Attenuated expression of 70-kDa heat shock protein in WI-38 human fibroblasts during aging in vitro

We examined the effects of cellular aging on the expression of the heat sho ck-inducible HSP70 gene in WI-38 diploid human fibroblasts serially passage d in vitro. The senescence of the cells was established by evaluating popul ation doubling level, cell density at confluency, and cell morphology along with the detection of senescence-associated beta-galactosidase activity (h istochemically detectable at pH 6), a reliable marker of aging in low-densi ty cultures. A marked decrease in the synthesis and accumulation of the ind ucible HSP70 protein was observed in serum-fed late passage cells exposed t o a severe heat shock (30 min at 45 degrees C) in comparison to early passa ge cells. However, the degree of HSF-DNA binding monitored by gel retardati on assay was similar in both early and late passage cells. Similarly, North ern blotting analysis indicated that comparable amounts of inducible HSP70 mRNA were present in the total RNA fraction, in the total polyadenylated RN A fraction, or in the nuclear polyadenylated RNA fraction extracted from bo th early and late passage cells. In contrast, much less inducible HSP70 mRN A was detected in the total cytoplasmic RNA fraction or in the polyadenylat ed cytoplasmic RNA fraction of late passage cells. Thus age-related differe nces in heat-induced HSP70 synthesis and accumulation observed in serum-fed WI-38 cells appeared to result from an impairment in the posttranscription al processing of the HSP70 mRNA at a level following the polyadenylation st ep and preceding translocation from the nucleus to the cytoplasm. When HF w ere serum deprived for 20 h before heat shock, the induction of HSP70 mRNA was less than 30% reduced in early passage cells in comparison to serum-fed cells; however, the level of HSP70 mRNA was markedly lover 80%) decreased in serum-deprived late passage cells. This result indicated that the presen ce of serum has a strong influence on heat shock-induced HSP70 gene express ion in human fibroblasts aging in vitro, (C) 1999 Academic Press.