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°C, the [Ca2+]f of individual cells ranged between 50 and 150 nm, with a mean of 120 nm. Raising the temperature to 41°C for 1 h resulted in a slight reversible decrease (10–20%) in the mean [Ca2+]f. At 44°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°C for 2–6 h after heating for 1 h at 44°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°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°C or 44°C were observed with cells incubated at 10, 1.0, and 0 [plus 1.0 mm ethylene glycol bis(β-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.
Research supported by USPHS Grants R01-CA38639 and P30 AM34928.