Analysis by Confocal Microscopy of the Behavior of Heat Shock Protein 70 within the Nucleus and of a Nuclear Matrix Polypeptide during Prolonged Heat Shock Response in HeLa Cells

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M_r = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42°C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.     

Induction of four heat-shock proteins and their mRNAs in rat after whole-body hyperthermia

When the body temperature of rats was brought to 42 degrees C, four heat-shock proteins, with molecular weights of 70,000, 71,000, 85,000, and 100,000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), were induced in various tissues of the rats. The hsp 70 was strongly induced by hyperthermia, and its accumulation was detected by Coomassie blue staining. The hsp 71 was abundant in various tissues of rats that were not heat-shocked. Analysis of translation products of liver mRNAs from heat-shocked rats also showed increased synthesis of the four heat-shock proteins, indicating that these hsp-mRNAs were induced after hyperthermia. Induction of the hsp-mRNAs was transient after hyperthermia. The four heat-shock proteins produced in various tissues after hyperthermia may be involved in homeostatic control in the heat-shock response of the rat.     

Induction of stress response and differential expression of 70 kDa stress proteins by sodium fluoride in hela and rat brain tumor 9l cells

We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-κB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221–231, 1998. © 1998 Wiley-Liss, Inc.