Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Abstract

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

Apoptotic Changes Precede Mitochondrial Dysfunction in Red Cell‐type Pyruvate Kinase Mutant Mouse Erythroleukemia Cell Lines

“…Max-Audit et al used erythroblasts isolated from human fetal liver for erythroid differentiation studies. 13) They demonstrated that total PK activity was much higher in basophilic than orthochromatic erythroblasts, and that the M2-PK activity markedly decreased, whereas the R-PK activity was only slightly modified during differentiation. These observations seemed similar to our present findings, and imply that R-PK gene mutation is unlikely to affect the expression of other PK isozyme genes in erythroid precursors, and that the SLC cells may develop decreased PK activity and decreased ATP content after R-PK becomes predominant owing to erythrodifferentiation.…”

“…Mammalian PK has four isozymes 6,7) ; R-PK is expressed almost exclusively in mature red blood cells (RBCs), 8) and the liver-type PK (L-PK) is transcribed from an alternative promoter of the PKLR gene. 9,10) In contrast with mature RBCs, erythroid precursors as well as other hematopoietic cells express a different isozyme of PK (muscle type 2 (M2)-PK), [11][12][13] which is encoded by the PKM gene. The PKM gene encodes both M1-PK and M2-PK, which are produced from a common primary transcript by alternative splicing.…”

Novel type of red blood cell pyruvate kinase hyperactivity predicts a remote regulatory locus involved in PKLR gene expression

“…In line with these findings a heterozygous missense mutation in PKLR was later identified [10]. In the second family [8] increased PK activity was attributed to persistent expression of the fetal isozyme PK-M2 [11,12].…”

Addressing the diagnostic gaps in pyruvate kinase deficiency: Consensus recommendations on the diagnosis of pyruvate kinase deficiency

“…[21][22][23] PK-M2 is the principal form in leukocytes, platelets, lung, spleen, kidney, and adipose tissue. In the liver, PK-M2 represents a minor component since, as the hepatocytes mature, the predominant isoenzyme becomes PK-L. During erythroid differentiation, the M2 isoenzyme is progressively replaced by the erythrocyte isoform PK-R. [24][25][26] PK is a homotetramer in almost all organisms, 27 although it may exist in different forms, from monomer to decamer. 28 A high degree of structural homology among PKs from different species has been reported based on published crystal structures [29][30][31][32] including human erythrocytes.…”

“…However, reticulocytosis is not proportional to the severity of hemolysis, likely due to a decreased erythropoietic drive since the oxygen delivery to tissues is relatively improved by the increase in 2, 3-DPG and because younger PK defective erythrocytes are selectively sequestered by the spleen. 25,26,[73][74][75] Splenectomy therefore results in a conspicuous rise of reticulocytes even if the anemia becomes less severe. Unconjugated bilirubin concentration is very often increased, but usually <5 mg/dL, and may show a slight rise after splenectomy.…”