USING PROTEASE BIOMARKERS TO MEASURE VIABILITY AND

ANDREW NILES1, MICHAEL SCURRIA2, LAURENT BERNAD2, BRIAN MCNAMARA1, KAY RASHKA1, DEBORAH LANGE1, PAM GUTHMILLER1 AND TERRY RISS11PROMEGA CORPORATION, 2PROMEGA BIOSCIENCES, INC. Introduction Although several biomarkers have been described and 2,500 Fluorescent LDH employed for measuring viability and cytotoxicity in CytoTox-Glo™ Assay 2,000 culture, none is without technical fault. We recently identified two new biomarker profiles for viability and 1,500 r2 = 0.9996 cytotoxicity that circumvent many historical assay chemistry

CYTOTOXICITY limitations and greatly facilitate multiplex measurements 1,000 (1). These markers(a) have proteolytic activities associated

Signal-to-Noise Ratio 500 with cell death or viability and can be measured in r2 = 0.9987 multiplex using either a single luminogenic substrate with 0 sequential reads, a luminogenic substrate in combination 0 2,500 5,000 7,500 10,000 with a fluorogenic substrate, or with two different Dead Cells/Well 6973MA fluorogenic substrates (2–4). Regardless of format, the assays for these markers generate large dynamic ranges Figure 1. The CytoTox-Glo™ Assay detects small changes in viability with excellent linearity, providing unprecedented sensitivity because of its high sensitivity. This graph shows the superior signal-to- noise ratios of the CytoTox-Glo™ Assay compared to a fluorescent in high-density formats (Figure 1). LDH assay.

Dead-Cell Qualify for a FREE SAMPLE of Protease the MultiTox-Fluor Assay at:

www.promega.com/ Dead-Cell MultiTox-Glo Assay Protease multitoxfluor_cn019/ MultiTox-Fluor Assay LDH

Dead-Cell Protease LDH LDH

LDH

CytoTox-Glo™ Assay CytoTox-Fluor™ Assay or 2° NECROSIS

The CytoTox-Glo™ Cytotoxicity Assay is the most sensitive method AAF + for measuring cytotoxicity. The assay quantifies the extracellular Dead-Cell Ultra-Glo™ Protease S N COOH + ATP Glo activity of an intracellular protease (dead-cell protease) when the rLuciferase protease is released from membrane-compromised cells. N S luciferin AAF S N COOH

N S 5350MI CytoTox-Glo™ Assay Chemistry.

CELL NOTES ISSUE 19 2007 16 www.promega.com Multiplex Cytotoxicity Assays

MultiTox Assay Overview CHALLENGE: Find enzymatic biomarkers for viability and cytotoxicity that are not modulated by any stimulus other than cytotoxicity and can discriminate betwen viable and nonviable Live Cell Live Cell populations in a proportional manner. Active Live-Cell Active Live-Cell EXPERIMENT: We focused our peptide-based, protease activity Protease Active Dead-Cell Protease Active Dead-Cell screen on proteases with constitutive, homeostatic function. Protease Protease Substrates for known inducible proteases (e.g., caspases, Nucleus Nucleus granzymes, calpain, tryptase, etc.) were avoided until counterscreening. Each test fluorogenic substrate was exposed to a limiting dilution series of viable and nonviable 1) Treat cells with thee cells to determine if it could select between viable and potential cytotoxictotoxicc agent.agent. dead cells.

RESULTS: Two proteolytic profiles emerged from cell-based Live Cell Active Dead-Cell screening (Table 1): Active Live-Cell Protease • An activity restricted to viable cells using Gly-Phe-AFC. This Protease Active Dead-Cell Dead Cell activity (likely from distinct aminopeptidases) was Protease significantly reduced in equivalent numbers of nonviable Nucleus Protease cells due to enzymatic lability. Inactive Live-Cell

Protease • An activity restricted to nonviable cells was measured using Ala-Ala-Phe-AMC. This activity is also likely due to 2) Add substrates.tes. housekeeping aminopeptidases and is also detected with GF– LIVE-CELLELL SUBSTRATE:STRATE: AAF– DEAD-CELLDEAD- CELL SUBSTRATE: cell-permeant flfluorogenic cell-imcell-impepermeant substrate substrates using other fluorescent (rhodamine 110) or substrate fof r thehli live-cell ll fof r thehd dead-cell d ll protease luminogenic detection groups. protease (Gly-Phe-AFCoumarin) (Ala-Ala-Phe-rhodamine 110 or Ala-Ala-Phe-Aminoluciferin) CONCLUSION: Both activities were dependent on membrane integrity and were independent of other external stimuli such Active Dead-Cell as proteasome inhibition or caspase induction (prior to Active Live-Cell GF+ Protease secondary necrosis caused by these treatments). Protease AAF+ Live-cell protease substrate can cross Nucleus the cell membrane; Dead Cell dead cell protease substrate cannot. Live Cell Active Dead-Cell Inactive Live-Cell Table 1. Signals Obtained with Various Substrates During the Protease Protease Primary Screen. Substrate Viability Cytotoxicity 3) Measure fluorescence/luminescence. Z-XXX-AMC None* None Z-XXXX-AMC None None Z-XXXXX-AMC None None 100% Live Cells 50%0%% Live / 5050%% Dead CelCellsls 100%100% DeadDead CCellsells GF-AMC +++ None GF-AFC +++++ None bis-GF-R110 None None Live Cell Live Cell Live Cell DeadDead CCellell Dead Cell Dead Cell AAF-AMC None ++ Nucleus NNucleucleusu s Nucleus bis-AAF-R110 None +++++ CYTOTOXICITY AAF-aminoluciferin None +++++ X-AMC + None

2 2 2 XX-AMC + None *None 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. 1 1 1 6581MB 0 0 0 (Left) The MultiTox Assays use differential protease biomarker detection to Live Dead Live Dead Live Dead quantify both the number of live and dead cells in a single well. By measuring Cells Cells Cells Cells Cells Cells both viability parameters in the same well, many sources of variability are Fluorescence at 520nm (rhodamine 110-red) or 505nm (coumarin-blue) controlled, resulting in more consistent data.

www.promega.com 17 CELL NOTES ISSUE 19 2007 Multiplex Cytotoxicity Assays

Figure 2. Potency profiles for cell viability and cytotoxicity can be Figure 3. These protease-based assays yield appropriate IC50 values for easily obtained from a variety of cell types. a variety of test compounds.

A. SW620 (colorectal adenocarcinoma) A. Viability EC = 8.4 nM Viability EC = 3.1 µM 50 50 Cytotoxicity EC = 7.5 nM Cytotoxicity EC = 3.3 µM 50 60,000 50 25,000 15,000 6,000 50,000 20,000 40,000 10,000 15,000 4,000 30,000 10,000 20,000 5,000 2,000 10,000 5,000 Fluorescence (RFU) Luminescence (RLU) 0 0 0 0 –7 –6 –5 –4 (RFU) Fluorescence Viability –9 –8 –7 –6 Cytotoxicity Fluorescence (RLU)

CYTOTOXICITY log [ionomycin] M 10 log10 [camptothecin] M

B. SH-SY5Y (neuroblastoma) B. Viability EC50 = 1.8 nM Viability EC50 = 3.68 µM Cytotoxicity EC = 1.2 nM Cytotoxicity EC = 4.69 µM 50 50,000 50 12,500 15,000 6,000 40,000 10,000 10,000 30,000 7,500 scence (RFU) 4,000 20,000 5,000 5,000 2,000 10,000 2,500 Fluorescence (RFU) Luminescence (RFU) 0 0 0 0 –7 –6 –5 –4 Fluore Viability –10 –9 –8 –7 –6 Cytotoxicity Fluorescence (RFU) Fluorescence Cytotoxicity log [ionomycin] M 10 log10 [paclitaxel] M

C. DU-145 (prostate carcinoma) C. Viability EC50 = 10.6 nM Viability EC = 1.7 µM 50 Cytotoxicity EC50 = N.D. 20,000 Cytotoxicity EC50 = 2.0 µM 20,000 10,000 1,500 15,000 15,000 7,500 1,200 escence (RLU) 10,000

10,000 5,000 escence (RFU) 900 5,000 5,000 2,500

0 600

0 0 Lumin Viability –9 –8 –7 –6 Viability Fluor Viability –7 –6 –5 –4 Cytotoxicity Luminescence (RLU) Cytotoxicity Fluorescence (RFU) log10 [colchicine] M log10 [ionomycin] M 6929MA 6931MA

CHALLENGE: Find ubiquitous and highly conserved markers of CHALLENGE: Find markers that are stable enough in culture to cytotoxicity and viability. be measured in reasonable time frames to accurately determine viability. EXPERIMENT: We tested the presence and abundance of our novel proteolytic biomarkers using cell lines representing the EXPERIMENT: We tested the effects of several standard diversity within the National Cancer Institute-60 (NCI-60) cytotoxicity-inducing compounds in a broad titration series collection (Figure 2; human colon, neuron and prostate during 24- and 48-hour exposure periods. shown). This panel included cells isolated from blood, brain, RESULTS: We observed the following: colon, breast, skin, ovary, prostate, lung, and kidney tissues. • The “live-cell” marker has no half-life constraints and RESULTS: We observed the following: typically delivers two asymptotes for accurate • All cell lines tested contained “live-cell” and “dead-cell” viability/potency determinations. proteases at a level useful for viability or cytotoxicity assays (less than 200 viable or nonviable cell sensitivity in limiting • The ability to detect the “dead-cell” protease marker during dilution). longer incubations is greatly dependent upon the kinetics of cell death. • The relative abundance of the two protease-marker activities varied slightly among cell lines with a generally CONCLUSION: Underestimation of cytotoxicity due to positive correlation, depending on cellular volume. biomarker degradation must be considered, but kinetics and mechanism of cell death dictate the usefulness of this CONCLUSION: The proteolytic biomarkers used in these biomarker during extended incubations. Reduction of initial viability and cytotoxicity assays have been detected human compound concentration or incubation periods often resolves and nonhuman mammalian cell lines (data not shown). this issue (Figure 3).

CELL NOTES ISSUE 19 2007 18 www.promega.com Multiplex Cytotoxicity Assays

Figure 4. The flexible formats of these protease-based assays allow CHALLENGE: Develop assays that perform well in a variety researchers to overcome most compound interference. of formats that limit compound interferences.

A. EXPERIMENT: We tested the performance of our protease GF-AFC channel Z´ = 0.812 biomarkers in three formats: multiplexed fluorescent 100,000 R110 channel Z´ = 0.935 (MultiTox-Fluor Assay(a)), multiplexed luminescent/fluorescent R110 (MultiTox-Glo Assay(a,b,c)), and luminescent cytotoxicity interference AFC (CytoTox-Glo™ Assay(a,b,c)). The assays were compared using 10,000 interference a mock library containing compounds known to cause color quenching and wavelength-specific fluorescent and No AFC luminescent interference. In addition different cell numbers 1,000 interference

Fluorescence (RFU) were added per well, with different viabilities. Color quenching RESULTS: We showed that each of the three formats 100 0 16 32 48 64 80 96 performed equally for cytotoxicity but differently with respect Well Number to compound interference (Figure 4). B. Fluorescence channel Z´ = 0.72 • All three formats demonstrated commensurate changes in 100,000 Luminescence channel Z´ = 0.87 viability and cytotoxicity versus control when cytotoxicity No R110 AFC interference was present. 10,000 interference • All three chemistries “flagged” assay wells with more or less 1,000 cells than control wells.

Color • Fluorescence interference occurred in either AFC or R110 100 quenching Luciferase channels, but not in both, thus “flagging” the data point. intereference Luminescence was unaffected in both. Fluorescence or Luminescence 10 0 16 32 48 64 80 96 • Luminescence interference occurred with a luciferase Well Number inhibitor, but not with fluorescent compounds. C. Fluorescence was unaffected by the luciferase inhibitor. 1,000,000 Viability luminescence Z´ = 0.79 Cytotoxicity luminescence Z´ = 0.85 • Color quenching agents tested affected both fluorescent 100,000 and luminescent formats negatively, but dual measures No R110 No AFC allowed “flagging” of affected data points. interference 10,000 interference CONCLUSION: All three assays have relative merits with regard to their ability to “flag” potential assay interference (Table 2). 1,000 Color Luminescence (RLU) Luciferase quenching intereference 100 0 16 32 48 64 80 96 Well Number 6930MA

Table 2. Multiplex Viability and Cytotoxicity Screening Options. CYTOTOXICITY MultiTox-Fluor Multiplex Cytotoxicity Assay MultiTox-Glo Multiplex Cytotoxicity Assay CytoTox-Glo™ Cytotoxicity Assay

Single-addition reagent Two-step reagent addition Two-step reagent addition

Nonlytic Nonlytic Lytic second step

96-, 384-, 1536-well plate formats 96-, 384-, 1536-well plate formats 96-, 384-, 1536-well plate formats

Avoids known luminescence inhibitors Good “hedge” for unknown libraries Avoids fluorescence interference

Enhanced sensitivity from luminescence Enhanced sensitivity from luminescence Can be multiplexed with other luminescence assays format format

Flags problem data points Flags problem data points Flags problem data points

www.promega.com 19 CELL NOTES ISSUE 19 2007 Multiplex Cytotoxicity Assays

Summary References Several sensitive and robust protease biomaker assay 1. Niles, A. et al. (2007) Anal. Biochem. 366, 197–206. chemistry options allow you to choose the best assay for your 2. Niles, A. et al. (2006) Cell Notes 15, 11–5. chemical library, treatment regimens, and desired endpoint. 3. Niles, A. et al. (2006) Cell Notes 16, 12–5. The markers are constitutive and conserved and may be used to distinguish between changes in viability and cytotoxicity. 4. Niles, A. et al. (2007) Cell Notes 18, 15–20. Ultimately, flexibility within detection platforms allows researchers to balance multiplex features with throughput and Ordering Information improve data quality. Product Size Cat.# MultiTox-Fluor Multiplex Cytotoxicity Assay 10 ml G9200

CYTOTOXICITY Qualify for a FREE SAMPLE of the CytoTox-Fluor™ Cytotoxicity Assay 10 ml G9260 MultiTox-Fluor Assay at: CytoTox-Glo™ Cytotoxicity Assay 10 ml G9290 MultiTox-Glo Multiplex Cytotoxicity Assay 10 ml G9270 www.promega.com/multitoxfluor_cn019/ CellTiter-Fluor™ Cell Viability Assay 10 ml G6080 For laboratory use. For invitro use only. Additional Sizes Available. (a)Patent Pending.

(b)U.S. Pat. Nos. 6,602,677 and 7,241,584, Australian Pat. No. 754312 and other patents and See Us at These Meetings... patents pending. (c)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. American Society for Cell Biology 5,583,024, 5,674,713 and 5,700,673. Washington, D.C. USA CellTiter-Fluor, CytoTox-Fluor, CytoTox-Glo and Ultra-Glo are trademarks of Promega Corporation. December 1–5, 2007 www.ascb.org

Biochemistry and Molecular Biology 2007 Yokohama, Japan December 11–14, 2007 www.aeplan.co.jp/bmb2007/

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