During 4 hr after puromycin (PUR: 20 pg/ml) treatment, the synthesis of three major heat shock protein families (HSPs: M, = 110,000,87,000, and 70,000) was enhanced 1.5-fold relative to that of untreated cells, as studied by one-dimensional gel electrophoresis. The increase of unique HSPs, if studied with twodimensional gels, would probably be much greater. In parallel, thermotolerance was observed at isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5OC or 43OC, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 pg/mI) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 pglml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01-10 pg/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 pglml) treatment, the addition of cycloheximide (CHM: 10 pg/mI), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families.
This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43OC in the presence of C H M or PUR immediately after a 2-hr pretreatment with C H M or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1-2 hr, and did not have any obvious relationship to the synthesis of HSP familes. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after C H M or PUR treatment immediately before and during heating at 43OC, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5OC caused by heating at 43OC after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR.
The protective effect of cycloheximide (CHM) or puromycin (PUR) on hyperthermic killing of mammalian cells has been reported (Palzer and Heidelberger, 1973;Lin et al., 1984;Lee and Dewey, 1986) although the mechanism of heat protection by drug i s s t i l l unknown. However, Lee et al. (1987) suggest that heat protection caused by treatment with C H M or PUR before and during heat apparently involves a common mode of action not associated w i t h either changes in total levels of heat shock protein (HSP) families or synthesis of HSP families during drug treatment before and during heat. Nevertheless, Hightower (1980) found that intermediate concentrations of PUR pretreatment caused the stimulation of the synthesis of several polypeptides (110, 88, 72,71, and 23 kDa) which appeared to be the same as a set of HSPs. Furthermore, several researchers (Perlman