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Erythroleukemia cells acquire an alternative mitophagy capability.

Wang J, Fang Y, Yan L, Yuan N, Zhang S, Xu L, Nie M, Zhang X, Wang J - Sci Rep (2016)

Bottom Line: Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery.This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair.Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Collaborative Innovation Center of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Soochow University School of Medicine, Suzhou 215123, China.

ABSTRACT
Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor brefeldin A or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

No MeSH data available.


Related in: MedlinePlus

Atg7 deletion leads to G2/M arrest and reduced differentiation potential.(A) The proliferation curve of wild-type and Atg7−/− cells. (B) Bar graph represents the DNA histogram of G0/G1, S, G2/M phases of the cell cycle of wild-type and Atg7−/− cells. (C) Western blotting analysis of cell cycle related proteins (CCNA2, CCND2, CCND3, CCNE1, p53, p-p53, and p21) in wild-type and Atg7−/− cells. (D) Graphs showing the CFU counts and diameter of wild-type and Atg7−/− cells.
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f2: Atg7 deletion leads to G2/M arrest and reduced differentiation potential.(A) The proliferation curve of wild-type and Atg7−/− cells. (B) Bar graph represents the DNA histogram of G0/G1, S, G2/M phases of the cell cycle of wild-type and Atg7−/− cells. (C) Western blotting analysis of cell cycle related proteins (CCNA2, CCND2, CCND3, CCNE1, p53, p-p53, and p21) in wild-type and Atg7−/− cells. (D) Graphs showing the CFU counts and diameter of wild-type and Atg7−/− cells.

Mentions: To examine the impact of Atg7 deletion on leukemia cell proliferation, K562 cells were seeded in plates and cell numbers were counted at the indicated time. The Atg7−/− leukemia cells showed a progressive reduction in cell counts in the culture, and the overall growth curve displayed lower proliferation compared with that of Atg7 wild-type controls (Fig. 2A). Consistent with the cell growth data, cell cycle analysis showed a significant increase in Atg7−/− cells in the G2/M phase and a slight increase in Atg7−/− cells in the S phase (Fig. 2B). To analyze the molecular cause of the retarded growth resulting from the Atg7 deletion, cell cycle regulatory proteins were examined by immunoblotting. The results showed that knockout of Atg7 resulted in reduced levels of CCNA2, CCND2, CCND3, and CCNE1 and, significantly, an enhanced level of p21. Despite no change in the total p53 protein levels, phosphorylated p53 was increased in Atg7−/− cells. Phosphorylated CHK1 and CHK2, two upstream regulators of p53, were also upregulated (Fig. 2C). These data suggest that the Atg7 deletion-associated reductions in growth were caused by G2/M arrest, which is attributed to the downregulation of CCNA2, CCND2, CCND3, and CCNE1 and the activation of the CHK1/CHK2-p53-p21 pathway. Examination of the differentiation potential of the cells over 121 hours showed that Atg7 deletion decreased leukemia cell clone formation, as demonstrated by the colony formation unit (CFU) count, and the diameters of single clones were significantly decreased compared with those of the wild-type controls (Fig. 2D). Examination of apoptosis in wild-type cells and Atg7−/− K562 cells in cultures over the same period of time showed that there was no difference in apoptosis between the two groups (Figure S1A). Therefore, the reduction in colony size and the numbers observed in the CFU assay were a consequence of reduced differential potential but not of apoptosis.


Erythroleukemia cells acquire an alternative mitophagy capability.

Wang J, Fang Y, Yan L, Yuan N, Zhang S, Xu L, Nie M, Zhang X, Wang J - Sci Rep (2016)

Atg7 deletion leads to G2/M arrest and reduced differentiation potential.(A) The proliferation curve of wild-type and Atg7−/− cells. (B) Bar graph represents the DNA histogram of G0/G1, S, G2/M phases of the cell cycle of wild-type and Atg7−/− cells. (C) Western blotting analysis of cell cycle related proteins (CCNA2, CCND2, CCND3, CCNE1, p53, p-p53, and p21) in wild-type and Atg7−/− cells. (D) Graphs showing the CFU counts and diameter of wild-type and Atg7−/− cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835698&req=5

f2: Atg7 deletion leads to G2/M arrest and reduced differentiation potential.(A) The proliferation curve of wild-type and Atg7−/− cells. (B) Bar graph represents the DNA histogram of G0/G1, S, G2/M phases of the cell cycle of wild-type and Atg7−/− cells. (C) Western blotting analysis of cell cycle related proteins (CCNA2, CCND2, CCND3, CCNE1, p53, p-p53, and p21) in wild-type and Atg7−/− cells. (D) Graphs showing the CFU counts and diameter of wild-type and Atg7−/− cells.
Mentions: To examine the impact of Atg7 deletion on leukemia cell proliferation, K562 cells were seeded in plates and cell numbers were counted at the indicated time. The Atg7−/− leukemia cells showed a progressive reduction in cell counts in the culture, and the overall growth curve displayed lower proliferation compared with that of Atg7 wild-type controls (Fig. 2A). Consistent with the cell growth data, cell cycle analysis showed a significant increase in Atg7−/− cells in the G2/M phase and a slight increase in Atg7−/− cells in the S phase (Fig. 2B). To analyze the molecular cause of the retarded growth resulting from the Atg7 deletion, cell cycle regulatory proteins were examined by immunoblotting. The results showed that knockout of Atg7 resulted in reduced levels of CCNA2, CCND2, CCND3, and CCNE1 and, significantly, an enhanced level of p21. Despite no change in the total p53 protein levels, phosphorylated p53 was increased in Atg7−/− cells. Phosphorylated CHK1 and CHK2, two upstream regulators of p53, were also upregulated (Fig. 2C). These data suggest that the Atg7 deletion-associated reductions in growth were caused by G2/M arrest, which is attributed to the downregulation of CCNA2, CCND2, CCND3, and CCNE1 and the activation of the CHK1/CHK2-p53-p21 pathway. Examination of the differentiation potential of the cells over 121 hours showed that Atg7 deletion decreased leukemia cell clone formation, as demonstrated by the colony formation unit (CFU) count, and the diameters of single clones were significantly decreased compared with those of the wild-type controls (Fig. 2D). Examination of apoptosis in wild-type cells and Atg7−/− K562 cells in cultures over the same period of time showed that there was no difference in apoptosis between the two groups (Figure S1A). Therefore, the reduction in colony size and the numbers observed in the CFU assay were a consequence of reduced differential potential but not of apoptosis.

Bottom Line: Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery.This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair.Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Collaborative Innovation Center of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Soochow University School of Medicine, Suzhou 215123, China.

ABSTRACT
Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor brefeldin A or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

No MeSH data available.


Related in: MedlinePlus