INTEGRITY OF INTERMEDIATE FILAMENTS IS ASSOCIATED WITH THE DEVELOPMENT OF ACQUIRED THERMOTOLERANCE IN 9L RAT-BRAIN TUMOR-CELLS

Withangulatin A (WA), a newly discovered withanolide isolated from an antitumor Chinese herb, has been shown to be a vimentin intermediate f ilament-targeting drug by using immunofluorescence microscopy. Togethe r with cytochalasin D and colchicine, these drugs were employed to inv estigate the importance of vimentin intermediate fi laments, actin fil aments, and microtubules in the development of acquired thermotoleranc e in 9L rat brain tumor cells treated at 45 degrees C for 15 min (prim ing heat-shock). Acquired thermotolerance was abrogated in cells incub ated with WA before the priming heat-shock but it could be detected in cells treated with WA after the priming heat-shock. In contrast, cyto chalasin D and colchicine do not interfere with the development of the rmotolerance at all. The intracellular localizations of vimentin and t he constitutive heat-shock protein 70 (HSC70) in treated cells were ex amined by using immunofluorescence microscopy and detergent-extractabi lity studies. In cells treated with WA before the priming heat-shock, vimentin Ifs were tightly aggregated around the nucleus and unable to return to their normal organization after a recovery under normal grow ing conditions. In contrast, the IF network in cells treated with WA a fter the priming heat-shock was able to reorganize into filamentous fo rm after a recovery period, a behavior similar to that of the cells tr eated with heat-shock only. HSC70 was found to be co-localized with vi mentin during these changes. It is suggested that the integrity of int ermediate filaments is important for the development of thermotoleranc e and that HSC70 may be involved in this process by stabilizing the in termediate fi laments through direct or indirect binding. (C) 1995 Wil ey-Liss, Inc.