Execution of apoptosis in mammalian cells requires the coordinated action of several aspartate-specific cysteine proteases, recently named caspases, which are responsible for the cleavage of key enzymatic and structural substrates, resulting in the systematic and orderly disassembly of the dying cell. Caspases were first implicated in apoptosis by genetic analysis in the nematode C. elegans, in which the deletion of a single gene, CED-3, results in the abolition of all the 131 programmed cell deaths that would normally occur during the worm's development. The finding that the product of the CED-3 gene is strongly related to mammalian interleukin1b-converting enzyme (ICE), together with the observation that overexpression of ICE induces apoptosis, prompted an intensive search for new family members, that has led to the identification of at least ten related caspases. These proteins are characterized by an absolute specificity for Asp in the cleavage site+s P1 position, and they all contain a conserved QACXG (where X is R, Q or G) pentapeptide motif in the catalytic site. Phylogenetic analysis of the caspases revealed that they can be grouped into three subfamilies: an ICE subfamily, comprising caspases -1, -4 and -5 (ICE, TX and TY respectively), a CED-3/CPP32 subfamily, comprising caspases -3, -6, -7, -8, -9 and -10 (CPP32, Mch2, Mch3, FLICE, Mch6, Mch4), and an Ich-1/Nedd-2 subfamily. Accumulating evidence indicates that members of the ICE subfamily predominantly play a role in inflammation, whereas members of the CPP32 subfamily are largely involved in apoptosis. Caspases are synthesized as proenzymes, with an N-terminal prodomain and two subunits sometimes separated by a linker peptide; the inactive proenzymes are activated during apoptosis by cleavage at specific Asp residues to yield the mature form of the enzymes, which contain both large (p20) and small (p10) subunits complexed to form an active tetramer. The role of the prodomain may be regulatory, enabling some caspases to be specifically recruited in order to facilitate the execution of the death program. Alternatively spliced forms of many caspases may in part regulate the activity of the full-length enzymes, either by acting as dominant inhibitors or by forming inactive heteromeric complexes. The peptide-recognition elements found in endogenous substrates allowed the development of a wide variety of reversible and irreversible inhibitors for ICE family proteases: for example, the tetrapeptide aldehyde similar to the cleavage site in pro-interleukin1b, Ac-YVAD-CHO, is a potent caspase-1 inhibitor, while Ac-DEVD-CHO, which mimics CPP32 favourite cleavage site, is an excellent inhibitor of this caspase. Three naturally occurring protein inhibitors have also been described, which comprise cowpox virus CrmA, baculovirus p35 and a group of polypeptides belonging to the IAP (Inhibitor of Apoptosis) gene family. Physiological regulation of caspase activity predominantly occurs at the level of proenzyme processing and maturation in a proteolytic cascade where some caspases sequentially activate others and eventually activate themselves by intermolecular autoproteolysis. A hierarchic model has been proposed in which an -initiator protease+, such as caspase-8 or -10, gets activated by an apoptotic stimulus, then it activates an -amplifier+ protease such as caspase-1, which in turn activates a -machinery+ protease such as caspase-3 or -7. Similar events have been demonstrated in the Fas and in the TNFR1 models of apoptosis, in which receptor ligation results in the recruitment of FLICE/ caspase-8, which undergoes autocatalytic activation and is able to process and activate all known caspases, supporting the idea that it lies at the apex of an apoptotic cascade. The striking morphological changes that occur in cells undergoing apoptotic suicide are consequent to the proteolytic cleavage of a number of specific proteins, some of which have been identified: caspase targets include enzymes involved in genome function (such as PARP, DNA-dependent protein kinase, U1 small ribonucleoprotein and the 140 kDa component of DNA-replication complex C), regulators of the cell cycle progression (Rb, PKCd, mdm2), structural proteins (lamins, actin, fodrin, gelsonin), and a DNA-fragmentation factor (DFF) which mediates internucleosomal cleavage of DNA. While many intracellular targets of caspases have been recognized, the critical cellular substrates leading to cell death have not yet been identified, although this is a field of intensive research. The mechanisms by which caspases are regulated will be the subject of intense investigation in the next times, and this will advance the understanding of important physiological and pathological processes.

CPP32 is one of the key executioners of apoptosis, being responsible either partially or totally for the proteolytic cleavage of many cellular substrates, such as PARP (poly-ADP-ribose polymerase). Thus, CPP32 protease activity can be used to monitor apoptosis earlier than many other commonly used assays, i.e. those based on DNA fragmentation detection. One way to measure CPP32 activity is based on a model fluorescent substrate, Ac-DEVD-AFC (Acetyl- Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin), that mimics the known cleavage site of PARP (DEVD:G), for which CPP32 shows the highest affinity. The AFC conjugate normally emits blue light (lmax= 400 nm), but, upon proteolytic cleavage by CPP32 or closely related caspases, the free AFC emits a yellow-green fluorescence at 505 nm. Comparison of the fluorescence emission of an apoptotic sample with an uninduced control allows one to determine the fold-increase in protease activity. Similarly, it is possible to detect an increase in ICE/caspase-1 activity by using the fluorogenic tetrapeptide YVAD-AFC, for which ICE subfamily shows the highest preference. It is also possible to perform another version of the same assay by using a cromophore-labeled substrate, DEVD- or YVAD-pNA (p-nitroanilide), which allows spectrophoto- metric detection of the cromophore pNA after cleavage from the labeled tetrapeptide. Both assays are highly sensitive, convenient and very rapid, as the entire protocol can be performed in less than 2 hr.

• Fluorometer or 96-well plate reader
• CPP32 substrate (DEVD-AFC), 1 mM in DMSO
• ICE substrate (YVAD-AFC), 1 mM in DMSO
• CPP32 inhibitor (DEVD-CHO), 1 mM stock in water
• ICE inhibitor (YVAD-CHO), 1 mM stock in ethanol
• CPP32 substrate (DEVD-pNA), 1 mM in DMSO
• ICE substrate (YVAD-pNA), 1 mM in DMSO
• Cell Lysis Buffer (A1)
• 2x Reaction Buffer (A1)
• Spectrofotometer or 96-well plate reader

Induce apoptosis in cells by the desired method. To evaluate protease activity in terms of fold-increase, concurrently incubate a negative control (control #1) without induction.

1. Count cells and pellet 106 cells for each sample.
2. Wash cells once with cold PBS.
3. Resuspend cells in 50 µl of chilled lysis buffer and incubate on ice for 20 min.
4. Lyse the cells by 3-4 cycles of freezing and thawing.
5. Pellet insoluble material by centrifugation for 15 min at 14,000 rpm at 4°C. The clear supernatant can be used immediately or stored at -70°C for assay at a later time.
6. Optional: to verify that the signal detected is attributable to CPP32 protease activity, and to assess the fluorescence contribution of the subpopulation of apoptotic cells normally present in culture, a negative control #2 can be performed by treating an induced sample with the CPP32 inhibitor DEVD-CHO before incubation with the fluorescent substrate.
7. To do so, add 50 µl of 2x Reaction Buffer and 1 ml of CPP32 inhibitor (from 1 mM solution) to an induced sample lysate and incubate at 30°C for 30 min (if your are measuring ICE activity, do the same with YVAD-CHO). Keep the other lysates on ice during this time. Then proceed to step 9, adding reaction buffer to the samples that were held on ice, and continue.
8. Add 50 µl of 2x Reaction Buffer to each reaction.
9. Add to each tube 5 µl of CPP32 substrate (DEVD-AFC) or ICE substrate (YVAD-AFC) from 1 mM stock solution to make 50 µM final concentration. Incubate at 37°C for 1 hr.
10. Read the samples in a fluorometer equipped with a 400 nm excitation filter and a 505 nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. The typical fluorescence emission of apoptotic samples is 4-7 fold above that of the uninduced control sample.

NOTE: As an alternative to DEVD-AFC, it is possible to use the fluorogenic substrates Ac-DEVD-AMC (7-amino-4- methylcoumarin) or Ac-YVAD-AMC, both available from Bachem, Bubendorf, Switzerland. In this case it will be necessary to read the samples using an excitation wavelength of 360 nm and an emission wavelength of 480 nm.

 12. Essentially the same procedure can be performed with a chromogenic instead of a fluorogenic tetrapeptide substrate. Although this technique is slightly less sensitive, it does not require a fluorometer, but it can be performed with a normal spectrophotometer.

13. Perform the entire protocol as for the fluorescent assay, adding at step 10 DEVD-pNA (or YVAD-pNA) at 50 µM final concentration, instead of DEVD-AFC (or YVAD-AFC).

14. At the end of the reaction, read samples in a spectrophotometer at 400 or 405 nm (for a standard spectrophotometer, dilute samples to 1 ml with H2O and transfer them to a 1 ml cuvette). Due to background from light scattering in the spectrophotometer, when performing the colorimetric assay it is essential to make a blank control with an uninduced lysate to which substrate has not been added (lysate + Reaction Buffer). This control determines the background OD of the sample and should be subtracted from of the induced and uninduced sample before determining the fold-induction of protease activity.
 

3A. Commentary

 
3A.1. Troubleshooting

The caspase activity assay protocol is very simple and there are not many steps that can be done in a wrong way. Anyway, if you do not see the expected results, check out the following things: a) the fluorometer has the right filters/the spectrophotometer is reading at the right wavelength. b) you start with a population of healthy cells, and c) your apoptotic samples are really apoptotic (and not, for example, necrotic).

Poly-ADP-ribose polymerase (PARP) is the best characterized proteolytic substrate of caspases, being cleaved during the execution phase of apoptosis in many systems. Intact PARP (116 kDa) is cleaved to 24 kDa and 89 kDa fragments, representing the N-terminal DNA-binding domain and the C-terminal catalytic domain of the enzyme, respectively. Although many caspases, including caspase-2, -4, -6, -8, -9 and -10 can cleave PARP in vitro when added at high concentrations, it appears that caspase-3 and -7 are primarily responsible for PARP cleavage during apoptosis. Although the biological relevance of PARP cleavage is not clear, PARP cleavage is considered one of the most valuable indicators of apoptosis. Moreover, this procedure is in principle applicable to all known and potential substrates, provided that the specific cDNA is available. The time needed to perform the following protocol is around 8-9 hours (2 hr for in vitro translation, 2-3 hr for cell lysis and cleavage reaction, 4-5 hr to run and dry the gel), plus an overnight film exposure.

PARP cDNA under T3 or T7 promoter
TNT Coupled Reticulocyte Lysate System (Promega)
35S Methionine, 1,000 Ci/mmol (Amersham Corp.) SDS-PAGE apparatus and power supply
Gel dryer
Autoradiography films and cassette
Method for measurement of protein concentration
Cell Lysis Buffer
5x Reaction Buffer

1. Perform PARP transcription and translation using the TNT kit according to the manufacturer's recommendations. Usually 5 m l of the 50 m l final reaction mixture contain a sufficient amount of 35S-labeled PARP.
2. Induce apoptosis in cells by the desired method, keeping an uninduced sample for negative control.
3. Collect cells (0.5-3x106/sample), wash once with cold PBS and resuspend in 50 m l chilled Cell Lysis Buffer.
4. Keep 20 min on ice.
5. Proceed with lysis by 3-4 cycles of freezing and thawing.
6. Pellet insoluble material by centrifugation for 15 min at 14,000 rpm at 4°C.
7. After centrifugation, collect the supernatants and determine protein concentration of 2-5 m l, while keeping the rest of the lysates on ice. Remaining lysates can be frozen at -70°C for assay at a later time.
8. In a 0.5 ml reaction tube kept on ice, mix together 10 µg cell lysate, 4 µl 5x Reaction Buffer, 5 µl 35S-labeled PARP, and bring the final volume to 20 µl with H2O.
9. Incubate 1 hr at 37°C.
10. Stop the reaction by adding 5 µl of 5x Laemmli Sample Buffer.
11. Boil the samples for 3 min and load on 10% polyacrylamide gel (we recommend the use of mini-gel apparatus, such as Biorad Mini Protean II or Hoefer Mighty Small ).
12. Fix and dry the gel as usual, and expose it overnight at room temperature (take any Saran wrap off the gel before exposing).
13. Develop film and observe the presence of the 24 kDa and of the 89 kDa fragments in apoptotic samples.

 3B.1. Troubleshooting
 

a) Do not see any band on the film: possible problems with the in vitro translation. Check the result of the reaction by running 5-10 µl of the 50 µl final mixture on a 10% polyacrylamide gel: you should clearly see the 116 kDa product; if you don't, refer to the troubleshooting indications of the kit's manufacturer.

b) Do not see any cleavage product: make sure your cells are really apoptotic. Make a positive control of apoptosis.

c) See cleavage products also in uninduced sample: your cells may have a high rate of spontaneous apoptosis. Start up again with healthy cells.

This approach is based on the use of irreversible peptide inhibitors tagged with biotin, that allow identification of labeled caspases by affinity blotting. The procedure is simple, widely applicable, and very sensitive; furthermore, since one molecule of caspase binds only one molecule of affinity label, this approach can be used to quantify amounts of active caspases in the extract, given that the caspases are saturated with the label. With the use of a high concentration of an irreversible inhibitor as biotin-YVAD-amk (acyloxymethylketone), which is reactive only when bound to an active cysteine protease and gives a low background, it is possible to label all caspases activated in apoptotic cells. Cell lysates are then subjected to SDS-PAGE and the separated proteins are transferred to a membrane, which is then probed with avidin followed by biotin-HRP to visualize the labeled proteins by ECL. With this method it is possible to detect several caspases of molecular weight between 17 and 21 kDa, only in the presence of the affinity label and in cells undergoing apoptosis. For further identification of the labeled proteins, since large subunits of many caspases have similar molecular weights, it would be necessary to use 2D electrophoresis followed by avidin affinity blot, but, for common purposes, the 1D affinity blot gives sufficiently good results. The time needed to perform the following protocol is around 7-8 hours.

Biotin-YVAD-amk, 10 mM stock in DMSO
Method for measurement of protein concentration
SDS-PAGE apparatus and power supply
Blotting apparatus
Immobilon PSQ membrane (Millipore)
avidin-Neutralite (Molecular Probes)
biotinylated HRP (Molecular Probes)
ECL (Amersham corp.) or Super Signal (Pierce) Autoradiography films and cassette
MDB Buffer
KPM Buffer
PTB Buffer

1. Induce apoptosis in cells by the desired method; keep an uninduced sample for negative control.
2. Resuspend cells at 107/ml in KPM Buffer.
3. Pellet cells and snap-freeze the pellet in liquid nitrogen.
4. Dilute stock solution of biotin-YVAD-amk to a 20 mM solution in MDB Buffer and add an equal volume of this solution to the cell pellet.
5. Lyse the cells by 3 cycles of freezing-thawing.
6. Incubate the lysates at 37°C for 15 min.
7. Centrifuge the lysates for 20 min at 16,000xg at 4°C and determine protein concentration of the supernatant.
8. Mix 10 µg of each lysate with Laemmli sample buffer, heat at 100°C for 5 min and load on 15% SDS-PAGE.
9. Transfer the labeled proteins to PVDF Immobilon PSQ membrane for 1 hr at 100 V.
10. Soak the membrane in methanol and dry overnight at RT or for 15 min at 80°C.
11. Incubate the dried membrane in avidin-Neutralite at 1 mg/ml in PTB 1% BSA.
12. Wash with PTB and incubate in biotinylated HRP at 25 ng/ml in PTB 1% BSA.
13. Visualize the labeled proteins by ECL. This assay can detect as little as 0.1 ng of an active caspase, which is the amount present in 1 m g of lysate prepared from apoptotic Jurkat cells.

a) Do not see any band on the blot: make sure the protein transfer has been efficient by using an appropriated marker. Verify that your blotting reagents work properly, for example by using a biotinylated marker. Make sure that in your system caspases are efficiently activated, i. e. by using another detection method.

b) Have a high background labeling: check concentration of the labeled tetrapeptide.

The methods previously described are helpful to test the presence of activated caspases in cell lysates, but they provide little information on which caspases are activated in apoptotic cells. If it is necessary to characterize the specific protease activated in vivo by a given apoptotic stimulus it is necessary to immunoblot the total cell lysate using antibodies recognizing individual caspases. Because caspases are activated by auto- or hetero-proteolysis of the respective pro-enzyme, the appearance of bands with lower molecular weight, corresponding to the subunit of the active enzymes, indicates their activation. With the same method it is possible to analyze individual substrates cleaved in vivo by caspases. The same membrane can be reused to be tested with different antibodies, specific for other caspases or substrates, by stripping and reprobing. Most of the antibodies for caspases are commercially available, while it might be more difficult to find antibodies specific for not common or potential substrates.

SDS-PAGE apparatus and power supply
Blotting apparatus
Antibodies against caspases or substrates
Horseradish peroxidase (HRP)-conjugated anti-Ig antibody
ECL (Amersham)
Autoradiography films and cassette
Lysis Buffer (LB)
Buffer A
Transfer buffer
Washing solution
Blocking solution
 

1. Induce apoptosis in cells by the desired method; keep an uninduced sample for negative control.

2. Wash cells twice in cold PBS or medium w/o serum.

3. Resuspend cells at 4x107/ml in chilled LB buffer and keep on ice for 10-15 min.

4. Centrifuge cell lysates for 20 min at 18,000 rpm at 4°C. Take the supernatant, aliquote and use immediatly or freeze at -80°C.

5. Mix a fixed volume of total lysate (25 µl corresponding to 1x106cells is normally enough to detect caspases) with Laemmli sample buffer, heat at 100°C for 5 min and load on SDS-PAGE.

6. Wash the gel in buffer A twice for 20 min each.

7. Transfer the proteins on a nitrocellulose membrane using a blotting apparatus for 2 hr at 50-60 V. At the end check the transfer soaking the membrane with a Ponceau solution. Wash with water; if bands appear proteins have transferred well.

8. Bleach the membrane with PBS-Tween, washing several times.

9. Soak membrane with blocking solution 1 hr at RT in agitation or overnight at 4°C.

10. Wash membrane four times with PBS-Tween for 5 min each.

11. Incubate 1 hr membrane with the antibody previous diluted in PBS-Tween solution (see antibody technical instructions for Western blotting).

12. Wash again with PBS-Tween as in point 10 and incubate 1 hr with the HRP-conjugated secondary antibody diluted in PBS-Tween.

13. Mix equal volumes of the substrate solutions of ECL kit and put on the membrane only 1 sec.

14. Put quickly the membrane in a autoradiography cassette and expose on films at different times. Keep in mind that the chemiluminescence signal decays very rapidly (see ECL instructions)
 

3D. Commentary

3D.1. Troubleshooting

a) Do not see anything on the film: check all the ECL reagents and their expiry date.

b) Can not see any band recognized by your antibody: - your antibody is not good, use the specific purified protein as control - proteins in your cell lysate are not enough, use more lysate- protein in your cells is expressed at low levels

c) Do not see any cleavage product: make sure your cells are really apoptotic.
 

1. Cohen, G. M. 1977. Caspases: the executioners of apoptosis. Biochem. J. 326: 1.

 Appendix 2 (A2): Reagents
Antibodies against caspases or substrates
Several commercial sources

Autoradiography film

Avidin-Neutralite
Molecular Probes

Biotinylated HRP
Molecular Probes

Biotin-YVAD-amk
Biosyn, Ireland

DC protein assay 500-0116
Bio-Rad

Ac-DEVD-AMC
Bachem

DEVD-AFC
Enzyme Systems Inc

DEVD-CHO
Bachem

DEVD-pNA
Bachem

ECL
Amersham

HRP-conjugated anti-Ig antibody
Several commericial sources

Immobilon PSQ membrane
Millipore

35S Methionine 1,000 Ci/mMol
Amersham

Super Signal
Pierce

TNT Coupled Reticulocyte Lysate System
Promega

Ac-YVAD-AMC
Bachem

YVAD-AFC
Enzyme Systems Inc.

YVAD-CHO
Bachem

YVAD-pNA
Bachem
 

Appendix 3 (A3): Equipment
Fluorometer or 96-well plate reader

Spectrofotometer or 96-well plate reader

SDS-PAGE apparatus mod. Mini Protean II Biorad
or
SDS-PAGE apparatus mod. Mighty Small
Hoefer

Power supply

Gel dryer

Autoradiography cassette

 Plotting apparatus