title: Biochemical requirements for the expression of heat shock protein 72 kda in human breast cancer MCF-7 cells.
abstract: Heat shock alters the susceptibility of tumor cells to chemotherapeutic agents. Cultured breast cancer MCF-7 and MDA-MB-231 cells that express high levels of heat shock protein 70 and 27 kDa are resistant to treatment with certain anticancer drugs. These findings indicate that expression of HSPs can negatively regulate the effectiveness of cytotoxic drugs. We conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in human breast cancer MCF-7 cells exposed to heat shock by intracellular free Ca2+ and protein kinase C. Cells exposed to 44 degrees C for 20 min displayed increased expression of HSP-72 and GRP-94, that reached a maximum 4-5 h later and returned to baseline levels within 24 h. Levels of HSP-72 mRNA in cells exposed to heat shock increased, suggesting that the heat-induced increase in HSP-72 occurs at the transcriptional level. The synthesis of HSP-72 but not GRP-94 was inhibited when cells were exposed to heat shock in medium devoid of Ca2+ and attenuated by more than 50% when cells were pretreated with the calcium chelator BAPTA for 30 min prior to heat shock. HSP-72 synthesis was enhanced when cells were treated with the protein kinase C inhibitor, GF-109203X. These data indicate that Ca2+ and PKC are involved in regulation of HSP-72 synthesis. However, removal of external Ca2+ and treatment with BAPTA, GF-109203X, or exposure to sublethal heat shock protected cells from subsequent thermal injury. The intracellular free calcium concentration ([Ca2+]i) in resting fura-2-loaded MCF-7 cells was 156 +/- 16 nM (n = 29). Heat shock increased [Ca2+]i in a time- and temperature-dependent manner. Exposure of cells to 44 degrees C for 20 min increased [Ca2+]i by 234 +/- 13%, which subsequently returned to baseline levels within 120 min. Removal of external Ca2+ eliminated the increase, indicating that the increase in [Ca2+]i was due to Ca2+ influx. Pretreatment of the cells with BAPTA or GF-109203X for 30 min or a sublethal heat shock to allow HSP-72 overexpression led to an attenuation of the increase in [Ca2+]i by a subsequent heat shock. The results suggest that HSP-72 but not GRP-94 is regulated by [Ca2+]i and PKC activity. The cytoprotection produced by chelation of Ca2+, GF-109203X, or HSP-72 overexpression is probably due to their ability to attenuate the [Ca2+]i response to heating.
authors.affiliation: Department of Clinical Physiology, Division of Medicine, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Kiang
authors.forename: I D
authors.initials: ID
authors.lastname: Gist
authors.forename: G C
authors.initials: GC
authors.lastname: Tsokos
mesh_terms: D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002614:Chelating Agents / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D050884:HSP72 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011960:Receptors, Estrogen / Q000378:metabolism; D013449:Sulfonamides; D014407:Tumor Cells, Cultured
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D050884:HSP72 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007546:Isoquinolines; D011960:Receptors, Estrogen; D013449:Sulfonamides; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium