Effect of repetitive heat shock stimulation on TNF‐α induced apoptosis in clone 9 cells

Abstract

Heat shock proteins, a family of highly conserved and inducible stress proteins, protect cells from subsequent stronger stresses. In our previous studies, we have shown that single heat shock pretreatment of cultured cells induces HSP70 to against TNF-α, Tumor necrosis Factors-α, induced apoptosis. However, few study mentioned about that long-termed or repetitive preconditioning of stress does benefit to cell survival in facing a really fetal stress. Therefore, we want to know Dose HSP70 protect hepatic epithelial cell line (Clone 9 cell) against TNF-α toxicity more effective after repetitive heat shock pre-treatment. In this study, we designed a cell model of clone 9 cell and detected HSP70 and Heat shock factor-1, HSF-1, levels by Western blot analysis and RT-PCR. Apoptosis was evaluated by in situ TUNEL stain and Flow cytometry. In our results: (1) Single and double heat shock pretreatment of clone 9 cells induced protection against TNF-α induced apoptosis, but triple heat shock pretreatments. (2) There is no difference between single and repetitive heat shock pretreatment of clone 9 cells in HSP70 content of total cellular extract as well as cytoplasmic or nuclear extract. (3)Phosphorylation of HSF-1 decreased accordance with the frequency of heat shock pretreatment. (4) Single and double heat shock pretreatment of clone 9 cells up-regulated the mRNA of hsp70 but down-regulated in triple heat shocked clone 9 cell. We suggest that decline of Heat shock factor-1 activation followed by decreased induction of newly synthesized Hsp70 might lead to the disappearance of cytoprotection after more than three times of heat shock pretreatment.