Glycolytic inhibition by mutation of pyruvate kinase gene increases oxidative stress and causes apoptosis of a pyruvate kinase deficient cell line.
OBJECTIVE
SLC3 is a Friend erythroleukemic cell line established from the Pk-1(slc) 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.


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.


CONCLUSIONS
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.
Aisaki
Ken-ichi
K
Cellular and Molecular Toxicology Division, National Institute of Health and Sciences, Tokyo, Japan.
Aizawa
Shin
S
Fujii
Hisaichi
H
Kanno
Jun
J
Kanno
Hitoshi
H
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D045744:Cell Line, Tumor; D004912:Erythrocytes / Q000201:enzymology; D005434:Flow Cytometry; D020022:Genetic Predisposition to Disease; D006019:Glycolysis / Q000235:genetics*; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000502:physiology*; D011770:Pyruvate Kinase / Q000097:blood / Q000172:deficiency / Q000235:genetics*
D011770:Pyruvate Kinase