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Updated: May 15, 2026
Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry
Published on: June 6, 2014
Studying the enucleation process, DNA breakdown and telomerase activity of the K562 cell lines during erythroid
Abdolkhaleg Deezagi1, Mahkameh Abedi-Tashi
1Department of Biochemistry, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. deezagi@nigeb.ac.ir
Abstract:
During erythropoiesis, some organelles such as mitochondria and nucleus are lost by autophagy and enucleation processes in the presence of macrophages in vivo. In vitro production of erythrocytes has raised many questions about the mechanism of enucleation. The aim of this work was to study the DNA breakdown, enucleation, hemoglobin synthesis and telomerase activity of K562 cells during erythroid differentiation. For these purposes, K562 cells were induced to differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was analyzed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder electrophoresis and comet assay. The telomerase activity was evaluated by TRAP assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell growth about 50-60% in comparison to untreated control cells. The percentage of benzidine-positive cells was about 45% in the presence of sodium butyrate after 10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM sodium butyrate in comparison with untreated cells. DAPI staining did not reveal any evidence of internal lysis of the nucleus during erythroid differentiation at first wk, but this was obvious in the second wk. DNA laddering pattern was not observed in differentiated cells during 14 d. In comet assay, the percentage of DNA in tail, tail length, and tail moment were significantly different between untreated and treated cells (p < 0.05). Telomerase activity was inhibited up to 90.3% during erythroid differentiation of these cells.
Insights
K562 cells undergoing erythroid differentiation showed significant DNA breakdown and inhibited telomerase activity. Hemoglobin synthesis increased, but nuclear enucleation occurred later than expected, indicating complex in vitro erythropoiesis.
Area of Science:
- Cell Biology
- Hematology
- Molecular Biology
Background:
- Erythropoiesis involves organelle loss, including nucleus expulsion (enucleation), typically in vivo with macrophage assistance.
- In vitro erythrocyte production models raise questions about the precise mechanisms of enucleation and associated cellular changes.
Purpose of the Study:
- To investigate DNA breakdown, enucleation timing, hemoglobin synthesis, and telomerase activity during K562 cell erythroid differentiation.
- To compare the effects of erythropoietin + rhGM-CSF, DMSO, and sodium butyrate on these processes.
Main Methods:
- K562 cells induced to differentiate using erythropoietin + rhGM-CSF, DMSO, or sodium butyrate for 14 days.
- Assessed hemoglobin synthesis via benzidine staining and γ-globin gene expression (RT-PCR).
- Analyzed DNA breakdown using DAPI staining, DNA ladder electrophoresis, and comet assay; evaluated telomerase activity with TRAP assay.
Main Results:
- Sodium butyrate and DMSO inhibited K562 cell growth by 50-60%.
- Sodium butyrate induced ~45% benzidine-positive cells and a 1.5-fold increase in γ-globin expression within 96 hours.
- Nuclear lysis became evident in the second week; DNA laddering was absent, but comet assays showed significant DNA damage. Telomerase activity was inhibited by up to 90.3%.
Conclusions:
- In vitro erythroid differentiation of K562 cells involves delayed nuclear enucleation and significant DNA fragmentation.
- Erythroid differentiation in vitro is associated with substantial telomerase inhibition.
- These findings highlight complexities in replicating in vivo erythropoiesis in vitro.
