title: Regulation of heat shock protein 72 kDa and 90 kDa in human breast cancer MDA-MB-231 cells.
abstract: It has been shown that expression of HSPs can negatively regulate the effectiveness of cytotoxic drugs. In this study, we conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in human breast cancer MDA-MB-231 cells. Using [35S]methionine incorporation and Western immunoblots, we established that heat shock increased production of HSP-72 and -90. Cells exposed to 44 degrees C for 20 min displayed increased expression of HSP-72 and -90, that reached a maximum 3-7 h later and returned to baseline levels within 24 h. The synthesis of both HSP-72 and -90 was attenuated when cells were exposed to heat shock in medium devoid of Ca2+ or pretreated with the calcium chelator BAPTA for 30 min prior to heat shock. Similarly, synthesis of HSP-72 and -90 was inhibited when cells were treated with the protein kinase A inhibitor, H89. These data indicate that Ca2+ and PKA are involved in the regulation of HSP-72 and -90 protein synthesis. 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. Also, heat shock caused phosphorylation and translocation from the cytosol to the nucleus of heat shock factor 1 (HSF 1), a transcription factor for heat shock protein synthesis. Removal of external Ca2+ or treatment with a PKA inhibitor prevented the phosphorylation and the translocation of HSF 1. Cells overexpressing HSP-72 and -90 induced by exposure to a sublethal temperature displayed cytoprotection from thermal injury. Removal of external Ca2+ and treatment with BAPTA or H89 prior to exposure to sublethal heat shock that reduced the amount of HSP-72 and -90 production still protected cells from subsequent thermal injury. The intracellular free calcium concentration ([Ca2+]i) in resting fura-2-loaded MDA-MB-231 cells was 175+/- nM. 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 30 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 H89 but not GF-109203X for 30 min led to an attenuation of the increase in [Ca2+]i by a subsequent heat shock. The results suggest that HSP-72 and -90 are regulated by [Ca2+]i and PKA activity in MDA-MB-231 cells. Kiang JG, Gist ID, Tsokos GC: Regulation of Heat Shock Protein 72 kDa and 90 kDa in Human Breast Cancer MDA-MB-231 Cells.
authors.affiliation: Department of Cellular Injury, 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: D001692:Biological Transport; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019610:Cytoprotection; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D050884:HSP72 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011960:Receptors, Estrogen / Q000172:deficiency / Q000378:metabolism; D014157:Transcription Factors; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D050884:HSP72 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D011960:Receptors, Estrogen; D014157:Transcription Factors; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium