The hypothesis that the expression of heat shock proteins following a preliminary hyperthermic treatment is responsible for subsequent thermotolerance to a second heat treatment is examined. CHO cells were given a 12 min, 45 degrees C pretreatment and then incubated for varying intervals at 37 degrees C. The synthesis of certain intracellular proteins was monitored as a function of time post-incubation by using 35S-methionine incorporation as determined in SDS polyacrylamide gel electrophoresis. Cell survival was concurrently measured by challenging the cells with a second heat treatment (45 degrees C/27 min). Major heat shock proteins were observed at 68 000, 89 000 and 110 000 daltons. The synthesis of these proteins was significantly reduced in the presence of cyclohexamide. The total 35S-methionine incorporation into these proteins correlated well with the induction of survival resistance (thermotolerance). An approximate exponential relationship between survival and the amount of each of these proteins may occur. These and other heat shock proteins were also present, in a significantly reduced degree, in control (non-heat shocked) cells maintained under normal culture conditions at 37 degrees C. It is possible that heat shock proteins are responsible for the phenomenon of thermotolerance.
In this report we examine the effects of chronic anaerobic exposure and subsequent reoxygenation on protein synthesis patterns in Chinese hamster ovary cells. It is observed by two-dimensional gel electrophoresis (isoelectric focusing/NaDodSOW/PAGE) that the transition from an atmospheric environment to an anaerobic state transiently induces the major heat-shock proteins (at 68 and 89 kDa). As the period of anaerobiosis increases, this heat-shock induction disappears and a new set of proteins (at 76 and 97 kDa) is induced. By two-dimensional gel electrophoresis and partial proteolytic mapping, these new proteins, which are induced by anaerobic exposures exceeding 12 hr, are identical to 76 and 97 kDa (p76 and p97, respectively) proteins induced by extended periods of glucose deprivation (>14 hr) when oxygen is present. Furthermore, the induction of these proteins under anoxia occurs in the presence of glucose, and increasing the glucose content of the starting media does not affect the induction. When anaerobic p76 and p97 induced cells are returned to atmospheric oxygen, p76 and p97 are repressed, while the heat-shock proteins are again transiently induced. This work further suggests the importance of deprivation and release environments in controlling the expression of these two stress protein systems. It is suggested that their natural expression may be determined by comparable circumstances.In response to certain adverse environments, cells are known to express sets of proteins. While there have been several suggestions regarding the function of the induced proteins, they may be responsible for adjusting a cell to the new environment and, perhaps, in temporarily maintaining it once there. Hyperthermia and glucose deprivation are two well-recognized conditions that can elicit specific protein systems. Glucose deprivation has been shown to result in the induction of two major proteins of -97 and 76 kDa (1, 2), while hyperthermia is known to induce proteins of [68][69][70] 89, and 110 kDa (3,4). Anoxia is also an insult that has been shown to induce specific anaerobic shock proteins (5), and early studies in lower organisms have suggested that release from anoxia can elicit a heat-shock response (ref. 22; reviewed in ref. 6). Understanding the response of cells to temporary periods of anoxia has important applications in several areas of biology. In this report, we examine the effects of a transition to and release from a chronic anaerobic exposure on protein synthesis in Chinese hamster ovary cells. METHODSGlucose and Oxygen Deprivation. Chinese hamster ovary cells (CHO cells) were grown in Ham's F-10 medium containing 15% newborn calf serum (GIBCO). Cells (5 ml) were seeded at a density of 150,000 cells per ml to T25 flasks (Corning) and were used for experiments in a subconfluent state -48 hr later. To achieve glucose deprivation, 15% serum was added to F-10 medium prepared without glucose, resulting in a partially glucose deficient broth. During incubation, cells used the remaining glucose after...
This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.
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