title: Overexpression of HSP-70 attenuates increases in [Ca2+]i and protects human epidermoid A-431 cells after chemical hypoxia.
abstract: This laboratory previously reported that thermotolerance diminishes the NaCN-induced increase in intracellular free calcium concentrations ([Ca2+]i) in human epidermoid A-431 cells and that blocking this increase protects the cells from NaCN toxicity. In this study, we report that cell viability after exposure to NaCN (10 mM, 1 h) is enhanced by the overexpression of HSP-70 resulting from heat shock (45 degrees C, 10 min), treatment with a protein kinase C activator phorbol 12 myristate 13-acetate (PMA; 1 microM, 4 h), or HSP-70 cDNA transfection. Because the toxicity of NaCN is mediated by increases in [Ca2+]i, we sought to determine whether the overexpression of HSP-70 might protect the cells by altering the [Ca2+]i response induced by NaCN. Basal [Ca2+]i in vector-, HSF1 cDNA-, and HSP-70 cDNA-transfected cells was 114 +/- 11 (n = 11), 95 +/- 5 (n = 6), and 151 +/- 11 (n = 15) nM, respectively, suggesting that HSP-70 metabolism is associated with maintenance of resting [Ca2+]i. Removal of external Ca2+ reduced the resting [Ca2+]i in all of these cells. With external Ca2+ reduced the resting [Ca2+]i by 97 +/- 21% in vector-transfected cells and 111 +/- 5% in HSF1 vector-transfected cells but by only 27 +/- 8% in HSP-70 cDNA-transfected cells. Heat shock or PMA treatment of vector- or HSF1 cDNA-transfected cells to induce HSP-70 also attenuated the NaCN-induced increase in [Ca2+]i, perhaps because of a decrease in Vmax for the uptake of external Ca2+. Removal of external Ca2+ or treatment with inhibitors of Na+/Ca2+ exchangers eliminated the NaCN-induced increase in [Ca2+]i in HSP-70 cDNA-transfected cells, but ryanodine treatment did not. HSP-70 cDNA transfection also reduced Ca2+ mobilization stimulated by various Ca(2+)-mobilizing agents. The results suggest that HSP-70 overexpression protects cells from NaCN cytotoxicity, perhaps by attenuating the [Ca2+]i response.
authors.affiliation: Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Kiang
authors.forename: X Z
authors.initials: XZ
authors.lastname: Ding
authors.forename: D E
authors.initials: DE
authors.lastname: McClain
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000633:toxicity; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism; D012966:Sodium Cyanide / Q000633:toxicity; D019831:Sodium-Calcium Exchanger / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D019831:Sodium-Calcium Exchanger; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D012966:Sodium Cyanide; D002118:Calcium