Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27‐kDa heat‐shock protein

Abstract

The Drosophila melanogaster small heat‐shock protein, hsp27 (Dhsp27) belongs to a family of polypeptides which shares a sequence related to α‐crystallin and which protect cell against heat shock. Dhsp27 accumulates following heat shock and, in absence of stress, in the central nervous system, imaginal discs and the gonads of the developing fly. Two internal and adjacent deletion mutants in the conserved α‐crystallin domain of Dhsp27 were constructed. Expression vectors containing either the coding sequence of Dhsp27 or that of the two deletion mutants linked to the Simian‐Virus‐40 late promoter were used to transfect monkey COS cells. The transient expression of Dhsp27 was found to decrease the sensitivity of COS cells to heat and hydrogen‐peroxide stresses as judged by Trypan‐blue staining and indirect immunofluorescence analysis. Using this rapid test, we observed that a deletion of 62 amino acids, which lies at the 5′ end of the conserved α‐crystallin domain and covers the first 41 amino acids of this region had only a weak effect on the protective activity of Dhsp27. This suggests that the N‐terminal half of the conserved α‐crystallin domain may not be essential for the protective activity of the small hsp. In contrast, Dhsp27 was no more active when the last 42 amino acids of the α‐crystallin domain were deleted. Biochemical fractionation and indirect immunofluorescence analysis indicated that the protective function of Dhsp27 was localized at the level of the nucleus.

Resistance of Ehrlich tumor cells to apoptosis can be due to accumulation of heat shock proteins

“…For example, involvement of HSP70 in protection of ischemic brain was suggested [11], whereas transient ischemia can result in apoptotic death of neurons [28]. Both HSP70 and HSP27 were found to be protective against necrosis caused by H20 2 and TNF [29,30]. At the same time, it was shown that low doses of oxidants are able to bring about apoptotic death [4,31], therefore, it is very probable that HSPs may perform an important protective function against oxidant-induced apoptosis.…”

Inhibition of heat shock protein 27 (HspB1) tumorigenic functions by peptide aptamers

“…Alternatively, for detection of Hsp27 dimers, cells were lysed in nonreducing SDS buffer without dithioerythritol. Cell lysates were then subjected to SDS-polyacrylamide gel electrophoresis (Laemmli, 1970), and immunoblots were performed as previously described (Mehlen et al, 1993). The detection of immunoblots was performed with the ECL system (Amersham Corp.).…”

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

“…The small HSP family (sHSP) exhibit a monomeric molecular mass ranging between 9 and 42 kDa (Arrigo and Landry, 1994). 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).…”