IDENTIFICATION OF THE CAUSATIVE GENES RESPONSIBLE FOR ERYTHROID APOPTOSIS INDUCED BY GLYCOLYTIC INHIBITION

我们先前从红细胞丙酮酸激酶（R-PK）缺乏的小鼠模型中建立了Friend红白血病细胞SLC3。SLC3在常规传代过程中出现自发凋亡。当在葡萄糖剥夺或补充2-脱氧葡萄糖的条件下培养时，对照Friend细胞CBA2也出现凋亡。n -乙酰半胱氨酸（n -乙酰半胱氨酸，谷胱甘肽前体）预孵育可减少2-脱氧葡萄糖诱导的CBA2细胞凋亡，提示糖酵解抑制可增加红细胞的氧化应激，红细胞凋亡可能是由活性氧（ROS）诱导的。在5mM磷酸烯醇丙酮酸（PEP）中培养48小时，细胞内PEP和丙酮酸浓度分别增加了7倍和2倍。pep处理后的红细胞ROS明显降低，凋亡细胞数量比未处理的细胞减少约50%。这一观察结果表明，PEP的积累改善了糖酵解，因为突变体R-PK由于活性位点突变而具有较低的底物特异性。综上所述，我们得出结论，糖酵解抑制增加红细胞的氧化应激，激活促凋亡基因表达，导致细胞凋亡。

We previously established a Friend erythroleukemic cell, SLC3, from the mice model of red blood cell pyruvate kinase (R-PK) deficiency. SLC3 showed spontaneous apoptosis during routine passage. When cultured in a condition of glucose deprivation or supplementation with 2-deoxyglucose, a control Friend cell, CBA2, also showed apoptosis. Preincubation with N-acetyl cysteine, glutathione precursor, reduces apoptosis of CBA2 induced by 2-deoxyglucose, suggesting that glycolytic inhibition increases oxidative stress in erythroid cells and that erythroid apoptosis is likely to be induced by reactive oxygen species (ROS).When cultured in 5mM phosphoenolpyruvate (PEP) for 48 hours, intracellular concentration of PEP and pyruvate increased up to 7- and 2-times, respectively. ROS in the PEP-treated erythroid cells significantly decreased, and apoptotic cell number decreased about 50% of non-treated cells. This observation suggests that accumulation of PEP improves glycolysis since the mutant R-PK has lower substrate specificity due to the active site mutation. Taken together, we conclude that glycolytic inhibition increases oxidative stress in erythroid cells and activate proapoptotic gene expressions, leading to apoptosis.