Functional Differences between Mitochondrial Haplogroup T and Haplogroup H in HEK293 Cybrid Cells

<div><h3>Background</h3><p>Epidemiological case-control studies have revealed associations between mitochondrial haplogroups and the onset and/or progression of various multifactorial diseases. For instance, mitochondrial haplogroup T was previously shown to be associated with vascular diseases, including coronary artery disease and diabetic retinopathy. In contrast, haplogroup H, the most frequent haplogroup in Europe, is often found to be more prevalent in healthy control subjects than in patient study groups. However, justifications for the assumption that haplogroups are functionally distinct are rare. Therefore, we attempted to compare differences in mitochondrial function between haplogroup H and T cybrids.</p> <h3>Methodology/Principal Findings</h3><p>Mitochondrial haplogroup H and T cybrids were generated by fusion of HEK293 cells devoid of mitochondrial DNA with isolated thrombocytes of individuals with the respective haplogroups. These cybrid cells were analyzed for oxidative phosphorylation (OXPHOS) enzyme activities, mitochondrial DNA (mtDNA) copy number, growth rate and susceptibility to reactive oxygen species (ROS). We observed that haplogroup T cybrids have higher survival rate when challenged with hydrogen peroxide, indicating a higher capability to cope with oxidative stress.</p> <h3>Conclusions/Significance</h3><p>The results of this study show that functional differences exist between HEK293 cybrid cells which differ in mitochondrial genomic background.</p> </div

Growth curves of mitochondrial haplogroup-specific cybrid cells.

<p>The number of cells on the days given were normalized to the number of cells on day two and determined as growth rate. (A) Comparison of HEK H (n = 3; gray circles) and HEK T (n = 3; black squares) cybrids at days three to seven in glucose medium. (B) Comparison of HEK H (n = 3; gray circles) and HEK T (n = 3; black squares) cybrids at days three to seven in galactose medium. Mean values of growth rates are given; error bars: standard deviation; *p<0.05.</p

Enzymatic activities of citrate synthase and oxidative phosphorylation complexes I – V in haplogroup H and T cybrid cells.

a<p>Values are given as mean ± standard deviation (SD).</p>b<p>P-value: Independent samples t-test.</p>c<p>Reported previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone-0052367-g002" target="_blank">Figure 2</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone.0052367-Mueller2" target="_blank">[31]</a>.</p><p>Enzymatic activity measurements were made on isolated mitochondria of cells grown in glucose medium with antibiotics, and on cells with five to 15 passages after the cybridization process.</p

Results of TaqMan qPCR analysis of HEK H and HEK T cybrid competitive co-cultures.

<p>After 10, 20 and 30 days (d10, d20, d30) of co-culture, isolated DNA of the cell mixtures was analyzed using TaqMan qPCR. ΔC_t values were calculated by subtraction of the mean C_t value of the FAM signal (probe recognizing haplogroup T) from the mean C_t value of the VIC signal (probe recognizing haplogroup H). ΔΔC_t values were calculated by subtraction of the mean ΔC_t values of the original cell mixtures (n = 36; day zero) from the mean ΔC_t values of all co-cultures at days 10, 20 or 30 (n = 36; except for d30 in galactose: n = 35). Dominance of haplogroup H results in a negative ΔΔC_t value and is presented as gray bars, whereas dominance of haplogroup T results in a positive ΔΔC_t value and is presented as black bars. (A) ΔΔC_t values of competitive co-cultures cultivated in glucose medium, at days 10, 20 and 30. (B) ΔΔC_t values of competitive co-cultures cultivated in galactose medium, at days 10, 20 and 30. Mean ΔΔC_t values are given; error bars: standard deviation.</p

Figure 1. Phylogenetic tree of haplogroup H and T subsets.

<p>The phylogenetic tree was constructed according to phylotree.org <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone.0052367-vanOven1" target="_blank">[32]</a>. ^aC16296T did not appear in the mtDNA sequence of cybrid T1. ^bBases of the Revised Cambridge Reference Sequence that appear in HEK T cybrids as polymorphisms diagnostic for non-H haplogroups (m.73A>G, m.2706A>G, m.7028C>T, m.11719G>A and m.14766C>T).</p

Mitochondrial DNA copy number in cybrid cells.

<p>(A) Comparison of all cybrid clones cultivated in glucose medium and galactose medium. (B) Comparison of HEK H and HEK T cybrids cultivated in glucose medium. (C) Comparison of HEK H and HEK T cybrids cultivated in galactose medium. Mean values of copy numbers are given; error bars: standard deviation; *p<0.05.</p

Cell survival after treatment with H₂O₂.

<p>Cell survival was measured 24 hours after H₂O₂ treatment and calculated as a percentage of the ratio between treated and untreated cells (% cell survival). (A) Comparison of HEK H (n = 3; gray bars) and HEK T (n = 3; black bars) cybrids at 250 µM to 475 µM H₂O₂ in glucose medium without serum and without sodium pyruvate. (B) Comparison of HEK H (n = 3; gray bars) and HEK T (n = 3; black bars) cybrids at 100 µM to 250 µM H₂O₂ in galactose medium without serum and without sodium pyruvate. Mean values of % cell survival are given; error bars: standard deviation; *p<0.05.</p

New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and <i>In Vitro</i> Generation

<div><p>Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. <i>In vitro</i> induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.</p></div

Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model

<div><p>Introduction</p><p>Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer’s oxidative phosphorylation system.</p><p>Methods</p><p>Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2)]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content).</p><p>Results</p><p>Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention.</p><p>Conclusions</p><p>Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens. Therefore, we propose that a ketogenic diet and/or calorie restriction should be further evaluated as a possible adjuvant therapy for patients undergoing treatment for neuroblastoma.</p></div

Immunohistochemical (IHC) staining of mitochondrial OXPHOS complexes I-V and VDAC in SH-SY5Y xenograft tumors.

<p>IHC staining of NB sections were scored on a scale from 0–3 as described in the methods section. The CR-KD group showed a significant decrease in complex I staining. All other evaluated parameters were unaffected by dietary changes. Voltage-dependent ion channel (VDAC) protein levels are used as a surrogate marker of mitochondrial mass. Statistics: ANOVA (p <0.05) followed by two-tailed Dunnett’s test correcting for multiple comparisons. Diet groups are compared to the corresponding SD group.</p