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Effect of Cycloheximide or Puromycin on Induction of Thermotolerance by Heat in Chinese Hamster Ovary Cells: Dose Fractionation at 45.5°C¹

Radiation Oncology Research Laboratory, CED-200, University of California, San Francisco, California 94143

While studying the quantitative relationship between hyperthermia-induced heat shock proteins (HSPs) and thermotolerance (TT), we observed that heat induced a family of HSPs, particularly an HSP 70 family, that might be involved in the development of TT. When cells were heated for 10 min at 45.5°C, they became thermotolerant to a second heat exposure at 45.5°C, with a thermotolerance ratio of 5–6 at 10^-3 isosurvival at 12 h after heating. In parallel, during the 12-h interval, heat shock resulted in a 2-fold relative increase in the synthesis of three major HSP families (M_r = 110,000, 87,000, and 70,000). Rate of synthesis was expressed relative to total protein synthesis, as studied with one-dimensional polyacrylamide gels analyzed by counting radioactivity in selected protein bands. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. Furthermore, even though the development of TT was partially suppressed by treatment with cycloheximide (10 µg/ml) or puromycin (100 µg/ml) at concentrations that inhibited total protein synthesis by 96 or 99%, respectively, a family of HSP 70 was still preferentially synthesized. Nevertheless, when cells were labeled for 3 days, the total level of HSP families did not change either when TT developed after a triggering heat treatment or as the development of TT was partially inhibited by suppressing protein synthesis with cycloheximide or puromycin. Thus, TT could still occur when total levels of HSP families did not change and when synthesis of HSP families was less than in unheated control cells, which may imply that TT is unrelated to HSPs. However, the finding that the amount of TT increased with increased synthesis of both total protein and HSP families, as studied with different concentrations of cycloheximide or puromycin, suggests that heat-inducible proteins, in particular the observed preferential synthesis of the HSP 70 family, may be necessary for the development of TT.

¹ This research was supported by National Cancer Institute grants CA 31808 and CA 09215.

² To whom requests for reprints should be addressed, at Radiation Oncology Research Laboratory, CED-200, University of California, San Francisco, CA 94143.

Received 9/30/86. Revised 1/30/87. Revised 8/20/87. Accepted 8/25/87.

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