Abstract
We examined the effects of cellular aging on the regulation of heat shock gene expression in IMR-90 human diploid fibroblasts. Heat shock (42-43 degrees C) and canavanine (200-400 micrograms/ml) were used to evoke the heat shock response in these cells. We showed that heat shock induced the synthesis of proteins with apparent molecular weights of 98,000, 89,000, 78,000, 72,000, 64,000, 50,000 and 25,000, with heat shock protein (HSP) 89 and 72 being most prominent. Canavanine induced the synthesis of the four high molecular weight HSPs, particularly HSP 89 and HSP 78, without noticeably enhancing synthesis of the low molecular weight HSPs. We found that, while a similar series of HSPs were induced in the young and old cells, there was a marked decrease in the magnitude of this induction in the old cells. Using cells with defined population doubling levels, we observed a direct correlation of the inducibility of HSP synthesis and the replicative potential of the cells used. Analysis of the amount of translatable and hybridizable mRNA, by the methods of in vitro translation and Northern blot hybridization, demonstrated that the induction of HSPs synthesis can be accounted for by increases in their mRNA. Nuclear runoff transcription provided evidence that the decrease in inducible expression of the HSPs in aging IMR-90 cells was attributable to a transcriptional mechanism. This conclusion was substantiated by analysis of the hsp 70 promoter activity in transient expression assay of the hsp 70 promoter-chloramphenicol acetyltransferase construct. We propose that there is an age-associated dysfunction in the signaling mechanism of the heat shock response.
Highlights
64,000, 50,000 and 25,000, with heat shock protein structure, regulation, and function of the heat shock genes (HSP) 89 and 72 being most prominent
We showed that an incubation temperature of 42-43 "C gave an optimal induction of heat shock proteins (HSPs) in both the young and old cells without significantly affecting either the synthesis of other cellular proteins orcell viability
Of interest is the observation that canavanine significantly repressed the synthesis of actin and that this repression was more pronounced in the young cells than thatof the old cells.It may be noteworthy that thetime course of induction of HSP synthesis by canavanine was more protracted when compared with that of heat shock; a maximal induction of HSP synthesis was not observed until 21 h of incubation in the presence of 400 pg/ml canavanine in both the young and old IMR-90 cells
Summary
We examined the effects of cellular aging on the heat shock produces biological changes in living organisms regulation of heat shock gene expression in IMR-90 was first described by Ritossa (1962) andhas since been human diploid fibroblasts. 2) The transforming gene product crease in inducible expression of the HSPs in aging of ElA, which has been implicated in theimmortalization of IMR-90 cells wasattributableto a transcriptional primary cells in culture, was found to increase the transcripmechanism. This conclusion wassubstantiatedby tion of hsp 70 gene (Kao and Nevins, 1983; Nevins, 1982).
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