Attenuated expression of 70-kDa heat shock protein in WI-38 human fibroblasts during aging in vitro
Attenuated expression of 70-kDa heat shock protein in WI-38 human fibroblasts during aging in vitro
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DOI：
10.1006/excr.1999.4614
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发表时间：
1999-10-10
影响因子：
3.7
通讯作者：
Borghetti, AF
中科院分区：
文献类型：
--
作者：
Bonelli, MA;Alfieri, RR;Borghetti, AF
我们研究了细胞老化对体外连续传代的二倍体人成纤维细胞中热休克诱导的HSP70基因表达的影响。通过评估种群倍增水平、融合时的细胞密度和细胞形态以及衰老相关的β-半乳糖苷酶活性(在pH 6时组织化学可检测到的)来确定细胞的衰老。在暴露于严重热休克(45℃，30min)的传代晚期细胞中，观察到血清喂养的传代晚期细胞与传代早期细胞相比，诱导的HSP70蛋白的合成和积累明显减少。然而，凝胶延迟实验监测的HSF-DNA结合程度在早期和晚期传代细胞中是相似的。同样，Northern blotting分析表明，从早期和晚期细胞中提取的总RNA组分、总多腺化RNA组分或核多腺化RNA组分中都存在类似数量的可诱导HSP70 mRNA。相反，在传代后期细胞的总胞浆RNA组分或多腺化胞浆RNA组分中检测到的可诱导HSP70mRNA要少得多。因此，在血清喂养的WI-38细胞中观察到的与年龄相关的热诱导HSP70合成和积累的差异似乎是由于HSP70 mRNA转录后加工的损害，该水平发生在聚腺苷化步骤之后和从细胞核到细胞质的转位之前。热休克前去血清20h，早期传代细胞HSP70mRNA的表达较有血清培养的细胞减少不到30%，而无血清培养的晚期细胞HSP70mRNA的表达水平明显降低(>80%)。这一结果表明，血清的存在对热休克诱导的体外老化的人成纤维细胞中HSP70基因的表达有强烈的影响，(C)1999学术出版社。
We examined the effects of cellular aging on the expression of the heat shock-inducible HSP70 gene in WI-38 diploid human fibroblasts serially passaged in vitro. The senescence of the cells was established by evaluating population doubling level, cell density at confluency, and cell morphology along with the detection of senescence-associated beta-galactosidase activity (histochemically detectable at pH 6), a reliable marker of aging in low-density cultures. A marked decrease in the synthesis and accumulation of the inducible HSP70 protein was observed in serum-fed late passage cells exposed to a severe heat shock (30 min at 45 degrees C) in comparison to early passage cells. However, the degree of HSF-DNA binding monitored by gel retardation assay was similar in both early and late passage cells. Similarly, Northern blotting analysis indicated that comparable amounts of inducible HSP70 mRNA were present in the total RNA fraction, in the total polyadenylated RNA fraction, or in the nuclear polyadenylated RNA fraction extracted from both early and late passage cells. In contrast, much less inducible HSP70 mRNA was detected in the total cytoplasmic RNA fraction or in the polyadenylated cytoplasmic RNA fraction of late passage cells. Thus age-related differences in heat-induced HSP70 synthesis and accumulation observed in serum-fed WI-38 cells appeared to result from an impairment in the posttranscriptional processing of the HSP70 mRNA at a level following the polyadenylation step and preceding translocation from the nucleus to the cytoplasm. When HF were 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 presence of serum has a strong influence on heat shock-induced HSP70 gene expression in human fibroblasts aging in vitro, (C) 1999 Academic Press.