8-hydroxyguanine and Neuroblastoma

Mycoplasma contamination is a major concern for in vitro cell culture models as its resistance to most antibiotics, which makes the prevention and treatment of infection challenging. Furthermore, numerous studies show that Mycoplasma infection alters a variety of cellular processes, in a wide range of cell lines. However, there is a lack of information pertaining to the effects of Mycoplasma infection on genomic stability. In this study, a dopaminergic neuronal cell line (BE-M17), a popular in vitro model for Parkinson's disease, was used to evaluate the effect of Mycoplasma infection on genomic instability, and base excision repair (BER) activity, using single cell gel electrophoresis (the comet assay). The results showed that Mycoplasma infection induced oxidative stress in the absence of an inflammatory response, with markedly increased levels of DNA damage [strand breaks/alkali-labile sites (SB/ALS), and oxidised purines], compared to uninfected cells. The source of the oxidative stress may have been increased ROS generation, or attenuation of cellular antioxidant capacity (or a combination of both). Uninfected cells initially repaired SB/ALS more rapidly than infected cells, although SB/ALS were fully repaired in both uninfected and infected cells 2 h after H

Topics: Cell Line, Tumor; Comet Assay; DNA Breaks; DNA Damage; DNA Glycosylases; DNA Repair; Guanine; Humans; Hydrogen Peroxide; Mycoplasma; Neuroblastoma; Oxidative Stress; Propidium; Purines; Single-Cell Analysis; Staining and Labeling

DNA is a frequent target of oxidative damage, and DNA damage removal is therefore a crucial process in prevention of or recovery from degenerative diseases. DNA repair is an essential system for maintaining the inherited nucleotide sequence of genomic DNA over time. Cells engage in efficient DNA repair mechanisms, the activity of which can vary depending on the type of lesion and the developmental stage. Base excision repair (BER) and nucleotide excision repair (NER) are the major repair pathways addressed in this study. BER is the principal mechanism for repair of DNA oxidative lesions, while NER is the mechanism for repair of a variety of helix-distorting lesions such as those caused by UV radiation. Recent studies suggest that NER plays a cooperative role in removal of oxidative lesions. Little is known about the roles of DNA damage sensors and repair factors in terminally differentiated, non-proliferating cells such as neurons, which are vulnerable to oxidative damage from reactive oxygen species generated by endogenous or exogenous agents. We used the human neuroblastoma MSN cell model to investigate whether terminally differentiated neuronal cells respond to lesions cause in the DNA helix, such as UV-induced CPD and the major DNA oxidative lesion 8OHdG, and thereby clarify the role of NER capacity. We observed differences in DNA damage removal depending on the challenge insult and the differentiation state. Differentiated MSN cells, compared with undifferentiated cells, showed greater sensitivity to UVC and decreased DNA damage over time. In contrast, undifferentiated cells displayed genotoxicity induced by oxidative insult and tended to accumulate DNA damage and 8OHdG lesions over time. Our findings suggest the participation of GG-NER, TC-NER and BER proteins in the removal of 8-OHG and CPDs indicating a dynamic role in overall response to damage.

Topics: Analysis of Variance; Carboxy-Lyases; Cell Differentiation; Cell Line, Tumor; Cell Survival; Comet Assay; DNA Damage; DNA Glycosylases; DNA Repair Enzymes; DNA-Binding Proteins; Dose-Response Relationship, Drug; Dose-Response Relationship, Immunologic; Gene Expression Regulation; Guanine; Humans; Hydrogen Peroxide; Lamins; Lipid Peroxidation; Neuroblastoma; Oxidants; Proteins; Pyrimidines; Reactive Oxygen Species; Ultraviolet Rays

In mammals, the polyamines affect cell growth, differentiation, and apoptosis; their levels are increased in malignant and proliferating cells, thus justifying an interest in a chemotherapeutic approach to cancer. The flavoprotein SMO is the most recently characterized catabolic enzyme, preferentially oxidizing SPM to SPD, 3-aminopropanal and H(2)O(2). In this report, we describe a novel functional characterization of the recently cloned splice variant isoforms from mouse brain, encoding, among others, the nuclear co-localized spermine oxidase mSMOmu. The over-expression of the active isoforms mSMOalpha and mSMOmu, and the inactive mSMOdelta and mSMOgamma in mouse neuroblastoma cells, demonstrated the first evidence of the direct oxidative DNA damage by the SMO activities, either alone or, in a higher extent, when associated with radiation exposure, thus working as radio sensitizer. These effects were reverted by treatment with 50 muM and 100 muM doses of the inhibitor of SMO activity MDL 72,527. The over-expression of all SMO isoforms failed to influence the expression of the regulating enzymes of polyamines metabolism ODC and SSAT. Dealing with the unbalanced tissue specific SMO activities, these results could indicate a new direction to tailor chemotherapy-associated radiotherapy, improving dose-rate protocol and allowing the modulation of deleterious side effects on healthy tissues.

Topics: Animals; Apoptosis; DNA Damage; Guanine; Isoenzymes; Mice; Neuroblastoma; Oxidation-Reduction; Oxidoreductases Acting on CH-NH Group Donors; Polyamine Oxidase; Polyamines; Radiation Tolerance; Tumor Cells, Cultured

Patients with Down's syndrome (DS) show elevated levels of copper, zinc-containing superoxide dismutase (SOD1) and appear to have increased lipid peroxidation and oxidative damage to DNA as well as elevated glutathione peroxidase activity. Increasing SOD1 levels by gene transfection in NT-2 and SK-N-MC cell lines also led to a rise in glutathione peroxidase activity, but this was nevertheless accompanied by decreased proliferation rates, increased lipid peroxidation and protein carbonyls, and a trend to a rise in 8-hydroxyguanine and protein-bound 3-nitrotyrosine. Transfection of these cell lines with DNA encoding two mutant SOD1 enzymes (G37R and G85R) associated with familial amyotrophic lateral sclerosis (FALS), produced similar, but more severe changes, i.e. even lower growth rates, higher lipid peroxidation, 3-nitrotyrosine and protein carbonyl levels, decreased GSH levels, raised GSSG levels and higher glutathione peroxidase activities. Since G85R has little SOD activity, these changes cannot be related to increased O(2)(-) scavenging. In no case was SOD2 (mitochondrial Mn-SOD) level altered. Our cellular systems reproduce many of the biochemical changes observed in patients with DS or ALS, and in transgenic mice overexpressing mutant SOD1. They also show the potentially deleterious effects of SOD1 overexpression on cellular proliferation, which may be relevant to abnormal development in DS.

Topics: Aldehydes; Amyotrophic Lateral Sclerosis; Antioxidants; Cell Division; Cell Line; Cell Survival; Down Syndrome; Gene Expression; Glutathione; Glutathione Disulfide; Guanine; Humans; Ketones; Lipid Peroxidation; Mutation; Neuroblastoma; Oxidative Stress; Superoxide Dismutase; Superoxide Dismutase-1; Teratocarcinoma; Transfection; Tyrosine

Mutations in alpha-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of alpha-synuclein overexpression on the response of cells to various insults. Wild-type alpha-synuclein and alpha-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type alpha-synuclein delayed cell death induced by serum withdrawal or H(2)O(2), but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)). By contrast, wild-type alpha-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type alpha-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant alpha-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant alpha-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal alpha-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.

Topics: 1-Methyl-4-phenylpyridinium; Aldehydes; alpha-Synuclein; Cell Division; Cell Line; Cell Survival; Clone Cells; Culture Media, Serum-Free; Enzyme Inhibitors; Gene Expression; Glutathione; Guanine; Humans; Hydrogen Peroxide; Ketones; Lipid Peroxidation; Mitochondria; Mutation; Nerve Tissue Proteins; Neuroblastoma; Oxidants; Oxidative Stress; Parkinsonian Disorders; Synucleins; Teratocarcinoma; Transfection; Tyrosine

Mutations in Cu/Zn-superoxide dismutase (SOD1) are associated with some cases of familial amyotrophic lateral sclerosis (ALS). We overexpressed Bcl-2, wild-type SOD1 or mutant SOD1s (G37R and G85R) in NT-2 and SK-N-MC cells. Overexpression of Bcl-2 rendered cells more resistant to apoptosis induced by serum withdrawal, H2O2 or 4-hydroxy-2-trans-nonenal (HNE). Overexpression of Bcl-2 had little effect on levels of protein carbonyls, lipid peroxidation, 8-hydroxyguanine (8-OHG) or 3-nitrotyrosine. Serum withdrawal or H2O2 raised levels of protein carbonyls, lipid peroxidation, 8-OHG and 3-nitrotyrosine, changes that were attenuated in cells overexpressing Bcl-2. Overexpression of either SOD1 mutant tended to increase levels of lipid peroxidation, protein carbonyls, and 3-nitrotyrosine and accelerated viability loss induced by serum withdrawal, H2O2 or HNE, accompanied by greater rises in oxidative damage parameters. The effects of mutant SOD1s were attenuated by Bcl-2. By contrast, expression of wild-type SOD1 rendered cells more resistant to loss of viability induced by serum deprivation, HNE or H2O2. The levels of lipid peroxidation in wild-type SOD1 transfectants were elevated. Overexpression of mutant SOD1s makes cells more predisposed to undergo apoptosis in response to several insults. Our cellular systems appear to mimic events in patients with ALS or transgenic mice overexpressing mutant SOD1.

Topics: Aldehydes; Amino Acid Substitution; Cell Death; Cell Survival; Cross-Linking Reagents; Culture Media, Serum-Free; Genes, bcl-2; Guanine; Humans; Hydrogen Peroxide; Kinetics; Lipid Peroxidation; Motor Neuron Disease; Mutagenesis, Site-Directed; Neuroblastoma; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Teratocarcinoma; Tumor Cells, Cultured; Tyrosine