Vlachaki M T, Meyn R E
Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Int J Radiat Oncol Biol Phys. 1998 Aug 1;42(1):185-90. doi: 10.1016/s0360-3016(98)00203-x.
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 intracellular glutathione. Our hypothesis is that pretreatment of radiation-sensitive cells, known to lack bcl-2 expression, with antioxidants will reduce radiation-induced apoptosis. For this purpose, the apoptotic response to radiation and the intracellular levels of GSH were tested before and after pretreatment with 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.
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 with 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.
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.
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.
bcl - 2原癌基因的表达与辐射诱导的细胞凋亡抗性相关。有证据表明，bcl - 2蛋白通过抗氧化途径发挥作用，可能通过增加细胞内谷胱甘肽水平来阻断介导细胞凋亡的活性氧的作用。我们的假设是，用抗氧化剂预处理已知缺乏bcl - 2表达的辐射敏感细胞，将减少辐射诱导的细胞凋亡。为此，在两种小鼠淋巴瘤细胞系中，一种是抗辐射、表达bcl - 2的（LY - ar）细胞系，另一种是辐射敏感、不表达bcl - 2的（LY - as）细胞系，在用抗氧化剂预处理前后，检测了对辐射的凋亡反应以及细胞内谷胱甘肽水平。
在收集细胞前0、1、2、3和4小时对LY - ar和LY - as细胞进行辐照。使用荧光染料邻苯二甲醛测定还原型（GSH）和氧化型（GSSG）谷胱甘肽的细胞内水平。LY - as细胞在辐照后用谷胱甘肽乙酯处理1和2小时。通过DNA片段化分析测定凋亡反应。辐射剂量为2.5 Gy。
辐照后，LY - ar和LY - as细胞的凋亡率分别为10 - 20%和50 - 70%。与LY - as细胞相比，LY - ar细胞的细胞内GSH和GSSG水平分别高69.9%和91.9%，LY - ar和LY - as细胞中的GSH/GSSG比值分别为15.09和17.09。辐照后的前2小时内GSH水平没有变化；然而，在辐照后3小时和4小时分别降低了49%和84%，此时LY - as细胞已经发生了DNA片段化。用浓度为7 mM的谷胱甘肽乙酯处理LY - as细胞1和2小时，细胞内GSH水平分别增加了70%和231%。用谷胱甘肽乙酯处理LY - as细胞1和2小时，也使其辐照后的凋亡反应降低了25 - 50%。
与辐射敏感、不表达bcl - 2的细胞相比，抗辐射、表达bcl - 2的小鼠淋巴瘤细胞中GSH和GSSG水平升高。GSH水平仅在辐射敏感细胞辐照后下降，但这似乎发生在凋亡细胞死亡时。外源性硫醇增加细胞内GSH水平并抑制辐射诱导的细胞凋亡。总之，细胞内硫醇似乎参与保护细胞免于凋亡性细胞死亡。应进一步开展研究以鉴定通过降低细胞内硫醇可能导致对辐射敏感的物质。
