Henle K J, Nagle W A
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock.
Exp Cell Res. 1991 Oct;196(2):184-91. doi: 10.1016/0014-4827(91)90249-t.
Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.
哺乳动物细胞在急性高温热休克后，对38 - 43摄氏度的高温表现出更高的敏感性；这种现象被称为降温加热（SDH）效应。我们以细胞存活为终点，表征了SDH效应对以下方面的影响：（1）主要热休克蛋白HSP110、90、72/70、60的合成（35S - 氨基酸标记），（2）热诱导的蛋白质糖基化（3H - D - 甘露糖标记），以及（3）耐热性表达。分别检测了标记物在可溶性和不溶性细胞组分之间的分配情况。急性（10分钟，45摄氏度）和慢性（1 - 6小时，41.5摄氏度）高温均增加了高分子量热休克蛋白（HSP110、90和72/70）的合成，主要是在可溶性胞质溶胶组分中。与单独的慢性高温相比，SDH（10分钟，45摄氏度 + 1至6小时，41.5摄氏度）完全抑制了HSP110的标记，部分抑制了HSP90的标记，而对HSP72/70的合成几乎没有影响。在细胞存活水平上，SDH使41.5摄氏度时的细胞杀伤率提高了七倍，但仅使耐热性表达降低了两倍。这表明SDH致敏不是由HSP72/70合成的变化引起的，也不仅仅是由耐热性的抑制导致的。急性和慢性高温后，35S标记的HSP60和HSP50主要存在于细胞沉淀组分中。SDH完全抑制了HSP60和HSP50的35S标记。SDH也完全抑制了用3H - D - 甘露糖对GP50的标记。此外，SDH逐渐降低了N - 乙酰半乳糖胺基转移酶的活性。数据表明，SDH引起的热致敏伴随着热休克蛋白合成和蛋白质糖基化的复杂且具有选择性的抑制模式。对HSP110、HSP60和HSP50/GP50标记的深度抑制表明，这些可能与SDH致敏机制有关。
