HSP27 inhibits cytochrome c‐dependent activation of procaspase‐9

Abstract

We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide-induced cytotoxicity by preventing apoptosis. As observed for Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage and procaspase-3 activation. In contrast with Bcl-2, HSP27 overexpression does not prevent etoposide-induced cytochrome c release from the mitochondria. In a cell-free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase-3 in both control and bcl-2-transfected U937 cells but fails to activate procaspase-3 in HSP27-overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP-mediated activation of procaspase-3. Overexpression of HSP27 also prevents procaspase-9 activation. In the cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis by preventing cytochrome c and dATP-triggered activity of caspase-9, downstream of cytochrome c release.

Differential regulation of HSP27 oligomerization in tumor cells grown in vitro and in vivo

“…Interestingly, by overcoming phosphorylation-driven dissociation of larger oligomers, cell growth at con¯uence in vitro and in vivo restored the ability of HSP27 to inhibit cell death. We have recently shown that HSP27 could interfere with the ®nal pathway to apoptotic cell death by preventing procaspase-9 and procaspase-3 activation, downstream of cytochrome c release from the mitochondria (Garrido et al, 1999). Accordingly, the wild type HSP27 and the alanine mutant, that forms small and large oligomeric structures, but not the aspartate mutant that only forms small oligomers, prevented caspase-3 and -9 activation induced either by exposure of cultured cells to cisplatin or by treating cell-free extracts with cytochrome c and dATP.…”

“…We have recently shown that overexpression of wild type HSP27 inhibited etoposide-induced apoptosis by preventing cytochrome-c mediated activation of caspase-9 and caspase-3 in the U937 human leukemic cell line (Garrido et al, 1999). To determine whether this inhibition could be con®rmed in REG cells, we used a cell-free system (Garrido et al, 1999).…”

“…To determine whether this inhibition could be con®rmed in REG cells, we used a cell-free system (Garrido et al, 1999). Addition of cytochrome c (10 mM) and dATP (1 mM) to acellular extracts from control-transfected REG cells induced a time-dependent cleavage of DEVD-AFC and LEHD-AFC substrates ( Figure 3B).…”

“…Immunoblots were performed as described (Garrido et al, 1999). Brie¯y, whole cell extracts were prepared by lysing the cells with 2% SDS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , and NaCl/P i (pH 7.4) at 688C for 5 min.…”

“…Their overexpression enhances the survival of mammalian cells exposed to a variety of death stimuli including heat (Landry et al, 1989), oxidative stress (Hout et al, 1991;Mehlen et al, 1993), anticancer drugs (Hout et al, 1991;Garrido et al, 1996Garrido et al, , 1997, TNF-a (Mehlen et al, 1995a), anti-Fas agonistic antibodies and staurosporine (Mehlen et al, 1996a). The biochemical mechanisms proposed to account for the sHSP protective e ect include molecular chaperoning of non-native proteins (Jakob et al, 1993) or capping/decapping enzymes (Lavoie et al, 1993(Lavoie et al, , 1995, modulation of the cellular detoxifying machinery to cope with oxidative stress (Mehlen et al, 1996b) and interaction with the cytochrome c-mediated pathway of apoptosis by preventing caspase-9 activation (Garrido et al, 1999).…”