Radiation-induced apoptosis in different pH environments in vitro.
OBJECTIVE
The effect of environmental pH on the radiation-induced apoptosis in tumor cells in vitro was investigated.


METHODS
Mammary adenocarcinoma cells of A/J mice (SCK cells) were irradiated with gamma-rays using a 137Cs irradiator and incubated in media of different pHs. After incubation at 37 degrees C for 24-120 h the extent of apoptosis was determined using agarose gel electrophoresis, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, flow cytometry, and release of 3H from 3H-thymidine labeled cells. The clonogenicity of the cells irradiated in different pH medium was determined, and the progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry.


RESULTS
Irradiation with 2-12 Gy of gamma-rays induced apoptosis in SCK cells in pH 7.5 medium within 48 h as judged from the results of four different assays mentioned. Radiation-induced apoptosis declined as the medium pH was lowered from 7.5 to 6.4. Specifically, the radiation-induced degradation of DNA including the early DNA breaks, as determined with the TUNEL method, progressively declined as the medium pH was lowered so that little DNA fragmentation occurred 48 h after irradiation with 12 Gy in pH 6.6 medium. When the cells were irradiated and incubated for 48 h in pH 6.6 medium and the medium was replaced with pH 7.5 medium, DNA fragmentation promptly occurred. DNA fragmentation also occurred even in pH 6.6 medium when the cells were irradiated and maintained in pH 7.5 medium for 8 h or longer post-irradiation before incubation in pH 6.6 medium. The radiation-induced G2 arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium.


CONCLUSIONS
Radiation-induced apoptosis in SCK cells in vitro is reversibly suppressed in an acidic environment. Taking the results of four different assays together, it was concluded that early step(s) in the apoptotic pathway, probably the DNA break or upstream of DNA break, is reversibly halted by an acidic environment in irradiated cells. Radiation-induced G2 arrest is prolonged in an acidic environment indicating that the suppression of radiation-induced apoptosis and prolongation of radiation-induced G2 arrest in an acidic environment are related.
Lee
H S
HS
Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota Medical School, Minneapolis 55455, USA.
Park
H J
HJ
Lyons
J C
JC
Griffin
R J
RJ
Auger
E A
EA
Song
C W
CW
D000230:Adenocarcinoma / Q000473:pathology / Q000532:radiotherapy*; D000818:Animals; D017209:Apoptosis / Q000235:genetics* / Q000528:radiation effects; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000235:genetics / Q000528:radiation effects; D004587:Electrophoresis, Agar Gel; D005260:Female; D005434:Flow Cytometry; D006863:Hydrogen-Ion Concentration; D008325:Mammary Neoplasms, Experimental / Q000473:pathology / Q000532:radiotherapy*; D051379:Mice; D008805:Mice, Inbred A
D004273:DNA, Neoplasm