Intracellular free calcium concentrations in cell suspensions during hyperthermia

The aim of this study was to explore the possibility that heat-induced alterations in calcium homeostasis are the cause of hyperthermic cell killing. Therefore, the intracellular free calcium concentration ([Ca2+]i) was determined spectrofluorometrically, using the fluorescent calcium probe fura-2/acetoxy methylester (AM), at both physiological and hyperthermic temperatures in cell suspensions from six different tumor cell lines. For all cell lines fura-2 leakage appears to contribute to a change in the fluorescence signal and hence leads to a false indication of an increase in [Ca2+]i, especially at the hyperthermic temperature. Two methods were introduced that circumvent this problem and results in true values of [Ca2+]i. Also, measurements of [Ca2+]i in single cells using a fluorescent microscopical technique (not affected by dye leakage) were used for comparison. All three approaches show that a hyperthermic treatment that kills > 90% of the cells does not lead to changes in the [Ca2+]i in most cell lines. Therefore, heat-induced alterations of calcium homeostasis cannot be considered the general cause for hyperthermic cell killing.