Application of Cell-free Systems in Apoptosis Research

Some Facts about Cell-free systems, prepared for the

Apoptosis Group's "Journal / Informal Data Club"

12. October 1999 - University of Iowa, Iowa City, IA








Cells studied in living organisms = in vivo


Cells studied in culture = in situ


Cell-free system = in vitro

In a Cell-free system biological processes are studied using fractions of disrupted cells, e.g. cytoplasmic extracts or isolated organelles such as cell nuclei or mitochondria. The different isolated components can be combined in different ways:


activate cytoplasmic extracts with e.g. cytochrome c and examine caspase-activation or other soluble factors

activate extracts and observe apoptotic changes in the nuclei, e.g. DNA fragmentation, chromatin condensation, cleavage of nuclear proteins (e.g. PARP, fodrin, lamin)

Activate cytoplasmic extracts and observe changes in mitochondrial transmembrane potential, or cytochrome c release. Or induce mitochondria to release apoptogenic factors which induce activity in extracts

study mitochondria-derived apoptogenic factors and their effect on isolated nuclei



One of the first report about the observation of the apoptotic process in vitro:


Lazebnik et al., 1993, J. Cell Biol., 123(1): 7-22.

"Nuclear Events of Apoptosis in Vitro in Cell-free Mitotic Extracts: A Model System for Analysis of the Active Phase of Apoptosis".





Cell-free extract systems can be used as models for the active phase of apoptosis and provide a tool for the identification of factors involved in the apoptotic process.



Cell-free apoposis was then also described in Xenopus egg extracts in which the presence of heavy membrane fractions (mitochondria) were essential for the apoptotic activity: Newmeyer et al., 1994, Cell, 353-364.


A cell-free system for the study human cell apoptosis was described for the first time by


Martin et al., 1995, EMBO, 14(21): 5191-5200

"Cell-free reconstitution of Fas-, UV radiation- and ceramide-induced apoptosis" and


Enari et al., 1995, EMBO, 14(21): 5201-5208

"Apoptosis by a cytosolic extract from Fas-activated cells"



For Bcl-2 in cell-free systems see also : Cosulich et al., 1999, Curr. Biol., 9(3):147-150: "Bcl-2 regulates amplification of caspase activation by cytochrome c."

A PubMed search for 'cell-free system' and 'apoptosis' revealed 140 hits (Oct. 1999).


Cell-free systems have been used for:




Slee et al., 1999, J. Cell Biol., 144(2): 281-292

Ordering the Cytochrome c-initiated Caspase Cascade:

Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9-dependent Manner





Apaf-1 activates caspase-9. Caspase-9 activates caspases-3, and -7. Caspase-3 activates caspases-2 and -6. Caspase-6 activates caspapses-8 and -10.


The results of this nice paper are not totally consistent with results published e.g. by Orth et al, 1996, JBC, 271(35): 20977-80, and Mesner et al., 1999, JBC, 274(32): 22635-45. Orth et al. were also using Jurkat extracts to which they added active recombinant caspases and observed the cleavage of endogeneous caspases: in their case, recombinant caspase-6 was able to cleave endogeneous caspases-3 and -9, but recombinant caspase-9 did not cleave caspases-3- annd -6.

Mesner et al. were studying the cytochrome c-initiated caspase cascade in cell extracts from HL-60 cells: they observed activation of caspasess-3, -6, and -7, but not caspases-2, and -8. Mesner et al. discussed this difference as a cell type-deendent selectivity of activation pattern.

Do cell-free conditions reflect the processes in situ (or even in vivo) ?

Have a look at the paper of Mesner et al., 1999, JBC, 274(32): 22635-45: