Establishment of Human Cell Lines Lacking Mitochondrial DNA

Hashiguchi, Kazunari; Zhang-Akiyama, Qiu-Mei

doi:10.1007/978-1-59745-521-3_23

Cited by 110 publications

(100 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…28 We confirmed that these cells had lost mitochondria by measuring cellular mitochondrial DNA content by quantitative PCR, western blotting cell lysates for the mitochondrial protein ATPase inhibitory factor 1 (ATPIF1), and flow cytometry of cells stained with the mitochondrial specific dye MitoTracker Green ( Fig. 4C and D).…”

Section: Mitochondrial Reactive Oxygen Activates the Inflammasome Folmentioning

confidence: 55%

“…Ethidium bromide (500 ng/ml) was added to the medium for 15 d. 28 Depletion of mitochondrial DNA was evaluated by quantitative PCR for the mitochondrial gene mt-Co1, compared to nuclear DNA encoding 18S rRNA, protein gel blot to detect mitochondrial ATPIF1, and flow cytometry for measurement of mitochondrial mass using MitoTracker Green FM stain (Invitrogen, M7514).…”

Section: Protein Transfectionmentioning

confidence: 99%

See 1 more Smart Citation

Mitochondrial damage contributes toPseudomonas aeruginosaactivation of the inflammasome and is downregulated by autophagy

Jabir¹,

Hopkins²,

Ritchie³

et al. 2015

Autophagy

148

112

View full text Add to dashboard Cite

Abbreviations: Three-MA, 3-methyladenine; AIM2, absent in melanoma 2; ATG, autophagy related; ATPIF1, ATPase inhibitory factor 1; BID, BH3 interacting domain death agonist; BMDM, bone marrow-derived macrophages; BrdU, 5-bromo-2-deoxyuridine; CASP, caspase; GFP, green fluorescent protein; IL1B, interleukin 1, b; LC3B, microtubule-associated protein 1 light chain 3 b; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; Mito-TEMPO, (2-(2, 2, 6, 6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride; MT-CO1, mitochondrially encoded cytochrome c oxidase I; mtDNA, mitochondrial DNA; NAC, N-acetylcysteine; NAIP, NLR family apoptosis inhibitor; NGS, normal goat serum; NLR, nucleotide-binding domain, leucine-rich repeat containing; NLRC4, NLR family, CARD domain containing 4; NLRP3, NLR family, pyrin domain containing 3; PBS, phosphate-buffered saline; PINK1, PTEN induced putative kinase 1; Rn18s, 18S rRNA; T3SS, type III secretion system; TNF, tumor necrosis factor; TUBB5, tubulin, b 5 class I; Vav, vav 1 oncogene.The nucleotide-binding domain, leucine-rich repeat containing family caspase recruitment domain containing 4 (NLRC4) inflammasome can be activated by pathogenic bacteria via products translocated through the microbial type III secretion apparatus (T3SS). Recent work has shown that activation of the NLRP3 inflammasome is downregulated by autophagy, but the influence of autophagy on NLRC4 activation is unclear. We set out to determine how autophagy might influence this process, using the bacterium Pseudomonas aeruginosa, which activates the NLRC4 inflammasome via its T3SS. Infection resulted in T3SS-dependent mitochondrial damage with increased production of reactive oxygen intermediates and release of mitochondrial DNA. Inhibiting mitochondrial reactive oxygen release or degrading intracellular mitochondrial DNA abrogated NLRC4 inflammasome activation. Moreover, macrophages lacking mitochondria failed to activate NLRC4 following infection. Removal of damaged mitochondria by autophagy significantly attenuated NLRC4 inflammasome activation. Mitochondrial DNA bound specifically to NLRC4 immunoprecipitates and transfection of mitochondrial DNA directly activated the NLRC4 inflammasome; oxidation of the DNA enhanced this effect. Manipulation of autophagy altered the degree of inflammasome activation and inflammation in an in vivo model of P. aeruginosa infection. Our results reveal a novel mechanism contributing to NLRC4 activation by P. aeruginosa via mitochondrial damage and release of mitochondrial DNA triggered by the bacterial T3SS that is downregulated by autophagy.

show abstract

Section: Mitochondrial Reactive Oxygen Activates the Inflammasome Folmentioning

confidence: 55%

Section: Protein Transfectionmentioning

confidence: 99%

Mitochondrial damage contributes toPseudomonas aeruginosaactivation of the inflammasome and is downregulated by autophagy

Jabir¹,

Hopkins²,

Ritchie³

et al. 2015

Autophagy

148

112

View full text Add to dashboard Cite

show abstract

“…Because avocatin B is a lipid and leukemia cells possess mitochondrial and metabolic alterations that result in their dependence on fatty acid substrates for survival (2), we hypothesized that avocatin B's toxicity was related to its localization in mitochondria. To first test avocatin B's reliance on mitochondria for cytotoxicity, we generated leukemia cells lacking functional mitochondria by culturing Jurkat-T cells in media supplemented with 50 ng/mL of ethidium bromide, 100 mg/mL sodium pyruvate, and 50 mg/mL uridine, as previously described (30,31). Following 60 days of passaging only live cells, the presence of mitochondria were tested by flow cytometry following 10-nonyl acridine orange (NAO) staining and by Western blotting for mitochondrial specific proteins ND1 and adenine nucleotide translocator (ANT).…”

Section: Mitochondria and Cpt1 Are Functionally Important For Avocatimentioning

confidence: 99%

Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death

et al. 2015

View full text Add to dashboard Cite

Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function. Cancer Res; 75(12); 2478-88. Ó2015 AACR.

show abstract

“…HepG2 cells lacking mitochondrial DNA ( 0 cells) were generated by chronic exposure to low doses of ethidium bromide, as described by Hashiguchi and Zhang-Akiyama (34). 0 mitochondria can maintain electrochemical potential across the inner membrane by a mechanism coupled to ATP hydrolysis, making them net consumers of ATP in cells and an excellent tool for studying mitochondrial function (34). The cells were immediately expanded and frozen after being obtained and restarted every 3-4 months from a frozen vial of the same batch of cells, and no additional authentication was done on these cells.…”

Section: Cell Culturementioning

confidence: 99%

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Figarola

Singhal

Tompkins

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. Hepatocellular carcinoma (HCC)2 is the most common and severe form of liver cancer, accounting for about 80 -90% of primary liver cancers and 5% of all human cancers. More than 600,000 deaths are attributed to HCC every year with 2:1 ratio for men versus women (1, 2). HCC is a primary cancer of hepatocytes that most typically occurs in the setting of known risk factors, including cirrhosis and chronic hepatitis B virus or hepatitis C virus infections (2, 3), although recently, several lines of evidence suggest that type 2 diabetes is also an independent risk factor for HCC development (4). HCC is an aggressive tumor and represents a major health problem as its incidence is increasing. Systemic chemotherapies have proven ineffective against advanced HCC, so it typically leads to death within 6 -20 months (5). Hepatocarcinogenesis is a multistep process involving inflammation, hyperplasia, and dysplasia that finally leads to malignant transformation. In recent years, mitochondria have been found to provide a novel targeting site for new anticancer drugs (known as "mitocans") that ca...

show abstract

Establishment of Human Cell Lines Lacking Mitochondrial DNA

Cited by 110 publications

References 24 publications

Mitochondrial damage contributes toPseudomonas aeruginosaactivation of the inflammasome and is downregulated by autophagy

Mitochondrial damage contributes toPseudomonas aeruginosaactivation of the inflammasome and is downregulated by autophagy

Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Contact Info

Product

Resources

About