Page 182 - 2021_02-Haematologica-web

A. Sarkar et al.
of ROS due to leakage of electrons from complexes I and III of the electron transport chain,6 and dysregulation of fundamental cellular functions describe abnormal mito- chondria that are structurally and functionally different from their normal counterparts.7-9 Maintenance of mito- chondrial homeostasis in a cell is dependent on strict and highly dynamic mitochondrial fusion and fission cycles10,11 guided by two opposing events: reparation of damage by fusion and removal of damage by fission. In a unique sce- nario, a defective mitochondrion incapable of making ATP via F1-F0-ATPase, instead produces excessive amounts of ROS and is forced to consume ATP to generate membrane
Methods
Cell lines and primary lymphoma analysis
Cell lines are described in the Online Supplementary Methods. Primary B-cell lymphomas were obtained following informed consent from every patient on an Institutional Review Board- approved protocol and were in accordance to the declaration of Helsinki.
Reagents and antibodies
The reagents and antibodies used in this study are presented in Online Supplementary Table S1.
Ionizing radiation, intracellular staining and flow cytometry analysis
Ionizing radiation (IR) or neocarzinostatin was used to induce DNA damage. Two potent uncoupling agents, CCCP or FCCP, were used to induce mitophagy. Cells were stained with appro- priate dyes to determine mitophagy, total and mitochrondrial ROS (mROS) and acquired by flow cytometry (FCS) assay.
Plasmids, lentiviral infection and transfection
pcDNA3.1+ Flag-His-ATM wt (#31985) and pcDNA3.1+ Flag- His-ATM kd (#31986) were obtained from Addgene. GFP- Parkin, GFP-LC3 and GFP vector plasmids were gifts. Cells were transfected with Lipofectamine 2000 (Invitrogen) or Fugene6 (Promega). Lentiviral non-target short hairpin (sh)RNA and Mission shATM clones and lentivirus particles were prepared according to the manufacturer’s instructions (Sigma).
Immunoprecipitation and co-immunoprecipitation experiments
Standard immunoprecipitation and co-immunoprecipitation experiments were performed by transfecting plasmids in either HEK293T, WT-HeLa or A-T cells.
Measurement of nucleoside triphosphates
Intracellular nucleotides were determined by high perform- ance liquid chromatography analysis after perchloric acid extrac- tion.
Oxygen consumption analyses
Cellular oxygen consumption rate was measured by a stan- dard Seahorse assay (Seahorse Bioscience, Billerica, MA, USA).
Cell fractionation and immunoblot analysis
Cell fractionation was performed using either a NE-PER kit (Thermo Fisher) or Cell fractionation kit (Abcam; AB109719) fol- lowing the manufacturers’ guidelines. Protein lysates and immunoblots were prepared and protein bands were quantified using a LI-COR Odyssey CLx Infrared Imaging System.
Quantitative reverse-transcriptase polymerase chain reaction and mitochondrial DNA analysis
Total RNA was isolated using an RNeasy Mini Kit and the reverse transcriptase reaction was performed using a RevertAidTM H Minus First Strand cDNA Synthesis kit. Reverse transcriptase polymerase chain reaction (RT-PCR) was per- formed using the SYBR® Green PCR Master Mix. mtDNA copy number was analyzed in total DNA by quantitative PCR (qPCR) using mtDNA- and nuclear DNA-specific primers (PMC3769921) and the copy number was calculated as described before.28
potential (DΨ
Although low levels of mitochondrial damage can be repaired by complementation through the fusion process, an excessively damaged pool of mitochondria may endan- ger functional mitochondria during their coexistence, affecting the quality control of mitochondria.12,13 Cells have an inherent capacity to sense damaged mitochondria and selectively degrade these defective organelles by a process called mitochondrial autophagy or mitophagy.
), impeding normal metabolic function. m
During mitophagy, Pink1 accumulates on the outer membrane of depolarized mitochondria14 and recruits the cytosolic ubiquitin ligase Parkin and phosphorylates both Parkin and ubiquitin, resulting in Parkin activation. Activated Parkin in turn ubiquitylates scores of outer mitochondrial membrane proteins of depolarized mito- chondria followed by recruitment of multiple autophagy cargo adaptors, such as OPTN and NDP52. Finally all these cargos bind directly to LC3 in the autophagosome leading to degradation of the entire mitochondrion within autophagolysosomes.15
Ataxia telangiectasia mutated (ATM) is obligatory to ini- tiate cellular responses to DNA double-strand breaks and DNA repair to preserve genomic integrity, and loss of ATM results in genetic disorders characterized by neu- rodegeneration, immunodeficiency, and cancer.16-20 ATM also possesses non-nuclear functions associated with its cytoplasmic localization in various cell types21,22 and loss of ATM leads to increased accumulation of ROS and aber- rant mitochondria leading to abnormal mitochondrial homeostasis and may trigger cancer progression.16,23 Further, loss of ATM leads to global dysregulation of ribonucleotide reductase activity and abrogation of mito- chondrial biogenesis and mtDNA content.24 Cells from patients with ataxia telangiectasia (A-T) contain a greater mitochondrial mass and are defective in mitochondrial respiration compared to wild-type (WT) fibroblasts.25 However, none of the phenotypic abnormalities common- ly observed in ATM-deficient cells are explained by defects in canonical DNA damage response pathways, particularly in neurodegeneration, cancer predisposition, and premature aging.
Even though ATM is frequently mutated in cancer, a comprehensive study relating ATM dysfunction in mitophagy in cancer cells and in cancer patients is lacking. Mantle cell lymphoma (MCL) is a genetically unstable and fatal B-cell non-Hodgkin lymphoma, in which deletions or inactivating mutations in the ATM gene are frequently acquired (40-75% of cases).26,27 We demonstrate the role of ATM in mitophagy using MCL cell lines and primary cells obtained directly from patients, and show that ATM con- trols mitophagy through interaction with Parkin in a kinase-independent manner.
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