Cytoprotection and regulation of heat shock proteins induced by heat shock in human breast cancer T47-D cells: role of [Ca2+]i and protein kinases

Overexpression of heat shock protein 70 kDa alters the susceptibility of tumor cells to chemotherapeutic agents. We conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in heat shock-treated T47-D cells, a human breast cancer cell line that expresses estrogen receptors. Cells exposed to heat shock at 44 degreesC displayed increased expression of heat shock protein 72 kDa (HSP-72), glucose-regulated protein 78 kDa (GRP-78), and GRP-94 in a time-dependent manner, as shown by [35S]methionine incorporation and Western blotting experiments. The maximal rate of synthesis occurred between 2 and 4 h after heat shock. Removal of external Ca2+ inhibited the synthesis of the heat shock-induced GRP-78 but not of HSP-72 and GRP-94, whereas treatment of cells with BAPTA (a Ca2+ chelator) inhibited HSP-72 and GRP-78. Treatment with H89 (a protein kinase A inhibitor) blocked the heat shock-induced GRP-78 synthesis, whereas GF-109203X (a protein kinase C inhibitor) attenuated the heat shock-induced HSP-72 synthesis and completely blocked synthesis of GRP-78 but not of GRP-94. These results indicate that protein kinase C is involved in regulation of the heat shock-induced synthesis of HSP-72, whereas PKA and PKC are involved in the regulation of GRP-78 synthesis. Cells overexpressing HSP-72 and GRPs after heat shock displayed resistance against lethal temperature (47 degreesC for 50 min) -induced death, which was diminished after removal of external Ca2+ and treatment with GF-109203X. Heat shock increased intracellular free Ca2+ concentration ([Ca2+]i) in a temperature- and heating duration-dependent fashion, and the increase was inhibited in the absence of external [Ca2+]i and significantly reduced by pretreatment with H89 and GF-109203X. The results suggest that different pathways are involved in the induction of synthesis of HSP-72, GRP-78, and GRP-94 by heat shock. It is highly likely that only HSP-72 and GRP-78 are involved in the process of cytoprotection from the thermal injury.