This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Freeman, M. L. Articles by Meredith, M. J. Search for Related Content PubMed PubMed Citation Articles by Freeman, M. L. Articles by Meredith, M. J.

Modulation of Diamide Toxicity in Thermotolerant Cells by Inhibition of Protein Synthesis¹

Vanderbilt Center for Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 [M. L. F.], and Department of Biochemistry, School of Dentistry, Oregon Health Science University, Portland, Oregon 97201 [M. J. M.]

Chinese hamster ovary cells were exposed in vitro to various concentrations of diamide for 1 h at 37°C. This treatment resulted in a dose dependent increase in cytotoxicity. Cells were also heated at 43°C for 15 min, incubated at 37°C for 3 h, and then exposed to various concentrations of diamide. This heat shock has been shown previously to trigger the synthesis of heat shock proteins and the development of thermotolerance. Further, under these experimental conditions both were inhibited if protein synthesis was inhibited by exposure to cycloheximide (M. L. Freeman et al., Radiat. Res., 112: 195–203, 1987). Diamide toxicity was diminished in cells made thermotolerant by the 43°C/15-min heat shock. For example, at the highest dose used, 0.8 mM, survival increased from 0.93% to 6.1%. However, diamide toxicity was unaffected if the cells were exposed to diamide 3 h after a 43°C/60 min heat shock. This latter heat shock produced significant inhibition of protein synthesis whereas the 15-min heat shock did not (M. L. Freeman et al., Cancer Res., 48: 7033–7037, 1988). Further, a 43°C/15-min heat shock did not confer protection against diamide toxicity if the cells were simultaneously exposed to cycloheximide.

Exposure to 0.8 mM diamide was shown to oxidize specific cellular proteins as measured by 2-dimensional thiol blotting. However, the degree of protein thiol modification was not affected by a prior heat shock. Nor did the heat shock increase the intracellular concentration of glutathione or the activity of glutathione reductase. The diamide treatment caused specific, as opposed to general, protein thiol oxidation and heat shock did not prevent this.

It is hypothesized that it was the oxidation of protein thiols which led to cellular toxicity. Protein synthesis, triggered by heat shock, protected cells from the diamide toxicity without preventing protein thiol modification. These results suggest that the proteins synthesized after heat shock can provide protection against the consequences of aberrant proteins produced by thiol oxidation.

¹ This investigation was supported in part by USPHS/NIH Grants Ca 38079, ES 03272, and ES 00267.

² To whom requests for reprints should be addressed, at E-1200 MCN, Radiation Biology, Vanderbilt University Hospital, Nashville, TN 37232.

Received 7/29/88. Revised 2/15/89. Accepted 5/17/89.