A TD-700 fluorometric method for

CaspACE™

ACKNOWLEDGMENTS: We wish to acknowledge Promega Corporation for its work in acquiring the data and writing much of the text that supports this applications note.

1.0 INTRODUCTION

The CaspACE™ Assay System provides reagents for measuring the activity of the Caspase, or Interleukin-1b -Converting Enzyme (ICE/CED-3), family of cysteine aspartic acid-specific proteases. These proteases have been shown to play key roles in inflammation and apoptosis in mammalian cells^1-4. The CaspACE™ Assay System provides fluorogenic substrates and inhibitors that allow highly sensitive, quantitative measurement of both Caspase-1 (ICE) and Caspase-3 (CPP32) protease activities, an early regulatory event in the apoptotic cell death process. The use of the two selective substrates and inhibitors provided allows discrimination between ICE and CPP32 activities. This assay system may be used with purified enzyme preparations or cell extracts and can also be adapted for use in high throughput systems.

Activation of caspases occurs as a result of growth factor withdrawal, exposure to radiation or chemotherapeutic agents, or initiation of the Fas/Apo-1 receptor mediated cell death process. Active caspases participate in a cascade of cleavage events, which disable key homeostatic and repair enzymes and bring about systematic structural disassembly of dying cells. The biological substrates of caspases include poly-(ADP ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK), lamins, topoisomerases, Gas2, protein kinase C (PKC)delta, sterol regulatory element binding proteins (SREBP), U1-70kDa protein and Huntington protein^5-8.

To date, fourteen mammalian homologs of CED-3 (pro-apoptotic gene of C. elegans) have been described⁹. Among these, caspace-1 and caspace-3 subfamilies have been identified based on amino acid sequence and substrate and inhibitor specificities.

Caspace-1 (ICE) shows specificity for cleavage at the C-terminal side of the aspartate residue of the sequence YVAD (Tyr-Val-Ala-Asp) and is inhibited by the tetrapeptide inhibitor Ac-YVAD-CHO. Caspace-3 (CPP32) shows specificity for cleavage at the C-terminal side of the aspartate residue of the sequence DEVD (Asp-Glu-Val-Asp) and is inhibited by the tetrapeptide inhibitor Ac-DEVD-CHO².

2.0 PRINCIPLE

The fluorogenic substrates Ac-YVAD-AMC and Ac-DEVD-AMC for Caspase-1 (ICE) and Caspase 3 (CPP32), respectively, are provided in the CaspACEä Assay System and are labeled with the fluorochrome 7-amino-4-methyl coumarin (AMC). The substrates produce a blue fluorescence that can be detected by exposure to UV light at 360nm. AMC is released from these substrates upon cleavage by Caspace-1 (ICE) or Caspace 3 (CPP32)-like enzymes. Free AMC produces a yellow-green fluorescence that is monitored by a fluorometer at 460nm. The amount of yellow-green fluorescence produced upon cleavage is proportional to the amount of Caspace-1 (ICE) or Caspace-3 (CPP32) activity present in the sample¹⁰.

Two potent reversible aldehyde inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO of Caspace-1 (ICE) and Caspace-3 (CPP32), respectively, are provided in the CaspACEä Assay System. In cell lysates, the fluorogenic substrates may be susceptible to cleavage by other related proteinases; therefore, in order to assess the specific contribution of Caspace-1 (ICE) or Caspace-3 (CPP32) enzyme activity in crude cell extracts, assays are performed in the presence and absence of the appropriate inhibitors. The difference between the substrate cleavage activity levels in the presence and absence of inhibitor reflects the contribution of either Caspace-1 (ICE) or Caspace-3 (CPP32) enzyme activity.

3.0 MATERIALS REQUIRED

3.1 From Turner BioSystems:

• TD-700 Fluorometer with standard PMT and 10 × 10 mm cuvette adaptor (P/N 7000-009).
  
• Near UV Lamp (P/N 10-049).
  
• Excitation Filter, 365 nm (P/N 034-0365).
  
• Emission Filter, 455-500 nm P/N 10-104R-C).
  
• Minicell Adaptor Kit (P/N 7000-951) which includes 1 adaptor and 400 borosilicate glass minicells.
  

3.2 From Promega Corporation:

• CaspACE™ Assay System, Fluorometric (cat. # G3540). Store protected from light and moisture at -20° C. Avoid multiple freeze-thaw cycles. Store substrates and inhibitors in aliquots at -20° C. Substrate and inhibitor solutions are stable for at least six months from the date of purchase if stored and handled properly. Each system contains sufficient reagents to perform at least 320 reactions and includes the following items:
  400 µL ICE Substrate Ac-YVAD-AMC (10 mM)
  400 µL CPP32 Substrate Ac-DEVD-AMC (10 mM)
  400 µL ICE Inhibitor Ac-YVAD-CHO (10 mM)
  400 µL CPP32 Inhibitor Ac-DEVD-CHO (10 mM)
  5 mg AMC Standard (7-amino-4-methyl coumarin)
  2 x 30 mL ICE-Like Enzyme Assay Buffer
  1 Protocol
  

3.3 Supplied By User:

• 30° C Incubator
  
• Dimethyl Sulfoxide (DMSO)
  
• DTT, 100 mM
  
• Deionized Water
  
• Microfuge tubes
  

4.0 REAGENT PREPARATION

4.1 Substrates and Inhibitors: Thaw the 10 mM substrate and inhibitor stock solutions to room temperature and mix thoroughly. Dilute (1 part substrate or inhibitor to 3 parts DMSO) the desired amount of these solutions to 2.5 mM with DMSO before use.

4.2 DTT: Prepare a 1 M stock solution of DTT in deionized water. Aliquot and store at -20° C. Dilute 1:10 with deionized water to make a 100 mM solution before use.

4.3 ICE-Like Enzyme Assay Buffer: Thaw and mix thoroughly before use.

5.0 STANDARD PREPARATION

5.1 Preparation of AMC Stock Solutions:

5.1.1 Prepare a 10 mM stock solution of AMC by adding 2.85 mL DMSO to the vial containing 5 mg of AMC Standard supplied. To confirm the AMC concentration, dilute the 10 mM solution 1:400 in water and read the Absorbance at 354 nm. The concentration can be calculated using an extinction coefficient of 16 x 10^-3 M^-1/cm^-1 as follows:

5.1.2 Dilute the AMC stock solution to 1 mM. Perform serial dilutions of this 1 mM stock solution in DMSO by combining the volumes shown in Table 1 to make 100 µM, 10 µM, and 1 µM stock solutions.

Table 1: Preparation of AMC Stock Solutions

5.2 Preparation of AMC Calibration Standards
5.2.1 Using the AMC stock solutions described above, prepare 250 µL of each AMC Standard as described in Table 2.

Table 2: Components Required for AMC Calibration Curve

6.0 CALIBRATION

6.1 Set-up and turn on your fluorometer according to your Operating Manual. Allow fluorometer to warm up before proceeding to step 6.2.
6.2 Pipette 200 µL of the 5000 picomoles AMC/250 µL standard (see Table 2), into a minicell and calibrate your fluorometer in direct concentration mode following the guidelines listed in your Operating Manual. Select <5> for number of standards. For concentration, enter in a value 10x lower than the actual concentration (ie. 500 for first standard). This factor will be accounted for later. Repeat with the rest of the standards in order of decreasing concentration. Use the 0 Picomoles
AMC/250 µL standard (see Table 2) as your blank.

7.0 ASSAY FOR CPP32/ICE ACTIVITY IN CELL EXTRACTS

7.1 Assay Conditions: Use the following assay conditions to determine the specific activity of CPP32/ICE-like enzymes in cell extracts:

• substrate only (blank)
  
• substrate + enzyme source (assay)
  
• substrate + enzyme source + inhibitor (negative control)

For determination of the activity of purified ICE or CPP32 enzymes, negative control reactions are optional.
Examples of the preparation and analysis of cell extracts that can be used as positive controls are given in Section 8.

7.2 Notes:

7.2.1 For optimal results, it may be necessary to assay several concentrations of the sample. In the standard format, up to 10 µL of cell extract (75-100 µg total protein) may be added to each reaction. If necessary, the sample may be diluted in ICE-Like Enzyme Assay Buffer. Test each sample concentration under all three assay conditions. For example protocols for the preparation of cell extracts see Section 8.

7.2.2 The amount of cell extract used in assay and negative control reactions must be identical.

7.3 Standard Assay (250 µL reactions)

7.3.1 Prepare duplicate microfuge tubes for each of the three assay conditions as shown in Table 3.

7.3.2 Mix the contents of the tubes by vortexing gently. Incubate at 30°C for 30 minutes.

7.3.3 Add 5.0 µL of the appropriate 2.5 mM substrate to each tube. For CPP32 assays, use the CPP32 Substrate (Ac-DEVD-AMC); for ICE assays, use the ICE Substrate (Ac-YVAD-AMC).

Table 3: Standard Assay

*NOTE: For CPP32 assays, use the CPP32 Inhibitor (Ac-DEVD-CHO); for ICE assays, use the ICE Inhibitor (Ac-YVAD-CHO).

7.3.4 Incubate at 30°C for 60 minutes. Pipette 200 µL of each into a minicell. Measure the fluorescence of each reaction on your fluorometer. Fluorescence measurements must be completed within 2 hours of the addition of substrate in order to stay within the linear range of the assay. If desired, Steps 7.3.2 and 7.3.4 may be performed at 37°C.

8.0 POSITIVE CONTROLS

Apoptosis can be included in experimental systems by a variety of methods that lead to caspace activation. These include:

• Treatment of Fas of TNF receptor-bearing cells by cross-linking with agonistic antibodies^{11, 12}.
  
• Treatment of cells with DNA topoisomerase inhibitors, e.g., etoposide¹³, with the protein kinase inhibitor Staurosporin¹⁴ or with microtubule damaging agents such as paclitaxel¹⁵.
  
• Exposure of cells to genotoxic damage induced by ionizing radiation^15,16.

8.1 Example 1: Analysis of Caspase Activity in Jurkat Cells Treated with Anti-Fas Antibody
Materials to Be Supplied by the User (Solution compositions are provided in Section 10.)

• hypotonic cell lysis buffer
  
• Anti-FAS antibody
  

8.1.1 Grow Jurkat cells in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glutamine and 1% penicillin-streptomycin in a humidified, 5% CO₂ incubator at 37°C.
8.1.2 Adjust the cell density to 5 x 10⁵ cells/mL. Add 100 ng/mL of anti-Fas MAb (CH-11) and incubate for 4 hours at 37°C in a humidified, 5% CO₂ atmosphere. As a negative control, concurrently incubate a culture treated with PBS.
8.1.3 Harvest cells by centrifugation at 450 x g for 10 minutes at 4°C. Keep the cell pellet on ice. Wash 1X with ice cold PBS and resuspend in hypotonic cell lysis buffer at a concentration of 10⁸ cells/mL.
8.1.4 Lyse the cells by subjecting them to four cycles of freezing and thawing. Centrifuge the cell lysates at 16,000 x g for 20 minutes at 4°C and collect the supernatant fraction.
8.1.5 Measure CPP32 and/or ICE activity of anti-Fas antibody treated (positive control) and untreated (negative control) cell lysates as described in Section 5.2, Steps 1-4. Use extract from at least 1 x 10⁶ cells/assay.

8.2 Example 2: Analysis of Caspace Activity in THP-1 Cell Lysates
Cytosolic extracts prepared from the human monocytic leukemia cell line THP-1 (ATCC #TIB-202) may be used as a source of ICE and CPP32 enzymes (positive control).
8.2.1 Culture THP-1 in Iscove’s Modified DMEM containing 9% horse serum, 2 mM glutamine, 2 x 10^-5 M b -mercaptoethanol and 1% penicillin-streptomycin in a 10% CO₂ incubator at 37°C. Grow cells to a density of 5-6 x 10⁵ cells/ml.
8.2.2 Harvest the cells and make cell extracts as described in Steps 6.1.3 and 6.1.4.
8.2.3 Pre-activate the THP-1 cell extract by incubating at 37°C for 1 hour. Keep the pre-activated extract on ice until ready to use.
8.2.4 Measure the CPP32 and/or ICE activity of the pre-activated THP-1 cell lysate as described in Section 5.2. Use extract from at least 1 x 10⁶ cells per assay.
8.2.5 Measure the protein content of the cell extract.

9.0 CALCULATION OF ENZYME SPECIFIC ACTIVITY

9.1 Estimate the protein concentration of each cell extract using BSA as a standard¹⁷.
9.2 Use the following formula to calculate the activity of ICE-like enzymes present in a cell extract

10.0 ADDITIONAL APPLICATIONS OF ICE AND CPP32 INHIBITORS

Inhibitory constants (K_i values) for the ICE Inhibitor (Ac-YVAD-CHO) and for the CPP32 Inhibitor (Ac-DEVD-CHO) will vary depending on the particular ICE-like enzyme involved. For example, Ac-DEVD-CHO is a potent inhibitor of CPP32 (K_i < 1 nM) and a weak inhibitor of ICE (K_i = 10 µM). Similarly, Ac-YVAD-CHO is a potent inhibitor of ICE (K_i = 0.76 nM) and a significantly less effective inhibitor of CPP32 (K_i = 20 nM). The difference in potency of these inhibitors may be used to discriminate between ICE and CPP32 enzyme activities present in cell lysates in either biological substrate (e.g., poly-(ADP-ribose) polymerase) cleavage assays with apoptotic cell lysates^2,11 or in DNA fragmentation analysis after incubation of rat thymocyte nuclei with apoptotic cell lysates^{11, 18, 19}. Assays are performed in the presence and absence of a range of concentrations (5 nM-50 µM) of each inhibitor. The inhibitor that is capable of suppressing apoptosis at the lowest concentration is indicative of the enzyme present. For example, if the CPP32 Inhibitor can inhibit DNA fragmentation or PARP cleavage activity at a concentration of 5 nM and the ICE Inhibitor is only effective at a concentration of 50 µM, the enzyme present in the cell lysate is CPP32²⁰.
10.1 Note: Concentrations of CPP32 Inhibitor or ICE Inhibitor in the range of 120-150 µM are required to inhibit CPP32 or ICE enzyme activities in whole cell culture systems¹¹.

11.0 COMPOSITION OF BUFFERS AND SOLUTIONS

11.1 ICE-Like Enzyme Assay Buffer

• 312.5 mM HEPES (pH 7.5)
  
• 31.25% sucrose
  
• 0.3125% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1 propane-sulfonate)

11.2 Hypotonic cell lysis buffer

• 25 mM HEPES (pH 7.5)
  
• 5 mM MgCl2
  
• 5 mM EDTA
  
• 5 mM DTT
  
• 2 mM PMSF (phenyl methyl sulfonyl fluoride)
  
• 10 µg/mL Pepstatin A (Sigma Cat.# P4265)
  
• 10 µg/mL Leupeptin (Sigma Cat.# L2884)

12.0 REFERENCES
1. Thornberry, N.A. et al. (1992) Nature 356, 768.
2. Nicholson, D.W. et al. (1995) Nature 376, 37.
3. Tewari, M. et al. (1995) Cell 81, 801.
4. Fernandes-Alnemri, T. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 7464.
5. Vaux, D.L. and Strasser, A. (1996) Proc. Natl. Acad. Sci. USA 93, 2239.
6. Kumar, S. and Lavin, M.F. (1996) Cell Death and Differentiation 3, 255.
7. Nicholson, D.W. and Thornberry, N.A. (1997) TIBS 22, 299.
8. Rosen, A. (1996) Nature Genetics 13, 380.
9. Van de Craen, M. et al. (1998) Cell Death and Differentiation 5,838.
10. Thornberry, N.A. (1994) Meth. Enzymol. 244, 615.
11. Schlegel, J. et al. (1996) J. Biol. Chem. 271, 1841.
12. Janicke, R.U et al. (1996) EMBO J. 15, 6969.
13. MacFarlane, M. et al. (1997) J. Cell Biol. 137, 469.
14. Weil, M. et al. (1996) J. Cell. Biol. 133, 1054.
15. Storbel, T. et al. (1997) Oncogene 14, 2753.
16. Datta, R. et al. (1997) J. Biol. Chem. 272, 1965.
17. Bradford, M.M. (1976) Anal. Biochem. 72, 248.
18. Schlegel, J. et al. (1995) FEBS Lett. 364, 139.
19. Lazebnik, Y.A. et al. (1994) Nature 371, 346.
20. Shimizu, S. et al. (1996) Oncogene 12, 225.

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Internet: http://www.promega.com

Address:
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