Downregulation of apoptosis-inducing factor in harlequin mutant mice sensitizes the myocardium to oxidative stress-related cell death and pressure overload-induced decompensation

Apoptosis-inducing factor (AIF), or programmed cell death 8 (Pdcd8), is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal apoptosis induced by oxidative stress. Conversely, in vitro, AIF has been demonstrated to have a proapoptotic role when, on induction of the mitochondrial death pathway, AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. To determine the role of AIF in myocardial apoptotic processes, we examined cardiomyocytes from an AIF-deficient mouse mutant, Harlequin (Hq). Hq mutant cardiomyocytes demonstrated increased sensitivity to H2O2-induced cell death. Further, Hq hearts subjected to ischemia/reperfusion revealed more cardiac damage and, unlike wild-type mice, the amount of damage increased with the age of the animal. Aortic banding caused enhanced hypertrophy, increased cardiomyocyte apoptotic and necrotic cell death, and accelerated progression toward maladaptive left ventricular remodeling in Hq mutant mice compared with wild-type counterparts. These findings correlated with a reduced capacity of subsarcolemmal mitochondria from Hq mutant hearts to scavenge free radicals. Together, these data demonstrate a critical role for AIF as a cardiac antioxidant in the protection against oxidative stress-induced cell death and development of heart failure induced by pressure overload.     

Cardiomyocyte apoptosis induced by Galphaq signaling is mediated by permeability transition pore formation and activation of the mitochondrial death pathway

Expression of the wild-type alpha subunit of Gq stimulates phospholipase C and induces hypertrophy in cardiomyocytes. Addition of Gq-coupled receptor agonists additionally activates phospholipase C, as does expression of a constitutively active mutant form of Galphaq. Under these conditions, hypertrophy is rapidly succeeded by apoptotic cellular and molecular changes, including myofilament disorganization, loss of mitochondrial membrane potential, alterations in Bcl-2 family protein levels, DNA fragmentation, increased caspase activity ( approximately 4-fold), cytochrome c redistribution, and nuclear chromatin condensation in approximately 12% of the cells. We used various interventions to define the molecular relationships between these events and identify potential sites at which these features of apoptosis could be rescued. Treatment with caspase inhibitors prevented DNA fragmentation and promoted myocyte survival; however, cytochrome c release and loss of mitochondrial membrane potential still occurred. In contrast, treatment with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore, not only prevented DNA fragmentation and reduced nuclear chromatin condensation but also preserved mitochondrial membrane potential and limited cytochrome c redistribution to only approximately 2% of cells. These data demonstrate the central role of mitochondrial membrane potential in initiation of caspase activation and downstream apoptotic events and suggest that preservation of mitochondrial integrity is crucial for prolonging the life and function of cardiomyocytes exposed to pathological levels of stress.     

Caspase-3 gene knockout defines cell lineage specificity for programmed cell death signaling in the ovary

Previous studies have proposed the involvement of caspase-3, a downstream executioner enzyme common to many paradigms of programmed cell death (PCD), in mediating the apoptosis of both germ and somatic cells in the ovary. Herein we used caspase-3 gene knockout mice to directly test for the functional requirement of this protease in oocyte and/or granulosa cell demise. Using both in vivo and in vitro approaches, we determined that oocyte death initiated as a result of either developmental cues or pathological insults was unaffected by the absence of caspase-3. However, granulosa cells of degenerating antral follicles in both mouse and human ovaries showed a strong immunoreaction using an antibody raised against the cleaved (activated) form of caspase-3. Furthermore, caspase-3 mutant female mice possessed aberrant atretic follicles containing granulosa cells that failed to be eliminated by apoptosis, as confirmed by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling) analysis of DNA cleavage and 4',6-diamidino-2-phenylindole staining of nuclear morphology (pyknosis). These in vivo results were supported by findings from in vitro cultures of wild-type and caspase-3-deficient antral follicles or isolated granulosa cells. Contrasting the serum starvation-induced occurrence of apoptosis in wild-type granulosa cells, caspase-3-null granulosa cells deprived of hormonal support were TUNEL-negative, showed attenuated chromatin condensation by 4',6-diamidino-2-phenylindole staining and exhibited delayed internucleosomal DNA cleavage. Such ex vivo findings underscore the existence of a cell autonomous (granulosa cell intrinsic) defect in apoptosis execution resulting from caspase-3 deficiency. We conclude that caspase-3 is functionally required for granulosa cell apoptosis during follicular atresia, but that the enzyme is dispensable for germ cell apoptosis in the female.     

Role of endonuclease G in senescence-associated cell death of human endothelial cells

Mitotic cells in culture show a limited replicative potential and after extended subculturing undergo a terminal growth arrest termed cellular senescence. When cells reach the senescent phenotype, this is accompanied by a significant change in the cellular phenotype and massive changes in gene expression, including the upregulation of secreted factors. In human fibroblasts, senescent cells also acquire resistance to apoptosis. In contrary, in human endothelial cells, both replicative and stress-induced premature senescence is accompanied by increased cell death; however mechanisms of cell death are poorly explored. In this communication, we addressed the role of endonuclease G (EndoG), a mitochondrial mediator of caspase-independent cell death, in senescence-associated cell death of human endothelial cells. Using immunofluorescence microscopy, we found, that EndoG is localized in the mitochondria in young cells, but relocalizes to the nucleus upon senescence. When EndoG gene expression was downregulated by lentiviral shRNA vectors, we found a significant reduction in the replicative life span and a corresponding increase in cell death. We also observed a slight shift in the cell death phenotype from necrosis to apoptosis. Together these observations suggest an important role of EndoG in the senescence program of human endothelial cells.     

Prolactin suppresses glucocorticoid-induced thymocyte apoptosis in vivo

The hypothesis that prolactin (PRL) functions as an immunomodulator was based on studies showing lymphocyte PRL receptors, and its effects on growth, differentiation, and apoptosis in lymphoid cells. However, studies of PRL (PRL-/-) and PRL receptor knockout mice indicated that PRL was not required for immune system development or function under basal conditions. Because PRL maintains survival in glucocorticoid (GC)-treated Nb2-T lymphocytes in vitro, and PRL and GCs are elevated during stress, we investigated whether PRL protected T cells in vivo from GC-induced apoptosis. Adrenalectomized mice [PRL -/-, undetectable PRL; pituitary grafted PRL-/- (PRL-/-Graft), elevated PRL; and PRL+/-, normal PRL] were treated with dexamethasone (DEX) or PBS. Thymocytes and splenocytes were isolated and annexin V labeling of phosphatidylserine, DNA fragmentation, and caspase-3 activation were assessed as indices of apoptosis. Total thymocytes and CD4+ and CD8+ T cells obtained from DEX-treated PRL-/- mice exhibited significantly increased annexin V binding. In contrast, binding was not altered by DEX in PRL-/-Graft thymocytes. In addition, DEX induced classic DNA fragmentation in PRL-/- thymocytes. Elevated serum PRL reduced this effect. Thymocytes from DEX-treated PRL-/- mice exhibited increased caspase-3 activation, which was inhibited in cells from PRL-/-Graft mice. Finally, elevated expression of X-linked inhibitor of apoptosis, XIAP, was observed in thymi from DEX-treated PRL -/-Graft mice. This is the first demonstration that elevated PRL antagonizes apoptosis in thymocytes exposed to GCs in vivo. These observations suggest that, under conditions of increased GCs, such as during stress, elevated PRL functions physiologically to maintain survival and function of T-lymphocytes.     

Severe hepatic injury in interleukin 18 (IL-18) transgenic mice: a key role for IL-18 in regulating hepatocyte apoptosis in vivo

BACKGROUND: Interleukin 18 (IL-18) is a cytokine with pleiotropic activity that augments T helper 1 responses and cytotoxic activity of natural killer cells. METHODS: To assess the function of IL-18 in vivo, we generated IL-18 transgenic (IL-18 Tg) mice under the control of a CD2 promoter/enhancer construct. RESULTS: Macroscopically, IL-18 Tg mice showed reduced relative liver weight compared with wild-type littermates. TUNEL assays demonstrated increased hepatocyte apoptosis, and primary hepatocytes isolated from IL-18 Tg mice exhibited an increased spontaneous apoptosis rate. Furthermore, cross linking of Fas increased significantly the apoptosis rate in hepatocytes isolated from wild- type mice but to a much lesser extent in IL-18 Tg mice, suggesting spontaneous activation of the Fas pathway in the latter mice. In fact, in vivo blockade of Fas signal transduction by an adenovirus overexpressing the dominant negative form of the Fas associated death domain rescued hepatocytes from undergoing apoptosis. Finally, adoptive transfer of CD4(+) T cells from IL-18 Tg mice but not from wild-type littermates in SCID mice resulted in severe liver failure with massive periportal fibrosis due to hepatocyte apoptosis. CONCLUSION: IL-18 plays a fundamental role in regulating hepatocyte apoptosis. Furthermore, our transgenic model provides a novel tool to study the mechanisms of IL-18 dependent liver injury in vivo.     

Opening of the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Fas-mediated hepatic apoptosis in mice

Although Fas stimulation has been reported to cause outer mitochondrial membrane rupture in Jurkat cells, the mechanism of this effect is debated, and it is not known if outer membrane rupture also occurs in hepatocyte mitochondria. We studied the in vivo effects of Fas stimulation on ultrastructural lesions and mitochondrial function in mice. Four hours after administration of an agonistic anti-Fas antibody (8 microg/animal), caspase activity increased 5.4-fold. Nuclear DNA showed internucleosomal fragmentation, whereas supercoiled mitochondrial DNA was replaced by circular and linear forms. Mitochondrial cytochrome c was partly released into the cytosol. Ultrastructurally, mitochondrial lesions were observed in both apoptotic hepatocytes (with nuclear chromatin condensation/fragmentation) and nonapoptotic hepatocytes (without nuclear changes). In nonapoptotic cells, outer mitochondrial membrane rupture allowed herniation of the inner membrane and matrix through the outer membrane gap. In apoptotic hepatocytes, the matrix became electron-lucent and no longer protruded through the outer membrane gap. Mitochondria clustered around the nucleus, whereas rough endoplasmic reticulum cisternae became peripheral. In liver mitochondria isolated after Fas stimulation, the membrane potential decreased, whereas basal respiration increased. Pretreatment with either z-VAD-fmk (an inhibitor of caspases) or cyclosporin A (a permeability transition inhibitor) totally or mostly prevented mitochondrial outer membrane rupture, membrane potential decrease, cytochrome c release, and apoptosis. In conclusion, in vivo Fas stimulation causes caspase activation, mitochondrial permeability transition (decreasing the membrane potential and increasing basal respiration), mitochondrial matrix expansion (as shown by matrix herniation), outer mitochondrial membrane rupture, and cytochrome c release.     

Role of p38alpha MAPK in cardiac apoptosis and remodeling after myocardial infarction

Acute coronary occlusion results in ischemia-mediated death of cardiomyocytes. In the days and weeks following myocardial infarction (MI), left ventricular remodeling occurs that is characterized by persistent cardiomyocyte apoptosis, thinning and fibrosis at the site of infarction, ventricular chamber dilatation, and growth of remaining viable cardiomyocytes. The p38 mitogen-activated protein kinase (MAPK) signaling cascade has been implicated in the remodeling process. In this work, mice with cardiac-specific expression of a dominant negative mutant form of p38 MAPK (DN-p38alpha) were subjected to MI by occlusion of the left coronary artery. Acute ischemia area was determined by transthoracic echocardiography 2 h after MI surgery, and was found to be nearly identical in DN-p38 mice and their wild-type littermates. Seven days after MI, mice were subjected to repeat echocardiography and histological examination of infarct size. DN-p38 mice had markedly reduced infarct size and increased ventricular systolic function 7 days after MI when compared to wild-type littermates. In addition, DN-p38 mice had less cardiomyocyte apoptosis than wild-type mice in the infarct border zone. Recently, it was discovered that Bcl-X(L) deamidation occurs in vivo, and this results in Bcl-X(L) degradation that sensitizes cells to apoptosis by enhancing BAX activity. Bcl-X(L) deamidation was found to occur in the cardiac tissue of wild-type mice after MI, but was reduced in DN-p38 mice. These results establish that p38 MAPK activity is required for pathological remodeling after MI and suggest that p38 MAPK may promote cardiomyocyte apoptosis through Bcl-X(L) deamidation.     

Co-inheritance of alpha- and beta-thalassaemia in mice ameliorates thalassaemic phenotype

Beta-thalassaemia is an inherited disease caused by defective synthesis of the beta-globin chain of haemoglobin, leading to an imbalance in globin chains. Excess alpha-globin chains precipitate in erythroid progenitor cells resulting in cell death, ineffective erythropoiesis and severe anaemia. Decreased alpha-globin synthesis leads to milder symptoms, exemplified by individuals who co-inherit alpha-thalassaemia and beta-thalassaemia. In this study, we set out to investigate whether co-inheritance of alpha- and beta-thalassaemia in mice leads to reduced anaemia. Heterozygous murine beta-globin knockout (KO) mice (beta+/-) which display severe anaemia were mated with heterozygous alpha-globin KO mice (alpha++/--). The resulting progeny were genotyped and classed as wild-type WT (alpha++/++;beta+/+), heterozygous alpha-KO (alpha++/--;beta+/+), heterozygous beta-KO (alpha++/++;beta+/-) or double heterozygous (DH) alpha-KO/beta-KO (alpha++/--;beta+/-). Mice were bled and full blood examinations (FBE) performed. FBE results for heterozygous beta-KO mice (beta+/-) showed marked reductions in haemoglobin and haematocrit levels and significant increases in red cell distribution widths and reticulocyte counts compared to WT mice. In contrast, FBE results for DH alpha-KO/beta-KO mice showed near normal red blood cell indices. These results indicate that reduction of alpha-globin expression leads to correction of the globin chain imbalance in beta-thalassaemic mice and therefore an improved phenotype. The analysis of DH alpha-KO/beta-KO mice leads to the following conclusions: (1) co-inheritance of alpha- and beta-thalassaemia in mice improves the thalassaemic phenotype, identical to the situation in humans; (2) the heterozygous murine beta-globin KO mouse model is a suitable in vivo model to test for therapeutic knockdown of alpha-globin.     

Mmh/Ogg1 gene inactivation results in accumulation of 8-hydroxyguanine in mice

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine or 7, 8-dihydro-8-oxoguanine (8-OH-G). Products of the human MMH/OGG1 gene are known to catalyze in vitro the reactions repairing this DNA lesion. To analyze the function of Mmh in vivo, we generated a mouse line carrying a mutant Mmh allele by targeted gene disruption. Mmh homozygous mutant mice were found to have a physically normal appearance, but to have lost nicking activity in liver extracts for substrate DNA containing 8-OH-G, exhibiting a 3-fold increased accumulation of this adduct at 9 weeks of age compared with wild-type or heterozygous mice. Further elevation to 7-fold was observed in 14-week-old animals. Substantial increase of spontaneous mutation frequencies was clearly identified in Mmh mutant mice bearing transgenic gpt genes. These results indicate that exposure of DNA to endogenous oxidative species continuously produces the mutagenic adduct 8-OH-G in mice, and Mmh plays an essential role in repair of this DNA damage.     

Nitric oxide-induced genotoxicity,mitochondrial damage,and apoptosis in human lymphoblastoid cells expressing wild-type and mutant p53

Nitric oxide (NO(*)) is mutagenic and, under appropriate conditions of exposure, also induces apoptosis in many in vitro and in vivo experimental models. Biochemical and cellular mechanisms through which NO(*) induces apoptosis are incompletely understood, but involve p53/mitochondria-dependent signaling pathways. In this study, we exposed human lymphoblastoid cells harboring either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) to NO(*), delivered by diffusion through Silastic tubing. Cells were exposed for 2 h at constant rates of 100-533 nM/s, similar to levels estimated to occur in vivo in inflamed tissues. DNA double-strand breaks and fragmentation detected 8-48 h after NO(*) treatment were more extensive in TK6 cells than in WTK-1 cells, whereas NO(*)-induced mutant fractions in both HPRT and TK1 genes were significantly lower in TK6 cells than in WTK-1 cells (P < 0.01-0.05). Treatment of TK6 cells with NO(*) caused extensive apoptosis, but this response was delayed and greatly reduced in magnitude in WTK-1 cells. Mitochondrial membrane depolarization and cytochrome c release were induced in both cell types. However, elevation of apoptotic protease-activating factor-1 (Apaf-1) protein and reduction of X-chromosome-linked inhibitor of apoptosis (XIAP) protein were observed only in TK6 cells. These results indicate that p53 status is an important modulator of NO(*)-induced mutagenesis and apoptosis, and suggest that levels of the Apaf-1 and XIAP proteins, but not mitochondrial depolarization and cytochrome c release, are regulated by p53 in these human lymphoblastoid cells. Thus, Apaf-1 and XIAP may play important roles in the regulation of p53-mediated apoptotic responses.     

Mitochondrial electron transport chain activity, but not ATP synthesis, is required for drug-induced apoptosis in human leukaemic cells: a possible novel mechanism of regulating drug resistance

There is increasing evidence for an association between mitochondrial function and susceptibility to apoptosis. It has been shown that the vinblastine-resistant leukaemic cell line CEM/VLB₁₀₀ has a more active mitochondrial electron transport chain (ETC) than the parental CCRF-CEM cell line. Inhibition of mitochondrial DNA replication by ethidium bromide (EB) depleted the activity of the ETC and reduced cellular respiratory rate. Depletion of mitochondrial DNA was associated with increased resistance to vinblastine-induced apoptosis in both cell lines. In contrast, the highly specific inhibitor of the energy producing mitochondrial enzyme F₁F₀-ATPase, oligomycin, rendered CEM/VLB₁₀₀ cells more sensitive to vinblastine by inhibiting the energy-dependent P-glycoprotein (Pgp) pump, suggesting that the effect of EB is independent of energy generation and ATPase activity. Both mitochondrial ETC depletion and ATPase inhibition decreased vinblastine-induced cell cycle changes in the CCRF-CEM cell line, suggesting that cell cycle changes are dependent on ATP generation. However, EB-induced ETC depletion in CEM/VLB₁₀₀ cells inhibited apoptosis in response to high concentration of vinblastine, but not G₂M arrest. We suggest that: (1) over-expression of Pgp by drug-resistant cells may up-regulate mitochondrial energy production; (2) mitochondrial ETC activity is required for DNA fragmentation in response to vinblastine, but the mechanism is independent of Pgp activity and ATP generation; (3) down-regulation of mitochondrial ETC activity may confer resistance to vinblastine-induced apoptosis; (4) the mitochondrial ETC is involved in vinblastine-induced apoptosis downstream of microtubule disruption and cell cycle changes.     

Double mutations in klotho and osteoprotegerin gene loci rescued osteopetrotic phenotype

Klotho gene mutant mice (klotho mice, also called kl/kl) exhibit osteopetrosis in the metaphysis of femora and tibiae and die within 3 months. We previously showed by semiquantitative RT-PCR that osteoprotegerin (opg) expression levels in klotho mice were about 2-fold higher than those in wild-type mice in the bone marrow, spleen, and lung. To examine whether the high osteoprotegerin expression levels account for the osteopetrotic phenotype in the klotho homozygous mutant mice in vivo, we made double mutant mice by crossing klotho mutant and osteoprotegerin-deficient mice. Micro computed tomography analysis in the two-dimensional sagittal planes of the metaphyses and cross-sections of femoral midshaft revealed that the abnormally high fractional trabecular bone volume in klotho homozygous mice (kl/kl; 29.71%), which was about 4-fold higher compared with that of wild-type [klotho (+/+) opg (+/+)] mice (7.81%), was rescued by the coexistence of heterozygous mutation in opg gene locus (+/-; 8.36%). Single heterozygous mutation in the opg gene locus alone (without klotho mutation) did not show phenotype (trabecular bone volume, 5.84%; not significantly different from wild type). High levels of osteoprotegerin mRNA expression in the bone marrow in klotho mutant mice were reduced by the heterozygous mutation in the opg gene locus. Furthermore, high osteoprotegerin protein levels in klotho mutant mice were also reduced by the heterozygous mutations in opg gene locus. Thus, elevated levels of osteoprotegerin in mutant mice contribute at least in part to reveal the osteopetrotic phenotype in klotho mice.     

Temperature-sensitive replication of H5ts125 adenovirus DNA in vitro.

A soluble adenovirus DNA replication system has been prepared in extracts of HeLa cells infected with the temperature-sensitive mutant H5ts125. These nuclear extracts synthesize full-sized viral DNA at 30 degrees but are temperature sensitive in this function at 38 degrees. A 72,000-dalton DNA-binding protein purified from cells infected with wild-type virus complements the temperature-sensitive defect and restores viral DNA replication to 74% of normal values.     

Wild-type, but not mutant, human p53 proteins inhibit the replication activities of simian virus 40 large tumor antigen.

Murine p53 blocks many of the replication activities of simian virus 40 (SV40) large tumor antigen (T antigen) in vitro. As murine cells do not replicate SV40 DNA, it was of interest to determine how p53 from permissive human cells functions. Recombinant baculoviruses encoding either the wild-type form of human p53 or a mutant p53 cloned from a human tumor cell line were constructed, and p53 proteins were purified from infected insect cells. Surprisingly, we found that wild-type human p53 was as inhibitory to the ability of T antigen to mediate replication of an SV40 origin-containing (ori DNA) plasmid in vitro as was murine p53. Wild-type human p53 also blocked the DNA unwinding activity of T antigen, as did its murine counterpart. In contrast to murine and wild-type human p53, the mutant human p53 did not block ori DNA replication or DNA unwinding. Murine p53 formed a complex with mutant human p53 in vivo. Furthermore, mutant human p53 reduced the inhibition of SV40 ori DNA replication by murine p53 in vitro. These results provide a model for the way in which mutant p53 proteins can affect normal functions of p53.     

Apoptosis in congestive heart failure induced by viral myocarditis in mice

Summary It has recently been speculated that progressive deterioration of left ventricular function in chronic heart failure is due to the ongoing loss of viable cardiac myocytes. However, as there is little direct evidence of significant apoptosis contributing to the pathogenesis in cardiac myocytes in vivo, the significance of apoptosis in heart failure remains to be clarified. We investigated the role of apoptosis in heart failure induced by encephalomyocarditis virus myocarditis. DBA/2 mice were inoculated with the virus (day 0), then killed, and their hearts were extracted 3 to 28 days later. Internucleosomal DNA fragmentation, chromatin binding dye staining, and in situ terminal transferase deoxyuridine triphosphate (dUTP) endlabeling were used to detect apoptosis. Internucleosomal DNA fragmentation (DNA ladder) was clearly demonstrated on days 5 to 14 in the virusinfected hearts when myocardial necrosis and infiltration of mononuclear cells were prominent in the hearts. Apoptotic cells demonstrated morphological changes typical of apoptosis (condensation of chromatin and nuclear fragmentation). Both Fas antigen and Fas ligand immunoreactivity were detected in the infiltrating mononuclear cells. The in situ terminal transferase dUTP end-labeling method demonstrated condensed nuclei of infiltrating mononuclear cells on day 7. However, nuclei of cardiac myocytes surrounded by the cellular infiltration were absent. The main source of apoptotic cells in the heart in mice with viral myocarditis appeared to be the infiltrating mononuclear cells. This work was supported in part by a grant-in-aid for General Scientific Research, and a research grant from the Ministry of Health and Welfare, Japan.     

Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver

After several weeks of treatment, levels of alanine aminotransferase (ALT) increase in 50% of patients treated with tacrine for Alzheimer's disease. We looked for progressive effects on DNA to explain delayed toxicity. We first studied the in vitro effects of tacrine on DNA replication and topoisomerase-mediated DNA relaxation. We then treated mice with doses of tacrine reproducing the human daily dose on a body area basis and studied the effects of tacrine administration for up to 28 days on hepatic DNA, mitochondrial function, and cell death. In vitro, tacrine impaired DNA polymerase gamma-mediated DNA replication and also poisoned topoisomerases I and II to increase the relaxation of a supercoiled plasmid. In vivo, administration of tacrine markedly decreased incorporation of [(3)H]thymidine into mitochondrial DNA (mtDNA), progressively and severely depleted mtDNA, and partly unwound supercoiled mtDNA into circular mtDNA. Incorporation of [(3)H]thymidine into nuclear DNA (nDNA) was barely decreased, and nDNA levels were unchanged. After 12 to 28 days of treatment, administration of tacrine increased p53, Bax, mitochondrial permeability transition, cytosolic cytochrome c, and caspase-3 activity and triggered hepatocyte apoptosis and/or necrosis. In conclusion, the intercalating drug tacrine poisons topoisomerases and impairs DNA synthesis. Tacrine has been shown to accumulate within mitochondria, and it particularly targets mtDNA. After several weeks of treatment, the combination of severe mtDNA depletion and a genotoxic stress enhancing p53, Bax, and permeability transition trigger hepatocyte necrosis and/or apoptosis.     

Endonuclease G: a role for the enzyme in recombination and cellular proliferation

Our earlier studies had suggested that endonuclease G (EndoG), a member of the evolutionarily conserved DNA/RNA nonspecific betabetaalpha-Me-finger nuclease family, functioned in the a sequence-mediated segment inversion observed during herpes simplex virus 1 replication. To test this hypothesis, we used RNA interference to reduce the level of EndoG in mammalian cells in culture. Reduction of EndoG produced a small but statistically significant decrease in a sequence-mediated recombination, suggesting that EndoG does play a role in this process. We also observed that reduction in the level of EndoG resulted in a deficiency in cell proliferation. Cells with a reduced level of EndoG also showed changes in cell distribution in the cell cycle, producing a pattern characteristic of cells that have been arrested in the G(2) phase. These findings suggest that EndoG is required for normal cellular proliferation.     

p21Cip1 levels differentially regulate turnover of mature endothelial cells, endothelial progenitor cells, and in vivo neovascularization

p21(Cip1) (p21) controls cell cycle progression and apoptosis in mature endothelial cells (ECs) and regulates size and cycling of the hematopoietic progenitor cell pool. Because circulating endothelial progenitor cells (EPCs) contribute to postnatal neovascularization in addition to mature ECs, we investigated the regulation of ECs and EPCs in p21-deficient mice. Mature aortic EC proliferation was increased in homozygous p21(-/-) and heterozygous p21(+/-) mice, in which p21 protein levels are reduced to one third of wild-type (WT). In contrast, apoptosis sensitivity was increased by 3.5-fold only in p21(-/-), but not in p21(+/-) mice. Consistently, in vivo apoptosis of ECs within areas of neovascularization was elevated in p21(-/-) but not in p21(+/-) mice. EPC numbers were elevated 2-fold in p21(-/-) mice compared with WT (P<0.001), and clonal expansion capacity of EPCs was increased from 25+/-4 (WT) to 57+/-8 colony-forming units in p21(-/-) mice (P<0.005). EPC numbers and expansion were likewise increased in p21(+/-) mice. As the integrative endpoint, in vivo neovascularization reflecting all p21-affected parameters was increased over WT only in p21(+/-) (P<0.001), but not in p21(-/-) mice. In conclusion, reduced p21 protein levels of mice lacking one p21 allele are associated with increased proliferation of ECs and EPCs, whereas survival of ECs to apoptotic stimuli in vitro and in vivo is not impaired. Under these conditions, neovascularization was increased. In contrast, complete p21 deficiency did not result in an increased neovascularization despite increased mature EC and EPC proliferation. This may be due to the sensitization of ECs against apoptosis.