Induction of HSP70 is associated with vincristine resistance in heat-shocked 9L rat brain tumour cells

Abstract

SummaryThe most prominent cellular changes in heat-shock response are induction of HSPs synthesis and reorganisation of cytoskeleton. Vincristine was used as a tool to evaluate the integrity of microtubules in 9L rat brain tumour cells recovering from heat-shock treatment. Cells treated at 45°C for 15 min and recovered under normal growing condition became resistant to vincristine-inflicted cytotoxicity and microtubule destruction. Among all HSPs, the level of HSP70 and the degree of vincristine resistance are best correlated. HSP70and tubulin were found to be associated with each other as they were co-immunoprecipitated by either anti-HSP70 or anti-p-tubulin monoclonal antibody. The current studies establish for the first time that HSP70can complex with tubulin in cells and this association may stabilise the organisation of microtubules thus protect the heat-treated cells from vincristine damage. These findings are noteworthy in combining hyperthermia and chemotherapy in the management of malignant diseases.Heat-shock proteins (HSPs) are a small set of proteins induced in cells subjected to supraoptimal temperature and other related physiological stresses (Lindquist & Craig, 1988;Schlesinger, 1990;Schlesinger et al., 1990; Morimoto et al., 1990). The synthesis of HSPs is suggested to be related to the development of thermotolerance in cells that survived the initial heat-treatment (Li & Mak, 1985;Hahn & Li, 1990;Black & Subjeck, 1990). The HSPs are highly conservative and usually termed by their apparent molecular weights. Three classes of major HSPs, i.e., HSPl10, 90 and 70 are commonly detected in mammalian cells (Lindquist & Craig, 1988). HSP70 exists as a family in most organisms and has been the most extensively studied. In rodent cells, this family consists of two closely related proteins (>80% homology at the amino acid level) which have been known as the constitutive form of HSP70 (also known as HSC70, designated as HSP72 hereafter because of its slightly higher molecular weight) and the inducible form of HSP70 (designated as the HSP70 hereafter) (Hightower & White, 1981;Lee et al., 1991). HSP72 is slightly heat inducible, constitutively expressed and found at higher levels in growing cells than in resting cells (Pelham, 1986). This protein was found to be multifunctional. It functions as clathrin uncoating ATPase (Chappell et al., 1986;Deluca-Flaherty et al., 1990) and as molecular chaperone that binds to nascent polypeptides and maintains them in unfolded states, to facilitate their intracellular translocation (Deshaies et al., 1988;Chirico et al., 1988) and/or to accelerate their proper folding and oligomerisation (Beckmann et al., 1990;Pelham, 1990). Recently, HSP72 has been shown to be involved the in vivo assembly of microtubules (Gupta, 1990). On the other hand, HSP70 is highly heat inducible and hardly detectable under normal conditions. Its function is suggested to be similar to that of HSP72 because of the high degree of homology between these two proteins (Lindquist & Craig, 1988). In additi...

Characterisation of a vindesine-resistant human small-cell lung cancer cell line

“…There is a possibility that the 68 kDa immunoprecipitated protein is the 68 kDa microtubule associated protein (Lim et al, 1984) or a heat-shock protein 70 (Lee et al, 1992). However, the change in the amount of the 68 kDa immunoprecipitated protein was not reproducible in several experiments.…”

The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

“…Alternatively, Hsp70 has also been shown to interact directly with microtubules. Expression of Hsp70 protects 9L rat brain cells from vincristine-induced apoptosis (Lee et al, 1992). At present we do not know the critical targets by which non-lethal heat stress protects EW36 B-cells, although the reduction of Bcl-2 phosphorylation suggests that protection occurs at a point relatively early in vincristine-induced signaling.…”

“…However, heat stress also activates cytoprotective pathways in cells mediated by the induction of several heat stress proteins that act as molecular chaperones stabilizing proteins against damage and facilitating the refolding of denatured proteins. In this context, non-lethal heat stress has been shown to be protective against apoptosis induction by some agents (Lee et al, 1992;Samali and Cotter, 1996).…”

“…Studies have shown that pre-treatment with limited heat stress protects cells against apoptosis induced by subsequent, more extensive, heat stress as well as by certain classes of drugs (Lee et al, 1992;Mosser et al, 1997;Samali and Cotter, 1996). There are several possible mechanisms by which non-lethal heat stress may protect cells against drug-induced apoptosis.…”

Characterisation of a navelbine-resistant bladder carcinoma cell line cross-resistant to taxoids

“…Moreover, molecular chaperone proteins play an essential role in the in vivo assembly of microtubules (Gupta, 1990). Some of these proteins have been reported to induce cell resistance to vinca alkaloids (Huot et al, 1991;Lee et al, 1992), and these newly synthesised proteins have been found to bind to tubulin in these resistant cells (Lee et al, 1992). We have tested the effect of cycloheximide, an inhibitor of protein synthesis, on the resistance of J82-NVB cells to navelbine.…”