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 the influence of reduced extracellular pH on cellular radiation and/or heat sensitivity. 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 with respect to the tumor micromilieu. As far as mechanisms are concerned, many authors have taken the view that the intracellular rather than the extracellular pH is decisive at least for heat sensitivity, but only rodent cells have been studied so far and again the effects of chronically low pH in the incubation medium have been largely neglected. We therefore studied human melanoma cells after incubation for up to 6 days in media with different pH between 6.5 and 7.3.


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 sensitivity against 250 kV X-rays and hyperthermia at 43 degrees C were determined in the colony-forming assay. The intracellular pH was measured flow cytometrically using 5(and 6)-carboxyfluorescein. Calibration curves were established with cells incubated in different buffers containing nigericin to equilibrate intra- and extracellular pH.


RESULTS
Cell growth was optimal with pH 7.3 and 7.1 in the medium, somewhat 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 regulating this parameter very well; the flow cytometric measurements revealed that the intracellular pH was 7.2 +/- 0.2 irrespective of the extracellular pH in the range considered here.


CONCLUSIONS
In contrast to the almost generally accepted hypothesis that intracellular pH is decisive for the heat sensitivity, the human melanoma cells studied here became sensitive after a few days of incubation under acidic conditions without changes in the intracellular pH. Other factors seem to be influencing the cellular response to radiation and/or heat under chronically low pH.
Zölzer
F
F
Institut für Medizinische Strahlenbiologie, Universitätsklinikum Essen, Germany. friedo.zoelzer@uni-essen.de
Streffer
C
C
D002455:Cell Division / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects*; D003131:Combined Modality Therapy; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006979:Hyperthermia, Induced; D008545:Melanoma / Q000532:radiotherapy*; D012878:Skin Neoplasms / Q000532:radiotherapy*; D016896:Treatment Outcome; D014407:Tumor Cells, Cultured / Q000528:radiation effects*