Regulation of heat shock protein 72 kDa and 90 kDa in human breast cancer MDA-MB-231 cells

It has been shown that expression of HSPs can negatively regulate the effec tiveness of cytotoxic drugs. In this study, we conducted experiments to stu dy the regulation of expression of heat shock proteins (HSPs) in human brea st cancer MDA-MB-231 cells. Using [S-35]methionine incorporation and Wester n immunoblots, we established that heat shock increased production of HSP-7 2 and -90. Cells exposed to 44 degrees C for 20 min displayed increased exp ression 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 at tenuated 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 treate d with the protein kinase A inhibitor, H89. These data indicate that Ca2+ a nd PKA are involved in regulation of HSP-72 and -90 protein synthesis. Leve ls 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. A lso, heat shock caused phosphorylation and translocation from the cytosol t o the nucleus of heat shock factor 1 (HSF1), a transcription factor for hea t shock protein synthesis. Removal of external Ca2+ or treatment with a PKA inhibitor prevented the posphorylation and the translocation of HSF1. Cell s overexpressing HSP-72 and -90 induced by exposure to a sublethal temperat ure displayed cytoprotection from thermal injury. Removal of external Ca2and treament with BAPTA, or H89 prior to exposure to sublethal heat shock t hat reduced the amount of HSP-72 and -90 production still protected cells f romsubsequent thermal injury. The intracellular free calcium concentration ([Ca2+](i)) in resting fura-2-loaded MDA-MB-231 cells was 175 +/- 8 nM. Hea t shock increased [Ca2+](i) in a time-and temperature-dependent manner. Exp osure 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 [Ca 2+](i) was due to Ca2+ influx. Pretreatment of the cells with H89 but not G F-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 regula ted by [Ca2+](i) and PKA activity in MDA-MB-231 cells. Kiang JG, Gist ID, T sokos GC: Regulation of Heat Shock Protein 72 kDa and 90 kDa in Human Breas t Cancer MDA-MB-231 Cells.