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
review the literature describing the application of cell-free systems
- some examples of the successful application of cell-free systems in the elucidation and dissection of biochemical mechanisms during the apoptotic process discuss a recent paper, studying the cytochrome c-initiated Caspase Cascade with the title: " Ordering the Cytochrome c-initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9-dependent Manner, Slee et al., 1999, J. Cell Biol., 144(2): 281-292
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:
- Use cytoplasmic extracts alone:
activate cytoplasmic extracts with e.g. cytochrome c and examine caspase-activation or other soluble factors
- Cytoplasmic extracts + nuclei
activate extracts and observe apoptotic changes in the nuclei, e.g. DNA fragmentation, chromatin condensation, cleavage of nuclear proteins (e.g. PARP, fodrin, lamin)
- Cytoplasmic extracts + mitochondria
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".
- Lazebnik et al. were actually studying chromosome condensation during mitosis. They used cytoplasmic cell extracts from (aphidicolin-treated) mitotic chicken hepatoma cells, so called S/M extracts. As found later, aphidicolin- treated cells were in a state of latent apoptosis due to a Apoptosis Promoting Activity (APA).
- Isolated HeLa nuclei, incubated in the S/M extracts at 37°C, displayed a dramatic morphological changes: chromatin condensation around the nuclear periphery, shrinking, peripheral chromatin formed condensed masses, which finally blebbed off as pebble like bodies ("apoptotic bodies"?)
- The morphology of nuclei undergoing apoptotic program in vitro and in situ was very similar
- Nuclei in the S/M extracts also displayed the typical internucleosomal DNA fragmentation ("laddering"), effect of divalent cations
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"
- Human T-cell lines were induced to undergo apoptosis by UV irradiation or Fas-ligation
- cell extracts were prepared from the stimulated cells (only few cells should exhibit apoptotic features at the time of extract preparation) and isolated nuclei were incubated in those extracts (source of nuclei is not important)
- As a result, nuclei in extracts from UV or Fas-activated cells displayed the typical morphological changes and internucleosomal DNA fragmentation.
- Interestingly, upon addition to the activated extracts, Bcl-2 partially prevented nuclear apoptosis in the cell-free system (reported in both papers)!
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:
- Ordering the caspase-cascade (e.g.Orth et al., 1996, JBC, 271(35): 20977-80, and Pan et al., 1998, FEBS, 426: 151-154, and Slee etal., 1999, J. Cell Biol., 144(2): 281-292, and Sasada et al., 1997, Oncogene, 14:2741-52)
- Determination of caspase-substrate-specificity (e.g. Takahashi et al, 1996, PNAS, 93: 8395-400)
- Mechanistic studies of caspase-activation (Qin et al., 1999, Nature, 399: 49-557)
- Inhibitor studies (e.g. Mesner et al., 1999, JBC, 274(32): 22635-45, and Roy et al., 1997, EMBO, 16: 6914-25, and Cosulich et al., 1999, Curr. Biol., 9(3):147-150)
- Identification and characterization of factors involved in the apoptotic machinery, e.g. cytochrome c/dATP (Liu et al., 1996, Cell, 86: 147-57), ICAD (Enari et al, 1998, Nature, 391: 43-50, Sakahira et al., 1998, Nature, 391: 96-99), AIF (Fulda et al., 1998, JBC, 273(51): 33942-948, and Susin et al., 1999, Nature, 397: 441-446), and Acinus (Shara et al., 1999, Nature, 401: 168-172).
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
- they generated [35S]methionine-labeled caspases-1,-2,-3,-4,-5,-6,-7,-8,-9, and -10 by in vitro transcription/translation.
- added labeled caspases to cytochrome c-activated Jurkat extracts and monitored the processing of those caspases: all caspases were processed, except the ICE subfamily proteases (capases-1, -4, and -5).
- time-courses reveal comparably late processing of caspases-8 and -10 in comparison to caspases-2, -3, -6, -7, -9.
- Pulldown experiments using immobilzed GST-APAF-1 fusion-protein demonstrated that Apaf-1 exclusively interacts with caspase-9
- Immunodepletion of caspase-9 from the Jurkat extract abrogated the processing of all caspases in response to cytochrome c.
- Inhibitor studies with zDEVD-CHO and GS-CrmA sugested that caspases-8 and -10 are processed downstream from all other caspases and that caspases-3 and -7 are the caspases which are first activated by caspase-9.
- Depletion of caspase-3 from the extracts ablated cytochrome c-induced processing of caspases-2, -6, -8, and -10 but not caspase-7
- Depletion of caspase-7 did not have any effect on caspase-processing, whereas depletion of caspase-6 from the extracts resulted in a lack of caspase-8 and -10 processing.
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:
- Mesner et al. compared the subsets of activated caspases in cytochrome c activated HL-60 extracts with those from Etoposide-treated HL60 cells using a technique called affinity labeling.
- The subset of caspases activated was comparable in vitro and in situ (caspases-3, -6, -7), but there were differences in activated species of, e.g. caspase-3, possibly in terms of the phosphorylation status.