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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

Generation of Atg7 knockout K562 cell line with CRISPR/Cas9.(A) Schematic diagram of Atg7 gene coding region and the targeting loci of gRNA: CRISPR/Cas9. (B) DNA electrophoresis of Atg7 PCR products in wild-type and Atg7−/− K562 cells. (C) The DNA sequencing results of wild-type and Atg7−/− K562 cells. (D) Detection of ATG7, ATG5-ATG12 conjugation and the conversion of LC3-I to LC3-II by immunoblotting.
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f1: Generation of Atg7 knockout K562 cell line with CRISPR/Cas9.(A) Schematic diagram of Atg7 gene coding region and the targeting loci of gRNA: CRISPR/Cas9. (B) DNA electrophoresis of Atg7 PCR products in wild-type and Atg7−/− K562 cells. (C) The DNA sequencing results of wild-type and Atg7−/− K562 cells. (D) Detection of ATG7, ATG5-ATG12 conjugation and the conversion of LC3-I to LC3-II by immunoblotting.

Mentions: To explore the possible implications of alternative autophagy in human leukemic cells, we first knocked out a canonical autophagy-essential gene, Atg7, with CRISPR/Cas9 to disrupt the capacity for canonical autophagy in the human K562 erythroleukemia cell line (Fig. 1A). Cas9 plasmids with guide RNA targeting exon 12 of Atg7 were transfected into K562 cells and single cell clones were analyzed. Genomic PCR followed by electrophoresis showed a reduced band (Fig. 1B), and DNA sequencing further conformed the 145 bp deletion in Atg7−/− cells (Fig. 1C). Western blotting analysis showed that Atg7 expression was undetectable in Atg7−/− cells (Fig. 1D). The Atg7 protein is essential for canonical autophagy, and it mediates autophagic flux involving ATG5-ATG12 conjugation and the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II12111213. The failure of ATG5-ATG12 conjugation and LC3-I/LC3-II conversion further indicated the successful knockout of the Atg7 gene in K562 cells (Fig. 1D).


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)

Generation of Atg7 knockout K562 cell line with CRISPR/Cas9.(A) Schematic diagram of Atg7 gene coding region and the targeting loci of gRNA: CRISPR/Cas9. (B) DNA electrophoresis of Atg7 PCR products in wild-type and Atg7−/− K562 cells. (C) The DNA sequencing results of wild-type and Atg7−/− K562 cells. (D) Detection of ATG7, ATG5-ATG12 conjugation and the conversion of LC3-I to LC3-II by immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Generation of Atg7 knockout K562 cell line with CRISPR/Cas9.(A) Schematic diagram of Atg7 gene coding region and the targeting loci of gRNA: CRISPR/Cas9. (B) DNA electrophoresis of Atg7 PCR products in wild-type and Atg7−/− K562 cells. (C) The DNA sequencing results of wild-type and Atg7−/− K562 cells. (D) Detection of ATG7, ATG5-ATG12 conjugation and the conversion of LC3-I to LC3-II by immunoblotting.
Mentions: To explore the possible implications of alternative autophagy in human leukemic cells, we first knocked out a canonical autophagy-essential gene, Atg7, with CRISPR/Cas9 to disrupt the capacity for canonical autophagy in the human K562 erythroleukemia cell line (Fig. 1A). Cas9 plasmids with guide RNA targeting exon 12 of Atg7 were transfected into K562 cells and single cell clones were analyzed. Genomic PCR followed by electrophoresis showed a reduced band (Fig. 1B), and DNA sequencing further conformed the 145 bp deletion in Atg7−/− cells (Fig. 1C). Western blotting analysis showed that Atg7 expression was undetectable in Atg7−/− cells (Fig. 1D). The Atg7 protein is essential for canonical autophagy, and it mediates autophagic flux involving ATG5-ATG12 conjugation and the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II12111213. The failure of ATG5-ATG12 conjugation and LC3-I/LC3-II conversion further indicated the successful knockout of the Atg7 gene in K562 cells (Fig. 1D).

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