Free Radical Mediated Cytotoxicity of Desferrioxamine

Toxic effects of desferrioxamine (DFO) upon cell growth were assayed with continuous bromodeoxyur-idine (BrdU) labeling and bivariate ethidium bromide/Hoechst 33258 Row cytometry. At 5% oxygen DFO caused a dose-dependent inhibition of cell growth. which was potentiated at 20% oxygen. and by cumene hydroperoxide but not by paraquat. An irreversible arrest in the GZ phase of the cell cycle was the cell-kinetic mechanism underlying this growth inhibition. The G2 arrest was not dependent upon the BrdU concentration in the medium, thus ruling out a direct attack of a free radical on thymidine residues. The observed cytotoxicity of DFO cautions against its use in the treatment of conditions of elevated oxidative stress.     

Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome.

The clinical radiosensitizer bromodeoxyuridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: BrdU/Hoechst flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.     

Cell Kinetic Evidence Suggests Elevated Oxidative Stress in Cultured Cells of Bloom's Syndrome

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse disturbed cell proliferation of fibroblasts and lymphoblastoid cells from Bloom's syndrome (BS). Fibroblasts show poor activation, arrest in the G2 phase of the cell cycle along with a prolongation of the Gl phase. This pattern of perturbed cells proliferation is akin to that elicited in normal fibroblasts by 4-hydroxy-nonenal, a breakdown product of lipid peroxides. Treatment with vitamin E improved growth of BS fibroblasts more strongly than growth of normal fibroblasts. Lymphoblastoid cells from BS, to the contrary, experience only a minor arrest in the G2 phase after one round of bromodeoxyuridine incorporation, but are strongly inhibited during and after the second S phase. Thus, their cell cycle arrest is dependent upon BrdU incorporation, as has been found previously in normal cells exposed to elevated concentrations of oxygen or paraquat, a superoxide generating compound. These results suggest that BS cells may suffer from an elevated, endogenous generation of oxygen free radicals.     

Bromodeoxyuridine amplifies free-radical-mediated DNA damage.

Elevated oxygen concentrations and paraquat, a superoxide-generating compound, induce an arrest of cells in the G2 phase of the cell cycle, which can be enhanced by adding bromodeoxyuridine (BrdU) to the culture medium. Experiments with the lipophilic peroxide cumene hydroperoxide and the free-radical scavenger vitamin E demonstrate that the BrdU-dependent G2 arrest is not mediated by lipid peroxidation. The BrdU-dependency of arrest in the G2 phase can be used as a sensitive cell biological assay to detect DNA damage elicited by oxygen free radicals.     

Rotenone-induced G2/M cell cycle arrest and apoptosis in a human B lymphoma cell line PW.

Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.     

Mutagenic effects of brief exposure to bromodeoxyuridine on mouse FM3A cells

Abstract. The time- and dose-dependency of the mutagenic effects of bromodeoxyuridine (BrdU), a thymidine analogue used for cell kinetics studies in vivo and in vitro , were investigated in FM3A cells. Cells incubated with 50–1000 fin BrdU for 72 h showed some inhibition of growth. Cells cultured in BrdU-free medium for 3 d after a 30 min or 2 h exposure to BrdU showed no growth inhibition, while those previously exposed for 24 h to BrdU showed retarded growth. After a 30 min exposure, 60% of cells were labelled with BrdU; after 2 h 70%; and after 24 h almost 100%. After incubation in BrdU-free medium for 3 d (the time required for this cell line to express mutation), cells previously treated for 30 min or 2 h showed reduced BrdU positivity, whereas almost 100% of those treated for 24 h remained BrdU positive. The mutation rate, determined by the number of colonies resistant to ouabain (2 mM) and 6-thioguanine (10 μ) 3 d after exposure to BrdU, was not affected by a 30 min treatment with up to 1000 μ BrdU. Cells treated for 1 or 2 h showed increased resistance to ouabain after exposure to BrdU at concentrations above 100 μM; cells treated for 12 or 24 h showed an increased mutation rate at BrdU concentrations above 50 μM… The number of colonies resistant to 6-thioguanine did not increase in cells treated with BrdU at concentrations up to 1000 μM for 1, 12 or 24 h. We cannot conclude with certainty that brief exposure to BrdU does not modulate DNA to the point of mutation. This study may serve as a guideline for limiting the dose and time of exposure to BrdU for cell kinetics studies in vivo and in vitro.     

Inducible expression of 15-lipoxygenase-2 and 8-lipoxygenase inhibits cell growth via common signaling pathways

Human 15-lipoxygenase (LOX)-2 and mouse 8-LOX represent orthologous members of the LOX family but display different positional specificities and tissue distribution. To study the functional role of 15-LOX-2 and 8-LOX in keratinocytes, an inducible Tet-On gene expression system was established in the premalignant mouse keratinocyte cell line 308. Doxycycline (dox)-induced expression of enzymatically active 15-LOX-2 and 8-LOX led to an inhibition of cell growth that was associated with an inhibition of DNA synthesis, as shown by a 15-46% reduction of 5-bromo-2-deoxy-uridine (BrdU) incorporation. The inhibitory effects were increased in the presence of exogenous arachidonic acid. In contrast, addition of linoleic acid or the LOX inhibitor baicalein reversed the growth-inhibitory effects. Treatment of the cells with 15-hydroxyeicosatetraenoic acid (HETE) or 8-HETE resulted in a similar inhibition of BrdU incorporation, whereas 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE, in contrast, had no effects. Dox-induced keratinocytes showed increased levels of reactive oxygen species (ROS). The antioxidant N-acetyl-L-cysteine and a specific inhibitor of p38 mitogen-activated protein kinase, but not of extracellular signal-regulated kinase 1/2 or c-Jun N-terminal kinase/stress-activated kinases, completely abolished the LOX-induced growth inhibition, indicating a critical role of ROS and p38. Our data suggest that 15-LOX-2 and 8-LOX, although displaying different positional specificity, may use common signaling pathways to induce growth inhibition in premalignant epithelial cells.     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G0/G1期阻滞

卡铂(carboplatin, CBP)是一种抗肿瘤活性较强的化疗药物, 通过诱导细胞周期阻滞抑制肿瘤细胞生长, 但其诱导细胞周期阻滞的报告不甚一致. 本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响. MTS结果显示, 卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长, 联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强. 采用Giemsa染色法观察到, 卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化. 流式细胞术检测细胞周期发现, 随卡铂浓度的增高, S期阻滞作用增强; 抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用, 增加G1期阻滞作用, 而对G2/M期细胞影响不明显. Western印迹结果显示, 随卡铂浓度的增高, p-ERK1/2、Cdc2(Y15)和p Cdc2(T161)的表达逐渐升高, Cyclin E1和Cyclin B1的表达逐渐降低; 抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用, 上调Cdc2(Y15)的表达受阻, 抑制Cyclin B1的下调作用, 促进Cyclin E1的下调作用. 本研究结果提示, 卡铂通过抑制ERK1/2激活, 诱导人卵巢癌HO-8910细胞S和G1期阻滞, 抑制卵巢癌细胞生长.     

p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest

We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.     

Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells

The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RARalpha (CD336), RARbeta (CD2019), RARgamma (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RARbeta/gamma (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARalpha and beta agonists or RAR panagonist, but not the RARgamma agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RARaalpha/beta and RXR agonists. The RARgamma agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The RARbetagamma antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G1-S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RARalpha/beta ligands are effective inhibitors of squamous differentiation. On the contrary, RARgamma ligand appears to be inefficient in metaplasia inhibition, but the selective RARgamma agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.     

De novo synthesis of glutathione is required for both entry into and progression through the cell cycle

To study the putative role of de novo synthesis of glutathione (GSH) in the regulation of the cell cycle, we exposed NIH-3T3 cells to buthionine sulfoximine (BSO) and analysed cell cycle kinetics with continuous bromodeoxyuridine (BrdU) labeling and bivariate Hoechst 33258/ethidium bromide flow cytometry. Treating quiescent cells, which themselves had a low GSH content, with BSO did not affect subsequent entry into and progression through the cell cycle. Adding BSO during serum stimulation, however, provoked a dose-dependent inhibition of cell growth and a delayed increase in GSH level. The cell kinetic mechanism underlying BSO-induced growth inhibition is a diminished entry into the cell cycle and a permanent arrest in the S and G2 phase of the cell cycle. Our results are consistent with the hypothesis that GSH de novo synthesis is required for cell activation and proper S and G2 phase transit. © 1995 Wiley-Liss, Inc.     

Effect of 5-bromo-2'-deoxyuridine on growth and differentiation of cultured embryonic retinal pigment cells.

Chicken embryo retinal pigment cells cultured for 2 weeks in 1 or 10 microns BrdU accumulate only 25% of the amount of melanin found in controls. Growth is inhibited by 10 microns BrdU but not by 1 micron BrdU. Cells cultured with BrdU had decreased tyrosinase activity and lost the typical epithelial appearance of controls. Equimolar concentrations of deoxythymidine did not prevent the inhibition of melanogenesis due to BrdU but did prevent the growth inhibition of the higher concentration of BrdU (10 microns). Tenfold higher concentrations of deoxythymidine prevented inhibition of epithelial differentiation and melanogenesis. Ultrastructurally, BrdU produced an absence of melanosomes and disruption of the lamellar array of rough endoplasmic reticulum. The perinuclear arrangement of microfilaments became irregular. The cell surface was radically affected as membranes were no longer ruffled and cell junctions disappeared. Melanogenesis is a complex form of differentiated function, involving synthesis of tyrosinase and control of its activity, macromolecular synthesis of the melanosome, and the subsequent synthesis of melanin within the melanosome. BrdU appears to inhibit melanogenesis through a coordinated and simultaneous interference with these processes, suggesting that there may be a "program" of gene activity for melanogenesis that is regulated as a unit.     

Oxidation of deseferrioxamine to nitroxide free radical by activated human neutrophils

Human neutrophils activatd by PMA were found to induced the formation of a nitroxide radical from DFO. The presence of SOD was necessary to permit the formation of the DFO radical. The inactive phorbol ester did not induce DFO radical, and _sphinganine suppressed the radical produced by the active phorbol ester. Other cell stimuli (Zymocel and the chemotactic peptide) also induced the formation of the DFO radical, although radical concentration was very much lower than with PMA. Participation of ^.NO, ^,OH or ¹O₂ was ruled out by the inability of N^G-methyl-L-arginine, N^G-nitro-L-arginine, DMSO, mannitol, histidine, and methionine to inhibit the formation of DFO radical produced by PMA-activated cells. Furthermore, PMA-activated cells dod not produce detectable levels of NO₂⁻, as a stable oxidation product of ^.NO, and D₂, which enhances the lifetime of singlet oxygen, did not modify the intensity or the lifetime of DFO radical. The involvement of cell MPO was suggested by the inhibition of the DFO radical observed after treatment with catalase or with antihuman MPO antibodies. Also, HOCI was found to induce the DFO radical in cell-free reactions, but our data indicate that the reaction leading to DFO radical formation by neutrophils involves the reduction of MPO compound II back to active enzyme (ferric-MPO). Anti-inflammatory drugs strongly increased the DFO radical produced by activated neutrophils. On the contrary, none of these drugs was able to increase the DFO radical produced by HOCl. Histidine and methionine that inhibited the DFO radical intensity in cell-free reactions, were shown to act directly onm HOCl. Experiments with MPO-H₂O₂ in SOD- and Cl⁻-free conditions showed the formation of DFO radical and confirmed the hypothesis of the involvement of compound II. The conversion of compound II to ferric MPO by DFO optimized the enzymatic activity of neurophils, and in the presence of monochlorodimedon (compound II promoting agent) we measured an increased HOCl production. When DFO was modified by conjugation with hydroxyethyl starch, it lost the ability to produce the radical either by neutrophils or by MPO-H₂O₂ and did not increase HOCl production. The inability of these DFO derivatives to produce potentially toxic species migh explain their reported lower toxicity in vivo.     

Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.     

Hypoxia inhibition of adipocytogenesis in human bone marrow stromal cells requires transforming growth factor-beta/Smad3 signaling

Although hypoxia and transforming growth factor-beta (TGF-beta) inhibit differentiation of adipocytes from preadipocytes and bone marrow-derived cells in several species, the relationship between hypoxia and TGF-beta signaling in adipocytogenesis is unknown. In this study, we evaluated the mechanisms of inhibition of adipocyte differentiation by hypoxia and TGF-beta in human and murine marrow stromal cells (MSCs) and the role of TGF-beta/Smad signaling in the inhibition of adipocytogenesis by hypoxia. Both hypoxia-mimetic deferoxamine mesylate (DFO) and TGF-beta1 inhibited adipocyte differentiation (1.0% versus the control at 15 microm DFO and 1.4% versus the control at 1 ng/ml TGF-beta1) and adipocyte gene expression (peroxisome proliferator-activated receptor-gamma2 and lipoprotein lipase) in human MSCs after 21 days of treatment. Hypoxia (2% O(2)) and DFO (but not TGF-beta1) increased hypoxia-inducible factor-1alpha as shown by Western blotting. Macroarrays and Western and Northern blot analyses showed that hypoxia activated the TGF-beta/Smad signaling pathway and that both hypoxia and TGF-beta1 modulated adipocyte differentiation pathways such as the insulin-, peroxisome proliferator-activated receptor-gamma-, phosphatidylinositol 3-kinase-, and MAPK-associated signaling pathways. Studies with mouse marrow stromal cell lines derived from Smad3(+/+) or Smad3(-/-) mice revealed that the TGF-beta type I receptor (ALK-5) and its intracellular signaling molecule Smad3 were necessary for the inhibition of adipocyte differentiation by both TGF-beta and hypoxia-mimetic DFO. Thus, the TGF-beta/Smad signaling pathway is required for hypoxia-mediated inhibition of adipocyte differentiation in MSCs.     

Perturbation of growth and differentiation of Friend murine erythroleukemia cells by 5-bromodeoxyuridine incorporation in early S phase.

Cultured Friend murine erythroleukemia cells (Friend cells) are induced to undergo erythroid differentiation when grown in the presence of dimethylsulfoxide (DMSO) and other compounds. The effects of unifilar substitution of bromouracil (BU) for thymidine in the DNA (BU-DNA) of Friend cells were examined. Cells were grown in the presence of 5-bromodeoxy-uridine (BrdU) for one generation, then centrifuged and resuspended in medium containing DMSO without BrdU. These cells exhibited a delay in the appearance of heme-producing, benzidine-reative (B+) cells and a decreased rate of cell proliferation in comparison to the control not containing BU-DNA. A transient inhibition of entry into S phase was observed when control cells or cells containing BU-DNA were grown in the presence of DMSO) for 10 to 20 hours. This transient inhibition was increased in the BrdU culture. Thus BU-substitution in Friend cells alters other cellular functions in addition to erythroid differentiation. The rate of increase in the percent of cells committed to differentiate (those forming B+ colonies in plasma clots) was similar in the BrdU and control cultures until 40 to 50 hours. After this time, a delay in the appearance of committed cells was observed in the BrdU culture. The effect of BrdU on the appearance of B+ cells was more pronounced and occurred earlier than its effect on the rate of commitment. Therefore, the delay in the appearance of B+ cells in the BrdU culture was due primarily to perturbation of post-commitment events such as the accumulation of hemoglobin. We also examined the effect on growth and differentiation after BrdU was incorporated during different intervals of S phase in cells synchronized by centrifugal elutriation or by double thymidine block and hydroxyurea treatment. The delay in the appearance of B+ cells and inhibition of cell proliferation were only observed when BrdU was incorporated in the first half of S phase. BrdU (10 muM) had no effect on growth or differentiation when present during late S or G1 and G2. These results, using two very different methods to achieve cell synchrony, indicate that the effects of BrdU on growth and differentiation described above are due to its incorporation into DNA sequences replicating during early S.     

Effect of tissue culture variables on sister chromatid exchange in a nontransformed rat cell line

The frequency of sister chromatid exchange (SCE) was determined in a nontransformed diploid rat cell line, FR3T3 , under several tissue culture variables such as cultivation temperature, growth conditions of cells, and concentrations of 5-bromo-2'-deoxyuridine (BrdU). The conclusions to be drawn from these experiments are: (a) The cell growth and mechanisms(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5 degrees C. (b) The concentration limits for BrdU incorporation are 5 to 100 microM; baseline frequency is about 11 SCE/metaphase (constant up to 20 microM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 microM) cause a decrease of SCE rates below that found at 100 microM, presumably due to selective cell death. (d) Keeping cells growth arrested over a long period causes substantial SCE induction after replating. (e) Induced increase of SCEs probably occurs in this manner during the first cell cycle after release from growth arrest. It is no longer detectable after the fourth consecutive cell division.     

The diverse effects of 5'-bromodeoxyuridine on enzyme activities in cultured H35 hepatoma cells.

Reuber (H35) hepatoma cells were grown in medium containing 10(-5)M bromodeoxyuridine (BrdU), which was incorporated into their DNA. Cell growth rate was unaffected by BrdU for the first two generations, after which it was reduced by about 50%. The effect of BrdU incorporation on the activities of several enzymes with rapid turnover rates was examined to test the hypothesis that the synthesis of such enzymes will be preferentially inhibited by BrdU. Tyrosine amino-transferase (TAT) activity decreased by 70% within two generations whereas thymidine kinase activity remained at control values. PEP carboxykinase activity was unchanged during the first generation in BrdU-containing medium but, during the second, its activity increased by at least 30%. Ornithine decarboxylase levels decreased by about 50% only after two generations in the presence of BrdU. There appeared to be no simple relationship between turnover rates and the effect of BrdU on enzyme activity. Incorporation of BrdU was found to inhibit the induction of both TAT and PEP carboxykinase by dexamethasone and to enhance the inhibition of cell growth by this steroid. These results are discussed with respect to possible mechanisms of gene expression and development in both normal and neoplastic cells.