Reversible regulation of metastasis by ROS-generating mtDNA mutations

It has been controversial whether mtDNA mutations are responsible for oncogenic transformation (normal cells to develop tumors), and for malignant progression (tumor cells to develop metastases). To clarify this issue, we created trans-mitochondrial cybrids with mtDNA exchanged between mouse tumor cells that express different metastatic phenotypes. The G13997A mutation in the ND6 gene of mtDNA from high metastatic tumor cells reversibly controlled development of metastases by overproduction of reactive oxygen species (ROS), but did not control development of tumors. The mtDNA-mediated reversible control of metastasis reveals a novel function of mtDNA, and suggests that ROS scavengers may be therapeutically effective in suppressing metastasis.     

A Specific Nuclear DNA Background Is Required for High Frequency Lymphoma Development in Transmitochondrial Mice with G13997A mtDNA

We previously found that mouse mitochondrial DNA (mtDNA) with a G13997A mutation (G13997A mtDNA) controls not only the transformation of cultured lung carcinoma cells from poorly metastatic into highly metastatic cells, but also the transformation of lymphocytes into lymphomas in living C57BL/6 (B6) mice. Because the nuclear genetic background of the B6 strain makes the strain prone to develop lymphomas, here we examined whether G13997A mtDNA independently induces lymphoma development even in mice with the nuclear genetic background of the A/J strain, which is not prone to develop lymphomas. Our results showed that the B6 nuclear genetic background is required for frequent lymphoma development in mice with G13997A mtDNA. Moreover, G13997A mtDNA in mice did not enhance the malignant transformation of lung adenomas into adenocarcinomas or that of hepatocellular carcinomas from poorly metastatic into highly metastatic carcinomas. Therefore, G13997A mtDNA enhances the frequency of lymphoma development under the abnormalities in the B6 nuclear genome, and does not independently control tumor development and tumor progression.     

Enhanced glycolysis induced by mtDNA mutations does not regulate metastasis

We addressed the issue of whether enhanced glycolysis caused by mtDNA mutations independently induces metastasis in tumor cells using mtDNA transfer technology. The resultant trans-mitochondrial cybrids sharing the same nuclear background of poorly metastatic carcinoma P29 cells, P29mtA11 and P29mtDelta cybrids, possessed mtDNA with a G13997A mutation from highly metastatic carcinoma A11 cells and mtDNA with a 4696bp deletion mutation, respectively. The P29mtDelta cybrids expressed enhanced glycolysis, but did not express ROS overproduction and high metastatic potential, whereas P29mtA11 cybrids showed enhanced glycolysis, ROS overproduction, and high metastatic potential. Thus, enhanced glycolysis alone does not induce metastasis in the cybrids.     

Din7 and Mhr1 expression levels regulate double-strand-break–induced replication and recombination of mtDNA at ori5 in yeast

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ⁻ mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ⁻ cells and increased deletion mutagenesis at the ori5 region in ρ⁺ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.     

Generation of trans-mitochondrial mito-mice by the introduction of a pathogenic G13997A mtDNA from highly metastatic lung carcinoma cells

To investigate the effects of respiration defects on the disease phenotypes, we generated trans-mitochondrial mice (mito-mice) by introducing a mutated G13997A mtDNA, which specifically induces respiratory complex I defects and metastatic potentials in mouse tumor cells. First, we obtained ES cells and chimeric mice containing the G13997A mtDNA, and then we generated mito-mice carrying the G13997A mtDNA via its female germ line transmission. The three-month-old mito-mice showed complex I defects and lactate overproduction, but showed no other phenotypes related to mitochondrial diseases or tumor formation, suggesting that aging or additional nuclear abnormalities are required for expression of other phenotypes.     

Trading mtDNA uncovers its role in metastasis

It has been controversial for many years of whether mtDNA mutations are involved in phenotypes related to cancer due to the difficulty in excluding possible involvement of nuclear DNA mutations in these phenotypes. We addressed this issue by complete trading of mtDNAs between tumor cells expressing different metastatic phenotypes. Resultant trans-mitochondrial cybrids share the same nuclear background, but possess mtDNA from tumor cells expressing different metastatic phenotypes, and thus can be used to uncover the role of mtDNA in these phenotypes. The results showed that mtDNA controls development of metastasis in tumor cells, while tumor development is controlled by nuclear genome.Key words: pathogenic mtDNA mutations, respiration defects, enhanced glycolysis, ROS overproduction, rho-zero cells, mtDNA transfer technology, metastasisHuman mtDNAs with pathogenic mutations inducing significant respiration defects have been shown to be closely associated with mitochondrial diseases.^1,2 Although mitochondrial respiratory function is controlled by both nuclear and mitochondrial genomes, the pathogenicity of these mtDNA mutations has been proven by co-transmission of the mutant mtDNAs and mitochondrial respiration defects to mtDNA-less (ρ⁰) human cells: the resultant trans-mitochondrial cybrids sharing the same nuclear backgrouond showed respiration defects, only when they accumulated the mutated mtDNA from the patients.^3–6 Moreover, we generated transmitochondrial mito-mice sharing the same nuclear background, but carrying various proportions of mtDNA with a pathogenic mutation, and provided model systems for studying exactly how mtDNAs with pathogenic mutations are transmitted and distributed in tissues resulting in the pathogenesis of mitochondrial diseases that show various clinical phenotypes.^7–9With respect to the involvement of mtDNA in tumor phenotypes, it has been proposed that most chemical carcinogens bind preferentially to mtDNA rather than to nuclear DNA in mammalian cells,^10–12 and thus, mtDNA should be the major cellular target of chemical carcinogens, and resultant creation of mutations in mtDNAs is responsible for expression of tumor phenotypes.¹²Although, there has been no direct evidence for creation of mtDNA mutations by chemical carcinogens, and for their contribution to tumor development in mammalian cells, recent studies showed that somatic mtDNA mutations accumulated in human colorectal tumors¹³ and in various tumor types¹⁴ rather than in normal cells of the same subjects, probably by the clonal expansion of the mutated mtDNAs along with the repeated division of tumor cells. Many subsequent studies supported preferential accumulation of mutated mtDNAs in tumor cells,^15–18 suggesting that mutated mtDNAs in tumor cells have acquired replication advantages to be homoplasmic. However, these studies did not address the fundamental question of whether the mutated mtDNAs are involved in tumor development.Our previous studies directly addressed this issue using transmitochondrial cybrids obtained by mtDNA trading between normal and tumor cells, and provided convincing evidence that mutations in nuclear DNA, but not in mtDNA were involved in tumor development in the mouse^19,20 and in human cultured cells.^21,22 The possibility that these observations may represent some specific tumor cases can be excluded since there has been no statistical evidence for association of tumor development and pathogenic mtDNA mutations in the patients with mitochondrial diseases expressing respiration defects caused by pathogenic mutations in mtDNA. The possibility that some polymorphic mtDNA mutations that do not induce respiration defects, but somehow contribute to tumor development also can be excluded, because there has been no statistical evidence for the presence of maternal inheritance of tumor development in spite of the strictly maternal inheritance of mammalian mtDNA.^23,24Nonetheless, it was still possible that mtDNA mutations are involved in other processes than oncogenic transformation of normal cells to develop tumors, such as in malignant progression of tumor cells to develop a metastatic potential. Recent studies demonstrated that mitochondrial respiration defects in TCA-cycle enzymes caused by nuclear DNA mutations controls tumor phenotypes as a consequence of induction of a pseudo-hypoxic pathway under normoxia.^25–27 Thus, some mtDNA mutations also induce the pseudo-hypoxic pathway under normoxia by inducing mitochondrial respiration defects. However, there has been no direct evidence for involvement of mtDNA mutations in malignant progression or in the regulation of the pseudo-hypoxic pathway under normoxia, because of the difficulty in excluding possible contribution of nuclear DNA mutations in these processes.²⁸Recently, we addressed this issue using trans-mitochondrial cybrids²⁹ obtained by complete trading of mtDNAs between highly and poorly metastatic mouse lung carcinoma cells (Fig. 1). By this approach, we could provide convincing evidence for the control of malignant progression of tumor cells to develop metastatic potential by mtDNA:²⁹ all the trans-mitochondrial cybrids with mtDNA from highly metastatic tumor cells expressed high metastatic potential, while those with mtDNA from poorly metastatic tumor cells expressed low metastatic potential, irrespective of whether their nuclear genome was derived from highly or poorly metastatic tumor cells. The findings in our study²⁹ can be summarized as follows: (1) A missense G13997A mutation in the ND6 gene of mtDNA from highly metastatic lung tumor cells induces a complex I defect, and reversibly controls malignant progression of tumor cells to develop metastatic potential, but does not control oncogenic transformation of normal cells to develop tumors; (2) The complex I defect simultaneously induces enhanced glycolysis and ROS overproduction, but induction of metastasis is due to ROS overproduction; (3) ROS overproduction induces metastasis not by acceleration of genetic instability as usually proposed, but by reversible upregulation of nuclear-coded genes related to metastasis, such as Mcl-1; (4) ROS scavengers are therapeutically effective in suppressing mtDNA-mediated metastasis.Open in a separate windowFigure 1Scheme for the isolation of the trans-mitochondrial cybrids with completely exchanged mtDNA between parental cells expressing different metastatic phenotypes. Trading mtDNA shown in this scheme uncovered a role of mtDNA in metastasis. For trading mtDNA, parental P29 and A11 cells were treated with ditercalinium, an antitumor bis-intercalating agent, to isolate ρ⁰P29 and ρ⁰A11 cells (*), which have no mtDNA. Complete depletion of mtDNA was confirmed by PCR analysis. Enucleated cells of the mtDNA donors were prepared by their pretreatment with cytochalasin B and centrifugation. Resultant cytoplasts were fused with ρ⁰ cells by polyethylene glycol to obtain trans-mitochondrial cybrids. High metastatic potential is transferred to the P29mtA11 cybrids with the transfer of mtDNA from the A11 cells, and poor metastatic potential is transferred to the A11mtP29 cybrids with the transfer of mtDNA from the P29 cells. Involvement of cytoplasmic factors other than mtDNA from the A11 cells in expression of the high metastatic potential in the P29mtA11 cybrids can be ruled out by the observations that the A11mtP29 cybrids lost their high metastatic potential, even though they always contain cytoplasmic factors transcribed by the nuclear genes derived from the A11 cells.Thus, our study partly resolves the controversial issue on the relevance or irrelevance of mtDNA mutations in tumor development and/or tumor phenotypes by showing that mutations in mtDNA control development of metastasis in tumor cells.²⁹ Considering that complex I defects simultaneously induce enhanced glycolysis under normoxia (the Warburg effect) and ROS overproduction,²⁹ it remains possible that the Warburg effect alone can control metastasis independently from ROS overproduction. More recently, we examined this possibility by generating trans-mitochondrial cybrids with the deletion mutant mtDNA, which can be expected to induce overall respiration defects, and express enhanced glycolysis under normoxia, but not express ROS overproduction. The results showed that the Warburg effect alone did not control metastasis.     

PLD1 promotes reactive oxygen species production in vascular smooth muscle cells and injury-induced neointima formation

Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1^?/? VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1^?/? VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty.     

A Novobiocin Derivative,XN4, Inhibits the Proliferation of Chronic Myeloid Leukemia Cells by Inducing Oxidative DNA Damage

XN4 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). The inhibition of proliferation of K562 and K562/G01 cells was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide). The mRNA levels of NADPH oxidase 1-5 (Nox1-5) genes were evaluated by qRT-PCR. The levels of extracellular reactive oxygen species (ROS), DNA damage, apoptosis, and cell cycle progression were examined by flow cytometry (FCM). Protein levels were analyzed by immunoblotting. XN4 significantly inhibited the proliferation of K562 and K562/G01 cells, with IC₅₀ values of 3.75±0.07 µM and 2.63±0.43 µM, respectively. XN4 significantly increased the levels of Nox4 and Nox5 mRNA, stimulating the generation of intracellular ROS, inducing DNA damage and activating ATM-γ-H2AX signaling, which increased the number of cells in the S and G2/M phase of the cell cycle. Subsequently, XN4 induced apoptotic cell death by activating caspase-3 and PARP. Moreover, the above effects were all reversed by the ROS scavenger N-acetylcysteine (NAC). Additionally, XN4 can induce apoptosis in progenitor/stem cells isolated from CML patients’ bone marrow. In conclusion, XN4-induced DNA damage and cell apoptosis in CML cells is mediated by the generation of ROS.     

Robust DNA Damage Response and Elevated Reactive Oxygen Species in TINF2-Mutated Dyskeratosis Congenita Cells

Dyskeratosis Congenita (DC) is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF) syndromes, converges on the DNA damage response (DDR) pathway and subsequent elevation of reactive oxygen species (ROS). Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT), perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients’ cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC) for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold) and ROS (1.5-fold to 2-fold). Upon exposure to ionizing radiation (XRT), DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold). DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease). Together, our data supports a mechanism whereby telomerase deficiency and subsequent shortened telomeres initiate a DDR and create a pro-oxidant environment, especially in cells carrying the TINF2 mutations. Finally, the ameliorative effects of antioxidants in vitro suggest this could translate to therapeutic benefits in DC patients.     

N-acetylcysteine increases the frequency of bone marrow pro-B/pre-B cells, but does not reverse cigarette smoking-induced loss of this subset

Background

We previously showed that mice exposed to cigarette smoke for three weeks exhibit loss of bone marrow B cells at the Pro-B-to-pre-B cell transition, but the reason for this is unclear. The antioxidant N-acetylcysteine (NAC), a glutathione precursor, has been used as a chemopreventive agent to reduce adverse effects of cigarette smoke exposure on lung function. Here we determined whether smoke exposure impairs B cell development by inducing cell cycle arrest or apoptosis, and whether NAC treatment prevents smoking-induced loss of developing B cells.

Methodology/Principal Findings

Groups of normal mice were either exposed to filtered room air or cigarette smoke with or without concomitant NAC treatment for 5 days/week for three weeks. Bone marrow B cell developmental subsets were enumerated, and sorted pro-B (B220⁺CD43⁺) and pre-B (B220⁺CD43⁻) cell fractions were analyzed for cell cycle status and the percentage of apoptotic cells. We find that, compared to sham controls, smoke-exposed mice have ∼60% fewer pro-B/pre-B cells, regardless of NAC treatment. Interestingly, NAC-treated mice show a 21–38% increase in total bone marrow cellularity and lymphocyte frequency and about a 2-fold increase in the pro-B/pre-B cell subset, compared to sham-treated controls. No significant smoking- or NAC-dependent differences were detected in frequency of apoptotic cells or the percentage cells in the G1, S, or G2 phases of the cycle.

Conclusions/Significance

The failure of NAC treatment to prevent smoking-induced loss of bone marrow pre-B cells suggests that oxidative stress is not directly responsible for this loss. The unexpected expansion of the pro-B/pre-B cell subset in response to NAC treatment suggests oxidative stress normally contributes to cell loss at this developmental stage, and also reveals a potential side effect of therapeutic administration of NAC to prevent smoking-induced loss of lung function.     

Parathyroid Hormone Administration Improves Bone Marrow Microenvironment and Partially Rescues Haematopoietic Defects in Bmi1-Null Mice

The epigenetic regulator Bmi1 is key in haematopoietic stem cells, and its inactivation leads to defects in haematopoiesis. Parathyroid hormone (PTH), an important modulator of bone homeostasis, also regulates haematopoiesis, so we asked whether PTH administration improves bone marrow microenvironment and rescues the haematopoietic defects in Bmi1-null mice. The mice were treated with PTH1-34 (containing the first 34 residues of mature PTH), an anabolic drug currently used for treating osteoporosis, and compared with the vehicle-treated Bmi1 ^-/- and wild-type littermates in terms of skeletal and haematopoietic phenotypes. We found that the administration significantly increased all parameters related to osteoblastic bone formation and significantly reduced the adipocyte number and PPARγ expression. The bone marrow cellularity, numbers of haematopoietic progenitors and stem cells in the femur, and numbers of lymphocytes and other white blood cells in the peripheral blood all increased significantly when compared to vehicle-treated Bmi1^-/- mice. Moreover, the number of Jagged1-positive cells and percentage of Notch intracellular domain-positive bone marrow cells and protein expression levels of Jagged1 and NICD in bone tissue were also increased in Bmi1 ^-/- mice upon PTH1-34 administration,whereas the up-regulation of PTH on both Notch1 and Jagged1 gene expression was blocked by the Notch inhibitor DAPT administration. These results thus indicate that PTH administration activates the notch pathway and partially rescues haematopoietic defects in Bmi1-null mice, further suggesting that haematopoietic defects in the animals are not only a result of reduced self-renewal of haematopoietic stem cells but also due to impaired bone marrow microenvironment.     

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Introduction  Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H₂O₂, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Materials and methods  Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl⁺ cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl⁺ CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl⁺ cells. Conclusion  NAC enhances imatinib-induced apoptosis of Bcr-Abl⁺ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.     

p47phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging

Bone remodeling is age-dependently regulated and changes dramatically during the course of development. Progressive accumulation of reactive oxygen species (ROS) has been suspected to be the leading cause of many inflammatory and degenerative diseases, as well as an important factor underlying many effects of aging. In contrast, how reduced ROS signaling regulates inflammation and remodeling in bone remains unknown. Here, we utilized a p47^phox knock-out mouse model, in which an essential cytosolic co-activator of Nox2 is lost, to characterize bone metabolism at 6 weeks and 2 years of age. Compared with their age-matched wild type controls, loss of Nox2 function in p47^phox−/− mice resulted in age-related switch of bone mass and strength. Differences in bone mass were associated with increased bone formation in 6-week-old p47^phox−/− mice but decreased in 2-year-old p47^phox−/− mice. Despite decreases in ROS generation in bone marrow cells and p47^phox-Nox2 signaling in osteoblastic cells, 2-year-old p47^phox−/− mice showed increased senescence-associated secretory phenotype in bone compared with their wild type controls. These in vivo findings were mechanistically recapitulated in ex vivo cell culture of primary fetal calvarial cells from p47^phox−/− mice. These cells showed accelerated cell senescence pathway accompanied by increased inflammation. These data indicate that the observed age-related switch of bone mass in p47^phox-deficient mice occurs through an increased inflammatory milieu in bone and that p47^phox-Nox2-dependent physiological ROS signaling suppresses inflammation in aging.     

DNA Damage Responses and Oxidative Stress in Dyskeratosis Congenita

Dyskeratosis congenita (DC) is an inherited multisystem disorder of premature aging, cancer predisposition, and bone marrow failure caused by selective exhaustion of highly proliferative cell pools. DC patients also have a poor tolerance to chemo/radiotherapy and bone marrow transplantation. Although critically shortened telomeres and defective telomere maintenance contribute to DC pathology, other mechanisms likely exist. We investigate the link between telomere dysfunction and oxidative and DNA damage response pathways and assess the effects of antioxidants. In vitro studies employed T lymphocytes from DC subjects with a hTERC mutation and age-matched controls. Cells were treated with cytotoxic agents, including Paclitaxel, Etoposide, or ionizing radiation. Apoptosis and reactive oxygen species (ROS) were assessed by flow cytometry, and Western blotting was used to measure expression of DNA damage response (DDR) proteins, including total p53, p53S15, and p21^WAF. N-acetyl-cysteine (NAC), an antioxidant, was used to modulate cell growth and ROS. In stimulated culture, DC lymphocytes displayed a stressed phenotype, characterized by elevated levels of ROS, DDR and apoptotic markers as well as a proliferative defect that was more pronounced after exposure to cytotoxic agents. NAC partially ameliorated the growth disadvantage of DC cells and decreased radiation-induced apoptosis and oxidative stress. These findings suggest that oxidative stress may play a role in the pathogenesis of DC and that pharmacologic intervention to correct this pro-oxidant imbalance may prove useful in the clinical setting, potentially alleviating untoward toxicities associated with current cytotoxic treatments.     

<Emphasis Type="Italic">N</Emphasis>-Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-cysteine protects thyroid cells against DNA damage induced by external and internal irradiation

We evaluated the effect of the antioxidant N-acetyl-l-cysteine (NAC) on the levels of reactive oxygen species (ROS), DNA double strand breaks (DSB) and micronuclei (MN) induced by internal and external irradiation using a rat thyroid cell line PCCL3. In internal irradiation experiments, ROS and DSB levels increased immediately after ¹³¹I addition and then gradually declined, resulting in very high levels of MN at 24 and 48 h. NAC administration both pre- and also post-¹³¹I addition suppressed ROS, DSB and MN. In external irradiation experiments with a low dose (0.5 Gy), ROS and DSB increased shortly and could be prevented by NAC administration pre-, but not post-irradiation. In contrast, external irradiation with a high dose (5 Gy) increased ROS and DSB in a bimodal way: ROS and DSB levels increased immediately after irradiation, quickly returned to the basal levels and gradually rose again after >24 h. The second phase was in parallel with an increase in 4-hydroxy-2-nonenal. The number of MN induced by the second wave of ROS/DSB elevations was much higher than that by the first peak. In this situation, NAC administered pre- and post-irradiation comparably suppressed MN induced by a delayed ROS elevation. In conclusion, a prolonged ROS increase during internal irradiation and a delayed ROS increase after external irradiation with a high dose caused serious DNA damage, which were efficiently prevented by NAC. Thus, NAC administration even both after internal or external irradiation prevents ROS increase and eventual DNA damage.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Nanoceria protects from alterations in oxidative metabolism and calcium overloads induced by TNFα and cycloheximide in U937 cells: pharmacological potential of nanoparticles

The present study is aimed to determine the protective effect of a novel nanoparticle with antioxidant properties, nanoceria, on reactive oxygen species (ROS) production, and calcium signaling evoked by the tumor necrosis factor-alpha (TNFα) in combination with cycloheximide (CHX) on apoptosis in the human histiocytic lymphoma cell line U937. Our results show that treatment of U937 cells with 10 ng/mL TNFα in combination with 1 μg/mL CHX led to several Ca²⁺ alterations. These stimulatory effects on calcium signals were followed by intracellular ROS production and mitochondria membrane depolarization, as well as a time-dependent increase in caspase-8 and -9 activities. Our results show that the pretreatment with well known antioxidants such as trolox and N-acetyl cysteine (NAC) partially reduced the apoptotic effects due to the administration of TNFα plus cycloheximide. Furthermore, nanoceria had a stronger protective effect than trolox or NAC. Our findings also suggest that TNFα plus cycloheximide-induced apoptosis is dependent on alterations in cytosolic concentration of calcium [Ca²⁺]_c and ROS generation in human histiocytic U937 cells.     

Gene profile of myeloid-derived suppressive cells from the bone marrow of lysosomal acid lipase knock-out mice

Background

Lysosomal acid lipase (LAL) controls development and homeostasis of myeloid lineage cells. Loss of the lysosomal acid lipase (LAL) function leads to expansion of myeloid-derived suppressive cells (MDSCs) that cause myeloproliferative neoplasm.

Methodology/Principal Findings

Affymetrix GeneChip microarray analysis identified detailed intrinsic defects in Ly6G⁺ myeloid lineage cells of LAL knock-out (lal−/−) mice. Ingenuity Pathway Analysis revealed activation of the mammalian target of rapamycin (mTOR) signaling, which functions as a nutrient/energy/redox sensor, and controls cell growth, cell cycle entry, cell survival, and cell motility. Loss of the LAL function led to major alteration of large GTPase and small GTPase signal transduction pathways. lal−/− Ly6G⁺ myeloid cells in the bone marrow showed substantial increase of cell proliferation in association with up-regulation of cyclin and cyclin-dependent kinase (cdk) genes. The epigenetic microenvironment was significantly changed due to the increased expression of multiple histone cluster genes, centromere protein genes and chromosome modification genes. Gene expression of bioenergetic pathways, including glycolysis, aerobic glycolysis, mitochondrial oxidative phosphorylation, and respiratory chain proteins, was also increased, while the mitochondrial function was impaired in lal−/− Ly6G⁺ myeloid cells. The concentration of reactive oxygen species (ROS) was significantly increased accompanied by up-regulation of nitric oxide/ROS production genes in these cells.

Conclusions/Significance

This comprehensive gene profile study for the first time identifies and defines important gene pathways involved in the myeloid lineage cells towards MDSCs using lal−/− mouse model.