USING PROTEASE BIOMARKERS TO MEASURE VIABILITY AND CYTOTOXICITY

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1 UIG PROTEAE BIOMARKER TO MEAURE VIABILITY AD ADREW ILE, MICHAEL CURRIA, LAURET BERAD, BRIA MCAMARA, KAY RAHKA, DEBORAH LAGE, PAM GUTHMILLER AD TERRY RI PROMEGA CORPORATIO, PROMEGA BIOCIECE, I Introduction Although several biomarkers have been described and employed for measuring viability and cytotoxicity in cell culture, none is without technical fault. We recently identified two new biomarker profiles for viability and cytotoxicity that circumvent many historical assay chemistry limitations and greatly facilitate multiplex measurements (). These markers (a) have proteolytic activities associated with cell death or viability and can be measured in multiplex using either a single luminogenic substrate with sequential reads, a luminogenic substrate in combination with a fluorogenic substrate, or with two different fluorogenic substrates ( 4). Regardless of format, the assays for these markers generate large dynamic ranges with excellent linearity, providing unprecedented sensitivity in high-density formats (Figure ). ignal-to-oise Ratio,5,,5, 5 Fluorescent Assay CytoTox-Glo Assay r =.99 r =.9987,5 5, 7,5, /Well Figure. The CytoTox-Glo Assay detects small changes in viability because of its high sensitivity. This graph shows the superior signal-tonoise ratios of the CytoTox-Glo Assay compared to a fluorescent assay. 6973MA Qualify for a FREE AMPLE of the MultiTox-Fluor Assay at: multitoxfluor_cn9/ MultiTox-Glo Assay MultiTox-Fluor Assay CytoTox-Glo Assay CytoTox-Fluor Assay ECROI or ECROI The CytoTox-Glo Cytotoxicity Assay is the most sensitive method for measuring cytotoxicity. The assay quantifies the extracellular activity of an intracellular protease (dead-cell protease) when the protease is released from membrane-compromised cells. AAF + COOH + ATP Ultra-Glo rluciferase Glo AAF COOH luciferin 535MI CytoTox-Glo Assay Chemistry. CELL OTE IUE 9 7

2 MultiTox Assay Overview ) Treat cells with the potential cytotoxic c agent. CHALLEGE: Find enzymatic biomarkers for viability and cytotoxicity that are not modulated by any stimulus other than cytotoxicity and can discriminate betwen viable and nonviable populations in a proportional manner. EXPERIMET: We focused our peptide-based, protease activity screen on proteases with constitutive, homeostatic function. ubstrates for known inducible proteases (e.g., caspases, granzymes, calpain, tryptase, etc.) were avoided until counterscreening. Each test fluorogenic substrate was exposed to a limiting dilution series of viable and nonviable cells to determine if it could select between viable and dead cells. ) Add substrates. tes. GF LIVE-CELL ELL UBTRATE: TRATE: cell-permeant fluorogenic substrate for the live-cell ll protease (Gly-Phe-AFCoumarin) -cell protease substrate can cross the cell membrane; dead cell protease substrate cannot. 3) Measure fluorescence/luminescence. AAF DEAD-CELL UBTRATE: cell-impermeant substrate for the dead-cell d protease (Ala-Ala-Phe-rhodamine or Ala-Ala-Phe-Aminoluciferin) Inactive -Cell % s 5% / 5% ls % GF+ Inactive -Cell AAF+ Fluorescence at 5nm (rhodamine -red) or 55nm (coumarin-blue) 658MB REULT: Two proteolytic profiles emerged from cell-based screening (Table ): An activity restricted to viable cells using Gly-Phe-AF This activity (likely from distinct aminopeptidases) was significantly reduced in equivalent numbers of nonviable cells due to enzymatic lability. An activity restricted to nonviable cells was measured using Ala-Ala-Phe-AM This activity is also likely due to housekeeping aminopeptidases and is also detected with substrates using other fluorescent (rhodamine ) or luminogenic detection groups. COCLUIO: Both activities were dependent on membrane integrity and were independent of other external stimuli such as proteasome inhibition or caspase induction (prior to secondary necrosis caused by these treatments). Table. ignals Obtained with Various ubstrates During the Primary creen. ubstrate Viability Cytotoxicity Z-XXX-AMC one* one Z-XXXX-AMC one one Z-XXXXX-AMC one one GF-AMC +++ one GF-AFC one bis-gf-r one one AAF-AMC one ++ bis-aaf-r one AAF-aminoluciferin one X-AMC + one XX-AMC + one *one denotes no statistically significant activity over control population. + to denote the relative strength of the response. Xs denote the number of amino acids in the substrate. (Left) The MultiTox Assays use differential protease biomarker detection to quantify both the number of live and dead cells in a single well. By measuring both viability parameters in the same well, many sources of variability are controlled, resulting in more consistent data. 7 CELL OTE IUE 9 7

3 Figure. Potency profiles for cell viability and cytotoxicity can be easily obtained from a variety of cell types. Figure 3. These protease-based assays yield appropriate IC 5 values for a variety of test compounds. Cytotoxicity Fluorescence (RLU) 6, 5, 4, 3,,, W6 (colorectal adenocarcinoma) Viability EC 5 = 3. µm Cytotoxicity EC 5 = 3.3 µm log 5,, 5,, 5, Luminescence (RLU) 5,, 5, Viability EC 5 = 8.4 nm Cytotoxicity EC 5 = 7.5 nm log [camptothecin] M 6, 4,, Cytotoxicity 5, 4, 3,,, H-Y5Y (neuroblastoma) Viability EC 5 = 3.68 µm Cytotoxicity EC 5 = 4.69 µm log,5, 7,5 5,,5 Luminescence (RFU) 5,, 5, Viability EC 5 =.8 nm Cytotoxicity EC 5 =. nm log [paclitaxel] M 6, 4,, Cytotoxicity, 5,, 5, DU-45 (prostate carcinoma) Viability EC 5 =.7 µm Cytotoxicity EC 5 =. µm log, 7,5 5,,5 693MA Viability Luminescence (RLU), 5,, 5, Viability EC 5 =.6 nm Cytotoxicity EC 5 =.D log [colchicine] M,5, 9 6 Cytotoxicity Luminescence (RLU) 699MA CHALLEGE: Find ubiquitous and highly conserved markers of cytotoxicity and viability. EXPERIMET: We tested the presence and abundance of our novel proteolytic biomarkers using cell lines representing the diversity within the ational Cancer Institute-6 (CI-6) collection (Figure ; human colon, neuron and prostate shown). This panel included cells isolated from blood, brain, colon, breast, skin, ovary, prostate, lung, and kidney tissues. REULT: We observed the following: All cell lines tested contained live-cell and dead-cell proteases at a level useful for viability or cytotoxicity assays (less than viable or nonviable cell sensitivity in limiting dilution). The relative abundance of the two protease-marker activities varied slightly among cell lines with a generally positive correlation, depending on cellular volume. COCLUIO: The proteolytic biomarkers used in these viability and cytotoxicity assays have been detected human and nonhuman mammalian cell lines (data not shown). CHALLEGE: Find markers that are stable enough in culture to be measured in reasonable time frames to accurately determine viability. EXPERIMET: We tested the effects of several standard cytotoxicity-inducing compounds in a broad titration series during 4- and 48-hour exposure periods. REULT: We observed the following: The live-cell marker has no half-life constraints and typically delivers two asymptotes for accurate viability/potency determinations. The ability to detect the dead-cell protease marker during longer incubations is greatly dependent upon the kinetics of cell death. COCLUIO: Underestimation of cytotoxicity due to biomarker degradation must be considered, but kinetics and mechanism of cell death dictate the usefulness of this biomarker during extended incubations. Reduction of initial compound concentration or incubation periods often resolves this issue (Figure 3). CELL OTE IUE

4 Figure 4. The flexible formats of these protease-based assays allow researchers to overcome most compound. CHALLEGE: Develop assays that perform well in a variety of formats that limit compound s. Fluorescence or Luminescence,,,,,,,, Luminescence (RLU),,, R o AFC o R GF-AFC channel R channel Well umber Well umber Well umber Z =.8 Z =.935 Fluorescence channel Luminescence channel Viability luminescence Cytotoxicity luminescence o R 8 Luciferase intereference 8 Z =.79 Z =.85 Luciferase intereference 8 AFC Z =.7 Z =.87 AFC o AFC 693MA EXPERIMET: We tested the performance of our protease biomarkers in three formats: multiplexed fluorescent (MultiTox-Fluor Assay (a) ), multiplexed luminescent/fluorescent (MultiTox - Glo Assay (a,b,c) ), and luminescent cytotoxicity (CytoTox-Glo Assay (a,b,c) ). The assays were compared using a mock library containing compounds known to cause color and wavelength-specific fluorescent and luminescent. In addition different cell numbers were added per well, with different viabilities. REULT: We showed that each of the three formats performed equally for cytotoxicity but differently with respect to compound (Figure 4). All three formats demonstrated commensurate changes in viability and cytotoxicity versus control when cytotoxicity was present. All three chemistries flagged assay wells with more or less cells than control wells. Fluorescence occurred in either AFC or R channels, but not in both, thus flagging the data point. Luminescence was unaffected in both. Luminescence occurred with a luciferase inhibitor, but not with fluorescent compounds. Fluorescence was unaffected by the luciferase inhibitor. agents tested affected both fluorescent and luminescent formats negatively, but dual measures allowed flagging of affected data points. COCLUIO: All three assays have relative merits with regard to their ability to flag potential assay (Table ). Table. Multiplex Viability and Cytotoxicity creening Options. MultiTox-Fluor Multiplex Cytotoxicity Assay MultiTox-Glo Multiplex Cytotoxicity Assay CytoTox-Glo Cytotoxicity Assay ingle-addition reagent Two-step reagent addition Two-step reagent addition onlytic onlytic Lytic second step -, 384-, 536-well plate formats -, 384-, 536-well plate formats -, 384-, 536-well plate formats Avoids known luminescence inhibitors Good hedge for unknown libraries Avoids fluorescence Can be multiplexed with other luminescence assays Enhanced sensitivity from luminescence format Enhanced sensitivity from luminescence format Flags problem data points Flags problem data points Flags problem data points 9 CELL OTE IUE 9 7

5 ummary everal sensitive and robust protease biomaker assay chemistry options allow you to choose the best assay for your chemical library, treatment regimens, and desired endpoint. The markers are constitutive and conserved and may be used to distinguish between changes in viability and cytotoxicity. Ultimately, flexibility within detection platforms allows researchers to balance multiplex features with throughput and improve data quality. Qualify for a FREE AMPLE of the MultiTox-Fluor Assay at: References. iles, et al. (7) Anal. Biochem. 366, iles, et al. (6) Cell otes 5, iles, et al. (6) Cell otes, iles, et al. (7) Cell otes 8, 5. Ordering Information Product ize Cat.# MultiTox-Fluor Multiplex Cytotoxicity Assay ml G9 CytoTox-Fluor Cytotoxicity Assay ml G CytoTox-Glo Cytotoxicity Assay ml G99 MultiTox-Glo Multiplex Cytotoxicity Assay ml G97 CellTiter-Fluor Cell Viability Assay ml G68 For laboratory use. For invitro use only. Additional izes Available. (a)patent Pending. ee Us at These Meetings... American ociety for Cell Biology Washington, D. UA December 5, 7 Biochemistry and Molecular Biology 7 Yokohama, Japan December 4, 7 (b)u.. Pat. os. 6,6,677 and 7,4,584, Australian Pat. o and other patents and patents pending. (c)the method of recombinant expression of Coleoptera luciferase is covered by U.. Pat. os. 5,583,4, 5,674,73 and 5,7,673. CellTiter-Fluor, CytoTox-Fluor, CytoTox-Glo and Ultra-Glo are trademarks of Promega Corporation. Promega Corporation 8 Woods Hollow Road Madison, WI UA Tel: Fax: Toll-Free: Toll-Free Fax: Internet: Promega Biosciences, Inc. A Division of Promega Corporation an Luis Obispo, California Australia, ydney Tel: 9565 Fax: Freecall: Freefax: aus_custserv@au.promega.com China, Beijing Tel: (86) Fax: (86) promega@promega.com.cn France, Lyon Tel: (33) Fax: (33) umero Vert: fr_custserv@fr.promega.com Germany/Austria, Mannheim Tel: (+49) () 6 85 Fax: (+49) () 6 85 Free Phone: Free Fax de_custserv@promega.com Italy, Milan Tel: (39) Fax: (39) umero Verde: it_custserv@it.promega.com Japan, Tokyo Tel: (8) Fax: (8) jptechserv@jp.promega.com Latin America Region, Brazil Tel/Fax: (55 3) carla.abdo@promega.com Belgium/Luxembourg/ The etherlands, Leiden Tel: (+3) () Fax: (+3) () Free Tel BE: Free Fax BE: 8 97 Free Tel L: 8 9 Free Fax L: bnl_custserv@nl.promega.com Pacific Asia Region, ingapore Tel: (65) Fax: (65) sg_custserv@promega.com pain, Madrid Tel: Fax: esp_custserv@promega.com witzerland, Wallisellen Tel: (4) Fax: (4) ch_custserv@promega.com United Kingdom, outhampton Tel: (44) Fax: (44) Free Phone: ukcustserve@promega.com CELL OTE IUE 9 7