Limits...
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

Mitophagy remains functional when ATG7-dependent autophagy is defective.(A) Histogram showing the mitochondrial mass by flow cytometry in wild-type and Atg7−/− cells. (B) A graph showing the ROS level by flow cytometry in wild-type and Atg7−/− cells. (C) Representative macroautophagy in wild-type and Atg7−/− cells. (D) Quantification analysis of the mitochondria by flow cytometry in wild-type and Atg7−/− cells treated with 20 μM CCCP and 10 nM Baf-A1. (E) Detection of the expression mitochondrial protein TOMM20, ATG7 and the conversion of LC3-I to LC3-II in K562 cells treated by 20 μM CCCP and 10 nM Baf-A1 by immunoblotting. (F) Detection of the expression of key alternative autophagy related proteins (RAB9A, BECN1, ULK1 and VPS34) by immunoblotting in K562 cells treated with 20 μm Etoposide for 18 h. (G) Detection of the expression mitochondrial protein TOMM20, ATG7 and the conversion of LC3-I to LC3-II in wild-type and Atg7−/− K562 cells treated by 20 μM CCCP and 0.1 μg/mL BFA by immunoblotting. (H) The mitochondrial mass analysis in wild-type and Atg7−/− K562 cells treated by 20 μM CCCP and 0.1 μg/mL BFA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835698&req=5

f3: Mitophagy remains functional when ATG7-dependent autophagy is defective.(A) Histogram showing the mitochondrial mass by flow cytometry in wild-type and Atg7−/− cells. (B) A graph showing the ROS level by flow cytometry in wild-type and Atg7−/− cells. (C) Representative macroautophagy in wild-type and Atg7−/− cells. (D) Quantification analysis of the mitochondria by flow cytometry in wild-type and Atg7−/− cells treated with 20 μM CCCP and 10 nM Baf-A1. (E) Detection of the expression mitochondrial protein TOMM20, ATG7 and the conversion of LC3-I to LC3-II in K562 cells treated by 20 μM CCCP and 10 nM Baf-A1 by immunoblotting. (F) Detection of the expression of key alternative autophagy related proteins (RAB9A, BECN1, ULK1 and VPS34) by immunoblotting in K562 cells treated with 20 μm Etoposide for 18 h. (G) Detection of the expression mitochondrial protein TOMM20, ATG7 and the conversion of LC3-I to LC3-II in wild-type and Atg7−/− K562 cells treated by 20 μM CCCP and 0.1 μg/mL BFA by immunoblotting. (H) The mitochondrial mass analysis in wild-type and Atg7−/− K562 cells treated by 20 μM CCCP and 0.1 μg/mL BFA.

Mentions: The reduced cell growth caused by the Atg7 deletion prompted us to examine whether this defect in canonical autophagy causes increased intracellular stresses such as an increase in ROS. Unexpectedly, the results show that Atg7 deletion did not alter mitochondrial mass or ROS levels in K562 leukemia cells (Fig. 3A,B), suggesting that canonical autophagy-defective leukemia cells possibly retained an ability to controlling cellular ROS levels.


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)

Mitophagy remains functional when ATG7-dependent autophagy is defective.(A) Histogram showing the mitochondrial mass by flow cytometry in wild-type and Atg7−/− cells. (B) A graph showing the ROS level by flow cytometry in wild-type and Atg7−/− cells. (C) Representative macroautophagy in wild-type and Atg7−/− cells. (D) Quantification analysis of the mitochondria by flow cytometry in wild-type and Atg7−/− cells treated with 20 μM CCCP and 10 nM Baf-A1. (E) Detection of the expression mitochondrial protein TOMM20, ATG7 and the conversion of LC3-I to LC3-II in K562 cells treated by 20 μM CCCP and 10 nM Baf-A1 by immunoblotting. (F) Detection of the expression of key alternative autophagy related proteins (RAB9A, BECN1, ULK1 and VPS34) by immunoblotting in K562 cells treated with 20 μm Etoposide for 18 h. (G) Detection of the expression mitochondrial protein TOMM20, ATG7 and the conversion of LC3-I to LC3-II in wild-type and Atg7−/− K562 cells treated by 20 μM CCCP and 0.1 μg/mL BFA by immunoblotting. (H) The mitochondrial mass analysis in wild-type and Atg7−/− K562 cells treated by 20 μM CCCP and 0.1 μg/mL BFA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Mitophagy remains functional when ATG7-dependent autophagy is defective.(A) Histogram showing the mitochondrial mass by flow cytometry in wild-type and Atg7−/− cells. (B) A graph showing the ROS level by flow cytometry in wild-type and Atg7−/− cells. (C) Representative macroautophagy in wild-type and Atg7−/− cells. (D) Quantification analysis of the mitochondria by flow cytometry in wild-type and Atg7−/− cells treated with 20 μM CCCP and 10 nM Baf-A1. (E) Detection of the expression mitochondrial protein TOMM20, ATG7 and the conversion of LC3-I to LC3-II in K562 cells treated by 20 μM CCCP and 10 nM Baf-A1 by immunoblotting. (F) Detection of the expression of key alternative autophagy related proteins (RAB9A, BECN1, ULK1 and VPS34) by immunoblotting in K562 cells treated with 20 μm Etoposide for 18 h. (G) Detection of the expression mitochondrial protein TOMM20, ATG7 and the conversion of LC3-I to LC3-II in wild-type and Atg7−/− K562 cells treated by 20 μM CCCP and 0.1 μg/mL BFA by immunoblotting. (H) The mitochondrial mass analysis in wild-type and Atg7−/− K562 cells treated by 20 μM CCCP and 0.1 μg/mL BFA.
Mentions: The reduced cell growth caused by the Atg7 deletion prompted us to examine whether this defect in canonical autophagy causes increased intracellular stresses such as an increase in ROS. Unexpectedly, the results show that Atg7 deletion did not alter mitochondrial mass or ROS levels in K562 leukemia cells (Fig. 3A,B), suggesting that canonical autophagy-defective leukemia cells possibly retained an ability to controlling cellular ROS levels.

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