Tight association of autophagy and cell cycle in leukemia cells.
Autophagy
Cell cycle
Cell sorting
Cyto-ID
DRAQ5
Metabolic analysis
Journal
Cellular & molecular biology letters
ISSN: 1689-1392
Titre abrégé: Cell Mol Biol Lett
Pays: England
ID NLM: 9607427
Informations de publication
Date de publication:
05 Apr 2022
05 Apr 2022
Historique:
received:
14
01
2022
accepted:
24
03
2022
entrez:
6
4
2022
pubmed:
7
4
2022
medline:
8
4
2022
Statut:
epublish
Résumé
Autophagy plays an essential role in maintaining cellular homeostasis and in the response to cellular stress. Autophagy is also involved in cell cycle progression, yet the relationship between these processes is not clearly defined. In exploring this relationship, we observed that the inhibition of autophagy impaired the G2/M phase-arresting activity of etoposide but enhanced the G1 phase-arresting activity of palbociclib. We further investigated the connection of basal autophagy and cell cycle by utilizing the autophagosome tracer dye Cyto-ID in two ways. First, we established a double-labeling flow-cytometric procedure with Cyto-ID and the DNA probe DRAQ5, permitting the cell cycle phase-specific determination of autophagy in live cells. This approach demonstrated that different cell cycle phases were associated with different autophagy levels: G1-phase cells had the lowest level, and G2/M-phase cells had the highest one. Second, we developed a flow-cytometric cell-sorting procedure based on Cyto-ID that separates cell populations into fractions with low, medium, and high autophagy. Cell cycle analysis of Cyto-ID-sorted cells confirmed that the high-autophagy fraction contained a much higher percentage of G2/M-phase cells than the low-autophagy fraction. In addition, Cyto-ID-based cell sorting also proved to be useful for assessing other autophagy-related processes: extracellular flux analysis revealed metabolic differences between the cell populations, with higher autophagy being associated with higher respiration, higher mitochondrial ATP production, and higher glycolysis. This work provides clear evidence of high autophagy in G2/M-phase cells by establishing a novel cell sorting technique based on Cyto-ID.
Sections du résumé
BACKGROUND
BACKGROUND
Autophagy plays an essential role in maintaining cellular homeostasis and in the response to cellular stress. Autophagy is also involved in cell cycle progression, yet the relationship between these processes is not clearly defined.
RESULTS
RESULTS
In exploring this relationship, we observed that the inhibition of autophagy impaired the G2/M phase-arresting activity of etoposide but enhanced the G1 phase-arresting activity of palbociclib. We further investigated the connection of basal autophagy and cell cycle by utilizing the autophagosome tracer dye Cyto-ID in two ways. First, we established a double-labeling flow-cytometric procedure with Cyto-ID and the DNA probe DRAQ5, permitting the cell cycle phase-specific determination of autophagy in live cells. This approach demonstrated that different cell cycle phases were associated with different autophagy levels: G1-phase cells had the lowest level, and G2/M-phase cells had the highest one. Second, we developed a flow-cytometric cell-sorting procedure based on Cyto-ID that separates cell populations into fractions with low, medium, and high autophagy. Cell cycle analysis of Cyto-ID-sorted cells confirmed that the high-autophagy fraction contained a much higher percentage of G2/M-phase cells than the low-autophagy fraction. In addition, Cyto-ID-based cell sorting also proved to be useful for assessing other autophagy-related processes: extracellular flux analysis revealed metabolic differences between the cell populations, with higher autophagy being associated with higher respiration, higher mitochondrial ATP production, and higher glycolysis.
CONCLUSION
CONCLUSIONS
This work provides clear evidence of high autophagy in G2/M-phase cells by establishing a novel cell sorting technique based on Cyto-ID.
Identifiants
pubmed: 35382734
doi: 10.1186/s11658-022-00334-8
pii: 10.1186/s11658-022-00334-8
pmc: PMC8981689
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
32Informations de copyright
© 2022. The Author(s).
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