A Multiplex Assay Approach to Screening Cytoactive ... - EMD Millipore

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In order to improve screening efficiency, we have developed ... level of the individual cell and the overall cell population. ... forms that then act to propagate the apoptotic cascade.7-9 ... and allowed to adhere for 8 hours prior to proceeding ..... Mid-Apoptosis. Dying/Dead Cells. % Caspase-3+/PI–. 0. 10. 20. 30. 0.01. 0.1. 1.
Application Note: A Multiplex Assay Approach to Screening Cytoactive Compounds

A Multiplex Assay Approach to Screening Cytoactive Compounds for Cell Cycle Disruption and Apoptotic Activity on the Guava EasyCyte™ System Amedeo Cappione, Lisa Diver, and Katherine Gillis, Guava Technologies, Inc., Hayward, CA 94545

ABSTR AC T

I NTRO DUC TI O N

Control of cell cycle progression and initiation of the apoptotic cascade are tightly regulated processes complicated by the myriad of signaling pathways contributing to their regulation. Functional assays capable of interrogating both processes in live cells are critical for target validation and compound optimization. In order to improve screening efficiency, we have developed the microcapillary-based Guava EasyCyte system, which, when used in conjunction with Guava’s suite of functional assays, provides an ideal platform for cellular analyses. For over 200 test compounds analyzed, the following parameters were measured: (1) Caspases 3/7, and 8 enzyme activity; (2) changes in mitochondrial membrane potential; (3) translocation of phosphatidylserine residues to the cell surface; (4) membrane permeability; and (5) cell cycle distribution. The simultaneous interrogation of Caspase activity and cell cycle progression, along with the information regarding the relative frequencies of treated cells in the early, mid- or late stages of apoptosis offers a powerful tool for both “hit” compound identification and endpoint analysis. In addition, this strategy provides a more detailed understanding of the mechanism of action both at the level of the individual cell and the overall cell population.

Within the anti-cancer drug discovery arena, the ability to monitor changes in mitotic state and apoptotic induction is critical for the design of potent, yet highly selective, therapeutic compounds. The importance of cell cycle control is underscored by the fact that unrestricted growth following loss of G1 checkpoint control is a hallmark of many solid tumors. Under appropriate cues or otherwise, once directed to divide, resting (G0/G1 phase) cells must first replicate their entire chromosomal complement (S phase). With the DNA content doubled, G2/M phase cells are now poised for mitotic partitioning into two equivalent daughter cells. The Guava Cell Cycle assay provides a simple screening method for changes in cell cycle kinetics and DNA content through staining fixed/permeabilized samples with propidium iodide (PI), a fluorescent DNA intercalating agent.1 Apoptosis, or programmed cell death, is characterized by a progressive series of morphological and biochemical changes ultimately resulting in disassembly of the cell proper. These changes include the externalization of phosphatidylserine (PS) residues to the cell surface, loss of mitochondrial membrane potential coincident with release of Cytochrome C, fragmentation of nuclear chromatin, and final loss of membrane integrity.2-6 Caspases (cysteinyldirected aspartate-specific proteases) are a family of proteolytic enzymes that mediate key aspects of this process from initiating early signaling events (Caspase-2, -8, -9) to directing cellular breakdown through cleavage of structural proteins (Caspase-3, -6, -7). In response to proper stimuli, site-directed cleavage results in conversion of functionally inert pro-Caspase molecules to their active forms that then act to propagate the apoptotic cascade.7-9 © 2006 Guava Technologies, Inc. All rights reserved. | 

Application Note: Multiplex Assay Approach to Screening Cytoactive Compounds

The ability to obtain multi-parameter information on compound behavior in the context of a living cell quickly and easily is invaluable to the drug discovery process. Furthermore, with the use of Guava’s ondemand cytometry platform, data is collected on individual cells in an automated fashion, enabling users to detect sub-population effects and analyze different cell types or cell lineages in the same assay. Multiple parameters of cell function can be interrogated in the same assay, allowing researchers to detect changes in cell viability, apoptosis, and other cellular processes that can reveal aspects of a compound’s specificity, toxicity and mechanism of action. In the example presented in this Application Note, 200 cytoactive compounds were evaluated on the Guava EasyCyte microplate screening system for their ability to disrupt cell cycle progression by assessing cellular DNA content. Each compound was further profiled through an additional series of assays to assess cell viability, distinct apoptosis indicators (Caspase-3/7, Caspase-8, Annexin-V), and changes in mitochondrial membrane potential. Taken together, this multiplex data set provides a more detailed view on the behavior of each of the test compounds with respect to both apoptotic induction and cell cycle progression.

M E TH O DS Cells: Two human cell lines, the non-adherent T-cell derived Jurkat (ATCC, #TIB-152) and the adherent cervical cancer derived HeLa (ATCC, #CCL-2), were used for these studies. Prior to compound treatment, each line was maintained in log phase using medium designed for optimal growth. For Jurkats, RPMI 1640 medium (Cellgro, #MT 10-04-CV) supplemented with 10% FBS (Cellgro, #MT 35-010-CV), 2 mM l-glutamine (Cellgro, #MT 25-005-CV), 4.5 g/L glucose (Sigma, #G8769), and 1 mM sodium pyruvate (Cellgro, #MT-25-000 CL) was used in culture while HeLa cells were sustained in Eagle’s MEM medium (Cellgro, #MT 10-01-CV) supplemented with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, and 0.1 mM non-essential amino acids (Cellgro, #MT 25-025-CV). Prior to compound treatment, cultures were synchronized for 24 hours in complete medium minus serum. For screening experiments, Jurkat were seeded at 50,000 cells/well (150 mL total volume) in 96-well round bottom polystyrene microplates (BD-Falcon, #353070) just prior to induction. Following induction, cells were harvested by centrifugation and assayed as described below.

HeLa cells were seeded at 30,000 cells/well in 96-well flat bottom polystyrene plates (BD-Falcon, #352072) and allowed to adhere for 8 hours prior to proceeding with drug treatment. Following treatment, HeLa cells were detached using trypsin. The cells were collected by centrifugation, and then transferred to hydrogelcoated polystyrene plates (Corning Costar, #3474) to minimize reattachment while performing assays. Compounds: A panel of 200 cytotoxic, immunosuppressive, anti-proliferative, and anti-inflammatory compounds was obtained from MicroSource Discovery Systems, Inc. For initial screening, a 10 mM stock solution of each compound was diluted in 2 steps to 10 µM in cell culture medium just prior to use. Negative controls (0.1% DMSO) and positive controls for apoptosis (1 mM staurosporine) were also included on each plate assayed. Treated plates were incubated for 4 and 20 hours (for Jurkat cells) or 16 and 36 hours (for HeLa cells). For hit confirmation and potency determination, selected compounds were titrated out to the indicated concentrations. Guava Caspase-3/7 and Caspase-8 Assays: Both assays were performed according to manufacturer’s instructions. Following harvest, cells were resuspended in 100 µL media. To this 10 µL of 10X FLICA reagent, Caspase-3/7 specific (#4500-0290) or Caspase-8 specific (#4500-0300), was added and cells were incubated at 37 °C for 1 hour in a 5% CO2 incubator. Plates were removed, washed, and then labeled with propidium iodide for an additional 5 minutes. Samples (2000 cells/well) were acquired on a Guava EasyCyte system using ExpressPlus software. Guava MitoPotential™ Assay: The Guava MitoPotential Assay (#4500-0250) was conducted according to manufacturer’s instructions. The JC-1 vial was resuspended at a 100X concentration according to manufacturer’s instructions. Following harvest, cells were resuspended in 200 µL media. A volume of 2 µL 100X JC-1 solution and 2 µL 7-AAD was then added to each well. Plates were incubated for 30 minutes at 37 °C in a 5% CO2 incubator. Samples (2000 cells/well) were acquired on a Guava EasyCyte system.

© 2006 Guava Technologies, Inc. All rights reserved. | 

Application Note: Multiplex Assay Approach to Screening Cytoactive Compounds

Guava Nexin™ Assay: The Guava Nexin assay (#45000161) was conducted following the manufacturer’s instructions. Following harvest, cells were resuspended in 50 µL media. A volume of 150 µL staining solution (135 µL 1X apoptosis buffer, 10 µL Annexin V-PE, and 5 µL of 7-AAD) was then added to each well. The cells were incubated in the dark at RT for 20 minutes. Samples (2000 cells/well) were acquired on the Guava EasyCyte system. Guava Cell Cycle: Following induction, samples were harvested, washed in 1X PBS, and then fixed overnight at –20 °C in 70% ethanol. Cells were washed, stained for DNA content with propidium iodide, and acquired (3000 cells/well) according to the Guava Cell Cycle Protocol. A compound was considered to have induced cell cycle arrest if the percentages of cells in one of the phases of the cell cycle were three standard deviations (SD) above the average of the negative controls.

R esu lts The Guava Cell Cycle Assay was used in combination with Guava’s suite of apoptosis assays to screen a panel of small molecules for their ability to arrest cell division. In addition, the 200 cytoactive compounds plus control

Control (DMSO)

Floxuidine

Guava Cell Cycle Assay Once released from serum starvation and allowed to grow in complete medium, both Jurkat and HeLa cells were found primarily in the G0/G1 and G2/M phases. Treatment with drugs that block cell division led to changes in the frequency of cells in each phase relative to untreated cultures. Figure 1 shows DNA content histogram plots (PI staining) for untreated Jurkat cells as well as following exposure to three representative compounds. In each culture where cells have been arrested, the relative frequency of the arrested phase was increased over that seen in untreated samples. Treatment with floxuidine resulted in a 45% increase

Aklavine HCl

Nocodazole

PI



compounds were assessed at two time points for the induction of the following markers of apoptotic progression: (1) externalization of phosphatidylserine (PS) (Nexin Assay); (2) elevations in active Caspase enzymes (Caspase-3/7 and 8 Assays); and (3) loss of mitochondrial membrane potential (MitoPotential Assay). Each of these assays also contains a cell-impermeant DNA stain (either PI or 7-AAD) permitting distinction of latestage apoptotic/dying cells. Because not all drugs elicit similar responses from all cell types, two human lines, Jurkat and HeLa, were chosen for screening.

FSC G0/G1 55.0 S 17.4 G2/M 26.3

G0/G1 71.8 S 17.8 G2/M 10.4

G0/G1 19 S 64 G2/M 15

G0/G1 5.5 S 10.3 G2/M 82.7

PI Figure 1. Jurkat cells were processed for DNA content analysis using Guava Cell Cycle Assay. Representative data are shown for a control sample and three test compounds. The top panel shows the gating dot plot for selection of single cell events, while the lower panel presents DNA content histograms for each compound. G1/GO, S, and G2/M phases are individually color coded and the relative frequency of each fraction is shown.

© 2006 Guava Technologies, Inc. All rights reserved. | 

Application Note: Multiplex Assay Approach to Screening Cytoactive Compounds

Control (DMSO)

7AAD



3%

Camptothecin 41%

5-Azacytidine

Emetine

70%

94%

Annexin V Figure 2. Jurkat cells were incubated for 4 hours in either control medium (DMSO) or three test compounds Camptothecin, 5-Azacytidine and Emetine (10 mM final concentration). Following treatment, apoptotic progression was assessed using the Guava Nexin Assay. Subpopulations are defined in the dot plots as healthy (teal), early- (blue), or late-stage apoptotic (magenta) through combined Annexin V and 7-AAD staining. Values represent the frequency of total apoptotic cells in a given sample.

Guava Nexin Assay Annexin V is a Ca2+-dependent phospholipid binding protein with high affinity for phosphatidylserine (PS), a membrane bound component localized to the inner face of the cell membrane. An indicator of early-stage apoptosis is the detection of exposed PS residues that have translocated to the cell surface.10-12 The Guava Nexin Assay permits simultaneous detection of early apoptotic events based on Annexin V binding to exposed PS and late apoptotic/dead events through uptake of 7-AAD. From Figure 2, the vast majority of Jurkat cells in the untreated control were healthy and thus unstained for Annexin V and 7-AAD. By contrast, treatment by any of the three compounds resulted in marked apoptotic induction, with Emetine eliciting the strongest response (50% of apoptotic cells in late stages or dying). From the plots shown it is evident that differences in the potency and mechanism of action contribute to variations in apoptotic progression induced by each test compound. Guava Caspase Assays Caspase proteins are intracellular proteases actively involved in the degradation of a wide range of cellular components during the apoptotic cell death. The Guava Caspase-8 and Caspase-3/7 Assay kits each employ a fluorescently-tagged cell permeable Caspase-specific FLICA (Fluorescent Inhibitor of Caspases) reagent which binds specifically to active Caspase molecules. The FLICA

Figure 3. Example of Caspase-8 activity in HeLa cells, untreated (DMSO) or treated for 16 hours with test compounds. The assay uses dual staining for active Caspase-8 enzyme and membrane integrity (PI) to define 4 unique subsets in a culture undergoing apoptosis: healthy, non-committed (teal); early (blue) or late stage apoptotic (magenta); and dead cells (purple). The relative frequencies of each subset are displayed within each dot plot.

Control (DMSO) 3 2

PI

in the number of cells present in G0/G1 over that found in untreated samples. Aklavine HCl promoted S-phase arrest with 64% of cells in the S phase as compared to 17% in control wells. By contrast, exposure to Nocodazole caused arrest of cultures in G2/M with 83% of cells in this phase as compared to 26% in control cells.

92 3

Mitoxanthrone HCl 22 6 52 20

Amphotericin B 2 2 74 22

Caspase-8

reagent is used in concert with the cell impermeant dye PI to permit simultaneous evaluation of apoptosis as a function of Caspase activity and generalized cell death (membrane integrity). Figure 3 shows that while both Mitoxanthrone HCl and Amphotericin B induced apoptosis within 4 hours, the cytotoxic effects of Mitoxanthrone HCl were far greater, as demonstrated by significant numbers of PI-positive cells (28%).

© 2006 Guava Technologies, Inc. All rights reserved. | 

Application Note: Multiplex Assay Approach to Screening Cytoactive Compounds

JC-1 Orange



Control (DMSO)

Azathioprine

Cytochalasin A

Polarized

Depolarized

32%

75%

JC-1 Green JC-1 Orange

Figure 4. Example of mitochondrial membrane depolarization using Guava MitoPotential assay of Jurkat cells, untreated or treated with test compounds for 4 hours. The top panel of dot plots shows the change in JC-1 fluorescence coincident with depolarization (values represent frequency of depolarized cells). Dual staining with 7-AAD permits assessment of membrane depolarization in concert with active programmed cell death (bottom panel).

Live

Dying

4%

29%

7AAD

Guava MitoPotential Assay

Multiparameter Analysis of “Hit” Compounds

Collapse of the mitochondrial inner transmembrane potential is believed to be coincident with release of Cytochrome C into the surrounding cytosol, ultimately resulting in the activation of downstream events in the apoptotic cascade.13-15 The Guava MitoPotential Assay combines JC-1, a ratiometric dye that fluoresces either orange (polarized) or green (depolarized) depending upon membrane potential,16 with 7-AAD to assess membrane permeability changes commonly observed in late-stage apoptosis. This two-dye assay not only enables users to monitor the changes in mitochondrial membrane potential as a function of apoptotic state, it also allows for the identification of those physiological conditions where either cellular event may occur independently. Untreated Jurkat cells are predominantly healthy and, with respect to JC-1 fluorescence, in a polarized state (Figure 4). By contrast, exposure to either Azathioprine or Cytochalasin A for 4 hours resulted in a significant shift of mitochondrial membrane potential, with cultures being 32% and 75% depolarized, respectively. While each compound was effective at inducing depolarization, only wells treated with Cytochalasin A showed high levels of cell death. While this finding suggests that azathioprine possesses no apoptotic capability, significant numbers of 7-AAD positive staining cells were found in cultures treated for 20 hours. From these findings, it is apparent that the kinetics of Azathioprine action are slower than that of Cytochalasin A.

Tables 1 and 2 summarize the effects of various cytoactive compounds as measured by changes in cell cycle distribution (not arrested or blocked in either G0/G1, S-phase, or at the G2/M boundary), fluctuations in mitochondrial membrane potential (% depolarization),  Caspase-8 activity, Caspase-3/7 activity, and the frequency of cells undergoing apoptosis (phosphatidylserine translocation). Inductions were carried out on two cell lines, non-adherent Jurkat cells (Table 1) and adherent Hela cells (Table 2), and data was acquired at two time points: 4/20 hours and 16/36 hours, respectively. In all, 200 cytoactive compounds were tested. By the second induction point, 35% of the compound panel (70 of 200) exhibited some biological effect, although the action and/or magnitude of this influence varied greatly both compound to compound and also between cell types. For compounds that arrested Jurkat cells at G0/G1, 7 of 12 had little or no pro-apoptotic activity. Two compounds, Dichlorophene and Suramin, however did show a change in JC-1 fluorescence. Similarly, among the 11 small molecules that promoted a G2/M arrest in Jurkat cells, virtually no signs of apoptotic induction were detected after 4 hours. However, 55% (6 of 11) showed significant apoptotic progression following 24-hour treatment. For those compounds that blocked HeLa cell division at the G2/M boundary, 78% (14 of 18) also induced expression of early apoptotic markers at the early time point. In particular, test

© 2006 Guava Technologies, Inc. All rights reserved. | 

Application Note: Multiplex Assay Approach to Screening Cytoactive Compounds

Table 1. Summary of “Hit” Compound Results for Jurkat Cells Jurkat

16 Hour Induction % Depolarized

Compound

G0/G1 Blockers 5-Azacytidine 5-Fluoro-5'-Deoxyuridine Floxuidine Isoniazid Lindane Neomycin Sulfate Cytochalasin E Dichlorophene Suramin Dequalinium Chloride Antimycin A Grisofulvin S-Phase Blockers Diaziquone Acriflavinium Hydrochloride Pyrimethamine Thioguanine Aklavine Hydrochloride Cycloheximide Oxyquinoline Physostigmine Saliculate E-Capsaicin Vinblastine Sulfate Chlorambucil 2,4-Dinitrophenol Atractyloside Possium Colidtimethate sodium Carmofur G2/M Blockers Podofilox Nitromide Colchicine Mebendazole Chloroambucil Rotenone Fenbendazole Albendazole Clindamycin Hydrochloride Nocodasole Bacitracin Not Arrested Hexestrol Diethylcarbamazine Citrate Primaquine Diphosphate Emetine Nicolsamide Camptothecin Azathioprine Emodin Celastrol Chloroquine Diphosphate Methotexate Parthenolide Mechlorethamine Quinacrine Hydrochloride Berberine Chloride Picropodophyllotoxin Pyrithione Zinc Gentian Violet Hexachlorophene Merbromin Phenylmercuric Acetate Floxuridine Aclacinomycin A1 Alexidine Hydrochloride Lasalocid Sodium Benzyl Isothiocyanate Mitoxanthrone Hydrochloride Ouabain Cytochalasin A Etoposide Puromycin Hydrochloride Melphalan Cyclophosphamide Hydrate No Affect

< 20%

Caspase-8 +

Caspase-3/7 +

36 Hour Induction % Apoptotic

% Depolarized

Caspase-8 +

Caspase-3/7 +

% Apoptotic

* Autofluorescing

20-50%

50-80%

80-100%

© 2006 Guava Technologies, Inc. All rights reserved. | 

Application Note: Multiplex Assay Approach to Screening Cytoactive Compounds

Table 2. Summary of “Hit” Compound Results for HeLa Cells HeLa

16 Hour Induction % Depolarized

Compound

G0/G1 Blockers Fluorouracil 5-Fluoro-5'-Deoxyuridine Emetine Camptothecin Azathioprine Methotrexate Mycophenolic Acid Isoniazid Lindane Neomycin Sulfate Helenine Oleandomycin Phosphate Tinidazole Citrinin Dacinomycin S-Phase Blockers Chloramphenicol Streptozosin Diaziquone Acriflavinium Hydrochloride Floxuridine Thioguanine Parthenolide Aklavine Hydrochloride Cycloheximide Oxyquinoline Etoposide Pentamidine Isethionate Colidtimethate Sodium Carmofur G2/M Blockers Podofilox Trioxsalen Colchicine Sanguinarine Sulfate Mebendazole Monocrotaline Chloroambucil Rotenone Fenbendazole Albendazole Gentian Violet Hexetidine Nocodasole Ouabain Cytochalasin B Melphalan Cyclophosphamide Hydrate p -Fluorphenylalanine Not Arrested Primaquine Diphosphate Nicolsamide Emodin Celastrol Chloroquine Diphosphate Carmustine Mechlorethamine Quinacrine Hydrochloride Berberine Chloride Picropodophyllotoxin Pyrithione Zinc Hexachlorophene Phenylmercuric Acetate Floxuridine Aclacinomycin A1 Alexidine Hydrochloride Vinblastine Sulfate Dequalinium Chloride Benzyl Isothiocyanate Mitoxanthrone Hydrochloride 3,5-Dinitrocatechol 5-Azacytidine Cytochalasin A Puromycin Hydrochloride No Affect

< 20%

Caspase-8

+

36 Hour Induction +

Caspase-3/7

% Apoptotic

% Depolarized

+

Caspase-8

Caspase-3/7

+

% Apoptotic

*Autofluorescing

20-50%

50-80%

80-100%

© 2006 Guava Technologies, Inc. All rights reserved. | 

Application Note: Multiplex Assay Approach to Screening Cytoactive Compounds

compounds showed significant differences in the ability to induce cell cycle arrest in the two cell lines. In addition, some compounds elicited no effect on cell cycle regulation but showed significant capacity to alter membrane potential and/or apoptotic induction.

stages of apoptosis (Annexin V+/7-AAD–) was 3% while the frequency of dead/dying was 1-2%. For Jurkat cells, the percentage of early apoptotic cells was 86 and 44% at 4 and 24 hours, respectively, and of late apoptotic plus dead (cytotoxic) cells was 3 and 46% in the positive controls. With Jurkat cells, at 4 hours, 12 compounds induced progress to early apoptosis but not beyond that stage. At 24 hours, 30 induced progress to early apoptosis and 25 to late apoptosis or death. Similar frequencies and patterns were displayed by HeLa cells. The Z' factors for the Guava Nexin assay are >0.8 for detecting both early apoptotic cells as well as late apoptotic and dead cells.

Detecting Cytotoxic Activity and Apoptotic Induction in a Panel of Compounds As shown in Figure 5, compounds can be quickly and easily screened for apoptotic and cytotoxic activity using the Guava Nexin Assay. Among unstimulated controls (8 per 96-well plate) following 4 and 20-hour inductions, the percentage of Jurkat cells in the early

80 70

Jurkat

60 50 40 30 20 10 0

A01 B01 C01 D01 E01 F01 H01 G01 Well #

% Dying/Dead Cells (ANNV+/7-AAD+)

% Dying/Dead Cells (ANNV+/7-AAD+)

Cytotoxic Activity

Time Pt #1 Time Pt #2 Mean + 3SD

50

HeLa

40 30 20 10 0

A01 B01 C01 D01 E01 F01 H01 G01 Well #

100 90 80

Jurkat

70 60 50 40 30 20 10 0

A01 B01 C01 D01 E01 F01 H01 G01 Well #

% Early Apoptosis (AnnV+/7-AAD–)

% Early Apoptosis (AnnV+/7-AAD–)

Apoptotic Induction 60 50

HeLa

40 30 20 10 0

A01 B01 C01 D01 E01 F01 H01 G01 Well #

Figure 5. Detection of cytotoxic activity and apoptotic induction in a panel of cytoactive compunds. Jurkat cells were cultured in 96-well plates for either 4 hours (Time Pt #1, orange) or 20 hours (Time Pt #2, blue) and then analyzed for the presence of cells in early (apoptotic induction) or late stage apoptosis (cytotoxic activity). Similar experiments were performed on HeLa cells with the cells harvested then analyzed at 16 and 36 hours post induction, respectively.

© 2006 Guava Technologies, Inc. All rights reserved. | 

Application Note: Multiplex Assay Approach to Screening Cytoactive Compounds

“Hit” Compound Titration for EC50 Determination

S U M M ARY

Using Jurkat cells, six “Hit” compounds were retested to confirm their activity and determine their EC50 values for the various stages of apoptosis (Table 3). Cells were cultured for 20 hours in the presence of varying concentrations of each test compound then assessed for apoptotic progression using the Guava Nexin and Guava Caspase-3/7 assays. In Figure 6, each curve (and corresponding EC50 values in table) represent the mean ± SD for three independent trials. Results replicated what was observed in the previous screening experiment. Of the six, mitoxanthrone HCl demonstrated the strongest induction capacity at all stages while all others compounds showed a much slower accumulation of dead cells and those in latestage apoptosis. Interestingly, all six compounds tested executed their apoptotic induction, whether direct or indirectly, through activation of the Caspase-8 pathway.

The microplate-based Guava EasyCyte system, when used in conjunction with Guava’s suite of functional assays, provides the ideal platform for the multiplex approach to cellular analysis required during both initial target validation and compound assessment for drug discovery. As shown for the 200 compounds tested, this combination of assays can easily identify both cell cycle inhibitors and apoptotic inducers, as well as characterize their EC50 values and quantify the percentage of cells affected. The detection of sub-population effects for these compounds highlights the benefit of a platform capable of analyzing populations at the single cell level. Because of the difference in the types of apoptotic cells detected (early vs. mid vs. late for Guava Nexin, Guava Caspase-8 and Caspase-3/7, respectively), initial screening of compounds for cytoactive capacity may only require the use of a single reliable apoptosis assay. However, to better understand the mechanism of action for a given compound and to gain more information concerning potential side effects (potency, toxicity, specificity), the multi-assay approach can be applied.

Table 3. Summary of “Hit” Compound Titration* Compound

Early Apoptotic

Mid-Stage Apoptotic Late Apoptotic

Primaquine Diphosphate

8.55

> 50

> 50

5-Azacytidine

2.24

9.13

9.53

Emetine

0.83

4.35

6.49

Amphotericin B

8.26

9.66

> 50

Phenylmercuric Ac

5.69

33.48

> 50

Mitoxanthrone HCl

0.27

1.16

2.14

*EC50 values in μM

B

Early Apoptosis

C

Mid-Apoptosis

Dying/Dead Cells

30

30

60

25

25

20

20

50 40 30 20 10 0 0.01

0.1

1

10

100

Compound Concentration (µM)

Primaquine DiP

% PI+

70

% Caspase-3+/PI–

% Annexin V+/7AAD–

A

15

15

10

10

5

5

0 0.01

0.1

1

10

100

0 0.01

5-Azacytidine

Emetine

Amphotericin B

0.1

1

10

100

Compound Concentration (µM)

Compound Concentration (µM) Phenylmercuric Ac

Mitoxanthrone HCl

Figure 6. Titration of “Hit” compounds. Dose response curves presented in A and B were derived from the frequency of cells possessing active Caspase-8 and Caspase-3/7, respectivley; C shows the dose response for each compound as a function of 7-AAD uptake.

© 2006 Guava Technologies, Inc. All rights reserved. | 

R E FE R E N C E S 1. Ho, J. and Fishwild, D. Screening Cytoactive Compounds to determine Cell Cycle Arrest Using the Guava PCA-96 System, a MicroplateBased Benchtop Cell Analysis System. Guava Technologies, 2004. 2. Kerr, J.F.R., et al. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239-257. 3. Wyllie, A.H., et al. Cell Death: The significance of apoptosis. Int Rev Cytol 1980;68:251-306. 4. Wyllie, A.H. Apoptosis. Br J Cancer 1993;67:205-208. 5. Majno, G. and Joris, I. Apoptosis, oncosis, and necrosis: An overview of cell death. Am J Pathol 1995;146:3-15. 6. Rudin, C.M. and Thompson, C.B. Apoptosis and disease: Regulation and clinical relevance of programmed cell death. Ann Rev Med 1997;48:267-281. 7. Salvesen G.C., Dixit, V.M. Caspases: intracellular signaling by proteolysis. Cell 1997;91:443-446. 8. Slee, E.A., et al. Serial killers: ordering Caspase activation events in apoptosis. Cell Death and Differentiation 1999;6:1067-74. 9. Alnemri, E.S., et al. Human ICE/CED-3 protease nomenclature. Cell 1996;87:171.

For Research Use Only. Not for use in diagnostic procedures. © 2006 Guava Technologies Inc. All rights reserved. Guava is a registered trademarks of Guava Technologies, Inc. EasyCyte, MitoPotential, and Nexin are trademarks of Guava Technologies, Inc.

10. Fadok, V., et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992;48:2207-16. 11. Martin, S., et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995;182:1545-56. 12. Vermes, I., et al. A novel assay for apoptosis: Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J Immunol Methods 1995;184:39-51. 13. Ly, J., et al. The mitochondrial membrane potential in apoptosis, an update. Apoptosis 2003;8:115-28. 14. Zamzami, N., et al. Mitochondrial control of nuclear apoptosis. J Exp Med 1996;183:1533-44. 15. Gollapudi, S., et al. Changes in mitochondrial membrane potential and mitochondrial mass occur independent of the activation of Caspase-8 and Caspase-3 during CD95-mediated apoptosis in peripheral blood T cells. Int J Oncology 2003;22:587-600. 16. Reers, M., et al. Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymology 1995;260:406-17.

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