% [v/v] fetal calf serum (FCS; Cambrex) and 1% [v/v]antibiotic-antimycotic (Bio-Whittaker) at 37 °C, 5% CO2.
Generating Glutathione Standard Curve
To calibrate the test system, a glutathione standard curve wasgenerated according to the GSH-Glo™ Glutathione AssayTechnical Bulletin #TB369. Serial dilutions of a 5 mM stocksolution of glutathione were performed in PBS (range was0–5 µM), then 10 µl of these solutions was put in triplicatewells of 96-well luminometer plates; subsequently 100 µl ofGSH-Glo™ Reagent was added to each well, and the platewas incubated for 30 minutes at 22 °C in the dark.Afterwards, 100 µl of reconstituted Luciferin DetectionReagent was added to each well, and luminescence wasmeasured after 15 minutes of incubation. Luminescencemeasurements were performed using a 1-second integrationperiod.
For our setup, a plot of GSH concentration versusluminescence, measured as relative light units (RLU; Figure 2), was linear between 0.5 and 5 µM, as expected(correlation coefficient >0.99).
Determining Intracellular Glutathione Content of U937 Cells
U937 cells were used as a model system for mammalian cellsin suspension. Cells were harvested by centrifugation,washed, resolubilized and diluted in PBS; then 1,000–2,000cells in 50 µl were added to triplicate wells. The 2X GSH-Glo™ Reagent (50 µl) was added to each well, the plate wasmixed on an orbital shaker for 1 minute and incubated for30 minutes at 22 °C in the dark. Following incubation, 100 µlof prepared Luciferin Detection Reagent was added to eachwell, samples were mixed briefly on an orbital shaker, andluminescence was measured after 15 minutes of incubation.Luminescence measurements were performed using a1-second integration period.
For our setup, the linear range of the function of theintracellular GSH concentration and the correspondingluminescence units (RLUs) (Figure 3) was linear between2,000–8,000 cells (correlation coefficient >0.99).
Modulation of Cellular Glutathione Level
To reduce biosynthesis of GSH, U937 cells were treated with1 mM of buthionine-(S,R)-sulfoxime (BSO) for 24 hours; toinduce biosynthesis of the thiol, cells were exposed to 10 mMof N-acetylcysteine (NAC) for 48 hours. To quantify theglutathione content, 4,000 cells were added to each well, andanalyses were performed as described in the previous section.
We found that the intracellular gluthatione level of U937 cellswas strongly reduced after BSO treatment, whereas it clearlyincreased after incubation with NAC (Figure 4).
DETERMINING GSH CONTENT IN MAMMALIAN CELLS
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Figure 2. Function of GSH concentration and luminescence Y-axis:luminescence, X-axis: glutathione concentration. Relative S.D. <10%.
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Figure 3. Function of intracellular GSH concentration of U937 cells andluminescence. Y-axis: luminescence, X-axis: number of U937 cells per wellquantified by their intracellular glutathione content. Relative S.D. <12%.
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Summary
We confirmed that the GSH-Glo™ Assay produced linearresults in the GSH concentration range (0.5–5 µM) indicatedby Promega. We also determined the optimal range of U937cell numbers for a linear correlation between intracellular GSHconcentration and luminescence. Finally, we showed theapplicability of the assay system by comparing theluminescence of nontreated cells with the luminescence ofcells with an increased (NAC-treated) or reduced (BSO-treated) intracellular GSH content.
The GSH-Glo™ Assay system is an easy-to-use assay toquantify glutathione in our model. The assay could be usedas a screening system to assess effects of differentcompounds on ROS linked with GSH.
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Figure 4. Modulation of intracellular GSH concentration of U937 cells by BSO orNAC. Y-axis: luminescence, X-axis: samples (control: nontreated control, BSO:buthionine sulfoxime-treated cells, NAC: N-acetylcysteine-treated cells).
References
1. Davenport, D.M. and Wargovich M.J. (2005). Food Chem.Toxicol., 43, 1753–62.
2. Dröge, W. (2002) Physiol. Rev. 82, 47–95.
3. Kim, H.J. et al. (2006) Oncogene 25, 2785–94.
Protocols
GSH-Glo™ Glutathione Assay Technical Bulletin #TB369(www.promega.com/tbs/tb369/tb369.html)
Ordering Information
Product Size Cat.#
GSH-Glo™ Glutathione Assay 10 ml V6911
50 ml V6912
GSH-Glo is a trademark of Promega Corporation.
Products may be covered by pending or issued patents or may have certain limitations. Pleasevisit our Web site for more information.
www.promega.com 9 CELL NOTES ISSUE 22 2008
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ERRATUMThe article “Screen for Kinase Modulators in a High-Throughput Format with Promega Kinase Reagents” (pages21–24 in Cell Notes Issue 20) contained an error in thelegend for Figure 4. The Z´-factor values in the experimentdescribed were determined for the Kinase-Glo® Max Assay,not the Kinase-Glo® Plus Assay. The figure with correctedlegend is presented here.
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1 17 33 49 65 81 97 113
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Determining Z´-factor for Kinase-Glo® Max Assay run in a 384-well plate.Panel A. The assay was performed as described in Technical Bulletin #TB372with 0.2 units/well PKA and 10 µM ATP for five minutes at room temperature(solid symbols) or without PKA (open symbols). Panel B. The assay wasperformed using 0.2 units/well PKA and 100 µM ATP for 30 minutes at roomtemperature (solid symbols) or without PKA (open symbols). Assays wereperformed in 384-well plates in a final volume of 20 µl. Solid lines indicate themean, and the dotted lines indicate ± 3 S.D. Z´-factor values were 0.8 for both10 µM and 100 µM ATP.
Introduction
The advantages of bioluminescence are most commonlyharnessed for assay design in two ways. First, gene reporterassays monitor changes in the concentration of theluciferase enzyme for studies of gene regulation. Second,ATP assays correlate changes in ATP concentration withchanges in the light output of an ATP-dependent luciferasereaction for measuring biomass and ATPase activities (1). Ina recent trend, a third approach brings the exquisitesensitivity and selectivity of bioluminescence into the sphereof traditional enzymology. Here we describe thisbioluminescent technology, which offers a new assay choicefor a wide variety of enzymes that include proteases,metabolic enzymes and β-galactosidase.
Enzymology relies heavily on the detection methods used tomeasure enzyme-catalyzed reaction products. Massspectrometry, absorbance, radioactivity, fluorescence,chemiluminescence and bioluminescence are some of themost commonly used methods. Each method hasadvantages but differs substantially in terms of sensitivity,ease of use, cost and susceptibility to interference.Nevertheless, within its validated space, each methodeffectively correlates signal (e.g., peak area, counts, opticalsignal) with the enzyme activity being monitored. In mostapplications bioluminescence ranks very high in terms ofsensitivity and ease of use (Figure 1).
In bioluminescence, a luciferase catalyzes the oxidation of aluminescent substrate known as a luciferin. Firefly luciferase
reactions are ATP-dependent (2). Assays that use luciferaseare configured by coupling a variable parameter of interest toa limiting component of the luciferase reaction (1). In theapproach described here, luciferin is the limiting component.The assays are based on proluciferins, which do not reactwith luciferase until they are converted to a luciferin by theenzyme of interest and then detected in a second reactionwith luciferase (Figure 2). The intensity of light correlateswith the amount of luciferin produced and therefore with theactivity of the enzyme in the first reaction.
LUMINOGENIC ENZYME SUBSTRATES: THE BASIS FOR A NEWPARADIGM IN ASSAY DESIGNBY PONCHO L. MEISENHEIMER1, MARTHA A. O’BRIEN2, AND JAMES J. CALI2, 1PROMEGA BIOSCIENCES, INC., 2PROMEGA CORPORATION
Luciferin derivatives as luminogenic substrates provide the basis for a new paradigm in enzyme assay design that brings theadvantages of bioluminescence—superior sensitivity, resistance to interference, and ease of use—to the enzymology researcher.Here we highlight our wide selection of bioluminescent enzyme substrates for proteases, metabolic enzymes and β-galactosidase.
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bioluminescence
radiometry
Figure 1. In most applications bioluminescence ranks very high in terms ofsensitivity and ease of use.
7686
MA
LightN
S S
N O
O
R1
R2 HO
N
S S
N O
O
R2
H2N
N
S S
N O
O
H
Enzyme LDR
Proluciferin
Luciferin, Luciferin ester orAminoluciferin
I
II
III
Figure 2. Luciferin derivatives as luminogenic substrates provide a new paradigm in enzyme assay design. In these bioluminescent enzyme assays, luciferin is limiting.The assays are based on luciferin derivatives (I) that are substrates for enzymes that convert them to luciferins (II and III). Luciferin is then detected in a secondreaction in the presence of luciferin detection reagent (LDR), which contains Ultra-Glo™ luciferase. The amount of light correlates with the amount of luciferinproduced by the activity of the enzyme in the first reaction.
www.promega.com 11 CELL NOTES ISSUE 22 2008
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Luciferin derivatives as luminogenic substrates provide thebasis for a new paradigm in enzyme assay design, and certainadvantages are intrinsic to the approach. Background signalsare absent from most biological systems since luciferase andluciferin are found only in bioluminescent organisms.Furthermore, bioluminescent background is minimal comparedto fluorescent assay chemistries. With bioluminescence, thephoton emitter is brought to its excited state by the luciferaseenzyme instead of a lamp. With fluorescence-based assays,background caused by the fluorescence excitation lamprequires mitigation and includes: light scatter, fluorescentemission from unreacted probe, and fluorescence fromcofactors or test compounds. By eliminating the lamp,bioluminescent assays eliminate several sources of backgroundand interference unique to fluorescence-based assays.
We have synthesized an extensive collection of luminogenicsubstrates with selectivity for a wide range of enzymes(Tables 1 and 2). They are used in assays for proteases (3),β-galactosidase (4), metabolic enzymes (5) includingcytochrome P450 (CYP), monoamine oxidase (MAO), N-acetyl
transferase 2 (NAT2) and glutathione by way of glutathione-S-transferase activity (GST). The substrates are used incombination with a luciferin detection reagent (LDR) thatcontains a unique purified recombinant form of fireflyluciferase that was stabilized by directed evolution (6). TheLDR formulations provide glow-style luminescent signals withtypical half-lives of about five hours. Both cell-based andnoncell-based assays are performed in multiwell, add-onlyformats that are easily configured on automated platforms.
Protease Assays
Protease enzymes are prime candidates for adaptingluminogenic substrates because peptides can be attached viaa peptide bond to aminoluciferin to provide inactive luciferaseprosubstrates. Protease selectivity is dictated by peptidesequence (Table 1). Cleavage of the peptide by a proteasefirst yields free aminoluciferin, which then is the substrate forluciferase (7).
For proteases, a one-step coupled-enzyme assay wherehydrolysis occurs simultaneously with the luciferase
Table 1. Bioluminescent Enzyme Assays and Screening Systems.
Substrate Associated Assay and/or Screening System Assay or System Features
7733
MACOOH
N
S
S
NN
H
Z–DEVD
Caspase-Glo® 3/7 AssayCell-based or in vitro assay for caspases 3 and 7 (8),widely used for monitoring apoptosis.
7735
MACOOH
N
S
S
NN
H
Z–LETD
Caspase-Glo® 8 AssayCell-based or in vitro assay for caspase 8, includesproteasome inhibitor to improve specificity.
7734
MACOOH
N
S
S
NN
H
Z–LEHD
Caspase-Glo® 9 AssayCell-based or in vitro assay for caspase 9, includesproteasome inhibitor to improve specificity.
7738
MACOOH
N
S
S
NN
H
Z–VDVAD
Caspase-Glo® 2 Assay
In vitro assay for caspase-2, can be adapted to cell-based assay by including proteasome inhibitor andcaspase-3/7 inhibitor.
7736
MACOOH
N
S
S
NN
H
Z–VEID
Caspase-Glo® 6 AssayIn vitro assay for caspase-6, ideal substrate for high-throughput inhibitor screening.
7731
MACOOH
N
S
S
NN
H
GP
DPPIV-Glo™ Protease AssayIn vitro assay for DPPIV, ideal substrate for high-throughput inhibitor screening.
7737
MACOOH
N
S
S
NN
H
Suc–LLVY Calpain-Glo™ Protease Assay
Proteasome-Glo™ Chymotrypsin-Like Assay;Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay
In vitro assay for calpain, speed is advantageous formonitoring Ca2+-activated calpain, which is rapidlyautoinactivated.
Cell-based or in vitro assay for the chymotrypsin-likeproteasome activity, eliminates the need for lysatepreparation.
7729
MACOOH
N
S
S
NN
H
Z–LRR
Proteasome-Glo™ Trypsin-Like Assay;Proteasome-Glo™ Trypsin-Like Cell-Based Assay
Cell-based or in vitro assay for the trypsin-like protea-some activity, eliminates need for lysate preparation,inhibitor included to improve specificity.
7730
MACOOH
N
S
S
NN
H
Z–nLPnLDProteasome-Glo™ Caspase-Like Assay;Proteasome-Glo™ Caspase-Like Cell-Based Assay
Cell-based or in vitro assay for the caspase-like protea-some activity, eliminates the need for lysate prepara-tion.
7732
MACOOH
N
S
S
NN
H
AAF
CytoTox-Glo™, MultiTox-Glo Assays
Cell-based assay for marker protease of cell death,can be performed in multiplex with fluorogenic live-cellprotease marker assay (12).
Luciferin Detection Reagent (LDR) is provided with assay kits.
CELL NOTES ISSUE 22 2008 12 www.promega.com
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consumption of aminoluciferin improves sensitivity andefficiency (8). Adding the protease produces a luminescentsignal that increases until a steady state between the proteaseand luciferase is achieved, at which point the signal typicallyremains constant for several hours. Light output at steadystate reflects the amount of protease activity present, and testcompounds that increase or decrease the signal are scored asactivators or inhibitors, respectively. This coupled-enzymeformat has been applied to several proteases (Table 1), and inall cases, the assays are more sensitive than comparablefluorescent assays and more convenient for automated high-throughput applications (9).
Metabolic Enzyme Assays
For metabolic enzymes including CYPs, MAO, NAT2 and GST,a two-step endpoint design is typically used for
bioluminescent assays. These enzymes feature prominently inthe transformation, detoxification and elimination of numerousendogenous and xenobiotic chemicals and in adverse drug-drug interactions. In drug discovery it is important todetermine if and to what extent candidate compounds inhibitor induce these activities. In the two-step assays enzyme-dependent luciferin accumulation occurs in a first step,followed by addition of an LDR that stops the enzyme activityand detects luciferin as a luminogenic signal (5,10,11).
The CYP and MAO substrates are a series of D-luciferinderivatives that provide selectivity for one or more enzymes byvarying the nature of a cleavable group attached by an etherlinkage to D-luciferin (Figure 2 and Table 1). The reaction productis D-luciferin or a D-luciferin ester that is processed to D-luciferinby an esterase included in the LDR. The CYP and MAO assays
Table 1. Bioluminescent Enzyme Assays and Screening Systems (continued).
Substrate Associated Assay and/or Screening System Assay or System Features
7687
MAO
N
S S
N OH
O
S
NO2
O O
GSH-Glo™ Assay
Luminescent assay to detect and quantify reducedglutathione (GSH) in cells or in various biologicalsamples (13).
7688
MA
O
N
S S
N O
O
H2N MAO-Glo™ Assay
Luminescent assay to measure monoamine oxidase (MAO) activity from recombinant and nativesources (11). Useful for determining drug candidateinhibition of MAO.
7690
MA
O
N
S S
N OH
O
F
F
F
FFP450-Glo™ CYP3A4 Assay
Cell-based or in vitro assay for 3A isozymes of thecytochrome P450 family. Primarily used in cell-basedassays to measure induction of CYP3A by drug candi-date test compounds.
7691
MAO
N
S S
N OH
O
NN
Ph
P450-Glo™ CYP3A4 DMSO-Tolerant Assay; P450-Glo™ CYP3A4 Screening System, DMSO-Tolerant(Luciferin-PPXE)
Cell-based or in vitro assay for CYP3A. Assay is toler-ant to DMSO. Primarily used for screening drug can-didate inhibition of CYP3A.
7692
MA
O
N
S S
N OH
O
PhP450-Glo™ CYP3A4, P450-Glo™ CYP3A7 Assays(Luciferin-BE)
First-generation in vitro assay for CYP3A4 and 3A7isozymes of the P450 family.
7693
MAN
S S
N OH
O
P450-Glo™ CYP2C9 Assay; P450-Glo™ CYP2C9Screening System (Luciferin-H)
Cell-based or in vitro assay for CYP2C9. Extremelyselective for CYP2C9. Assay measures either induc-tion or inhibition of CYP2C9 by drug candidate testcompounds.
7694
MA
O
N
S S
N OH
O
ClP450-Glo™ CYP1A1, P450-Glo™ CYP1B1 Assays(Luciferin-CEE)
Cell-based or in vitro assay for CYP1A1. Measuresinduction or inhibition of CYP1A1 by test compounds.In vitro assay for CYP1B1.
7695
MA
O
N
S S
N OH
OP450-Glo™ CYP1A2, P450-Glo™ CYP2C8 Assays;P450-Glo™ CYP1A2 Screening System (Luciferin-ME)
In vitro assay for CYP1A2 or CYP2C8. Also, used incell-based assays to measure induction of CYP4A bydrug candidate test compounds.
7696
MA
O
N
S S
N OOH
O
P450-Glo™ CYP2D6 Assay; P450-Glo™ CYP2D6Screening System (Luciferin-ME-EGE)
In vitro assay for CYP2D6. Assay can be used tomeasure inhibition of CYP2D6 by drug candidate testcompounds.
7697
MAN
S S
N OOH
O
P450-Glo™ CYP2C19 Assay; P450-Glo™ CYP2C19Screening System (Luciferin-H-EGE)
In vitro assay for CYP2C19. Assay used to measure inhibition of CYP2C19 by drug candidatetest compounds.
7704
MAO
N
S S
N OH
O
O
OH
OH OH
HOBeta-Glo® Assay System (6-o-β-galactopyranosyl-luciferin)
Homogeneous method for quantitating β-galactosidase expression in cells.
Luciferin Detection Reagent (LDR) is provided with assay kits.
www.promega.com 13 CELL NOTES ISSUE 22 2008
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are exquisitely sensitive, and they detect enzyme inhibitors ascompounds that decrease light output with IC50 values thatcorrelate well with those from conventional assays (5). The CYPassays also are used in a cell-based approach for measuring CYPinduction by chemicals that increase light output.
Treatments that cause a decrease in cellular reducedglutathione levels (GSH) typically have a toxic effect. This canindicate the presence of a reactive electrophile, an inhibitor ofGSH synthesis, or conditions of oxidative stress. A luminescentassay for measuring GSH concentration in cell lysates isconfigured around a luciferin derivative that is a GST substratelinked to luciferin by a sulfonate bond. GST transfers itssubstrate to GSH with the displacement of luciferin. Lysatesare prepared from cultured cells by adding a reagent thatincludes GST and the luciferin derivative. The amount of GST-dependent luciferin produced is dependent on the GSHconcentration, so the amount of light produced when LDR isadded is proportional to the GSH concentration. Treatmentsthat reduce light output correlate with decreased GSH levels.
The biotransformation of many xenobiotic chemicals includesacetylation by N-acetyltransferase (NAT) enzymes. The
luminogenic substrate for NAT2 is an aminoluciferin derivativethat reacts weakly with luciferase to give a dim luminescentsignal (14). Acetylation of this substrate by NAT2 converts thisderivative to a strong substrate that produces bright signalswith luciferase in a two-step assay format. Of the two N-acetyltransferases expressed in humans, NAT1 and NAT2, theluminogenic substrate is highly selective for NAT2.Compounds that inhibit the light output of this assay may benoncompetitive inhibitors of NAT2 or NAT2 substrates actingas competitive inhibitors.
β-Galactosidase Assay
β-galactosidase is widely used as a reporter for gene expressionstudies and in complementation assays that monitor thefunctional assembly of two β-galactosidase fragments. 6-o-β-galactopyranosyl-luciferin (Table 1) is hydrolyzed by the β-galactosidase enzyme to produce D-luciferin that is detected ina one-step approach similar to a one-step protease assay.Although many assay systems for this enzyme exist incolorimetric and fluorescent formats, the luminogenic approachis the most sensitive and convenient (4).
Table 2. Luciferin Derivatives Available as Stand-Alone Substrates.
Structure Substrate Name Description
7689
MA
NH
N
S S
N OH
O
H2NLuciferin-NAT2 Luminogenic Substrate
N-acetyltransferase 2 (NAT2) converts this weakluciferase substrate to a strong luciferase substrate(14). Useful for determining drug candidate inhibitionof NAT2.
7698
MA
O
N
S S
N OH
O
O
N
S S
N OH
O
Luciferin-3A7 Luminogenic Substrate
The 3A7 isozyme of the CYP450 family selectivelyconverts this compound to luciferin. Useful for deter-mining drug candidate inhibition of CYP3A7.
7699
MA
OH
O
S
NN
O
Luciferin-4A11 Luminogenic Substrate
The 4A11 isozyme of the CYP450 family selectivelyconverts this compound to a known luciferase sub-strate. Useful for determining drug candidate inhibi-tion of CYP4A11 and CYP4A induction in a cell-basedassay.
7700
MA
O
N
S S
N OH
O
s Luciferin-4F2/3 Luminogenic Substrate
The 4F2 and 4F3 isozymes of the CYP450 familyselectively convert this compound to luciferin. Usefulfor measuring CYP4F2 and CYP4F3 activity and inhi-bition.
7701
MA
O
N
S S
N OH
O
Cl Luciferin-4F12 Luminogenic Substrate
The 4F12 isozyme of the CYP450 family selectivelyconverts this compound to luciferin. Useful for meas-uring CYP4F12 activity and inhibition.
7702
MA
O
N
S S
N OOH
O
O
O
Luciferin-2J2/4F12 Luminogenic Substrate
The 2J2 and 4F12 isozymes of the CYP450 familyconvert this compound to a luciferin. Useful for measuring CYP2J2 and CYP4F12 activity and inhibi-tion.
7703
MA
O
N
S S
N O
O
Luciferin-MultiCYP Generic Luminogenic Substrate
Several cytochrome P450 isozymes convert this com-pound to luciferin. Potentially useful for evaluatingliver samples for metabolic potential or P450 struc-ture or activity studies.
NNoottee:: These substrates are supplied as stand-alone lyophilized powders. They must be used in conjunction with Luciferase Detection Reagent in order to perform aluminescent assay. Please see the Product Information sheet supplied with each substrate for solubility information. See www.promega.com/enotes/applications.htmfor additional information on using these substrates in bioluminescent enzyme assays.
CELL NOTES ISSUE 22 2008 14 www.promega.com
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Summary
Luciferins, the light-generating substrates of firefly luciferaseare versatile scaffolds for the synthesis of luminogenic enzymesubstrates, as the present inventory of bioluminescentenzyme assays indicates. The assays bring benefits toenzymology that were previously limited mainly to assays thatuse luciferase as a genetic reporter or ATP sensor (2). Thefeatures include:
• Exquisite sensitivity.• Homogeneous formats.• Scalability to 96-, 384-, 1536- and 3456-well formats.• Simple luminescent readout.• No fluorescent interference.
References
1. Fan, F. and Wood, K.V. (2007) Assay Drug Dev. Tech. 5, 127–36.
2. Branchini, B.R. et al. (1998) Biochemistry 37, 15311–9.
3. Monsees, T. et al. (1994) Anal. Biochem. 221, 329–34.
4. Hannah, R. et al. (2003) Cell Notes, 6, 16–8.
5. Cali, J.J. et al. (2008) Exp. Op. Drug Metab. Toxicol. 44, 103–20.
6. Hall, M.P. et al. (1998) In: Bioluminescence andChemiluminescence: Perspectives for the 21st Century. Rode, A.et al. (eds.), John Wiley & Sons, Chichester 392–5.
7. White, E.H. et al. (1966) J. Am. Chem. Soc. 88, 2015 –9.
8. O’Brien, M.A. et al. (2005) J. Biomol. Screen. 10, 137–48.
9. O’Brien, M.A. (2006) In: Handbook of Assay Development inDrug Discovery. Minor, L.K. (ed.) CRC Press, Taylor and FrancisGroup (Boca Raton, FL) 125–39.
10. Cali, J.J. (2006) Exp. Op. Drug Metab. Toxicol. 2, 629–45.
11. Valley, M.P. et al. (2006) Anal. Bioch. 359, 238–46.
12. Niles, A.L. et al. (2007) Anal. Bioch. 366, 197–206.
13. Zhou, W. et al. (2006) Chem. Comm. 44, 4620–2.
14. Woodroofe, C.C. et al. (2008) Biochem. (in press).
Ordering Information
Product Size Cat.#
Caspase-Glo® 3/7 Assay 100 ml G8092
Caspase-Glo® 8 Assay 100 ml G8292
Caspase-Glo® 9 Assay 100 ml G8212
Caspase-Glo® 6 Assay 50 ml G0971
Caspase-Glo® 2 Assay 50 ml G0941
DPPIV-Glo™ Protease Assay 50 ml G8351
Calpain-Glo™ Protease Assay 50 ml G8502
Proteasome-Glo™ 3-SubstrateCell-Based Assay System 10 ml G1180
Proteasome-Glo™ Chymotrypsin-LikeCell-Based Assay 10 ml G8660For Laboratory Use. Additional sizes available.
Product Size Cat.#
Proteasome-Glo™ Trypsin-LikeCell-Based Assay 10 ml G8760
Proteasome-Glo™ Caspase-LikeCell-Based Assay 10 ml G8860
MultiTox-Glo Multiplex Cytotoxicity Assay 10 ml G9270
CytoTox-Glo™ Cytotoxicity Assay 10 ml G9290For Laboratory Use. Additional sizes available
Product Size Cat.#
P450-Glo™ CYP1A1 Assay 10 ml V8751
P450-Glo™ CYP1B1 Assay 10 ml V8761
P450-Glo™ CYP1A2 Assay 10 ml V8771
P450-Glo™ CYP2C8 Assay 10 ml V8781
P450-Glo™ CYP2C9 Assay 10 ml V8791
P450-Glo™ CYP3A4 Assay 10 ml V8801
P450-Glo™ CYP3A7 Assay 10 ml V8811
P450-Glo™ CYP2C19 Assay 10 ml V8881
P450-Glo™ CYP2D6 Assay 10 ml V8891
P450-Glo™ CYP3A4 Assay(Luciferin-PPXE) DMSO-Tolerant Assay 10 ml V8911
P450-Glo™ CYP3A4 Assay (Luciferin-PFBE)Cell-Based/Biochemical Assay 10 ml V8901Additional sizes available.
Product Size Cat.#
P450-Glo™ CYP1A2 Screening System 1,000 assays V9770
P450-Glo™ CYP2C9 Screening System 1,000 assays V9790
P450-Glo™ CYP3A4 Screening System 1,000 assays V9800
P450-Glo™ CYP2C19 Screening System 1,000 assays V9880
P450-Glo™ CYP2D6 Screening System 1,000 assays V9890
P450-Glo™ CYP3A4 Screening System (Luciferin-PPXE) DMSO-Tolerant 1,000 assays V9910Additional sizes available.
Product Size Cat.#
Luciferin-NAT2 Luminogenic Substrate 3mg P1721
Luciferin-3A7 Luminogenic Substrate 3mg P1741
Luciferin-4A11 Luminogenic Substrate 3mg P1621
Luciferin-4F2/3 Luminogenic Substrate 3mg P1651
Luciferin-4F12 Luminogenic Substrate 3mg P1661
Luciferin-2J2/4F12 Luminogenic Substrate 3mg P1671
Luciferin-MultiCYP Generic Luminogenic Substrate 3mg P1731
Product Size Cat.#
Luciferin Detection Reagent 10 ml V8920
Luciferase Detection Reagent with Esterase 10 ml V8930Additional sizes available.
Products may be covered by pending or issued patents or may have certain limitations. Pleasevisit our Web site for more information.
Beta-Glo and Caspase-Glo are registered trademarks of Promega Corporation. Calpain-Glo,CytoTox-Glo, DPPIV-Glo, GSH-Glo, MAO-Glo, P450-Glo, Proteasome-Glo and Ultra-Glo aretrademarks of Promega Corporation.
www.promega.com 15 CELL NOTES ISSUE 22 2008
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Introduction
The ability to repeat previously published observations inscientific research is important for confirming new discoveries.If previous data are not repeatable, it is doubtful that anyonewill continue to pursue that work. Much of biomedicalresearch—medicine, genetics, drug discovery, vaccinedevelopment, reconstructive medicine, basic science, HIVtesting/treatment, and cell biology—is done with cultured cellsobtained from major repositories such as American TypeCulture Collection (ATCC) or from fellow researchers. Anestimated 15–20% of the time, cells used in experiments havebeen misidentified or cross-contaminated with another cellline (1–3). ATCC, along with the Coriell Institute for MedicalResearch, European Collection of Cell Cultures (ECACC),Deutsche Sammlung von Mikrorganismen and Zellkulturen(DSMZ), and the Japanese Collection of Research Resources,all have received cell line submissions that, uponauthentication, were determined to have been misidentified bythe depositor (4,5). This poses a huge threat to the quality ofpublications and legitimacy of research findings producedfrom any of these cell cultures. For this reason, thesedepositories now authenticate cell line submissions andmonitor cross-contamination.
Historical Perspective on the Problem
The first human cancer cell line, HeLa, was developed in1952, and for the next 15 years, many more human celllines from different tissues continued to be developed (6). In1968, researchers discovered that many cultured cellsexhibited characteristics that did not match thecharacteristics of the original source. This was the firstevidence that particular methods of culturing cells couldproduce unpredictable changes to the cells ormisidentification. While the development of better steriletechniques helped decrease cross-contamination, few testswere available to determine which cells were alreadyaffected. Since then, however, standardized methods havebeen developed that are both quick and inexpensive toperform. While these improvements should have eliminatedmuch cellular cross-contamination, it remains a prominentissue. ATCC and other similar repositories now monitorcross-contamination and authenticate all cell lines that theydistribute, but most individual investigators do not adhere tothe same meticulous authentication processes. In fact, a2004 survey of approximately 500 biologists by GertrudeBuehring of the University of California, Berkeley, and hercolleagues showed that less than half of all researchers
regularly verify the identities of their cell lines using anystandard techniques, such as DNA fingerprinting by shorttandem repeat (STR) analysis (6). Without requiring that allcell lines be authenticated, misidentification will remain asignificant problem.
Authentication Saves Time and Money
Aside from the issue of inconsistent or questionable data,cross-contamination also wastes time and money. Forinstance, Mordechai Liscovitch, a cancer researcher in Israel,says that he and researchers in his lab spent three yearsworking on two breast cancer cell lines (MCF-7 and MCF-7/AdrR, now renamed NCI/ADR-RES) that they believed wererelated, only to discover later that the cell lines were actuallyunrelated. Although some researchers suspected themisidentification of these cell lines as early as 1998, theactual identity of these two cell lines was not confirmed untilrecently (4,7). NCI/ADR-RES is not derived from the breastcancer cell line MCF-7 as originally thought but rather fromthe ovarian cancer cell line OVCAR-8 (7). All three of thesecell lines are part of the NCI-60 panel of cell lines that areroutinely used in drug-screening applications (4).
The Liscovitch lab cancelled the publication of a manuscriptthat contained erroneous conclusions based on the mistakencell line identity; however, an unknown number of studieshave been published that contain conclusions based onmisidentified cell lines. Charles Patrick Reynolds of theUniversity of Southern California and the Children’s HospitalLos Angeles’ Institute for Pediatric Clinical Researchestablishes new pediatric cancer cell lines and tests potentialcancer drugs on existing lines. According to his estimations,
DOING GOOD SCIENCE: AUTHENTICATING CELL LINE IDENTITYJILL HARLEY DUNHAM1, PAM GUTHMILLER2, 1PH.D. CANDIDATE DEPARTMENT OF PHARMACOLOGY EMORY UNIVERSITY, ATLANTA, GA. USA,2PROMEGA CORPORATION
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up to 35–40% of previously published cell biology paperswould need to be retracted due to invalid data. Theseestimates have caused Roland Nardone at The CatholicUniversity of America to call for authentication both as acondition for receipt of grant funds from major agencies suchas the National Institutes of Health and American CancerSociety as well as for publication of cell culture-basedresearch in leading journals. He also is requesting educationfor technicians and scientists regarding prevention anddetection of cross-contamination, including relevantprofessional societies’ endorsements of these proposedpolicies and sponsored conferences and workshops tofacilitate adoption of the standards. Dr. Nardone’s belief thatthese changes are vital to scientific research has even led himto cocreate the Cell Line Authentication Global AwarenessMonth [May 2008], started by “an ad hoc group of scientistsbecause of the chaos and waste caused by rampantmisidentification and cross-contamination of cell lines.’
The problems of cross-contamination have been recognizedalready by a few organizations, such as the ATCC (7) and FDA,which requires that in-process materials, such as cell lines, thatare used to produce pharmaceuticals be tested for identity andpurity (8). Similarly, Nature recently mandated STR fingerprintdata for papers reporting new human embryonic stem cell lines
but not other lines (4). Although journals and funding agenciesrecognize the problems of cell line misidentification, they areuncertain how best to address them. When would researchersbe asked to confirm cell line identity, before or after peerreview? Where would the resources come from to confirmauthors’ assertions of cell line identity?
In addition to wasted time and money, inconsistent ornonreproducible findings, and retraction of publications, apotential for health consequences also exists in misidentified celllines. Drugs, vaccines and other biomedicines all are createdbased on findings in the lab, often times via cell culture.Products made using misleading or false data can cause majordelays in the production and availability of treatments for avariety of diseases. The longer it takes for a treatment to bedeveloped, the more people these diseases affect.
STR-Based Methods Provide Easy, Quick Cell ID
All of these issues can be prevented with inexpensive andnow standard procedures used to authenticate cell lines, butthe procedures must be performed. Roderick MacLeod andhis colleagues at DSMZ, German Collection of Microorganismsand Cell Cultures, have found that about 90% of scientistsignore or refuse a cell bank’s request to send in new lines,preventing the establishment of cell line DNA fingerprints forfuture attempts at verification (5,7). Researchers need to beeducated early on in their research to learn how to detectintra- and inter-species cross-contamination, as well as why itis so important to do so.
Many methods, such as isoenzyme analysis, karyotyping,human lymphocyte antigen (HLA) typing, and characterizationof amplified fragment length polymorphisms (AFLP), havebeen used to identify cross-contamination in cell culture.
7426
MA
100 200 300 400
Fluorescein:
JOE:
TMR:
CXR:Size (bp)
TH01 D21S11
D5S818
vWA TPOXA
A = Amelogenin
D13S317 D7S820 D16S539 CSF1PO
7265
TA
Figure 1. Panel A. Allele ranges for the Cell ID™ System. STR fragmentsamplified by the Cell ID™ System are labeled with different dyes and areseparated by capillary electrophoresis based on size. A size standard isincluded in each sample to determine the size of the fragments. JOE-labeledloci are shown in gray. Fluorescein-labeled loci are shown in white. The CXR-labeled Internal Lane Standard 600 fragments are represented by black bars.Panel B. K562 cell line DNA profile. After amplification with the Cell ID™System and subsequent detection on a capillary electrophoresis instrument,the resulting sample data are displayed as a series of dye-labeled allele peaks.
Figure 2. Determination of cell line contamination using the Cell ID™ System.Panel A. HEK293 cell line STR profile. Panel B. STR profile of HEK293 Cell Linewith 29% HeLa cell line contamination. Panel C. HeLa cell line STR profile.DNA was extracted from 104 cells using the Maxwell® 16 Cell LEV DNAPurification Kit then amplified with the Cell ID™ System. Amplified productswere detected on a capillary electrophoresis instrument. For simplicity, on theJOE-labeled allele profiles are shown.
7814
TA
AA..
BB..
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However, a superior method is STR-profiling, well establishedin the field of DNA-based forensic identification. At the ATCC,STR analysis is performed using multiplex PCR (PromegaPowerPlex® 1.2 System) to simultaneously amplify eight STRloci plus Amelogenin for gender determination (10,11). Aunique pattern of repeating DNA is generated for each humancell line analyzed, so the DNA profile of each new stock isverified by comparing to the baseline profile. STR profilingalready has prevented the further distribution of six differentcell lines at ATCC after it revealed the presence of Ychromosome-specific amplification products in cell lines thatare derived from females. The research community hopesthat STR profiling will provide a global reference technique todetect and eliminate cell line contamination.
Cell ID™ System Allows STR-Based Authentication of Your Cell Line
Promega is a leader in providing STR-profiling systems forforensic and paternity applications, and the PowerPlex® 1.2STR analysis system has become the “gold standard” tool usedby cell culture facilities to authenticate cell lines. To support theneed for simpler methods to authenticate cell lines, the CellID™ System(a,b) was developed and offers an improved systemthat includes the reagents required to successfully and simplyidentify and authenticate human cell lines as well as detectintra-species cell line cross-contamination.
The Cell ID™ System uses STR analysis of specific, highlypolymorphic loci in the human genome through simultaneousamplification and three-color detection of ten loci (nine STRloci and Amelogenin for gender identification; includingD21S11, THO1, TPOX, vWA, Amelogenin, CSF1PO, D16S539,D7S820, D13S317 and D5S818). These loci collectivelyprovide a genetic profile with a random match probability of
1 in 2.92 × 109. The system includes a hot-start Taq DNApolymerase for convenient room temperature reactionassembly. Following amplification, samples are analyzed bycapillary electrophoresis in a single injection in conjunctionwith provided standards to assist in determining allele sizesfor the different loci (Figure 2). The genetic profile isdetermined using allele-calling software.
Most research scientists have access to institute core facilitiesand service companies that have the instrumentation (capillaryelectrophoresis), software and experience to perform cell lineprofiling, even if they do not have the same capability withintheir own labs. General recommendations in the literaturesuggest that investigators authenticate an early passage (firstweek of culture) of their cells to establish the identity of the cellline. Cells should be authenticated again before freezing, onceevery two months that the culture is actively growing, and beforepublication. If a lab is using more than one cell line, all linesshould be tested initially to rule out cross-contamination (8).
Summary
Because of the importance of cell culture to biomedicalresearch and technology, proper cell line authentication is ineveryone’s best interest. However, cross-contaminationcontinues to be a problem. With the increasing number of newcell lines and the high rate of cell culture use in labsworldwide, significant gaps have been created in basicprinciples of quality control (i.e., cell line authentication). Fromresearch articles published with misidentified cell lines andresultant questionable results, to stem cell lines and other linesdestined for clinical uses, cross-contamination affects sciencein all realms—from lab bench to clinic. Without significantchange to the handling and treatment of cell cultures, it willbecome only a larger and more serious issue.
References
1. Drexler, H.G., Dirks, W.G. and MacLeod, R.A.F. (1999) Leukemia1133, 1601–7.
2. Drexler, H. G. et al. (2001) Blood 98, 3495–6.
3. Cabrera, C.M. et al. (2006) Cytotechnology 51, 45–50.
4. Chatterjee, R. (2007) Science 315, 928–31.
5. MacLeod, R.A.F. et al. (1999) Int. J. Cancer 83, 555–63.
6. Buehring, G.C., Eby, E.A. and Eby, M.J. (2004) In Vitro Cell. Dev.Biol. 40, 211–5.
7. Liscovitch, M. and Ravid, D. (2007) Cancer Lett. 245, 350–2.
8. FDA. General Requirements for Laboratory Controls. 21 CFR211.160 and 21 CFR 610.18.
9. ATCC Connection Newsletter (2007) 27, 2–4.
10. ATCC Connection Newsletter (2000) 21, 1–2.
11. Masters, J.R. et al. (2001) Proc. Natl. Acad .Sci. USA 98,8012–7.
Protocol
Cell ID™ System Technical Manual #TM074(www.promega.com/tbs/tm074/tm074.html)
Ordering Information
Product Size Cat.#
Cell ID™ System 50 reactions G9500For Research Use Only. Not for use in diagnostic procedures.
(a)This product is sold under licensing arrangements with the USB Corporation for Forensic andGenetic Identity Applications Fields specifically excluding tissue typing related to transplantationor other medical procedures. Further licensing information may be obtained by contacting theUSB Corporation, 26111 Miles Road, Cleveland, OH 44128. (b)This product is sold under licensing arrangements with Stratagene for Forensic and Genetic IdentityApplications Fields specifically excluding tissue typing related to transplantation or other medicalprocedures. Further licensing information may be obtained by contacting the Business DevelopmentDepartment, Stratagene California, 11011 North Torrey Pines Road, La Jolla, CA 92037.
Products may be covered by pending or issued patents or may have certain limitations. Please visitour Web site for more information.
Maxwell and PowerPlex are registered trademarks of Promega Corporation. Cell ID is a trademark ofPromega Corporation.
Introduction
To stimulate cells to proliferate, differentiate or undergoapoptosis, extracellular signals need be relayed to thenucleus where they alter the pattern of gene expression. Oneof the main mechanisms for relaying such signals involves agroup of proteins called the mitogen-activated protein kinases(MAPK). These enzymes are proline-directed serine/threoninekinases that are activated by dual phosphorylation ofthreonine and tyrosine residues in response to diverseextracellular stimuli. Small molecules that inhibit MAPKs areimportant for understanding cell signaling.
MAPK Pathways
Six MAP kinase signaling pathways have been identified inmammals (ERK1/2, ERK3/4, ERK5, ERK7/8, JNK1/2/3 andp38/ERK6; 1). Despite being complex in nature, thesepathways can be divided into three main steps involving thesequential activation of MAPK kinase kinase (MAPKKK),MAPK kinase (MAPKK) and MAPK (Figure 1). At least fourmembers of the MAPK family have been identified[extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun-amino-terminal kinase (JNK), p38 and ERK5; 2].
ERK1 and ERK2 (ERK1/2) are isoforms of the “classical”MAPK and are activated by MAP/ERK kinase 1 (MEK1) andMEK2 (MEK1/2), which are members of the MAPKK family.ERK1/2 and MEK1/2 are acutely stimulated by growth anddifferentiation factors in pathways mediated by receptortyrosine kinases, heterotrimeric G protein-coupled receptorsor cytokine receptors, primarily through p21Ras-coupledmechanisms. MEK or ERK activity is upregulated in responseto cell stimulation by phosphorylation at residues locatedwithin the activation lip of each kinase. In the case of MEK,phosphorylation at two serine residues by upstream proteinkinases, Raf-1, c-Mos or MAPK kinase kinase, leads tomaximal enzyme activation. Subsequently, MEK1/2 activatesERK1/2 by phosphorylating regulatory threonine and tyrosineresidues. Thus, MEKs fall within a relatively rare class ofprotein kinases with dual specificity toward Ser/Thr and Tyrresidues on exogenous substrates (3). Activated ERK1/2phosphorylates many substrates including transcriptionfactors, such as Elk1 and c-Myc, and protein kinases, suchas ribosomal S6 kinase (RSK). Subsequently, immediateearly genes, such as c-Fos, are induced. ERK1/2 is thereforean important contributor to cell proliferation (4).
Tools for Studying MAP Kinases
Biochemical reagents that are highly specific and highlyselective for individual enzymes in the pathway are essential
to understanding the MAPK cascade. Such reagents haveenabled scientists to distinguish among the main MAP kinasepathways and to understand the role of various MAP kinasesin cellular regulation by extracellular stimuli. The availability ofSB203580, a specific inhibitor of p38, has made it possibleto identify effects mediated by or requiring active p38 proteinkinase (5). Likewise, another inhibitor, PD098059, whichinhibits MEK1/2 activation, has been used to study ERK-mediated responses (6).
Promega also offers the inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4 bis[2-aminophenylthio] butadiene; Figure 1), achemically synthesized organic, white, solid compound thatis soluble in dimethyl sulfoxide (DMSO). U0126 was initiallyrecognized as an inhibitor of cellular AP-1 transactivation incell-based reporter assays (7). It specifically inhibits MEK1activity in vitro, as well as activation of ERK1/2 by MEK1/2
STUDYING MAP KINASE SIGNALING WITH A SMALL-MOLECULEINHIBITOR, U0126SIMON T.M. ALLARD, PROMEGA CORPORATION
CELL NOTES ISSUE 22 2008 18 www.promega.com
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Figure 1. Activation of three different MAPK signaling cascades by differentextracellular stimuli. The ERK, JNK and p38 cascades all contain the sameseries of three kinases. A MEK Kinase (MEKK) phosphorylates and activates aMAP Kinase Kinase (MEK), then MEK phosphorylates and activates a MAPKinase (MAPK).
www.promega.com 19 CELL NOTES ISSUE 22 2008
MEK INHIBITOR U0126
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in vivo. The compound appears to inhibit MEK1 directly incontrast to the widely used inhibitor PD098059, whichinhibits MEK1/2 activation indirectly by binding to the inactiveenzyme thereby preventing its activation by Raf kinase.
Because of this, PD098059 may not be useful for inhibitingendogenously active MEK1/2. However, U0126 is capable ofdirectly inhibiting activated MEK1 and preventingendogenously active MEK1/2 from phosphorylating andactivating ERK1/2 (Figure 1). Therefore, the compoundU0126 acts one step further downstream of PD098059 andblocks MEK1/2 activity (8). The inhibitor U0126 thus servesas a useful tool to investigate the effect of various signals onthe activation of MAPK in cells and further delineation of theMAPK pathways in cellular regulation.
7801
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Figure 2. The small molecule MEK1/2 inhibitor U0126.
References
1. Hawkins, T.A. et al. (2008) BMC Dev. Biol. 8, 42.
2. Nishimoto, S. and Nishida, E. (2006) EMBO Rep. 7, 782–6.
3. Ahn, N.G. et al. (1999) Promega Notes 71, 4–7.
4. Lewis, T.S., Shapiro, P.S. and Ahn, N.G. (1998) Adv. Cancer Res.74, 49–139.
5. Cuenda, A. et al. (1995) FEBS Lett. 364, 229.
6. Dudley, D.T. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7686.
7. Favata, M. et al. (1998) J. Biol. Chem. 273, 18623.
8. Goueli, S.A. et al. (1998) Promega Notes 69, 6–9.
Ordering Information
Product Size Cat.#
MEK Inhibitor U0126 5 mg (5 × 1 mg) V1121
Peer-Reviewed Articles Citing Use of U0126 Inhibitor
Joo, J.H. and Jetten, A.M. (2008) NF-κB-dependenttranscriptional activation in lung carcinoma cells by farnesolinvolves p65/RelA(Ser276) phosphorylation via the MEK-MSK1signaling pathway. J. Biol. Chem. 283, 16391–9.
This article showed that expression of several immuneresponse genes could be induced in lung adenocarcinomaH460 cells by treatment with farnesol and that this inductionproceeds through an NF-κB pathway. To determine whichMAPKs were involved in the activation of these genes, theauthors used the MEK Inhibitor U0126 and showed that itinhibited the expression of several of the immune responsegenes as well as specifically inhibited the phosphorylation ofp65 on Ser276.
Maekawa, M. et al. (2007) Requirement for ERK MAP kinasein mouse preimplantation development. Development 134,2751–9.
The authors of this study investigated the role of ERK1/2kinase signaling in the preimplantation developmental eventsthat precede compaction and blastocyst formation (2-cell to 8-cell stages). The MEK Inhibitor U0126 was added to 2-cellstage mouse embryos, and embryos were observed to arrestat the 4-cell stage. The arrest was reversible, but experimentsindicated that ERK1/2 signaling was required for M-phaseprogression early cell cycles during mammalian development.
Case, M. et al. (2008) Mutation of genes affecting the RASpathway is common in childhood acute lymphoblasticleukemia. Cancer Res. 68, 6803–9.
The authors of this study investigated the relationship ofsomatic mutations that deregulate the RAS-RAF-MEK-ERKpathway and Acute Lymphoblastic Leukemia (ALL) and itsprogression to relapse in some patients. They show that suchmutations are common in ALL and in its recurrence.Furthermore, lymphoblasts from patients with mutations thatwere associated with upregulation of ERK showed increasedcytotoxicity over wild-type controls when treated with MEKInhibitor U0126, suggesting that specific ERK inhibitors mayeventually yield useful therapeutics.
Are you interested in seeing how other researchers have usedU0126 to investigate the role of MAPK signaling in theirexperimental systems?
Visit the Promega Citations Database, an online resourcehighlighting peer-reviewed articles that cite the use ofPromega products in all areas of life science research.
www.promega.com/citations/
CELL NOTES ISSUE 22 2008 20 www.promega.com
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