Hsp90 Inhibition Accelerates Cell Lysis

Abstract

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent-and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.The 90 kDa heat shock protein (Hsp90) 1 is a central part of a chaperone meshwork chaperoning a large number of substrate proteins (1-5). Besides being a partner of a large number of co-chaperones and substrates, Hsp90 binds to filamentous actin and tubulin (6 -8) and the involvement of the cytoskeleton in the traffic of Hsp90 substrates has also been demonstrated (5, 9). Together with other chaperones, like Hsp27 and Hsp70, Hsp90 is involved in cytoprotection (10 -12).Cell lysis is one of the most commonly used methods to test cellular integrity. Moreover, lysis rate anomalies (13,14) together with diffusional anomalies (15, 16) were used as important arguments for the organization of the cytoplasm. Since cellular integrity is preserved after a partial cell lysis to a large extent (13,14), partial lysis provides a highly sensitized, but still somewhat organized cellular system, where the contribution of various components to both the cytoplasmic organization and cellular stability can be studied.The original aim of the present study was to examine whether Hsp90 inhibition induces any change in the rate of cell lysis induced by mild detergent treatment or hypotonic shock. The rationale behind these experiments was to test, whether Hsp90, a cytoprotective chaperone, binding to "thousand-andone" substrates and other proteins is involved in the maintenance of cellular integrity (17), and whether its inhibition renders cells more "lysis-prone." The first experiments were very promising: geldan...

Evidence that hsp90 Is Involved in the Altered Interactions ofAcanthamoeba castellaniiVariants with Bacteria

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Live Cell Imaging of Outward and Inward Vesiculation Induced by the Complement C5b-9 Complex

“…Second line defense is provided by intracellular resistance mechanisms that are still not characterized. Elevation of intracellular Ca 2ϩ concentration (10,11), protein phosphorylation by PKC and ERK (12)(13)(14), synthesis of new proteins (15), altered lipid metabolism (16), and heat shock proteins (17,18) have all been implicated in protection. Cells protect themselves also by elimination of the MAC from the plasma membrane (19 -21).…”

Enhancement of complement-induced cell lysis: a novel mechanism for the anticancer effects of Hsp90 inhibitors

“…Interestingly, later experiments of the above studies [32] demonstrated that the geldanamycin-induced additional lysis of Jurkat cells strongly depended on the experimental conditions, namely, if cells were mildly shaken during the experiment or not. At this time the first results of geldanamycin-induced superoxide generation appeared.…”

“…In recent studies [31,32] we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin and novobiocin induced a significant acceleration of detergent-and hypotonic shock-induced cell lysis both in red blood cells and in the Jurkat T lymphoid cell line.…”

“…These results turned our attention to examine the contribution of superoxide-related versus Hsp-related events to diminished cellular integrity after Hsp90 inhibition. Using various Hsp90 inhibitors as well as newly developed anti-Hsp90 hammerhead ribozymes [32] we demonstrated that besides an increase in membrane fragility by geldanamycin-induced superoxides, inhibition or lack of Hsp90 alone also results in a compromised cellular integrity. The contribution of superoxide-induced lipid peroxidation and the bona fide Hsp90 inhibition to the enhanced cellular lysis was roughly 50% each, irrespectively from the fact, if we measured the extent of superoxide-mediated events by the addition of reducing agents or that of Hsp90-dependent events by ribozyme treatment [32].…”