Radiation and/or hyperthermia sensitivity of human melanoma cells grown for several days in media with reduced pH

Background: There are a number of reports in the literature dealing with th e influence of reduced extracellular pH on cellular radiation and/or heat s ensitivity. The focus of these earlier studies has been on the effects of a short-term incubation under acidic conditions, whereas much less attention has been given to chronic acidosis which would seem to be more relevant wi th respect to the tumor micromilieu. As far as mechanisms are concerned, ma ny authors have taken the view that the intracellular rather than the extra cellular pH is decisive at least for heat sensitivity, but only rodent cell s have been studied so far and again the effects of chronically low pH in t he incubation medium have been largely neglected. We therefore studied huma n melanoma cells after incubation for up to 6 days in media with different pH between 6.5 and 7.3. Material and Methods: MeWo cells were used throughout. Cells were incubated for 0, 3 or 6 days in media with pH 6.5. 6.7, 6.9, 7.1 or 7.3. The sensiti vity against 250 kV X-rays and hyperthermia at 43 degrees C were determined in the colony-forming assay. The intracellular pH was measured flow cytome trically using 5(and 6)-carboxyfluorescein. Calibration curves were establi shed with cells incubated in different buffers containing nigericin to equi librate intra- and extracellular pH. Results: Cell growth was optimal with pH 7.3 and 7.1 in the medium, somewha t reduced at pH 6.9, and largely inhibited at pH 6.7 and 6.5. Radiation and /or hyperthermia sensitivities were noticeably increased after several days of incubation at reduced pH: the surviving fraction after 4 Gy and 1 h at 43 degrees C either alone or in combination being a factor of 2 to 4 lower at pH 6.5 than at 7.3. These changes in sensitivity could not be correlated with changes in the intracellular pH. Cells seemed to be capable of regula ting this parameter very well: the flow cytometric measurements revealed th at the intracellular pH was 7.2 +/- 0.2 irrespective of the extracellular p H in the range considered here. Conclusions: In contrast to the almost generally accepted hypothesis that i ntracellular pH is decisive for the heat sensitivity, the human melanoma ce lls studied here became sensitive after a few days of incubation under acid ic conditions without changes in the intracellular pH. Other factors seem t o be influencing the cellular response to radiation and/or heat under chron ically low pH.