Decreased Expression of Heat Shock Protein 70 mRNA and Protein in WI-38 Human Fibroblasts Aging in ‘vitro C. CAMPANINI, P. G. PETRONINI, R. ALFIERI, AND A. F. BORGHETTI Istituto di Patologia Generale Universitadegli Studi di Parma Via Gramsci, 14 Parma, Italy Heat shock proteins (HSPs) are a family of highly conserved and universally expressed proteins that are induced in all organisms and cultured cells by hyperthermia and a variety of other stressors.’ It has been suggested that the function of induced HSPs is to prevent the damage caused to the cell by the accumulation of proteins whose structure and folding have been perturbed under stress conditions.* Recently it was proposed that under physiological conditions the 70-kD HSPs might act as molecular “chaperones” by participating in posttranslational protein assembly and transl~cation.~-~ Human diploid cell strains have a finite lifetime in vitro, and it has been suggested that senescence in culture reflects aging in vivo. Human diploid fibroblasts exhibit increased doubling time during the life span of the cell population, until they arrest g r ~ w t hIt. ~has also been shown that the expression of HSP70, the major stress-induced protein, is cell-cycle regulated? In addition, it is generally accepted that physiological aging is associated with a reduced response to stress and an accumulation of abnormal protein^.^ We examined the effect of cellular aging in vitro on HSP70 protein synthesis and accumulation and on HSP70 mRNA level in WI-38 human fibroblasts exposed to severe heat shock, 30 minutes at 45”C, and then incubated under recovery conditions. Our results indicate that exposure of WI-38 cells with high replicative potential to hyperthermia induces the synthesis of at least five HSPs. With aging in culture, however, both the concentrations and the number of induced HSPs were markedly decreased. To investigate if differences exist in the expression of HSP70, both inducible and constitutive isoforms, as a function of cell aging, the levels of these two HSPs were analyzed by Western blotting. This analysis revealed no difference in the level of constitutive HSP70 expressed by young and senescent cells. However, in senescent cells a decreased basal level and reduced synthesis of inducible HSP70 occur together with a delayed accumulation of this protein. The amount of mRNA encoding for the inducible HSP70 was also analyzed by Northern blotting. Hybridization was carried out using a plasmid containing a portion of the gene encoding the inducible HSP70. This analysis shows that the level of hybridizable mRNA is undetectable both in young and in senescent control cells. It also reveals a marked induction of mRNA 2 hours after heat shock. The amount of hybridizable mRNA was greatly reduced 4 hours later. This analysis also shows that at the peak of its induction, an attenuated level of mRNA encoding for HSP70 occurs in senescent WI-38 fibroblasts. In conclusion, our data indicate that in senescent cells the attenuated induction of HSP70 protein synthesis can be accounted for by a decrease in their mRNA level, 442

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and they are in agreement with the results obtained by Liu el d 8in a different cell strain and during long-lasting and mild hyperthermic treatment. REFERENCES

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