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

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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.