Possible involvement of ubiquitin function and ATP requirement in the development of thermotolerance in mammalian cells

Thermotolerance under chronic exposure to moderate hyperthermia at 41 degrees C was hardly induced in the mouse temperature-sensitive mutant ts85 cells, in contrast to the parental wild-type FM3A cells. Thermotolerance was induced at a reduced level in the mutant cells compared with the wild-type cells by incubation at 33 degrees C (permissive temperature), but not at 39 degrees C (non-permissive temperature), after a brief exposure at 44 degrees C. Under conditions where protein synthesis was inhibited by cycloheximide at 41 degrees C, significant amounts of thermotolerance developed in FM3A cells. FM3A cells depleted of cellular ATP by treatment with 2.4-dinitrophenol and 2-deoxyglucose were not sensitized to thermal cell killing at 44 degrees C, when drug-treated cells were washed and exposed to hyperthermia in drug-free growth medium, where cellular ATP rapidly recovered. However, the cells deprived of ATP under the treatment at 41 degrees C failed to develop thermotolerance, indicating a requirement of ATP for thermotolerance development. The decay of thermotolerance was not affected by ATP levels after it was developed. The degradation of abnormal cellular proteins which contained amino acid analogues was promoted at 33 degrees C relative to normal protein degradation in FM3A and ts85 cells. Both normal and abnormal proteins were degraded at a reduced rate at 43 degrees C. Pretreatment of cells at 41 degrees C decreased the rate of degradation of abnormal proteins at 33 degrees C by 20% in FM3A cells and by about 100% in ts85 cells. Pretreatment of cells at 41 degrees C increased significantly the conjugation of 125I-labeled ubiquitin to cellular endogenous proteins in extracts of FM3A cells, but decreased the conjugation in extracts of ts85 cells. The data presented here, in conjunction with the observations by others that the ts85 cell is a mutant defective in the ubiquitination of cellular proteins at nonpermissive temperatures, suggest that the ATP-dependent ubiquitination may be crucial for the development of thermotolerance.