ABSTRACT
Comparison of the frequency of occurrence of restriction fragment length polymorphisms in control human DNAs and DNAs from infants dying from sudden infant death syndrome has indicated no significant difference in the case of restriction fragment length polymorphisms associated with the heat shock protein genes hsp70 and hsp90. A highly significant difference was detected, however, in the case of the specific restriction fragment length polymorphisms detected by an hsp60 gene probe in MspI digests.
Subject(s)
Heat-Shock Proteins/genetics , Polymorphism, Restriction Fragment Length , Sudden Infant Death/genetics , Chaperonin 60/genetics , DNA/genetics , HSP70 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/genetics , Humans , InfantSubject(s)
Arabidopsis/physiology , Oryza/physiology , Oxidative Stress , Sodium Chloride , Acclimatization , Climate , Cold Temperature , Glutathione/analogs & derivatives , Glutathione/metabolism , Glutathione Disulfide , Hydrogen Peroxide/metabolism , Mitochondria/metabolism , Superoxide Dismutase/metabolism , Tropical ClimateABSTRACT
Exposure of HeLa cells in monolayer culture to increasing concentrations of exogenously added H2O2 causes damage to cellular DNA. When the DNA is subsequently isolated from the non-apoptotic cells remaining in such cultures, evidence was obtained to suggest that the DNA damage elicited in intact cells was non-random and that certain nucleotide sequences associated with, or related to, the genes for heat shock protein 60 and catalase were more susceptible to damage than others. In contrast, these particular sequences were not specifically susceptible to damage when naked human DNA was exposed directly to H2O2 in vitro. On an overall comparative basis, sequences in the genes encoding catalase, alpha-1 antitrypsin and beta-actin appear more vulnerable to H2O2 in vivo, than sequences in H-ras and the P53 gene which seem surprisingly resistant.
Subject(s)
DNA Damage , DNA, Neoplasm/drug effects , Hydrogen Peroxide/toxicity , Actins/genetics , Base Sequence , Catalase/genetics , DNA/chemistry , DNA/drug effects , DNA/isolation & purification , DNA Probes , DNA, Neoplasm/chemistry , DNA, Neoplasm/isolation & purification , Dose-Response Relationship, Drug , Electrophoresis, Agar Gel , Female , Genes, p53 , Genes, ras , HeLa Cells , Humans , Placenta , Pregnancy , alpha 1-Antitrypsin/geneticsABSTRACT
Addition of H2O2 at 100 microM, or 1 mM, to the culture medium of BHK-21 fibroblasts results in increased intracellular levels of H2O2. Whilst exposure of BHK-21 cells to lower levels of H2O2 (1 microM) actually stimulates proliferation, these higher oxidant concentrations not only depress proliferation rates but also lead to an increase in the appearance of apoptotic-like cells in the cultures. Other agents such as inhibitors of glutathione peroxidase and catalase, or mimics of superoxide dismutase, which also bring about elevated cellular levels of H2O2 in BHK-21 cells, similarly lead to decreased proliferation and an apparent increase in cells with apoptopic features. Thus intracellular conditions which are considered more prooxidant than normal, appear to favour apoptosis over proliferation in BHK-21 fibroblasts. Additionally these abnormal cellular conditions also appear to favour excessive DNA replication, in remaining non-apoptotic cells.
Subject(s)
Apoptosis/physiology , Cell Division/physiology , Hydrogen Peroxide/metabolism , Amitrole/pharmacology , Animals , Apoptosis/drug effects , Cell Adhesion , Cell Death/genetics , Cell Division/drug effects , Cell Line , Cricetinae , Culture Media , DNA/chemistry , Electrophoresis, Agar Gel , Fibroblasts/drug effects , Fibroblasts/metabolism , Hydrogen Peroxide/pharmacology , Microscopy, Electron , Nucleosomes , Salicylates/pharmacology , Succimer/analogs & derivatives , Succimer/chemistry , Succimer/pharmacologyABSTRACT
Intracellular levels of H2O2 in BHK-21 cells are not static but decline progressively with cell growth. Exposure of cells to inhibitors of catalase, or glutathione peroxidase, not only diminishes this decline but also depresses rates of cell proliferation, suggesting important growth regulatory roles for those antioxidant enzymes. Other agents which also diminish the growth-associated decline in intracellular levels of H2O2, such as the superoxide dismutase mimic, copper II-(3,5-diisopropylsalicylate)2, or docosahexaenoic acid, also reduced cell proliferation. In contrast, proliferation can be stimulated by the addition of 1 microM exogenous H2O2 to the culture medium. Under these conditions, however, intracellular levels of H2O2 are unaffected, whereas there is a reduction in intracellular levels of glutathione. It is argued that critical balances between intracellular levels of both H2O2 and glutathione are of significance in relation both to growth stimulation and inhibition. In addition growth stimulatory concentrations of H2O2, whilst initially leading to increased intracellular levels of lipid peroxidation breakdown products, appear to "trigger" their metabolism, possibly through aldehyde dehydrogenase, whose activity is also stimulated by H2O2.
Subject(s)
Cell Division/physiology , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Lipid Peroxidation , Aldehyde Dehydrogenase/metabolism , Amitrole/pharmacology , Animals , Cell Division/drug effects , Cell Line , Cricetinae , Culture Media, Serum-Free , Docosahexaenoic Acids/pharmacology , Kidney , Kinetics , Lipid Peroxidation/drug effects , Lipid Peroxides/metabolism , Salicylates/pharmacology , Thiomalates/pharmacology , Time FactorsABSTRACT
A wide variety of normal and malignant cell types generate and release superoxide or hydrogen peroxide in vitro either in response to specific cytokine/growth factor stimulus or constitutively in the case of tumour cells. These species at submicromolar levels appear to act as novel intra and intercellular "messengers" capable of promoting growth responses in culture. The mechanisms may involve direct interaction with specific receptors or oxidation of growth signal transduction molecules such as protein kinases, protein phosphatases, transcription factors, or transcription factor inhibitors. It is also possible that hydrogen peroxide may modulate the redox state and activity of these important signal transduction proteins indirectly through changes in cellular levels of GSH and GSSG. Critical balances appear to exist in relation to cell proliferation on one hand and lipid peroxidation and cell death on the other. Progression to a more prooxidant state whilst initially leading to enhanced proliferative responses results subsequently in increased cell death.
Subject(s)
Cell Division/drug effects , Hydrogen Peroxide/metabolism , Superoxides/metabolism , Animals , Cell Death , Cells, Cultured , Cricetinae , Free Radicals , Glutathione/pharmacology , Humans , Hydrogen-Ion Concentration , Mice , Platelet-Derived Growth Factor/pharmacology , RatsABSTRACT
In BHK-21 cells (baby hamster kidney fibroblasts) cellularly generated active oxygen species such as hydrogen peroxide and superoxide appear to be important growth regulatory signals as judged from the growth inhibitory effects of catalase, superoxide dismutase and superoxide dismutase mimics. These active oxygen species may contribute a novel redox system of regulatory control superimposed upon established growth signal pathways. This may be achieved by direct oxidative modification of cell regulatory proteins such as transcription factors or protein kinases or indirectly through, for example alterations in levels of glutathione (GSH). This latter possibility is suggested from observations that catalase, or superoxide dismutase treatment of BHK-21 cells brings about increased cellular levels of GSH. However during the normal growth phase cellular levels of GSH actually decline although this effect can be partly reversed by N-acetylcysteine and by mercaptosuccinate which also impair proliferation of these cells.
Subject(s)
Glutathione/metabolism , Hydrogen Peroxide/metabolism , Reactive Oxygen Species/metabolism , Superoxides/metabolism , Acetylcysteine/pharmacology , Animals , Catalase/metabolism , Cell Division/physiology , Cell Line , Cricetinae , Fibroblasts/cytology , Fibroblasts/metabolism , HeLa Cells , Humans , Kidney/cytology , Kidney/metabolism , Superoxide Dismutase/metabolism , Thiomalates/pharmacologyABSTRACT
Reactive oxygen species (ROS) have been implicated in the pathogenesis of pregnancy-induced hypertension (PIH). A genetic factor is also thought to be associated with the disease. The aim of the present study was to investigate whether decreased superoxide dismutase (SOD) activity in PIH resulted from gene abnormalities. Fourteen patients with PIH were enrolled in the study. Normal pregnant women and normal nonpregnant women served as controls. Genomic DNA and mRNA were isolated from white cells and subjected to Southern and Northern blot analysis with a 600 bp CuZn-SOD probe. SOD activity was also determined in the white blood cells and red blood cells. The results showed that SOD activity was significantly reduced in patients with PIH compared to both control groups. There were no significant differences in the size of the CuZn-SOD gene and its expression between the patients with PIH and the controls. This study confirmed that there was a decreased SOD activity in PIH but revealed neither major structural changes in the genomic DNA nor mRNA size of CuZn-SOD. Our results suggest that the decreased SOD levels in PIH are not due to abnormalities in the CuZn-SOD gene and are an acquired phenomenon which occurs during the development of the disease.
Subject(s)
Hypertension/enzymology , Pregnancy Complications, Cardiovascular/enzymology , Superoxide Dismutase/genetics , Adult , Blotting, Northern , DNA/blood , Female , Humans , Leukocytes/chemistry , Pregnancy , RNA, Messenger/blood , Superoxide Dismutase/bloodABSTRACT
alpha-Tocopherol (alpha-T) uptake and its relationship to cell proliferation and lipid peroxidation was studied in a baby hamster kidney cell line (BHK-21/C13) and its polyoma virus-transformed malignant counterpart (BHK-21/PyY cells). The principal findings were as follows. (a) The level of lipid peroxidation judged by malondialdehyde (MDA) measurement by HPLC, was higher in the transformed cells than in the nontransformed cells. Oxidative stress by 374 mM Fe3+/10 mM ADP caused a significant increase in the level of MDA of a similar magnitude in both cell types. Addition of 7, 14, and 21 mM alpha-T caused no diminution of the MDA level in the unstressed cells and abolished the increase in MDA seen in the stressed cells. (b) The endogenous level of alpha-T in the transformed cells was lower than in the nontransformed cells and all of the measurable alpha-T in these cells was destroyed by the oxidative stress. Supplementation of the cells with alpha-T caused an increase in the level of alpha-T that was proportional to the level of inclusion of alpha-T in the medium. (c) Growth was stimulated by 7 and 14 mM alpha-T but not by the higher levels of inclusion in the medium. The growth stimulation was much larger in the transformed cells (163% of growth in the unsupplemented medium) than in the nontransformed cells (120%). (d) These results demonstrate that, in this cell system, the growth-stimulating ability of alpha-T is unrelated to the ability of alpha-T to control lipid peroxidation and that the level of peroxidation is increased in the malignant state. The difference between the findings reported here and earlier work showing increased levels of alpha-T and decreased levels of peroxidation in transformed malignant cells is discussed and possible explanations for it are advanced.
Subject(s)
Anticarcinogenic Agents/metabolism , Anticarcinogenic Agents/pharmacology , Lipid Peroxidation/drug effects , Vitamin E/metabolism , Vitamin E/pharmacology , Animals , Biological Transport , Cell Division/drug effects , Cell Line , Cell Transformation, Neoplastic , Cricetinae , Kidney , Malondialdehyde/analysis , PolyomavirusABSTRACT
alpha-Tocopherol (alpha-T) uptake and its relationship to cell proliferation and lipid peroxidation was studied in a baby hamster kidney cell line (BHK-21/C13) and its polyoma virus-transformed malignant counterpart (BHK-21/PyY cells). The principal findings were as follows. (i) The level of lipid peroxidation, judged by malondialdehyde (MDA) measurement by HPLC, was higher in the transformed cells than in the nontransformed cells. Oxidative stress by 374 microM Fe3+/10 mM ADP caused a significant increase in the level of MDA of a similar magnitude in both cell types. Addition of 7, 14, and 21 microM alpha-T caused no diminution of the MDA level in the unstressed cells and abolished the rise in MDA seen in the stressed cells. (ii) The endogenous level of alpha-T in the transformed cells was lower than in the nontransformed cells and all the measurable alpha-T in these cells was destroyed by the oxidative stress. Supplementation of the cells with alpha-T caused a rise in the level of alpha-T proportional to the level of inclusion of alpha-T in the medium. (iii) alpha-Tocopheryl quinone in the transformed cells was unaffected by oxidative stress and in the nontransformed cells stress caused a large increase in this metabolite when alpha-T was included at the 21 microM level. (iv) Growth was stimulated by 7 and 14 microM alpha-T but not by the higher level of inclusion in the medium. The growth stimulation was much larger in the transformed cells (163% of growth in the unsupplemented medium) than in the nontransformed cells (120%). (v) These results demonstrate that, in this cell system, the growth-stimulating ability of alpha-T is unrelated to the ability of alpha-T to control lipid peroxidation and that the level of peroxidation is increased in the malignant state.
Subject(s)
Cell Division/drug effects , Cell Transformation, Viral , Kidney/metabolism , Lipid Peroxidation/drug effects , Vitamin E/metabolism , Adenosine Diphosphate/pharmacology , Animals , Cell Line , Cell Line, Transformed , Chromatography, High Pressure Liquid , Cricetinae , Ferric Compounds/pharmacology , Kidney/drug effects , Malondialdehyde/metabolism , Polyomavirus , Vitamin E/pharmacologySubject(s)
Heat-Shock Proteins/physiology , Amino Acid Sequence , Animals , Autoimmune Diseases/physiopathology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Chaperonins , Eukaryotic Cells/physiology , Gene Expression Regulation , Genes , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/classification , Heat-Shock Proteins/genetics , Hot Temperature , Humans , Hyperthermia, Induced , Mammals/physiology , Molecular Sequence Data , Prokaryotic Cells/physiology , Proteins/genetics , Proteins/physiology , Signal Transduction , Stress, Physiological/physiopathologyABSTRACT
In HeLa cells evidence is provided that active oxygen species such as hydrogen peroxide and superoxide at low levels are important growth regulatory signals. They may constitute a novel regulatory redox system of control superimposed upon the established cell growth signal transduction pathways. Whilst for example hydrogen peroxide can be added exogenously to elicit growth responses in these cells, it is clear that cellularly generated superoxide and hydrogen peroxide are important. Experiments with superoxide dismutase, superoxide dismutase mimics and inhibitors of both superoxide dismutase and xanthine oxidase suggest that superoxide generated intracellularly and superoxide released extracellularly are both relevant to growth control in HeLa cells.
Subject(s)
Cell Division , Reactive Oxygen Species/metabolism , Allopurinol/pharmacology , Catalase/pharmacology , Ditiocarb/pharmacology , HeLa Cells/cytology , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Oxidation-Reduction , Oxypurinol/pharmacology , Reactive Oxygen Species/pharmacology , Salicylates/pharmacology , Signal Transduction , Superoxide Dismutase/antagonists & inhibitors , Superoxide Dismutase/metabolism , Superoxides/metabolism , Superoxides/pharmacology , Tetrazolium Salts/metabolism , Tetrazolium Salts/pharmacology , Thiazoles/metabolism , Thiazoles/pharmacology , Xanthine Oxidase/antagonists & inhibitors , Xanthine Oxidase/metabolismABSTRACT
Experiments have been carried out to explore the use of a tetrazolium salt, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide in the detection of intracellularly generated superoxide in HeLa cells. From the use of a low molecular weight lipophilic mimic of superoxide dismutase, as well as superoxide dismutase, and inhibitors of superoxide dismutase, it is suggested that at least 20-30% of the intracellular reduction of MTT is due to superoxide. Whilst this may arise from mitochondria another possible intracellular source in HeLa cells may be xanthine oxidase. The overall rate of intracellular MTT reduction in HeLa cells is inversely dependent on levels of serum in the culture medium. Serum components with a modulatory role in this context are those with antioxidant function. Reduced MTT is also detectable extracellularly in cultures of HeLa cells and at least 80% of this is due to superoxide. Use of inhibitors suggest that whilst a small proportion (30%) may arise through an NADPH-oxidase type enzyme, other sources of extracellular superoxide in HeLa cells remain a possibility.
Subject(s)
Superoxides/metabolism , Tetrazolium Salts/metabolism , Thiazoles/metabolism , Allopurinol/pharmacology , Anti-Infective Agents/pharmacology , Biphenyl Compounds/pharmacology , Coloring Agents , Culture Media , Culture Media, Serum-Free , Formazans , HeLa Cells , Humans , Kinetics , Onium Compounds/pharmacology , Oxidation-Reduction , Oxypurinol/pharmacology , Superoxide Dismutase/metabolism , Superoxide Dismutase/pharmacology , Vitamin E/pharmacologyABSTRACT
Studies on glutathione (GSH) metabolism in an established baby hamster kidney fibroblast cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY) have revealed a significant stimulation of intracellular GSH peroxidase (GSHpx) activity (selenium-independent plus selenium-dependent) by alpha-tocopherol supplementation (14 microM). This stimulation was found to be much greater in the transformed cells. Other GSH-requiring enzyme activities (i.e. GSH reductase and GSH S-transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx. In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by oxidative stress. However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting that some protection was afforded by the alpha-tocopherol.
Subject(s)
Fibroblasts/enzymology , Glutathione Peroxidase/drug effects , Vitamin E/pharmacology , Animals , Cell Line, Transformed , Cricetinae , Fibroblasts/drug effects , Glutathione/metabolism , Glutathione Reductase/drug effects , Glutathione Transferase/drug effects , Tumor Cells, CulturedABSTRACT
Studies on glutathione metabolism in an established baby hamster kidney cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY), have revealed a significant stimulation of intracellular glutathione peroxidase activity (Se-independent plus Se-dependent) by alpha-tocopherol supplementation (14 microM). This stimulation was found to be much greater in the transformed cells. Other GSH-requiring enzyme activities (namely glutathione reductase and glutathione transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx. In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by an oxidative stress. However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting a protection afforded by the alpha-tocopherol.
Subject(s)
Cell Transformation, Neoplastic/drug effects , Glutathione Peroxidase/metabolism , Vitamin E/pharmacology , Animals , Cell Line , Cricetinae , Fibroblasts/drug effects , Fibroblasts/enzymology , Kidney , Oxidation-Reduction , Polyomavirus/geneticsABSTRACT
The effects of oxidant stress were studied in immortalised hamster (BHK-21) and rat (208F) cell lines before and after transformation to the malignant state with polyoma virus, or activated H-ras, respectively. Whilst intracellular superoxide production was detectable in both transformed and immortalised cells the rate was somewhat higher in the transformed cells which have lower levels of superoxide dismutase. Because growth of transformed cells was particularly depressed in the presence of MTT, a tetrazolium compound reduced by superoxide, the possible role of active oxygen species in the promotion of cell growth was examined. Low levels of hydrogen peroxide were stimulatory towards both immortalised and transformed cells. In the case of H-ras transformed rat cells, paraquat was also stimulatory provided serum was present in the growth medium. In the absence of serum, paraquat was notably inhibitory but inhibition could be alleviated by addition of low concentrations of alpha-tocopherol (10(-8)M) to the serum-depleted medium. Although depletion of serum from the growth medium also leads to lower cell proliferation, subsequent experiments showed that alpha-tocopherol addition to serum-free medium was sufficient to restimulate growth. In the case of transformed cells, yields of cells were even greater than that encountered in the presence of 10% serum. Thus whilst certain active oxygen species (e.g. hydrogen peroxide) may have a role in promoting the growth of transformed and immortalised cells the necessity for antioxidant protection is important.
Subject(s)
Cell Transformation, Neoplastic/drug effects , Oxygen/metabolism , Tetrazolium Salts/pharmacology , Thiazoles/pharmacology , Animals , Antigens, Polyomavirus Transforming/physiology , Cell Division/drug effects , Cell Line , Cell Line, Transformed , Cell Transformation, Neoplastic/metabolism , Cricetinae , Culture Media/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Free Radicals , HeLa Cells/drug effects , Hydrogen Peroxide/metabolism , Mesocricetus , Oncogene Protein p21(ras)/physiology , Oxidation-Reduction , Paraquat/pharmacology , Rats , Superoxides/metabolism , Vitamin E/pharmacologyABSTRACT
Antioxidants such as mannitol, butylated hydroxytoluene and alpha-tocopherol enhance the growth of polyoma virus transformed and non-transformed BHK-21 cells. In the case of mannitol this is observed even in the absence of added calf serum. In part these effects may operate to protect cellular growth control mechanisms. On the other hand oxidants such as H2O2 and t-butyl hydroperoxide can inhibit growth and overall cellular protein synthesis, through mechanisms that are likely to involve radicals. In the case of H2O2 the inhibitory effects can nevertheless be reduced by 'prestressing' the cells with mild heat or with H2O2 itself. Paradoxically very low concentrations (10(-8) M) of H2O2 or t-butyl hydroperoxide can actually stimulate cell growth, even in the absence of serum. These stimulatory effects however do not appear to involve radicals as they are enhanced by inclusion of mannitol or DMSO in the medium.
Subject(s)
Cell Division/drug effects , Hydrogen Peroxide/pharmacology , Oxidation-Reduction , Peroxides/pharmacology , Amino Acids/metabolism , Animals , Butylated Hydroxytoluene/pharmacology , Catalase/metabolism , Cell Line , Mannitol/pharmacology , Methionine/metabolism , Protein Biosynthesis , Vitamin E/pharmacology , tert-ButylhydroperoxideABSTRACT
The question of whether free radicals or free radical-related species play a role in the modulation of mammalian cell proliferation is examined. Although a positive role for free radicals as specific components of mitogenic pathways is not apparent it is clear that certain free radical-derived species can have a significant modulatory influence on components of major growth signal transduction mechanisms. Free radical-derived species are also involved in the production of prostaglandins which themselves can modulate cell growth. Free radicals themselves appear to have a down regulatory effect on cell proliferation inasmuch as protection from oxidative stress enhances cell proliferation. On the other hand, in certain cases low levels of active oxygen species can enhance cell proliferation.
Subject(s)
Cell Division , Free Radicals , Animals , Arachidonic Acids/physiology , Homeostasis , Mammals , Second Messenger Systems , Signal TransductionABSTRACT
That some of the effects of hyperthermia on HeLa cell protein synthesis may involve free radical activity is suggested by experiments with free radical scavengers. A possible source of damaging free radicals induced by heat may lie in peroxidative events within lipid-containing membranes. These can be blocked by EGTA. Other experiments with t-butylhydroperoxide indicate that cycloheximide can also neutralise peroxidative effects. These effects are discussed in the light of observations that calcium depletion (or EGTA treatment) can induce thermotolerance and that cycloheximide does not prevent the development of thermotolerance.