Glycolytic inhibition by mutation of pyruvate kinase gene increases oxidative stress and causes apoptosis of a pyruvate kinase deficient cell line

• Published in: Experimental hematology Vol. 35; no. 8; pp. 1190 - 1200
• Main Authors: Aisaki, Ken-ichi, Aizawa, Shin, Fujii, Hisaichi, Kanno, Jun, Kanno, Hitoshi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Inc 01.08.2007
• Subjects: Advanced Basic Science; Animals; Apoptosis; Cell Line; Cell Line, Tumor; Erythrocytes - enzymology; Flow Cytometry; Genetic Predisposition to Disease; Glycolysis - genetics; Hematology, Oncology and Palliative Medicine; Leukemia, Erythroblastic, Acute; Mice; Mutation; Oligonucleotide Array Sequence Analysis; Oxidative Stress - physiology; Pyruvate Kinase - blood; Pyruvate Kinase - deficiency; Pyruvate Kinase - genetics
• ISSN: 0301-472X; 1873-2399
• DOI: 10.1016/j.exphem.2007.05.005

Objective SLC3 is a Friend erythroleukemic cell line established from the Pk-1slc mouse, a mouse model of red blood cell type-pyruvate kinase (R-PK) deficiency. This study was aimed to elucidate the mechanisms attributing to apoptosis induced by R-PK deficiency. Materials and Methods SLC3 and a control Friend cell line, CBA2, were cultured in a condition of glucose deprivation or supplementation with 2-deoxyglucose, and apoptosis was detected by annexin V. We established two stable transfectants of SLC3 cells with human R-PK cDNA, and examined the effect of R-PK on an apoptotic feature by cell cycle analysis. Intracellular oxidation was measured with 2′,7′-dichlorofluorescin diacetate. DNA microarray analysis was performed to examine gene-expression profiles between the two transfectants and parental SLC3. Results SLC3 was more susceptible than CBA2 to apoptosis induced by glycolytic inhibition. The forced expression of R-PK significantly decreased cells at the sub G0 /G1 stage in an expression-level dependent manner. Microarray analysis showed that proapoptotic genes, such as Bad , Bnip3, and Bnip3l, were downregulated in the transfectants. In addition, peroxiredoxin 1 ( Prdx1 ) and other antioxidant genes, such as Cat , Txnrd1 , and Glrx1 were also downregulated. A significant decrease of dichlorofluorescein fluorescence was observed by R-PK expression. Preincubation with a glutathione precursor showed a significant decrease of apoptosis. Conclusion These results indicated that glycolytic inhibition by R-PK gene mutation augmented oxidative stress in the Friend erythroleukemia cell, leading to activation of hypoxia-inducible factor-1 as well as downstream proapoptotic gene expression. Thus, R-PK plays an important role as an antioxidant during erythroid differentiation.