RADIATION-INDUCED APOPTOSIS IN DIFFERENT PH ENVIRONMENTS IN-VITRO

Purpose: The effect of environmental pH on the radiation-induced apopt osis in tumor cells in vitro was investigated. Methods and Materials: Mammary adenocarcinoma cells of A/J mice (SCK cells) were irradiated w ith gamma-rays using a Cs-137 irradiator and incubated in media of dif ferent pHs, After incubation at 37 degrees C for 24-120 h the extent o f apoptosis was determined using agarose gel electrophoresis, TdT-medi ated dUTP-biotin nick end labeling (TUNEL) staining, flow cytometry, a nd release of H-3 from H-3-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 diff erent pHs was also determined with how cytometry, Results: Irradiation with 2-12 Gy of gamma-rays induced apoptosis in SCK cells in pH 7.5 m edium within 48 h as judged from the results of four different assays mentioned, Radiation-induced apoptosis declined as the medium pH was l owered from 7.5 to 6.4, Specifically, the radiation-induced degradatio n of DNA including the early DNA breaks, as determined with the TUNEL method, progressively declined as the medium pH was lowered so that li ttle DNA fragmentation occurred 48 h after irradiation with 12 Gy in p H 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 fra gmentation 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 G(2) arrest in pH 6.6 medium lasted mark edly longer than that in pH 7.5 medium, Conclusion: Radiation-induced apoptosis in SCR 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 th e DNA break or upstream of DNA break, is reversibly halted by an acidi c environment in irradiated cells, Radiation-induced G(2) arrest is pr olonged in an acidic environment indicating that the suppression of ra diation-induced apoptosis and prolongation of radiation-induced G(2) a rrest in an acidic environment are related. (C) 1997 Elsevier Science Inc.