Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating.

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.