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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

The RAB9A-dependent alternative mitophagy is essential for buffering cellular stresses when canonical autophagy is abolished.(A) Quantification analysis of the apoptosis rate in wild-type and Atg7−/− K562 cells treated with CCCP when Rab9A was knocked down. (B) Quantification analysis of the ROS level in wild-type and Atg7−/− K562 cells treated with CCCP when Rab9A was knocked down. (C) The expression of γ-H2AX detected by flow cytometry in wild-type, Atg7−/− and Atg7−/−-shRab9A K562 cells post 3 Gy ionizing radiation. (D) Comet assay of wild-type, Atg7−/−, wild-type shRab9A and Atg7−/−-shRab9A cells 1 h post 3 Gy ionizing radiation and quantification analysis of the tail length of comet assay of wild-type, Atg7−/−, wild-type shRab9A and Atg7−/−-shRab9A cells 3h post 3Gy ionizing radiation. (E) Detection of the expression of DNA damage repair related proteins (RAD50, RAD51, KU70, KU80, MRE11) and the conversion of LC3-I to LC3-II by immunoblotting in K562 cells post 3 Gy ionizing radiation.
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f5: The RAB9A-dependent alternative mitophagy is essential for buffering cellular stresses when canonical autophagy is abolished.(A) Quantification analysis of the apoptosis rate in wild-type and Atg7−/− K562 cells treated with CCCP when Rab9A was knocked down. (B) Quantification analysis of the ROS level in wild-type and Atg7−/− K562 cells treated with CCCP when Rab9A was knocked down. (C) The expression of γ-H2AX detected by flow cytometry in wild-type, Atg7−/− and Atg7−/−-shRab9A K562 cells post 3 Gy ionizing radiation. (D) Comet assay of wild-type, Atg7−/−, wild-type shRab9A and Atg7−/−-shRab9A cells 1 h post 3 Gy ionizing radiation and quantification analysis of the tail length of comet assay of wild-type, Atg7−/−, wild-type shRab9A and Atg7−/−-shRab9A cells 3h post 3Gy ionizing radiation. (E) Detection of the expression of DNA damage repair related proteins (RAD50, RAD51, KU70, KU80, MRE11) and the conversion of LC3-I to LC3-II by immunoblotting in K562 cells post 3 Gy ionizing radiation.

Mentions: To understand the role of alternative autophagy in the protection of leukemia cells from external and internal stresses, we challenged K562 cells with CCCP. We found that after 18 h of co-culture with CCCP, the apoptosis rate was significantly elevated when Rab9A was knocked down in both the wild-type and Atg7-knockout cells. Interestingly, compared with that of the wild-type shRab9A, the apoptosis rate was further increased in the Atg7 deleted-shRAB9A cells (Fig. 5A), suggesting an important role for RAB9A-dependent alternative mitophagy in suppressing apoptotic cell death. Similarly, the ROS levels were increased in both the wild-type and Atg7−/− K562 cells, and this increase was further enhanced when Rab9A was knocked down (Fig. 5B), suggesting that RAB9A-dependent alternative mitophagy also contributed to the reduction of ROS.


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)

The RAB9A-dependent alternative mitophagy is essential for buffering cellular stresses when canonical autophagy is abolished.(A) Quantification analysis of the apoptosis rate in wild-type and Atg7−/− K562 cells treated with CCCP when Rab9A was knocked down. (B) Quantification analysis of the ROS level in wild-type and Atg7−/− K562 cells treated with CCCP when Rab9A was knocked down. (C) The expression of γ-H2AX detected by flow cytometry in wild-type, Atg7−/− and Atg7−/−-shRab9A K562 cells post 3 Gy ionizing radiation. (D) Comet assay of wild-type, Atg7−/−, wild-type shRab9A and Atg7−/−-shRab9A cells 1 h post 3 Gy ionizing radiation and quantification analysis of the tail length of comet assay of wild-type, Atg7−/−, wild-type shRab9A and Atg7−/−-shRab9A cells 3h post 3Gy ionizing radiation. (E) Detection of the expression of DNA damage repair related proteins (RAD50, RAD51, KU70, KU80, MRE11) and the conversion of LC3-I to LC3-II by immunoblotting in K562 cells post 3 Gy ionizing radiation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: The RAB9A-dependent alternative mitophagy is essential for buffering cellular stresses when canonical autophagy is abolished.(A) Quantification analysis of the apoptosis rate in wild-type and Atg7−/− K562 cells treated with CCCP when Rab9A was knocked down. (B) Quantification analysis of the ROS level in wild-type and Atg7−/− K562 cells treated with CCCP when Rab9A was knocked down. (C) The expression of γ-H2AX detected by flow cytometry in wild-type, Atg7−/− and Atg7−/−-shRab9A K562 cells post 3 Gy ionizing radiation. (D) Comet assay of wild-type, Atg7−/−, wild-type shRab9A and Atg7−/−-shRab9A cells 1 h post 3 Gy ionizing radiation and quantification analysis of the tail length of comet assay of wild-type, Atg7−/−, wild-type shRab9A and Atg7−/−-shRab9A cells 3h post 3Gy ionizing radiation. (E) Detection of the expression of DNA damage repair related proteins (RAD50, RAD51, KU70, KU80, MRE11) and the conversion of LC3-I to LC3-II by immunoblotting in K562 cells post 3 Gy ionizing radiation.
Mentions: To understand the role of alternative autophagy in the protection of leukemia cells from external and internal stresses, we challenged K562 cells with CCCP. We found that after 18 h of co-culture with CCCP, the apoptosis rate was significantly elevated when Rab9A was knocked down in both the wild-type and Atg7-knockout cells. Interestingly, compared with that of the wild-type shRab9A, the apoptosis rate was further increased in the Atg7 deleted-shRAB9A cells (Fig. 5A), suggesting an important role for RAB9A-dependent alternative mitophagy in suppressing apoptotic cell death. Similarly, the ROS levels were increased in both the wild-type and Atg7−/− K562 cells, and this increase was further enhanced when Rab9A was knocked down (Fig. 5B), suggesting that RAB9A-dependent alternative mitophagy also contributed to the reduction of ROS.

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