HYPERTHERMIA EFFECTS ON CYTOSOLIC [CA2+] - ANALYSIS AT THE SINGLE CELL LEVEL BY DIGITIZED IMAGING MICROSCOPY AND CELL-SURVIVAL

Digitized video-intensified fluorescence microscopy with the Ca2+- sensitive fluorescent dye fura-2 was used to measure cytosolic free Ca2+ ([Ca2+]f) in HT-29 human colon cancer cells. At 37-degrees-C, the [Ca2+]f of individual cells ranged between 50 and 150 nM, with a mean of 120 nM. Raising the temperature to 41-degrees-C for 1 h resulted in a slight reversible decrease (10-20%) in the mean [Ca2+]f. At 44-degrees-C for 1 h, most (>80%) cells exhibited a [Ca2+]f greater than 200 nM. This heat-induced rise in [Ca2+]f was not immediate but commenced after a lag time of 30 min. Postincubation at 37-degrees-C for 2-6 h after heating for 1 h at 44-degrees-C resulted in a recovery of the basal [Ca2+]f in some but not all cells. A linear relationship was determined between percentage of cell killing and the number of cells with [Ca2+]f of >200 nM after 37-degrees-C post-heating incubation. Manipulation of extracellular [Ca2+] between 0.1 and 10 mM during heating did not modify the heat-induced changes in [Ca2+]f. No significant differences in survival at 37-degrees-C or 44-degrees-C were observed with cells incubated at 10, 1.0, and 0 [plus 1.0 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] mM extracellular Ca2+. The Ca2+ channel blockers verapamil and nifedipine did not protect cells from heat treatment. These results suggest that irreversible heat-induced changes in intracellular Ca2+ homeostasis mechanisms may be a critical factor in heat cytotoxicity.