Caspases,Paracaspases, and Metacaspases Methods and Protocols

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Several theoretical models have been postulated aiming to clarify

the molecular mechanism by which initiator caspases become acti-

vated [ 3 ]. Although these models still suffer from lack of support-

ive experimental evidences it is clear that initiator zymogens possess

intrinsic enzymatic activity generated from clustering or dimeriza-

tion per se, which in turn allows for autoprocessing of the fi rst

caspase in the apoptotic cascade. In summary, irrespectively of cas-

pase type analyzed, detection of processed fragments by western

blotting is undoubtedly a strong indication for enzymatic activity.

On the other hand, the absence of these fragments may not neces-

sarily mean that an initiator caspase is catalytically inactive.

As an alternative to SDS-PAGE, active caspases can also be visual-

ized by immunocytochemistry (Fig. 2 ). However, to date, only a

limited number of antibodies suitable for immunostaining are

available for specifi c detection of active caspases.

Execution of apoptotic cell death is mediated by caspase cleavage of

various vital proteins. To date, there are more than 500 caspase

substrates presented in a continuously growing list [ 4 , 5 ]. Several of

these substrates have a characterized function in propagation of the

1.2 Immuno-
cytochemical
Detection of Active
Caspases

1.3 Immunoblot
Analysis of Caspase
Substrate Cleavage

Fig. 2 Detection of processed caspase-3 in formaldehyde-fi xed cells by immunocytochemistry. HCT116 cells
were cultured on coverslips in the absence and in the presence of 5-FU (768 μM) for 24 h. Immunodetection
of active caspase-3 was then performed as outlined in the main text (60 × magnifi cation)

Caspases in Mammalian Cell Cultures