Tumor Cell Sensitivity to Hyperthermia as a Function of Extracellular and Intracellular pH23

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Abstract
Mammalian cells subjected to environmental acidification exhibit enhanced sensitivity to heat. To test the postulate that intracellular pH changes are responsible for this effect, BP-8 murine sarcoma cells were acidified in three buffer systems, and the degree of thermal death was correlated to the degree of extracellular and intracellular acidification. Intracellular pH was measured by loading cells with 6-carboxyfluorescein and estimating the pH from the shape of the pH-dependent absorption spectrum. The relationship between intracellular and extracellular pH depended on the buffer used. Cells acidified in 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid plus 2-(N-morpholino)-ethanesulfonic acid (HEPES-MES) buffer retained an internal pH of 0.4–0.5 pH units above the buffer pH. Addition of 5,5-dimethyl-2,4-oxazolidinedione (DMO) to the HEPES-MES buffer or acidification of bicarbonate-buffered media with CO2 induced rapid equilibration between internal and external pH. The degree of heat sensitization achieved by lowering the medium pH appeared to be proportional to the changes in internal cell pH. Lowering of the medium pH from 7.4 to 6.5 caused pronounced thermal sensitization in cells suspended in bicarbonate-buffered media or in media containing DMO. The zwitterionic buffers HEPES and MES, which were less effective in lowering intracellular pH, also were less effective in enhancing thermal death, and the medium pH had to be lowered to pH 6.0 to increase thermal sensitization to the level observed in cells maintained in DMO or bicarbonate-buffered media at pH 6.5. These findings suggested that intracellular rather than extracellular pH changes are responsible for the phenomenon of heat sensitization at reduced environmental pH.