Mizuno S, Ishii A, Murakami Y, Akagawa H
Department of Bioactive Molecules, National Institute of Health, Tokyo, Japan.
Cell Struct Funct. 1997 Feb;22(1):7-13. doi: 10.1247/csf.22.7.
When a clone of Chinese hamster ovary (CHO) cells transfected with a plasmid containing a luciferase reporter gene under the control of the human heat shock protein (hsp) 70 gene promoter was treated with cycloheximide during heat exposure at 42 and 43 degrees C for 15 to 100 minutes and then incubated at 37 degrees C after removal of cycloheximide, reporter gene expression was suppressed by the protein synthesis inhibitor only at small heat shock doses (i.e., heat shock of less than 40 minutes at 42 degrees C and 15 minutes at 43 degrees C). A similar stress dose-dependent suppression of reporter gene expression by cycloheximide was also demonstrated by treatment with sodium arsenite at 37 degrees C. However, dexamethasone-dependent reporter gene expression in a different CHO clone was not inhibited after the inducer treatment for different times in the presence of cycloheximide. In addition, synthesis of most cellular proteins (except for hsp) was not affected after heat shock treatment with cycloheximide. The results suggested that the cycloheximide inhibition of gene expression is specific to hsp gene expression induced by limited stress doses. Furthermore, a prior 42 degrees C heat shock treatment for 30 minutes induced a decreased responsiveness (tolerance) to a second 42 degrees C heat treatment for hsp gene expression, but tolerance did not develop in cells exposed to the first heat shock in the presence of cycloheximide. These results confirm previous findings that induction of hsp gene expression by stress is balanced by the severity of stress and rate of protein synthesis. They also support the proposed model of autoregulation of hsp gene expression by levels of free hsp70.
当用含有在人热休克蛋白（hsp）70基因启动子控制下的荧光素酶报告基因的质粒转染的中国仓鼠卵巢（CHO）细胞克隆，在42和43摄氏度热暴露15至100分钟期间用环己酰亚胺处理，然后在去除环己酰亚胺后于37摄氏度孵育时，报告基因表达仅在小热休克剂量（即，42摄氏度下少于40分钟和43摄氏度下15分钟的热休克）下被蛋白质合成抑制剂抑制。在37摄氏度用亚砷酸钠处理也证明了环己酰亚胺对报告基因表达的类似应激剂量依赖性抑制。然而，在环己酰亚胺存在下用诱导剂处理不同时间后，不同CHO克隆中地塞米松依赖性报告基因表达未被抑制。此外，用环己酰亚胺进行热休克处理后，大多数细胞蛋白质（hsp除外）的合成未受影响。结果表明，环己酰亚胺对基因表达的抑制对有限应激剂量诱导的hsp基因表达具有特异性。此外，42摄氏度热休克处理30分钟会导致对hsp基因表达的第二次42摄氏度热处理的反应性（耐受性）降低，但在环己酰亚胺存在下暴露于第一次热休克的细胞中未产生耐受性。这些结果证实了先前的发现，即应激诱导的hsp基因表达由应激的严重程度和蛋白质合成速率平衡。它们还支持了由游离hsp70水平对hsp基因表达进行自动调节的提出的模型。
