Alterations in specific and general protein synthesis after heat shock in heat-sensitive mutants of CHO cells and their wild-type counterparts.
The rates of general and specific protein synthesis were studied in two heat-sensitive strains of CHO cells (Harvey and Bedford, Radiat. Res. 113, 526-542, 1988), both of which show a reduced ability to develop thermotolerance following an initial 45 degrees C heat shock. After various labeling periods with [35S]methionine, wild-type and mutant labeled proteins were separated by one- and two-dimensional polyacrylamide gel electrophoresis. Autoradiograms showed differences in levels of synthesis of several proteins after a 45 degrees C heat shock. In particular, these were in the hsp-70 group referred to as hsp-70a, b, and c, having molecular weights of 76, 73, and 72 kDa and isoelectric focusing pH values of 5.7, 5.5, and 5.7, respectively. Of particular note were changes in the hsp-70c region of the autoradiograms. We found that there was perhaps a low level of synthesis of hsp-70c in unheated wild-type cells but none was detectable in the mutant lines. After an isosurvival (approximately 10%) pulse of 45 degrees C heat there was a gradual increase in the synthesis of hsp-70c for wild-type but a smaller increase for the heat-sensitive strain 36 (HS-36) cells. In contrast, for HS-23 cells there was a very large initial increase by 5 to 7 h after the heat pulse and then a rapid decrease to undetectable levels by 11 to 13 h. The inhibition and recovery of general protein synthesis for both mutant and wild-type cells was also measured following various heat treatments at 45 degrees C. We observed that inhibition and resumption to a "normal" rate of protein synthesis for HS-23 cells paralleled the same response observed for the wild-type 10B2 cells. In sharp contrast, the time for recovery from the inhibition of protein synthesis for HS-36 cells was severely reduced for all heating times tested. Our results show that the period of delay before resumption of protein synthesis after heating does not always correlate with heat sensitivity or the degree of thermotolerance development. Several explanations for these observations are possible. One is that while synthesis of certain heat-shock proteins may indeed be responsible for the development of thermotolerance, the timing of the synthesis of these proteins in relation to the period of inhibition of general protein synthesis is crucial to such development.(ABSTRACT TRUNCATED AT 400 WORDS)
Harvey
W F
WF
Department of Radiology and Radiation Biology, Colorado State University, Fort Collins 80523.
Bedford
J S
JS
Li
G C
GC
D000064:Acclimatization / Q000502:physiology*; D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006358:Hot Temperature; D066298:In Vitro Techniques; D009154:Mutation
D006360:Heat-Shock Proteins