Bending and breaking the code: dynamic changes in promoter integrity may underlie a new mechanism regulating gene expression.
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Animals↗
Biomedical subjects
Glenn L Wilson
Publications and source records attributed to Glenn L Wilson.
Protection of INS-1 cells from free fatty acid-induced apoptosis by targeting hOGG1 to mitochondria.
Chronic exposure to elevated levels of free fatty acids (FFAs) impairs pancreatic beta-cell function and contributes to the decline of insulin secretion in type 2 diabetes. Previously, we reported that FFAs caused increased nitric oxide (NO) production, which damaged mitochondrial DNA (mtDNA) and ultimately led to apoptosis in INS-1 cells. To firmly establish the link between FFA-generated mtDNA damage and apoptosis, we stably transfected INS-1 cells with an expression vector containing the gene for the DNA repair enzyme human 8-oxoguanine DNA glycosylase/apurinic lyase (hOGG1) downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase. Successful integration of MTS-OGG1 into the INS-1 cellular genome was confirmed by Southern blot analysis. Western blots and enzyme activity assays revealed that hOGG1 was targeted to mitochondria and the recombinant enzyme was active. MTS-OGG1 cells showed a significant decrease in FFA-induced mtDNA damage compared with vector-only transfectants. Additionally, hOGG1 overexpression in mitochondria decreased FFA-induced inhibition of ATP production and protected INS-1 cells from apoptosis. These results indicate that mtDNA damage plays a pivotal role in FFA-induced beta-cell dysfunction and apoptosis. Therefore, targeting DNA repair enzymes into beta-cell mitochondria could be a potential therapeutic strategy for preventing or delaying the onset of type 2 diabetes symptoms.
Animals↗
Role of nitric oxide-induced mtDNA damage in mitochondrial dysfunction and apoptosis.
An increasing body of evidence suggests that nitric oxide (NO) can be cytotoxic and induce apoptosis. NO can also be genotoxic and cause DNA damage and mutations. It has been shown that NO damages mitochondrial DNA (mtDNA) to a greater extent than nuclear DNA. Previously, we reported that conditional targeting of the DNA repair protein hOGG1 into mitochondria using a mitochondria targeting sequence (MTS) augmented mtDNA repair of oxidative damage and enhanced cellular survival. To determine whether enhanced repair resulting from augmented expression of hOGG1 could also protect against the deleterious effects of NO, we used HeLa TetOff/MTS-OGG1-transfected cells to conditionally express hOGG1 in mitochondria. The effects of additional hOGG1 expression on repair of NO-induced mtDNA damage and cell survival were evaluated. These cells, along with vector transfectants, in either the presence or absence of doxycycline (Dox), were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate). Functional studies revealed that cells expressing recombinant hOGG1 were more proficient at repairing NO-induced mtDNA damage, which led to increased cellular survival following NO exposure. Moreover, the results described here show that conditional expression of hOGG1 in mitochondria decreases NO-induced inhibition of ATP production and protects cells from NO-induced apoptosis.
Apoptosis↗
Oxidative stress-induced apoptosis in neurons correlates with mitochondrial DNA base excision repair pathway imbalance.
Neurodegeneration can occur as a result of endogenous oxidative stress. Primary cerebellar granule cells were used in this study to determine if mitochondrial DNA (mtDNA) repair deficiencies correlate with oxidative stress-induced apoptosis in neuronal cells. Granule cells exhibited a significantly higher intracellular oxidative state compared with primary astrocytes as well as increases in reductants, such as glutathione, and redox sensitive signaling molecules, such as AP endonuclease/redox effector factor-1. Cerebellar granule cultures also exhibited an increased susceptibility to exogenous oxidative stress. Menadione (50 muM) produced twice as many lesions in granule cell mtDNA compared with astrocytes, and granule cell mtDNA repair was significantly less efficient. A decreased capacity to repair oxidative mtDNA damage correlates strongly with mitochondrial initiated apoptosis in these neuronal cultures. Interestingly, the mitochondrial activities of initiators for base excision repair (BER), the bifunctional glycosylase/AP lyases as well as AP endonuclease, were significantly higher in cerebellar granule cells compared with astrocytes. The increased mitochondrial AP endonuclease activity in combination with decreased polymerase gamma activity may cause an imbalance in oxidative BER leading to an increased production and persistence of mtDNA damage in neurons when treated with menadione. This study provides evidence linking neuronal mtDNA repair capacity with oxidative stress-related neurodegeneration.
Animals↗
Cytokines induce nitric oxide-mediated mtDNA damage and apoptosis in oligodendrocytes. Protective role of targeting 8-oxoguanine glycosylase to mitochondria.
Nitric oxide (NO) that is produced by inducible NO synthase (iNOS) in glial cells is thought to contribute significantly to the pathogenesis of multiple sclerosis. Oligodendrocytes can be stimulated to express iNOS by inflammatory cytokines, which are known to accumulate in the multiple sclerotic brain. The potentially pathological levels of NO produced under these circumstances can target a wide spectrum of intracellular components. We hypothesized that one of the critical targets for damage that leads to disease is mtDNA. In this study, we found that cytokines, in particular a combination of tumor necrosis factor-alpha (50 ng/ml) and IFNgamma (25 ng/ml), cause elevated NO production in primary cultures of rat oligodendrocytes. Western blot analysis revealed a strong enhancement of iNOS expression 48 h after cytokine treatment. Within the same time period, NO-mediated mtDNA damage was shown by Southern blot analysis and by ligation-mediated PCR. Targeting the DNA repair enzyme human 8-oxoguanine DNA glycosylase (hOGG1) to the mitochondria of oligodendrocytes had a protective effect against this cytokine-mediated mtDNA damage. Moreover, it was shown that mitochondrial transport sequence hOGG1-transfected oligodendrocytes had fewer apoptotic cells compared with cells containing vector only following treatment with the cytokines. Subsequent experiments revealed that targeting hOGG1 to mitochondria reduces the activation of caspase-9, showing that this recombinant protein works to reduce apoptosis that is occurring through a mitochondria-based pathway.
Animals↗
Involvement of mtDNA damage in free fatty acid-induced apoptosis.
A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.
Adenosine Triphosphate↗
TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells.
The protein transduction domain (PTD) from the HIV-1 TAT protein has been widely utilized to deliver biologically active macromolecules, including full-length proteins, into a variety of cell types in vitro and in vivo. Without additional targeting signals, the intracellular localization of the proteins delivered in this fashion appears to be cytoplasmic, nuclear or, as recently reported, endosomal. In this study, we show that the presence of the mitochondrial targeting signal (MTS) from hMnSOD on the N-terminus of TAT-fusion proteins directs them into mitochondria of breast cancer cells. We generated and purified fusion proteins containing GFP (MTS-GFP-TAT) or Exonuclease III (MTS-ExoIII-TAT) from Escherichia coli. The results of Western blots of subcellular fractions and fluorescent microscopic analyses revealed efficient protein transduction and mitochondrial localization of the fusion proteins. Specific exonuclease activity was found in the mitochondrial extracts isolated from MTS-ExoIII-TAT transduced cells. This increased exonuclease activity reduced the repair of mtDNA damage following oxidative stress. This diminished mtDNA repair led to a decrease in survival of breast cancer cells. Thus, the present study demonstrates the applicability of this new approach for intramitochondrial targeting of TAT-fusion proteins capable of modulating mitochondrial function and cell survival.
Breast Neoplasms↗
Contribution of mitochondrial DNA repair to cell resistance from oxidative stress.
Numerous studies have revealed that a part of the cellular response to chronic oxidative stress involves increased antioxidant capacity. However, another defense mechanism that has received less attention is DNA repair. Because of the important homeostatic role of mitochondria and the exquisite sensitivity of mitochondrial DNA (mtDNA) to oxidative damage, we hypothesized that mtDNA repair plays an important role in the protection against oxidative stress. To test this hypothesis mtDNA damage and repair was evaluated in normal HA1 Chinese hamster fibroblasts and oxidative stress-resistant variants isolated following chronic exposure to H2O2 or 95% O2. Reactive oxygen species were generated enzymatically using xanthine oxidase and hypoxanthine. When treated with xanthine oxidase reduced levels of initial mtDNA damage and enhanced mtDNA repair were observed in the cells from the oxidative stress-resistant variants, relative to the parental cell line. This enhanced mtDNA repair correlated with an increase in mitochondrial apurinic/apyrimidinic endonuclease activity in both H2O2- and O2-resistant HA1 variants. This is the first report showing enhanced mtDNA repair in the cellular response to chronic oxidative stress. These results provide further evidence for the crucial role that mtDNA repair pathways play in protecting cells against the deleterious effects of reactive oxygen species.
Animals↗
Oxidants in signal transduction: impact on DNA integrity and gene expression.
Physiological stimuli using reactive oxygen species (ROS) as second messengers caused nucleotide-specific base modifications in the hypoxic response element of the VEGF gene in lung vascular cells, with the 3' guanine of the HIF-1 DNA recognition sequence uniformly targeted. Modeling this effect by replacing the targeted guanine with an abasic site increased incorporation of HIF-1 and the bi-functional DNA repair enzyme and transcriptional coactivator, Ref-1/Ape1, into the transcriptional complex and engendered more robust reporter gene expression. Oxidants generated in the context of physiological signaling thus affect nuclear DNA integrity and may facilitate gene expression by optimizing DNA-protein interactions.
Angiotensin II↗
Genomic threats from physiological signals in lung vascular cells.
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Animals↗
Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress.
Mitochondrial DNA (mtDNA) is exposed to reactive oxygen species (ROS) produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions, including oxidized pyrimidines, in mtDNA may lead to structural genomic alterations, mitochondrial dysfunction and associated degenerative diseases. In Escherichia coli, oxidative pyrimidines are repaired by endonuclease III (EndoIII) and endonuclease VIII (EndoVIII). To determine whether the overexpression of two bacterial glycosylase/AP lyases which predominantly remove oxidized pyrimidines from DNA, could improve mtDNA repair and cell survival, we constructed vectors containing sequences for the EndoIII and EndoVIII downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase (MnSOD) and placed them under the control of the tetracycline (Tet)-response element. Successful integrations of MTS-EndoIII or MTS-EndoVIII into the HeLa Tet-On genome were confirmed by Southern blot. Western blots of mitochondrial extracts from MTS-EndoIII and MTS-EndoVIII clones revealed that the recombinant proteins are targeted into mitochondria and their expressions are doxycycline (Dox) dependent. Enzyme activity assays and mtDNA repair studies showed that the Dox-dependent expressions of MTS-EndoIII and MTS-EndoVIII are functional, and both MTS-EndoIII and MTS-EndoVIII (Dox+) clones were significantly more proficient at repair of oxidative damage in their mtDNA. This enhanced repair led to increased cellular resistance to oxidative stress.
Cell Survival↗
Ref-1/Ape is critical for formation of the hypoxia-inducible transcriptional complex on the hypoxic response element of the rat pulmonary artery endothelial cell VEGF gene.
The co-transcription factor and DNA repair enzyme, Redox effector factor-1/apurinic/apyrimidinic endonuclease (Ref-1/Ape), facilitates DNA binding and transcriptional activity of a number of transactivating factors, including those governing hypoxia-induced gene expression HIF-1. It is not known, however, whether Ref-1/Ape is a component of the hypoxic transcriptional complex. Electrophoretic mobility shift assays failed to detect direct DNA binding of Ref-1/Ape to either the HIF-1 or AP1 DNA recognition sequences present in the hypoxic response element of the VEGF gene. However, immunodepletion of Ref-1/Ape from nuclear extract prevented DNA binding of ATF/CREB and HIF-1 to the HIF-1 DNA recognition sequence. DNA affinity-precipitation analyses showed that Ref-1/Ape was part of the multiprotein transcriptional complex forming on a 64-mer sequence encompassing a minimal hypoxic response element. Immunodepletion of Ref-1/Ape prevented probe association with HIF-1, p300, ATF, and CREB. Co-immunoprecipitation experiments indicated that Ref-1/Ape present in nuclear extract interacted with HIF-1 and p300 but not ATF/CREB. However, when Ref-1/Ape was immunoprecipitated from the oligonucleotide probe, both HIF-1 and p300 remained probe-associated while ATF/CREB co-immunoprecipitated. These findings suggest that Ref-1/Ape is a critical component of the hypoxia-inducible transcriptional complex forming on the VEGF gene's hypoxic response element and that the presence of Ref-1/Ape in the complex is required for the apparent high affinity association between HIF-1 and its DNA recognition sequence.
Activating Transcription Factor 1↗
Mitochondrial DNA and aging.
Among the numerous theories that explain the process of aging, the mitochondrial theory of aging has received the most attention. This theory states that electrons leaking from the ETC (electron transfer chain) reduce molecular oxygen to form O2*- (superoxide anion radicals). O2*-, through both enzymic and non-enzymic reactions, can cause the generation of other ROS (reactive oxygen species). The ensuing state of oxidative stress results in damage to ETC components and mtDNA (mitochondrial DNA), thus increasing further the production of ROS. Ultimately, this 'vicious cycle' leads to a physiological decline in function, or aging. This review focuses on recent developments in aging research related to the role played by mtDNA. Both supportive and contradictory evidence is discussed.
Aged↗
alpha-complementation-enabled T7 expression vectors and their use for the expression of recombinant polypeptides for protein transduction experiments.
Over the past few years protein transduction has emerged as a powerful means for the delivery of proteins into cultured cells and into whole mice. This method is based on the ability of proteins containing protein transduction domains (PTDs), short stretches of 9-16 predominantly basic amino acids, to traverse the cytoplasmic membrane and accumulate inside cells in a time- and dose-dependent fashion. The number of PTDs, both natural and synthetic, is constantly expanding, as is the need to test newly discovered PTDs for their ability to mediate the internalization of the corresponding fusion proteins. Here we describe a strategy and methodology that can be used for the construction of vectors for the T7 RNA polymerase-driven expression of PTD fusions. The cloning in these vectors is facilitated by alpha-complementation. Also, these vectors are small in size (less than 3 kbp) and express influenza virus hemagglutinin tag as well as His tag as part of the fusion for immunological identification and purification respectively of expressed proteins.
Bacteriophage T7↗
The expression of Exonuclease III from E. coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress.
The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment. One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA). The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage. Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells. Successful repair of DNA is achieved through a precise and highly regulated multistep process. Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency. To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231. Following transfection, specific exonuclease activity was found in mitochondrial extracts. In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine. mtDNA repair was evaluated using quantitative Southern blot analysis. The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS. This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress.
Blotting, Southern↗
Targeting human 8-oxoguanine glycosylase to mitochondria of oligodendrocytes protects against menadione-induced oxidative stress.
Within the central nervous system (CNS), there is a differential susceptibility among cell types to certain pathological conditions believed to involve oxidative stress. Oligodendrocytes are extremely sensitive to oxidative stress, which correlates with a decreased ability to repair damage in mitochondrial DNA (mtDNA), as we have shown previously. To determine whether there is a causal relationship, studies were carried out to correct the deficit in repair of the oxidative damage in mtDNA in cultured oligodendrocytes. A vector containing a mitochondrial transport sequence (MTS) upstream of the sequence for human 8-oxoguanine-DNA glycosylase (OGG) was transfected into the cells. The efficiency of transfection and the localization of recombinant protein were determined by fluorescence microscopy and by Western blot analysis. Subsequent mtDNA repair studies, employing 100 micro M menadione to produce reactive oxygen species, showed a significant enhancement in repair of oxidative lesions in mtDNA of MTS-OGG transfected oligodendrocytes compared with cells transfected with vector only. Experiments were also conducted to determine the effect of changing mtDNA repair capacity on menadione-induced apoptosis in oligodendrocytes. These experiments show that targeting the OGG repair enzyme to mitochondria reduces the release of cytochrome c from the intermitochondrial space and the activation of caspase 9 in oligodendrocytes after exposure to menadione. Therefore, targeting of DNA repair enzymes to mitochondria appears to be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.
Animals↗
Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria.
Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.
Animals↗
Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death.
In rat cultured pulmonary arterial (PA), microvascular, and venous endothelial cells (ECs), the rate of mitochondrial (mt) DNA repair is predictive of the severity of xanthine oxidase (XO)-induced mtDNA damage and the sensitivity to XO-mediated cell death. To examine the importance of mtDNA damage and repair more directly, we determined the impact of mitochondrial overexpression of the DNA repair enzyme, Ogg1, on XO-induced mtDNA damage and cell death in PAECs. PAECs were transiently transfected with an Ogg1-mitochondrial targeting sequence construct. Mitochondria-selective overexpression of the transgene product was confirmed microscopically by the observation that immunoreactive Ogg1 colocalized with a mitochondria-specific tracer and, with an oligonucleotide cleavage assay, by a selective enhancement of mitochondrial Ogg1 activity. Overexpression of Ogg1 protected against both XO-induced mtDNA damage, determined by quantitative Southern analysis, and cell death as assessed by trypan blue exclusion and MTS assays. These findings show that mtDNA damage is a direct cause of cell death in XO-treated PAECs.
Animals↗