Characterization of cold-induced heat shock protein expression in neonatal rat cardiomyocytes

Cardiac surgery is usually performed under conditions of cardioplegicischemic arrest. To protect the heart during the ischemic period, themyocardium is exposed to varying degrees of hypothermia. Althoughhyperthermia is known to induce the heat shock response, the moleculareffects of hypothermia on the myocardium have not been investigated. We havestudied the effect of hypothermia on the induction of heat shock proteins inprimary cultures of neonatal cardiomyocytes. Cold stress in cardiomyocytesinduced a 6 fold increase in the heat shock protein HSP70 as compared tocontrol. Increased HSP70 protein levels correlated with induction of HSP70mRNAs. Maximal levels of HSP70 protein appeared 4-6 h following recoveryfrom cold shock, indicating the transient nature of the response. Inductionof HSP25 mRNA was also observed in cold-shocked cardiomyocytes, even thoughincreased HSP25 protein levels were not detected. Our results indicate thathypothermia is capable of inducing the heat shock response in neonatalcardiomyocytes.     

Effect of the serine protease inhibitor N-tosyl-l-phenylalanine-chloromethyl ketone (TPCK) on MCF-7 mammary tumour cells growth and differentiation

Previous studies from this and other laboratories indicated that the oestrogen-regulated heat shock protein HSP27 is involved in the control of MCF-7 cells growth and differentiation, as it also appears to be in other cell types, including osteoblasts and HL-60 cells. In the latter instance, induction of differentiation is associated with the downregulation of myeloblastin, a serine protease now identified as proteinase 3 (hence its designation as PR3/Mbn), mirrored by an increase in the cellular content of the small heat shock protein HSP27, a substrate to this enzyme. Besides, antisense inhibition of PR3/Mbn production sufficed for inducing HL-60 cells monocytic differentiation. This prompted us to examine the hypothesis that a post-translational control on HSP27 levels (and by this on differentiation) by a serine protease might also be operating in human mammary tumour cells. As part of our attempt to evaluate this hypothesis, the present work consisted of testing the effects of a treatment of MCF-7 cells with the serine protease inhibitor N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). Our data show that this resulted in a four-fold increase in HSP27 content, associated with a 2.5-fold decrease in growth rate, the formation of cytoplasmic vesicles and increased secretion of 52 kDa peptides, identified by Western immunoblot as the isoforms of the oestrogen-regulated protein, cathepsin D. TPCK only affected growth in MDAMB-231 cells (in which HSP27 levels are very low and remained below MCF-7 cells basal levels after treatment) and failed to affect L929 cells, in which the hsp27 gene is silent. This provides circumstantial support for the assumption that effects of TPCK on the MCF-7 cells phenotype are linked to the associated increase in HSP27 content. Our recent demonstration that MCF-7 cells do in fact express PR3/Mbn fits with our concept and opens the way to test it directly, using antisense strategy.     

Higher induction of heat shock protein 72 by heat stress in cisplatin-resistant than in cisplatin-sensitive cancer cells

Induction of the heat shock proteins (HSPs) is involved in the increased resistance to cancer therapies such as chemotherapy and hyperthermia. We used two human ovarian cancer cell lines; a cisplatin (CDDP)-sensitive line A2780 and its CDDP-resistant derivative, A2780CP. The concentration of intracellular glutathione (GSH) is higher (2.7-fold increase) in A2780CP cells than in A2780 cells. A mild treatment with a heat stress (42 degrees C for 30 min) induced synthesis of both the heat shock protein 72 (Hsp72) mRNA and the HSP72 protein in A2780CP cells, but not in A2780 cells. In contrast, a severe heat stress (45 degrees C for 30 min) increased synthesis of the HSP72 protein in the two cell lines. The induced level of the HSP72 protein by the severe treatment was higher in A2780CP than in A2780 cells. The gel mobility shift assay showed that DNA binding activities of the heat shock factor (HSF) in the two cell lines were induced similarly and significantly by the mild heat stress. Immunocytochemistry using an anti HSF1 antibody also indicated that mild heat stress activated the HSF1 translocation from the cytosol to the nucleus similarly in the both cell lines. Pretreatment of CDDP-sensitive A2780 cells with N-acetyl-L-cysteine, a precursor of GSH, effectively enhanced induction of the Hsp72 mRNA by the mild heat stress. The present findings demonstrate that induction of the Hsp72 mRNA by the mild heat stress was more extensive in CDDP-resistant A2780CP cells. It is likely that the higher GSH concentration in A2780CP cells plays an important role in promoting Hsp72 gene expression induced by the mild heat stress probably through processes downstream of activation of HSF-DNA binding.     

Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis

The effects of cellular antioxidant capacity on hyperthermia (HT)-induced apoptosis and production of antiapoptotic heat shock proteins (HSPs) were investigated in HL-60 cells and in HL-60AR cells that are characterized by an elevated endogenous catalase activity. Exposure of both cell lines to 43 degrees C for 1 h initiated apoptosis. Apoptosis peaked at 3-6 h after heat exposure in the HL-60 cells. Whereas HL-60AR cells were partially protected against HT-induced apoptosis at these early time points, maximal levels of apoptosis were detected later, i.e. 12-18 h after heat exposure. This differential induction of apoptosis was directly correlated to the induction of the antiapoptotic HSP27 and HSP70. In particular, in the HL-60 cells HSP27 was significantly induced at 12-18 h after exposure to 43 degrees C when apoptosis dropped. In contrast, coinciding with the late onset of apoptosis in HL-60AR cells at that time HL-60AR cells lacked a similar HSP response. In line with the higher antioxidant capacity HL-60AR cells accumulated reactive oxygen species to a lesser degree than HL-60 cells after heat treatment. Protection from HT-induced apoptosis as well as diminished heat-induced HSP27 expression was also observed after cotreatment of HL-60 cells with 43 degrees C and catalase but not with superoxide dismutase. These data emphasize the pivotal role of reactive oxygen species for HT induced pro- and antiapoptotic pathways.     

Sphingosine 1-Phosphate Regulates Heat Shock Protein 27 Induction by a p38 MAP Kinase-Dependent Mechanism in Aortic Smooth Muscle Cells

In an aortic smooth muscle cell line, A10 cells, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein. Sphingosine 1-phosphate significantly induced the accumulation of HSP27 in a pertussis toxin-sensitive manner. The effect was dose-dependent in the range between 0.1 and 30 microM. Sphingosine 1-phosphate stimulated an increase in the levels of mRNA for HSP27. Sphingosine 1-phosphate stimulated both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase activation. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, did not affect sphingosine 1-phosphate-stimulated HSP27 induction. In contrast, SB203580, an inhibitor of p38 MAP kinase, reduced sphingosine 1-phosphate-induced HSP27 induction. SB203580 reduced the levels of mRNA for HSP27 induced by sphingosine 1-phosphate. These results indicate that sphingosine 1-phosphate stimulates the induction of HSP27 via p38 MAP kinase activation in aortic smooth muscle cells.     

Magnetic field exposure enhances mRNA expression of σ32 in E. coli

The mechanism of interaction between weak electromagnetic fields and cells is not understood. As a result, the health effect(s) induced by exposure to these fields remains unclear. In addition to questions relating to the site of initial magnetic field (MF) interactions, the nature of the cell's response to these perturbations is also unclear. We examined the hypothesis that the cells respond to MFs in a manner similar to other environmental stressors such as heat. Using the bacterium Escherichia coli, we examined the mRNA levels of σ³², a protein that interacts with RNA polymerase to help it recognize a variety of stress promoters in the cell. Our data show that the intracellular level of σ³² mRNA is enhanced following a 15-min exposure to a 60 Hz, 1.1 mT magnetic field. J. Cell. Biochem. 68:1–7, 1998. © 1998 Wiley-Liss, Inc.     

The effect of 100 mT SMF on activation of the hsp70 promoter in a heat shock/luciferase reporter system

Human exposure to magnetic fields, increased through use of new technologies like magnetic resonance imaging (MRI), has prompted investigations into possible effects of static magnetic fields (SMFs) on cellular processes. However, controversy still remains between many studies, which likely results from a lack of uniformity across experimental parameters, including the length of magnetic field exposure, the strength of the magnetic field, and the cell type or organism under investigation. The purpose of this research was to monitor effects of SMF exposure using real‐time luminescence photometry. The study investigated the potential interaction of a 100 mT SMF on a heat shock protein (hsp70)/luciferase reporter construct in stably transfected NIH3T3 cells. Changes in heat shock promoter activation following 100 mT SMF exposure were analyzed and detected as bioluminescence in real‐time. Two heat parameters were considered in combination with sham‐ and 100 mT‐exposed experiments: no heat or 1,800 s heat. As expected, there was a significant increase in bioluminescence in response to 1,800 s of heat alone. However, no significant difference in average hsp70 promoter activation between sham and 100 mT experiments was observed for no heat or 1,800 s heat experiments. Therefore, a 100 mT SMF was shown to have no effect on the activation of the heat shock protein promoter during SMF exposure or when SMF exposure was combined with a heat insult. J. Cell. Biochem. 108: 956–962, 2009. © 2009 Wiley‐Liss, Inc.