Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c

Abstract

A cell-free system based on cytosols of normally growing cells is established that reproduces aspects of the apoptotic program in vitro. The apoptotic program is initiated by addition of dATP. Fractionation of cytosol yielded a 15 kDa protein that is required for in vitro apoptosis. The absorption spectrum and protein sequence revealed that this protein is cytochrome c. Elimination of cytochrome c from cytosol by immunodepletion, or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Adding back cytochrome c to the cytochrome c-depleted extracts restored their apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.

Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3

“…Whereas other studies have demonstrated that release of cyt c from mitochondria induces a cas-3-mediated apoptotic cascade (Li et al, 1997;Liu et al, 1996;Zou et al, 1997), we asked if heat-shock treatment inhibits cyt cmediated activation of cas-3. As demonstrated previously for other cell types, expression of Hsp27 was induced in 293T cells at 6 ± 8 h after mild heat shock (Figure 1a).…”

“…Previous studies have demonstrated that release of cyt c from mitochondria to cytosol is an important step in inducing cas-3-mediated apoptosis (Li et al, 1997;Liu et al, 1996;Zou et al, 1997). To assess whether Hsp27 functions upstream or downstream to cyt c release from mitochondria, L929/pRc and L929/Hsp27 cells were treated with various agents, such as methylmethane sulphonate (MMS), 1-b-D-arabinofuranosyl-cytosine (ara-C) or staurosporine (STSP), that are known to cause the release of mitochondrial cyt c (Kharbanda et al, 1997a), and cytosols were analysed by immunoblotting (IB) with anti-cyt c. The results demonstrate that MMS treatment of both L929/pRc and L929/Hsp27 cells is associated with the release of cyt c ( Figure 3a, upper panel).…”

“…Cyt c forms a complex with Apaf-1 and cas-9, and is necessary at least in part for activation of cas-3 (Li et al, 1997;Liu et al, 1996;Zou et al, 1997). Since Hsp27 functions downstream to cyt c, Apaf-1 and cas-9, we next determined whether Hsp27 associates with cas-3 and thereby inhibits its activity.…”

“…Moreover, Hsp27 functions as an inhibitor of cas-3 activation by associating with and limiting the availability of cas-3 for cleavage by active cas-9. The release of cyt c from mitochondria is induced by exposure to STSP, diverse DNA damaging agents and ischemia (Kharbanda et al, 1997a;Kluck et al, 1997;Li et al, 1997;Liu et al, 1996;Yang et al, 1997;Zou et al, 1997;Narula et al, 1999). Other studies have shown that cyt c release is inhibited by the anti-apoptotic Bcl-2 and Bcl-x L proteins (Kharbanda et al, 1997a;Yang et al, 1997).…”

Dissociation of DNA damage and mitochondrial injury caused by hydrogen peroxide in SV-40 transformed lung epithelial cells

“…In certain situations, death stimuli induce an increase in the permeability of the outer mitochondrial membrane that releases apoptosis inducers, such as cytochrome c [39] and apoptosis-inducing factor (AIF) [40]. In our study, the ΔΨm was quantified by the flow cytometric analysis of JC-1-stained cells.…”