Astro research fellowship: The role of BCL-2 and glutathione in an antioxidant pathway to prevent radiation-induced apoptosis

Purpose: The expression of the bcl-2 proto-oncogene has been associated with resistance to radiation-induced apoptosis. There is evidence that the bcl-2 protein acts in an antioxidant pathway to block the effects of reactive oxygen species that mediate apoptosis possibly by increasing the levels of inh acellular glutathione. Our hypothesis is that pretreatment of radiation-sensitive cells, known to lack bcl-2 expression, with antioxidants sill reduce radiation-induced apoptosis. For this purpose, the apoptotic response to radiation and the intracellular levels of GSH were tested before end after pretreatment,vith antioxidants in two murine lymphoma cell lines, a radiation-resistant, bcl-2- expressing (LY-ar) line and a radiation-sensitive, non-bcl-2-expressing (LY-as) line. Methods and Materials: LY-ar and LY-as cells were irradiated at 0,1,2,3, and 4 hours before collection. The intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione were determined by the use of the fluorescent dye o-phthalaldehyde. LY-as cells were treated,vith GSH ethyl-ester for 1 and 2 hours after irradiation. Apoptotic response was measured by the DNA fragmentation assay. The radiation dose was 2.5 Gy. Results: After irradiation, the apoptotic rate of LY-ar and LY-as cells was 10-20% and 50-70% respectively. LY-ar cells had higher intracellular GSH and GSSG levels compared to LY-as cells by 69.9% and 91.9% respectively and the GSH/GSSG ratio in LY-ar and LY-as cells was 15.09 and 17.09 respectively. GSH levels did not change during the first 2 hours after irradiation; however, there was a 49% and 84% reduction at 3 and 4 hours after irradiation, respectively, times at which the LY-as cells have already fragmented their DNA. Treatment of LY-as cells with GSH ethyl-ester at a concentration of 7 mM for 1 and 2 hours resulted in 70% and 231% increases in the intracellular GSH levels respectively. Treatment of LY-as cells with GSH ethyl-ester for 1 and 2 hours also conferred a 25-50% decrease in their apoptotic response after irradiation. Conclusions: GSH and GSSG levels are elevated in radiation-resistant, bcl-2-expressing murine lymphoma cells compared to radiation-sensitive, non-bcl-2-expressing cells. GSH levels decline only in radiation-sensitive cells after irradiation but this appears to occur at the time of apoptotic cell death. Exogenous thiols increase intracellular GSH levels and repress radiation-induced apoptosis. In conclusion, intracellular thiols appear to be involved in protecting cells from apoptotic cell death. Further investigation should be directed in identifying substances which by lowering intracellular thiols may result in sensitization to radiation. (C) 1998 Elsevier Science Inc.