Hsp90 Inhibition Accelerates Cell Lysis: ANTI-Hsp90 RIBOZYME REVEALS A COMPLEX MECHANISM OF Hsp90 INHIBITORS INVOLVING BOTH SUPEROXIDE- AND Hsp90-DEPENDENT EVENTS

Amere Subbarao Sreedhar,Katalin Mihály,Bálint Pató,Tamás Schnaider,Attila Steták,Katalin Kis-Petik,Judit Fidy,Tibor Simonics,Anna Maráz,Péter Csermely

doi:10.1074/jbc.m301371200

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.

Highlights

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells
Cell lysis after hypoxia and complement attack was 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
One of the major findings of the present report is that the Hsp90 inhibitor, geldanamycin has a dual action in damaging cellular integrity: roughly half of its effects come from an accelerated superoxide production, but the other half of increased cellular fragility is a direct consequence of diminished Hsp90 function

Summary

Introduction

The 90 kDa heat shock protein, Hsp, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. The original aim of the present study was to examine whether Hsp 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 Hsp, 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.”. Later experiments demonstrated that the extent of geldanamycin-induced enhancement of cell lysis was dependent on the experimental conditions, namely, if cells were shaken during the experiment or not At this time the first results of geldanamycin-induced superoxide generation appeared [20, 21].

Objectives

Results

Conclusion