Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.     

Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.     

Cytosolic DNA triggers mitochondrial apoptosis via DNA damage signaling proteins independently of AIM2 and RNA polymerase III

A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA. This cell death induced by foreign DNA is independent of caspase-8 and carries features of mitochondrial apoptosis: dependence on BAX, APAF-1, and caspase-9. Although it does not require the IFN pathway known to be triggered by infection with MVA or transfected DNA via polymerase III and retinoid acid-induced gene I-like helicases, it shows a strong dependence on components of the DNA damage signaling pathway: cytosolic delivery of DNA or infection with MVA leads to phosphorylation of p53 (serines 15 and 46) and autophosphorylation of ataxia telangiectasia mutated (ATM); depleting p53 or ATM with small interfering RNA or inhibiting the ATM/ATM-related kinase family by caffeine strongly reduces apoptosis. Taken together, our findings suggest that a pathway activating DNA damage signaling plays an important independent role in detecting intracellular foreign DNA, thereby complementing the induction of IFN and activation of the AIM2 inflammasome.     

High-level transgene expression by homologous recombination-mediated gene transfer

Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expression.     

Prion protein protects against DNA damage induced by paraquat in cultured cells

Exposure of cells to paraquat leads to production of superoxide anion (O2*-). This reacts with hydrogen peroxide to give the hydroxyl radical (*OH), leading to lipid peroxidation and cell death. In this study, we investigated the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using an established model system, rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity.     

Hsp105alpha suppresses the aggregation of truncated androgen receptor with expanded CAG repeats and cell toxicity

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The N-terminal fragment of AR containing the expanded polyglutamine tract aggregates in cytoplasm and/or in nucleus and induces cell death. Some chaperones such as Hsp40 and Hsp70 have been identified as important regulators of polyglutamine aggregation and/or cell death in neuronal cells. Recently, Hsp105alpha, expressed at especially high levels in mammalian brain, has been shown to suppress apoptosis in neuronal cells and prevent the aggregation of protein caused by heat shock in vitro. However, its role in polyglutamine-mediated cell death and toxicity has not been studied. In the present study, we examined the effects of Hsp105alpha on the aggregation and cell toxicity caused by expansion of the polyglutamine tract using a cellular model of SBMA. The transient expression of truncated ARs (tARs) containing an expanded polyglutamine tract caused aggregates to form in COS-7 and SK-N-SH cells and concomitantly apoptosis in the cells with the nuclear aggregates. When Hsp105alpha was overexpressed with tAR97 in the cells, Hsp105alpha was colocalized to aggregates of tAR97, and the aggregation and cell toxicity caused by expansion of the polyglutamine tract were markedly reduced. Both beta-sheet and alpha-helix domains, but not the ATPase domain, of Hsp105alpha were necessary to suppress the formation of aggregates in vivo and in vitro. Furthermore, Hsp105alpha was found to localize in nuclear inclusions formed by ARs containing an expanded polyglutamine tract in tissues of patients and transgenic mice with SBMA. These findings suggest that overexpression of Hsp105alpha suppresses cell death caused by expansion of the polyglutamine tract without chaperone activity, and the enhanced expression of the essential domains of Hsp105alpha in brain may provide an effective therapeutic approach for CAG repeat diseases.     

Homologous recombination between transfected DNAs.

An extensive analysis of the fate and structure of polyomavirus-plasmid recombinant molecules transfected into Rat-1 cells has revealed that the DNA often becomes integrated within transformed cell DNA in a head-to-tail tandem arrangement. This occurs independently of the replicative capacity of the transforming DNA and is facilitated by the use of large quantities of DNA during transfection. These observations have led us to suggest that head-to-tail tandems are formed by homologous recombination between transfected DNAs either before or after integration within cellular DNA. To test this hypothesis, we have measured the transforming activity of pairs of mutant, nontransforming, recombinant plasmid DNAs that carry different lesions in the transforming gene of polyomavirus. The results show that, although the individual mutant DNAs are incapable of transformation, transfection with pairs of mutant DNAs leads to the formation of transformed cells at high frequency. Moreover, there is a direct relationship between the distance between the lesions in pairs of mutant DNAs and their transforming activity. Finally, analyses of the structures of integrated recombinant plasmid DNAs and the viral proteins within independent transformed cells prove that recombination occurs between the mutant genomes to generate a wild-type transforming gene.     

A Fatal Combination: A Thymidylate Synthase Inhibitor with DNA Damaging Activity

2′-deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2′-deoxythymidine 5′-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.     

The interaction between mobile DNAs and their hosts in a fluctuating environment

The interaction between mobile DNA sequences and their hosts raises important questions in the context of hosts which reproduce clonally with only rare horizontal transmission between clones. The activity of some mobile DNAs as reversible mutators of genes raises the possibility that, in a fluctuating environment, cells may gain an advantage if they have mobile DNAs which mutate genes whose inactivation is favoured in one of the environments that the population encounters. Here we analyse a model of this process and ask what would be the optimal rate of transposition in a population whose elements are maintained by this mechanism. We also examine the impact of horizontal transfer on such a population. With movement of elements between cells, we can imagine elements with differing rates of transposition and host cells with differing rates of transposition. We find that evolution in the population of elements favours a rapid rate of transposition, and evolution of the host cells favours cells in which this rapid rate of element-dependent transposition results in an optimal rate of transposition per cell. However, when horizontal transfer rates are high, some unexpected features of the model are observed. In particular, a polymorphism between cell types (some with an optimal rate of transposition and some with no transposition at all from endogenous elements) can be stably maintained. We consider the possible biological predictions of this analysis.     

A recB recC sbcB recJ host prevents recA-independent deletions in recombinant cosmid DNA propagated in Escherichia coli.

Segments of DNA are deleted from recombinant cosmid DNAs with high frequency during propagation in standard recA Escherichia coli hosts. An attempt has been made to derive an appropriate strain of E. coli, suitable for cosmid cloning, in which such deletions do not occur. We examined the effects of a series of host recombinational mutations on the deletion process, using six independent recombinant cosmids that carry inserts of mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying the recombinant cosmids were cultured serially in liquid medium, and the recombinant cosmid DNAs were extracted from the host cells and analyzed by agarose gel electrophoresis and by gene transfer of the DNAs into cultured mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or recN) quadruple combination of host mutations prevented the deletion of DNA segments. The recombinant cosmid DNAs propagated in E. coli hosts that carried this combination of mutations were functionally as well as structurally intact. We propose that the recJ (and/or recN) gene is involved in some aspect of the events that lead to deletions of cosmid DNA in a recB recC sbcB genetic background.     

Endonuclease G mediates endothelial cell death induced by carbamylated LDL

End-stage kidney disease is a terminal stage of chronic kidney disease, which is associated with a high incidence of cardiovascular disease. Cardiovascular disease frequently results from endothelial injury caused by carbamylated LDL (cLDL), the product of LDL modification by urea-derived cyanate. Our previous data suggested that cLDL induces mitogen-activated protein kinase-dependent mitotic DNA fragmentation and cell death. However, the mechanism of this pathway is unknown. The current study demonstrated that cLDL-induced endothelial mitotic cell death is independent of caspase-3. The expression of endonuclease G (EndoG), the nuclease implicated in caspase-independent DNA fragmentation, was significantly increased in response to cLDL exposure to the cells. The inhibition of EndoG by RNAi protected cLDL-induced DNA fragmentation, whereas the overexpression of EndoG induced more DNA fragmentation in endothelial cells. Ex vivo experiments with primary endothelial cells isolated from wild-type (WT) and EndoG knockout (KO) mice demonstrated that EndoG KO cells are partially protected against cLDL toxicity compared with WT cells. To determine cLDL toxicity in vivo, we administered cLDL or native LDL (nLDL) intravenously to the WT and EndoG KO mice and then measured floating endothelial cells in blood using flow cytometry. The results showed an increased number of floating endothelial cells after cLDL versus nLDL injection in WT mice but not in EndoG KO mice. Finally, the inhibitors of MEK-ERK1/2 and JNK-c-jun pathways decreased cLDL-induced EndoG overexpression and DNA fragmentation. In summary, our data suggest that cLDL-induced endothelial toxicity is caspase independent and results from EndoG-dependent DNA fragmentation.     

Sequence-specific toxicity of transfected retroviral DNA.

Experimental gene transfer and viral infections can result in the accumulation of unintegrated DNA in target cells. The effects of such accumulation on target cell metabolism have not been directly studied. The experiments reported in this paper show that transfection of cloned retroviral long-terminal-repeat (LTR) DNA, or of a variety of eukaryotic promoters, into proliferating HeLa cells results in rapid, sequence-specific, and dose-dependent cell death. Plasmids containing the Rous sarcoma virus LTR or the human immunodeficiency virus LTR cloned in pUC-related plasmids are 5 to 10 times more toxic than pUC19. The demonstrated sensitivity of eukaryotic cells to exogenously introduced DNA has important implications for the interpretation of gene transfer experiments and may be relevant to the pathogenic mechanisms in the course of retroviral infections such as AIDS.     

Streptococcus mutans lipoteichoic acid-induced apoptosis in cultured dental pulp cells from human deciduous teeth

Herein, we suggest that Streptococcus mutans lipoteichoic acid-induced death of dental pulp cells on human deciduous teeth is caused by apoptosis. We provide evidence for the causal role of apoptosis in this process by demonstrating an increase in the proportion of fragmented DNA in such dental pulp cells, which results in a ladder pattern of DNA fragmentation. Additionally, Streptococcus mutans lipoteichoic acid-induced apoptotic cell death is suppressed by caspase inhibitor. Collectively, these findings suggest that Streptococcus mutans lipoteichoic acids may cause apoptosis in human dental pulp cells, and serve as an important factor in pulpitis.     

Variables Affecting In Vivo Performance of High-Capacity Adenovirus Vectors

In high-capacity adenovirus (HC-Ad) vectors the size and/or composition of the vector genome influences vector stability during production and the expression profile following gene transfer. Typically, an HC-Ad vector will contain both a gene or an expression cassette and stuffer DNA that is required to balance the final vector genome to a size of between 27 and 36 kb. To gain an improved understanding of factors that may influence gene expression from HC-Ad vectors, we have generated a series of vectors that carry different combinations of human alpha-1 antitrypsin (hAAT) expression constructs and stuffer DNAs. Expression in vitro did not predict in vivo performance: all vectors expressed hAAT at similar levels when tested in cell culture. Hepatic expression was evaluated following in vivo gene transfer in C57BL/6J mice. hAAT levels obtained from genomic DNA were significantly higher than levels achieved with small cDNA expression cassettes. Expression was independent of the orientation and only marginally influenced by the location of the expression cassette within the vector genome. The use of lambda stuffer DNA resulted in low-level but stable expression for at least 3 months when higher doses were applied. A potential matrix attachment region element was identified within the hAAT gene and caused a 10-fold increase in expression when introduced in an HC-Ad vector genome carrying a phosphoglycerate kinase (pgk) hAAT cDNA construct. We also illustrate the influence of the promoter on anti-hAAT antibody formation in C57BL/6J mice: a human cytomegalovirus but not a pgk promoter resulted in an anti-hAAT antibody response. Thus, the overall design of HC-Ad vectors may significantly influence amounts and duration of gene expression at different levels.     

Adenoviral-induced islet cell cytotoxicity is not counteracted by Bcl-2 overexpression

BACKGROUND: The ability to transfer immunoregulatory, cytoprotective, or anti-apoptotic genes into pancreatic islet cells may allow enhanced resistance against the autoimmune destruction of these cells in type 1 diabetes. We describe here an inducible transduction system for expression of the anti-apoptotic bcl-2 gene in insulin-producing cells as a potential tool for protecting against beta-cell death. MATERIALS AND METHODS: Isolated pancreatic rat islet cells or rat insulinoma (RINm5F) cells were transduced using a progesterone antagonist (RU 486) inducible adenoviral vector system, expressing the bcl-2 gene. Bcl-2 overexpression was measured by Western blot assays and flow cytometry analysis. Following exposure to cytokines or to the mitochondrial uncoupler FCCP, cell survival was determined using fluorescence and electron microscopy, and a colorimetric assay (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]- 2H-tetrazolium-5-carboxanilide [XTT]-based) for cell viability. The mitochondrial membrane potential ((m)) was assessed using the lipophilic cationic membrane potential-sensitive dye JC-1. RESULTS: The adenoviral gene transfer system induced Bcl-2 expression in more than 70% of beta-cells and the protein expression levels were successfully regulated in response to varying concentrations of progesterone antagonist RU 486. Exposure of islet cells to proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma, or to the mitochondrial uncoupler FCCP resulted in disruption of the mitochondrial membrane potential ((m)) and beta-cell death. Bcl-2 overexpression stabilized (m) and prevented cell death in RINm5F cells but not in islet cells. In addition, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis. CONCLUSIONS: The RU 486-regulated adenoviral system can achieve an efficient control of gene transfer at relatively low doses of the adenoviral vector. However, Bcl-2 overexpression in islet cells did not prevent adenoviral- or cytokine-induced toxicity, suggesting that the specific death pathway involved in adenoviral toxicity in beta-cells may bypass the mitochondrial permeability transition event.     

Cytotoxic activity of 2',2'-difluorodeoxycytidine, 5-aza-2'-deoxycytidine and cytosine arabinoside in cells transduced with deoxycytidine kinase gene

Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2'-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2',2'-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death.     

bri3, a novel gene, participates in tumor necrosis factor-alpha-induced cell death

bri3 was identified to be a novel gene up-regulated in TNF-treated cells with suppressed subtractive hybridization (SSH) in our laboratory. Previous studies showed that overexpression of BRI3 induced apoptosis in L929 cells. To further study the function of bri3, we disrupted its expression by expressing bri3 antisense RNA. The antisense RNA promoted resistance to TNF-induced cell death by more than 1000-fold in L929 cells, suggesting the involvement of BRI3 in TNF-induced cell death in this cell line. Analysis of cell death caused by other apoptotic inducers showed that the effect of BRI3 antisense RNA is highly specific to TNF-induced cell death. Taken together, bri3 appears to play an important role in TNF-induced cell death. Finally, we reported here that BRI3 may be localized to lysosome and function through lysosome.     

Lack of site specific recombination of exogenous DNA in mouse L cells

Plasmids were constructed containing the HSV thymidine kinase gene and two copies of X. borealis 5S rDNA. Mouse L TK- cells were transformed with these DNAs, with selection for the TK+ gene. Transformed cells were then analyzed by Southern blot hybridization and hybridization in situ to determine whether integration of the exogenous DNA occurred at regions of chromosomal homology i.e., at the 5S rDNA regions. Four cell lines were analyzed by Southern blots. Differences in restriction endonuclease specificity strongly suggested that integration was at a different site in each cell line. Two cell lines were further analyzed by hybridization in situ; each showed a single integration site, both different from each other and different from the mouse L cell 5S rDNA sites. Therefore, the presence of two copies of the 5S rDNA gene in the DNA introduced by gene transfer and approximately 300-350 copies of the mouse 5S rDNA gene was not sufficient in these experiments to produce homologous integration into a specific site.