Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation.

The 'housekeeping' enzyme Cu/Zn-superoxide dismutase (SOD-1) is encoded by a gene residing on human chromosome 21, at the region 21q22 known to be involved in Down's syndrome. The SOD-1 gene and the SOD-1 cDNA were introduced into mouse L-cells and human HeLa cells, respectively as part of recombinant plasmids containing the neoR selectable marker. Human and mouse transformants were obtained that expressed elevated levels (up to 6-fold) of authentic, enzymatically active human SOD-1. This enabled us to examine the consequences of hSOD-1 gene dosage, apart from gene dosage effects contributed by other genes residing on chromosome 21. Human and mouse cell clones that overproduce the hSOD-1 had altered properties; they were more resistant to paraquat than the parental cells and showed an increase in lipid peroxidation. The data are consistent with the possibility that gene dosage of hSOD-1 contributes to some of the clinical symptoms associated with Down's syndrome.     

Gene dosage of CuZnSOD and Down''s syndrome: diminished prostaglandin synthesis in human trisomy 21, transfected cells and transgenic mice.

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investigate the possible involvement of CuZnSOD gene dosage in perturbation of prostaglandin biosynthesis we analyzed transfected cells and transgenic mice that express elevated levels of human CuZnSOD. It was found that the synthesis of prostaglandin E2 (PGE2) was diminished in transfected PC12-CuZnSOD cells as well as in fibroblasts from DS patients. Primary cells derived from transgenic CuZnSOD mice showed similar reduction. Impaired biosynthesis of prostaglandins was not confined to cells grown in culture since secretion of PGE2 and PGD2 by kidney and cerebellum of transgenic CuZnSOD was significantly lower than in non-transgenic littermate mice. These findings strongly suggest that overexpression of the CuZnSOD gene induces a demotion in PGE2 and PGD2 formation and establish a connection between alteration of prostaglandin biosynthesis in trisomy 21 cells and gene dosage of CuZnSOD.     

Brachymesophalangia-5 without cone-epiphysis mid-5 in down's syndrome

Brachymesophalangia-5 proved to be far more frequent in 212 cases of Down's syndrome karyotype (i.e., 21%) than in 14,197 survey volunteers of European ancestry (1.4%). However, none of 28 juvenile Down's syndrome patients with brachymesophalangia-5 exhibited a cone-epiphysis on mid-5, as against the 47% that would be expected. Apparently the manifestation of brachymesophalangia-5 in the 47,G + karyotype is not simply a dosage effect associated with trisomy of chromosome 21.     

Superoxide dismutase 1 modulates expression of transferrin receptor

Copper–zinc superoxide dismutase (SOD1) plays a protective role against the toxicity of superoxide, and studies in Saccharomyces cerevisiae and in Drosophila have suggested an additional role for SOD1 in iron metabolism. We have studied the effect of the modulation of SOD1 levels on iron metabolism in a cultured human glial cell line and in a mouse motoneuronal cell line. We observed that levels of the transferrin receptor and the iron regulatory protein 1 were modulated in response to altered intracellular levels of superoxide dismutase activity, carried either by wild-type SOD1 or by an SOD-active amyotrophic lateral sclerosis (ALS) mutant enzyme, G93A-SOD1, but not by a superoxide dismutase inactive ALS mutant, H46R-SOD1. Ferritin expression was also increased by wild-type SOD1 overexpression, but not by mutant SOD1s. We propose that changes in superoxide levels due to alteration of SOD1 activity affect iron metabolism in glial and neuronal cells from higher eukaryotes and that this may be relevant to diseases of the nervous system.     

DNA damage, superoxide, and mutant K-ras in human lung adenocarcinoma cells

DNA single-strand breaks (quantitative comet assay) were assessed to indicate ongoing genetic instability in a panel of human lung adenocarcinoma cell lines. Of these, 19/20 showed more DNA damage than a nontransformed cell line from human peripheral lung epithelium, HPL1D. DNA damage was significantly greater in those derived from pleural effusates vs those from lymph node metastases. DNA strand breaks correlated positively with superoxide (nitroblue tetrazolium reduction assay), and negatively with amount of OGG1, a repair enzyme for oxidative DNA damage. Levels of CuZn superoxide dismutase varied moderately among the lines and did not correlate with other parameters. A role for mutant K-ras through generation of reactive oxygen species was examined. Cells with mutant K-ras had significantly lower amounts of manganese superoxide dismutase (MnSOD) vs those with wild-type K-ras, but MnSOD protein correlated positively with superoxide levels. In a subset of cell lines with similar levels of MnSOD, comparable to those in HPL1D cells, K-ras activity correlated positively with levels of both superoxide and DNA strand breaks. These results suggest that persistent DNA damage in some lung adenocarcinoma cells may be caused by superoxide resulting from mutant K-ras activity, and that OGG1 is important for prevention of this damage.     

Induction of superoxide dismutase and cytotoxicity by manganese in human breast cancer cells.

MnCl2 induced manganese-containing superoxide dismutase (MnSOD) expression (mRNA, immunoreactive protein, and enzyme activity) in human breast cancer Hs578T cells. The induction of MnSOD immunoreactive protein in Hs578T cells was inhibited by tiron (a metal chelator and superoxide scavenger), pyruvate (a hydrogen peroxide scavenger), or 2-deoxy-d-glucose (DG, an inhibitor of glycolysis and the hexose monophosphate shunt), but not by 5,5-dimethyl-1-pyrroline-1-oxide (a superoxide scavenger), N-acetyl cysteine (a scavenger for reactive oxygen species and precursor of glutathione), diphenylene iodonium (an inhibitor of flavoproteins such as NADPH oxidase and nitric oxide synthase), or SOD (a superoxide scavenger). Northern blotting demonstrated that tiron or DG affected at the mRNA level, while pyruvate affected Mn-induced MnSOD expression at both the mRNA and protein levels. These results demonstrate that Mn can induce MnSOD expression in cultured human breast cancer cells. Mn also induced apoptosis and necrosis in these cells. Since inhibitors of Mn-induced MnSOD induction did not affect cell viability, MnSOD induction is probably not the cause of the Mn-induced cell killing.     

Rapid detection of Down's syndrome using quantitative real-time PCR (qPCR) targeting segmental duplications on chromosomes 21 and 11

Objective

Development of a qPCR test for the detection of trisomy 21 using segmental duplications.

Methods

Segmental duplications in the TTC3 gene on chromosome 21 and the KDM2A gene on chromosome 11 were selected as molecular markers for the diagnostic qPCR assay. A set of consensus primers selected from the conserved regions of these segmental duplications were used to amplify internal diverse sequences that were detected and quantified with different probes labeled with distinct fluorescence. The copy numbers of these two fragments were determined based on the ΔCq values of qPCR. The results of qPCR for prenatal and neonatal screening of Down's syndrome were compared with the conventional karyotype analysis by testing 82 normal individuals and 50 subjects with Down's syndrome.

Results

The ΔCq values of segmental duplications on chr21 and 11 ranged between 0.33 and 0.75 in normal individuals, and between 0.91 and 1.18 in subjects with Down's syndrome. The ΔCq values of these two segmental duplications clearly discriminated Down's syndrome from normal individuals (P < 0.001). Furthermore, the qPCR results were consistent with karyotype analysis.

Conclusion

Our qPCR can be used for rapid prenatal and neonatal screening of Down's syndrome.     

A FINE STRUCTURAL STUDY OF THE HIPPOCAMPUS AND DORSAL ROOT GANGLION IN MOUSE TRISOMY 16, A MODEL OF DOWN'S SYNDROME

Mouse trisomy 16 (Ts16) appears to provide an animal model of Down's syndrome in that a portion of mouse chromosome 16 is syntenic with part of human chromosome 21. Trisomy 21 in human beings leads to the mental retardation of Down's syndrome and in middle age, to some presenile anatomic and clinical features of Alzheimer's disease. Neural tissue from aging Ts16 mice is unavailable, however, as Ts16 mouse embryos die late in utero. We studied these embryos looking at the ultrastructure of neurons from the hippocampus and dorsal root ganglion in normal control mice embryos (diploid) and in Ts16 late embryonic litter mates after day 15 of gestation. The organelles in the Ts16 neurons looked similar to those in control neurons, fixed and processed under similar conditions. No obvious neuropathological structures were observed. These results, when compared to reports on electrophysiological abnormalities of cultured fetal Ts16 neurons and on abnormalities in neurotransmitter markers in the Ts16 fetal brain, lead us to suggest that the mental retardation of Down's syndrome is likely to result from functional and chemical defects not directly related to abnormal neuronal ultrastructure. When related to fine structural studies of transplanted embryonic Ts16 hippocampus which have been maintained for long periods of time, these results indicate that the trisomic mouse brain would not be useful as a structural model for Down's syndrome and hence presenile Alzheimer's disease, as it is not associated with any detectable morphological abnormality.     

Induction of superoxide dismutase, chromosomal aberrations and sister-chromatid exchanges by paraquat in Chinese hamster fibroblasts

We have recently shown that Bloom syndrome fibroblasts have elevated levels of superoxide dismutase activity compared to those of normal fibroblasts. Based on this observation we decided to test whether an increased rate of superoxide radical production could be responsible for the induction of superoxide dismutase and of chromosomal aberrations and sister-chromatid exchanges characteristic of Bloom syndrome. Utilizing the superoxide-generating herbicide paraquat in Chinese hamster fibroblasts, we assayed the cells for dismutase activity, chromosomal aberrations and sister-chromatid exchanges. All 3 parameters investigated demonstrated a dose-dependent increase with paraquat and, consequently, with the superoxide produced. Since the induction of the enzyme is mediated by its substrate, the superoxide anion radical, we concluded that the increased dismutase activity (in Bloom syndrome and paraquat-treated cells) may be a secondary manifestation of an overall imbalance in oxygen metabolism and that this elevated enzymatic activity is insufficient to detoxify the high superoxide levels, which results in elevated levels of chromosomal damage.     

Ethanol induces the generation of reactive free radicals by neural crest cells in vitro

Developmental craniofacial anomalies related to the neural crest derived ectomesenchymal cell population are associated with fetal alcohol syndrome (FAS). Information regarding any potential relationship between ethanol, free radicals, and the viability, proliferation, etc., of isolated neural crest cells was sought. The hypersensitivity of neural crest cells to ethanol was observed. This drug severely depressed cell viability while simultaneously inducing the generation of such reactive oxygen intermediates (ROI) as superoxide, hydrogen peroxide, and hydroxyl anions. Addition of the free radical scavenging enzyme superoxide dismutase to the culture medium significantly reversed these effects of ethanol. The cytotoxicity of ethanol was further confirmed by the release of radiolabeled chromium (51Cr) from cells prelabeled prior to ethanol treatment. This effect was also depressed by the addition of superoxide dismutase. Interestingly, an assay for superoxide dismutase activity showed that neural crest cells may be devoid of this enzyme. The latter may help to explain the overt sensitivity of these cells to such a broad spectrum of teratogens, many of which can either dissociate directly into ROI, or cause the radicalization of biological structures and molecules. Plasmalemmal lipids (via lipid peroxidation) and DNA are at an especially high risk from uncontrolled ROI. Changes in neural crest cell surface morphology, i.e., loss of microvilli, formation of xeiotic blebs, as well as the "leakage" of radiolabeled Cr from prelabeled cells, would seem to show that ethanol, as a result of induced free radical formation, alters the physiology and biochemistry of the cell membrane. These findings however, should not exclude other potential sites for ETOH-induced cell injury related to free radicals, especially the nuclei (DNA), mitochondria, organelle membranes, and the cytoskeleton.     

α-Tocopherol is an effective Phase II enzyme inducer: protective effects on acrolein-induced oxidative stress and mitochondrial dysfunction in human retinal pigment epithelial cells

Vitamin E has long been identified as a major lipid-soluble chain-breaking antioxidant in mammals. α-Tocopherol is a vitamin E component and the major form in the human body. We propose that, besides its direct chain-breaking antioxidant activity, α-tocopherol may exert an indirect antioxidant activity by enhancing the cell's antioxidant system as a Phase II enzyme inducer. We investigated α-tocopherol's inducing effect on Phase II enzymes and its protective effect on acrolein-induced toxicity in a human retinal pigment epithelial (RPE) cell line, ARPE-19. Acrolein, a major component of cigarette smoke and also a product of lipid peroxidation, at 75 μmol/L over 24 h, caused significant loss of ARPE-19 cell viability, increased oxidative damage, decreased antioxidant defense, inactivation of the Keap1/Nrf2 pathway, and mitochondrial dysfunction. ARPE-19 cells have been used as a model of smoking- and age-related macular degeneration. Pretreatment with α-tocopherol activated the Keap1/Nrf2 pathway by increasing Nrf2 expression and inducing its translocation to the nucleus. Consequently, the expression and/or activity of the following Phase II enzymes increased: glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase 1, heme-oxygenase 1, glutathione S-transferase and superoxide dismutase; total antioxidant capacity and glutathione also increased. This antioxidant defense enhancement protected ARPE-19 cells from an acrolein-induced decrease in cell viability, lowered reactive oxygen species and protein oxidation levels, and improved mitochondrial function. These results suggest that α-tocopherol protects ARPE-19 cells from acrolein-induced cellular toxicity, not only as a chain-breaking antioxidant, but also as a Phase II enzyme inducer.     

Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [³H]DNA in alkaline sucrose gradients or by direct assay of the amount of [³H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.     

A diagnostic survey of infants referred for chromosome analysis in the neonatal period.

Examination and assessment of 140 liveborn and stillborn infants referred within two weeks of birth for chromosome analysis showed that 48 had Down''s syndrome, 12 other chromosome abnormalities, 17 single gene disorders, 18 recognisable anomalads, 8 recognisable syndromes of unknown aetiology, and the remainder were undiagnosed. Of the non-Down''s cases that were diagnosed, 21% had a chromosomal abnormality. These results suggest that a request for chromosome analysis in the newborn period should be viewed as one step in syndrome identification.     

Altered expression of hypothetical proteins in hippocampus of transgenic mice overexpressing human Cu/Zn-superoxide dismutase 1

Background  

Cu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in the metabolism of reactive oxygen species (ROS) and pathogenetically relevant for several disease states including Down syndrome (DS; trisomy 21). Systematically studying protein expression in human brain and animal models of DS we decided to carry out "protein hunting" for hypothetical proteins, i.e. proteins that have been predicted based upon nucleic sequences only, in a transgenic mouse model overexpressing human SOD1.     

Role of reactive oxygen species in cells overexpressing manganese superoxide dismutase: mechanism for induction of radioresistance

Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide anions (O(2)(-)) into hydrogen peroxide (H(2)O(2)). We altered the intracellular status of reactive oxygen species by introducing human MnSOD cDNA into the human ovarian cancer cell line SK-OV-3. The overexpression of MnSOD inhibited cell growth and induced a concomitant increase in the level of H(2)O(2) in SK-OV-3 cells. The cells overexpressing MnSOD were more resistant to irradiation than parental cells. MnSOD overexpression shortened the G(2)-M duration in irradiated cells. Either inhibition of p38 mitogen-activated protein kinase (p38MAPK) or scavenging free radicals blocked the induction of radioresistance by MnSOD and also abolished the shortening of the G(2)-M duration with concomitant inhibition of p38MAPK phosphorylation. Irradiation increased the generation of H(2)O(2) even more in these transfectants. These results suggest that the accumulated H(2)O(2) potentiated the activation of p38MAPK after irradiation in cells overexpressing MnSOD, which led to the protection of cells from irradiation-mediated cell death through the G(2)-M checkpoint. SK-OV-3 cells had no constitutive expression of p53, and the overexpression of MnSOD and/or irradiation did not induce p53 or p21(WAF1), which causes cell cycle arrest. Thus, our results suggest that MnSOD alters the cell cycle progression of irradiated cells independently of p53 and p21(WAF1).