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 associate d with resistance to radiation-induced apoptosis. There is evidence th at the bcl-2 protein acts in an antioxidant pathway to block the effec ts of reactive oxygen species that mediate apoptosis possibly by incre asing the levels of inh acellular glutathione. Our hypothesis is that pretreatment of radiation-sensitive cells, known to lack bcl-2 express ion, with antioxidants sill reduce radiation-induced apoptosis. For th is purpose, the apoptotic response to radiation and the intracellular levels of GSH were tested before end after pretreatment,vith antioxida nts in two murine lymphoma cell lines, a radiation-resistant, bcl-2- e xpressing (LY-ar) line and a radiation-sensitive, non-bcl-2-expressing (LY-as) line. Methods and Materials: LY-ar and LY-as cells were irrad iated at 0,1,2,3, and 4 hours before collection. The intracellular lev els of reduced (GSH) and oxidized (GSSG) glutathione were determined b y the use of the fluorescent dye o-phthalaldehyde. LY-as cells were tr eated,vith GSH ethyl-eater for 1 and 2 hours after irradiation. Apopto tic response was measured by the DNA fragmentation assay. The radiatio n dose was 2.5 Gy. Results: After irradiation, the apoptotic rate of L Y-ar and LY-as cells was 10-20% and 50-70% respectively. LY-ar cells h ad 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-a s cells was 15.09 and 17.09 respectively. GSH levels did not change du ring 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 GS H levels respectively. Treatment of LY-as cells with GSH ethyl-ester f or 1 and 2 hours also conferred a 25-50% decrease in their apoptotic r esponse after irradiation. Conclusions: GSH and GSSG levels are elevat ed in radiation-resistant, bcl-2-expressing murine lymphoma cells comp ared to radiation-sensitive, non-bcl-2-expressing cells. GSH levels de cline only in radiation-sensitive cells after irradiation but this app ears to occur at the time of apoptotic cell death. Exogenous thiols in crease intracellular GSH levels and repress radiation-induced apoptosi s. In conclusion, intracellular thiols appear to be involved in protec ting cells from apoptotic cell death. Further investigation should be directed in identifying substances which by lowering intracellular thi ols may result in sensitization to radiation. (C) 1998 Elsevier Scienc e Inc.