DNA oxidative damage during differentiation of HL-60 human promyelocytic leukemia cells.

DNA oxidative damage was measured in human promyelocytic leukemia HL-60 cells, in the same cells committed to granulocytic differentiation with dimethyl sulfoxide (DMSO) or all-trans-retinoic acid (RA) and in mature human peripheral granulocytes (HPG). DNA damage was evaluated as single strand breaks and 8-OHdG adducts, measured by single cell electrophoresis or by monoclonal antibodies, respectively. The basal levels of either marker of DNA damage were higher in undifferentiated HL-60 cells than in HPG and DMSO- or RA-differentiated cells. Treatment with H(2)O(2) increased 8-OHdG formation in all cells, but the levels of DNA damage remained higher in undifferentiated cells as compared to the differentiated ones. Three lines of evidence suggested that the higher levels of DNA damage observed in undifferentiated cells were at least in part attributable to a reduced detoxification of reactive oxygen species (ROS). First, undifferentiated cells were shown to accumulate higher levels of dichlorodihydrofluorescein-detectable ROS than HPG and DMSO- or RA-differentiated cells. Second, undifferentiated HL-60 cells were characterized by reduced levels of GSH and lower GSH/GSSG ratios as compared to the differentiated cells. Third, pretreatment of undifferentiated HL-60 cells with antioxidants such as alpha-tocopherol or beta-carotene suppressed the elevation of ROS and the formation of 8-OHdG induced by H(2)O(2). Further evidence for the importance of the oxidant/antioxidant balance was obtained by modulating the iron-catalyzed decomposition of H(2)O(2) to hydroxyl radicals in undifferentiated HL-60 cells. In fact, pretreatment with FeSO(4) increased the formation of 8-OHdG induced by H(2)O(2), whereas pretreatment with the iron chelator deferoxamine produced the opposite effect. These results illustrate correlations between the oxidant/antioxidant balance and DNA damage and suggest that the capability of a cell population to withstand oxidative stress and DNA damage may depend on its degree of differentiation.

Effect of extracellular magnesium on topoisomerase II activity and expression in human leukemia HL-60 cells.

Topoisomerase II (TopoII) is a Mg-dependent enzyme involved in topological modifications of DNA that are crucial to the regulation of cell proliferation and possibly differentiation. To investigate the role of Mg availability in the modulation of TopoII in whole cells, we studied enzyme activity and expression in HL-60 cells grown in the presence of decreasing amounts of extracellular Mg (0.5, 0.03, and 0.01 mM MgSO(4)). In comparison to cells grown in 0.5 mM Mg, cells grown in 0.03 mM Mg exhibited a decrease in TopoII activity, as evidenced by reduced induction of DNA/TopoII cleavable complexes and apoptosis by etoposide and teniposide. Enzyme activity was restored by the readdition of Mg (0.5 and 1.5 mM) in the incubation medium, confirming that this effect was indeed modulated by extracellular Mg. Restriction of Mg to 0.01 mM was associated with a dramatic decrease in TopoII activity resembling that observed in HL-60 cells differentiated by dimethyl sulfoxide treatment. The restriction of Mg, while decreasing enzyme activity, was found to upregulate TopoII protein expression, determined by Western blot analysis. The increase of TopoII protein levels was correlative with the degree of Mg deprivation. Collectively, these results indicate that extracellular levels of Mg may control availability of intracellular Mg, thus affecting the regulation of TopoII activity/expression and downstream processes of cell proliferation and/or differentiation.

In vitro evaluation of the mutagenic and carcinogenic power of high purity zirconia ceramic.

Tetragonal zirconia polycrystal (TZP) is a new interesting ceramic for the manufacture of medical devices. Its wide use in orthopedic and odontoiatric implants was limited till now by the high chemical and radiochemical impurities of the raw materials. Purification processes now available allow to obtain high purity ceramic grade powders suitable for TZP ceramics manufacture, even if their possible mutagenic and transforming effects are still unclear. The aim of this work is to study in vitro the mutagenic and oncogenic effects of a new zirconia ceramic stabilized by yttria (Y-TZP). This ceramic was sintered from high purity powders obtained by a process developed under a project carried out within the Brite EuRam programme. For comparison, ceramics made from unpurified zirconia powder were also tested. Fibroblasts irradiated by a linear accelerator were used as positive control. The results obtained show that Y-TZP ceramic does not elicit either mutagenic or transforming effect on C3H/10T(1/2) (10T(1/2)) cells and demonstrate that ceramic from high purity powders can be considered suitable for biomedical applications from the point of view of the effects of its radioactive impurity content.

Changes in magnesium content and subcellular distribution during retinoic acid-induced differentiation of HL60 cells.

Magnesium (Mg) is required for cellular proliferation; however, the differences in subcellular regulation of Mg between proliferating and differentiated cells has not been determined. We used electron probe microanalysis (EPMA) to investigate the subcellular distribution of Mg in HL60 cells (a promyelocytic leukemia cell line) before and after retinoic acid (RA)-induced differentiation. Most intracellular Mg is bound to ATP and the Mg-ATP complex regulates several metabolic enzymes. We also compared alterations in Mg content following differentiation with the changes in ATP and ADP levels. Using atomic absorption spectrophotometry, we observed a significant decrease (-20%) in cellular Mg content in RA-differentiated HL60 cells. To investigate which intracellular compartments were involved in these changes, we analyzed subcellular elemental composition in freeze-dried cryosections of rapidly frozen undifferentiated and differentiated HL60 cells by EPMA. Following differentiation of HL60 cells, we observed an 18% decrease in Mg content in both the cytoplasm (regions of the cell excluding mitochondria and nuclei) and mitochondria. There was also a significant (40%) decrease in cytoplasmic Ca content after RA-induced differentiation. Nuclear Mg concentration was not significantly different between differentiated and undifferentiated HL60 cells, although differentiation was accompanied by a 30% decrease in the nuclear K/Na ratio. After differentiation, cellular ATP and ADP content decreased by 31 and 40%, respectively. We conclude that during exit from the cell cycle, Mg redistributes within cells and that the decrease in cytoplasmic and mitochondrial Mg is accompanied by a decrease in ATP and ADP content.

Differentiation of HL-60 promyelocytic leukemia cells is accompanied by a modification of magnesium homeostasis.

Magnesium homeostasis in HL-60 promyelocytic leukemia cells was compared to that in neutrophyl-like HL-60 cells obtained by 1.3% DMSO treatment. Magnesium homeostasis was studied by the characterization of magnesium efflux, the identification of intracellular magnesium pools, and the regulation of intracellular ionized Mg2+. In both undifferentiated and neutrophyl-like HL-60 cells, magnesium efflux occurred via the Na-Mg antiporter which was inhibited by imipramine and stimulated by db cAMP and forskolin. Receptor-mediated signals such as ATP, IFN-alpha, or PGE1, which can trigger cAMP-dependent magnesium efflux, were ineffective in undifferentiated HL-60 cells but induced 60-70% increase of magnesium efflux in neutrophyl-like HL-60 cells. Selective membrane permeabilization by the cation ionophore A23187 induced a large magnesium release when cells were treated with rotenone. In both cell populations, the addition of glucose to rotenone-treated cells restored magnesium release to the control level. Permeabilization by 0.005% digitonin provoked the release of 90% cell total magnesium in both cell types. Intracellular [Mg2+]i was 0.15 and 0.26 mM in undifferentiated and neutrophyl-like HL-60 cells, respectively. Stimuli that triggered magnesium efflux, such as db cAMP in undifferentiated and IFN-alpha in neutrophyl-like HL-60 cells, induced a slow but consistent increase of [Mg2+]i which was independent from Ca2+ movements. Overall, these data indicate that magnesium homeostasis is regulated by receptor-mediated magnesium efflux which was modified during differentiation of HL-60 cells. Stimulation of magnesium efflux is paralleled by an increase of [Mg2+]i which reflects a release of magnesium from the bound cation pool.

Y-TZP ceramics for artificial joint replacements.

Due to their excellent mechanical properties, Yttria-stabilized Tetragonal Zirconia Polycrystal ceramics (Y-TZP) are used in ball heads for Total Hip Replacements. It is known that Y-TZP materials may show strength degradation due to ageing or to hydrothermal treatment. Also high wear of UHMWPE sockets coupled to steam sterilized Y-TZP ball heads after a short implantation period was recently reported. This effect may be related to ball head surface phase transformation, due to corrosive attack. The aim of this study is the evaluation of Y-TZP ceramics stability. Y-TZP made out of Yttria coated powders were aged at 140 degrees C under 0.2 MPa water pressure, in Ringer's solution at 37 degrees C, in NZW rabbits. Samples made out Yttria coated powders show lower strength degradation than samples made out coprecipitated powders, and UHMWPE discs coupled to Y-TZP rings made out coated powders do not show increase in wear after repeated sterilization cycles of the ceramic rings.

Magnesium restriction induces granulocytic differentiation and expression of p27Kip1 in human leukemic HL-60 cells.

When cultured in Mg restricted medium, human leukemic HL-60 cells develop morphological and functional granulocytic differentiation. In 0.03 mM Mg, cells display the distinctive features of differentiation, without appreciable inhibition of proliferation. In 0.01 mM Mg, cells show terminal differentiation, accompanied by clear inhibition of proliferation. Such cells accumulate in the G0/G1 phase and subsequently die via apoptosis, similar to HL-60 cells that have been induced to differentiate by DMSO. These phenotypic changes are associated with a marked increase in the expression level of the cyclin dependent kinase inhibitor p27Kip1. Cyclin E expression is also slightly increased in Mg restricted cells, whereas no changes are observed in the expression level of cyclin D1. We also show that during differentiation cell total Mg decreases, whereas [Mg2+]i increases in both Mg-depleted and DMSO-treated cells. These data suggest that the maturation process is paralleled by a redistribution of intracellular Mg, leading to a shift from the bound to the free form. These changes could modulate the kinetics of Mg-dependent enzyme(s) that are involved in the control of the differentiation pathway. We propose that this model may represent an useful tool for the study of the mechanisms of cell differentiation and related events, such as aging and death.

Regulation of magnesium efflux from rat spleen lymphocytes.

Rat spleen lymphocytes (RSL) incubated at 37 degrees C in Mg-free medium (O-trans conditions) exibited Mg2+ efflux with apparent velocity of 0.2 nmol/mg protein/min. After 30 min, this process accounted for the mobilization of about 15% of cell total Mg2+. Half of the Mg2+ efflux depended on extracellular Na+ and was stimulated by cAMP. IFN-alpha significantly enhanced Mg2+ efflux under O-trans conditions as well as in the presence of physiological extracellular Mg2+. Pretreatment of RSL with indomethacin completely abolished IFN-alpha-induced Mg2+ efflux, suggesting a crucial role for cyclooxygenase-dependent arachidonate metabolism. On the other hand, pretreatment of RSL with the PKA inhibitor (Rp)8-Br-cAMPS prevented IFN-alpha stimulation of Mg2+ efflux, indicating the involvement of cAMP. Consistently, both IFN-alpha and exogenous PGE1 increased cAMP from 50 to 125 pmol/mg protein. Altogether these results show that IFN-alpha stimulates Mg2+ efflux by activating arachidonate metabolism and synthesis of prostaglandins. By influencing adenylcyclase activity, PGEs can eventually promote cAMP-dependent Mg2+ efflux, possibly through the activity of a Na-Mg antiport. In RSL, therefore, magnesium movements can be under the control of IFN-alpha and, perhaps, of other cytokines, suggesting the involvement of Mg2+ in cell response to receptor-mediated stimuli.

Norepinephrine protects mice from acute lethal doses of carboplatin.

We demonstrated in previous studies that adrenergic agents may affect hematopoiesis via high- and low-affinity alpha1-adrenoceptors present on bone marrow (BM) cells [1-3]. Here we show that norepinephrine administration in mice rescued hematopoiesis from the toxic effect of the non-cell cycle specific chemotherapeutic agent, carboplatin. Protection of granulocyte/macrophage colony-forming units (GM-CFUs) was already apparent only a few hours after carboplatin and norepinephrine administration. On day 3, hematopoietic rescue was reflected by higher leukocyte and platelet counts. At its most effective dose (3 mg/kg, subcutaneously injected), norepinephrine protected 77% of the mice previously injected intravenously with 200 mg/kg of carboplatin (LD 100: 170 mg/kg). Simultaneous administration of the alpha1-adrenoceptor antagonist prazosin reduced the percentage of surviving mice to 30%, indicating that alpha1-adrenoceptors mediated most of the norepinephrine-induced hematopoietic rescue. Consistently, prazosin administration also reduced blood counts and GM-CFUs. In vitro, norepinephrine (1 microM) rescued GM-CFUs in BM cells, although this effect was counteracted by low concentrations (0.1-10 nM) of prazosin. Our findings indicate a previously undescribed novel mechanism of hematopoietic regulation and may find application in preventing the myeloablative effect of anticancer treatments.

Regulation of intracellular magnesium in ascites cells: involvement of different regulatory pathways.

Extracellular ATP causes 40% stimulation of Mg2+ efflux from Ehrlich ascites tumor cells (EATC) incubated under 0-trans conditions. ATP also causes a threefold increase of arachidonic acid (AA) metabolite release from [3H]AA-preloaded EATC, indicating that, under these experimental conditions, it induces phospholipase A2 (PLA2) activation. ATP-induced Mg2+ efflux can be prevented by cyclooxygenase inhibition with indomethacin or lysine acetylsalicylate, but not by 5-lipooxygenase inhibition with BWA4C. Mg2+ efflux is also directly stimulated by exogenous AA in a concentration-dependent manner. This phenomenon involves PKA as it is virtually abolished by the specific inhibitor 8-bromoadenosine-3',5'-cyclic monophosphothioate. While stimulating Mg2+ efflux, exogenous AA also increases cAMP content of EATC and this effect can be prevented by cyclooxygenase inhibition. Measurements in mag-fura-2-loaded EATC reveal that stimulation of Mg2+ efflux does not correlate with significant fluctuations of [Mg2+]i. This suggests that Mg2+ efflux is compensated for by mobilization of bound Mg2+, leaving [Mg2+]i unaltered. Altogether these data indicate that Mg2+ efflux can be modulated by extracellular stimuli capable of activating PLA2. This modulation is triggered by cyclooxygenase products which, by activating adenylcyclase, determine an elevation of cAMP. This intracellular messenger upregulates Na-dependent Mg2+ efflux.

cAMP activates magnesium efflux via the Na/Mg antiporter in ascites cells.

In intact ascites cells magnesium efflux is substantially stimulated by cAMP analogues and by low concentrations of prostaglandins (PGE1 and PGE2) which increases the synthesis of cAMP. cAMP stimulates magnesium efflux by directly stimulating the Na-Mg antiporter rather than determining a magnesium release from mitochondria as reported for other cell types. Protein kinase inhibitors abolish the cAMP stimulated magnesium efflux suggesting that the stimulation is mediated by the phosphorylation of the carrier protein, increasing its affinity for intracellular magnesium. These data provide evidence that receptor-mediated stimuli eventually leading to cell activation are directly connected to the regulation of intracellular magnesium.

Hematopoietic rescue via T-cell-dependent, endogenous granulocyte-macrophage colony-stimulating factor induced by the pineal neurohormone melatonin in tumor-bearing mice.

We investigated whether melatonin can affect tumor growth and/or hematopoiesis in mice transplanted with Lewis lung carcinoma and treated with cyclophosphamide or etoposide. These agents were injected i.p. for 5 days at two different cumulative doses (cyclophosphamide, 40 and 160 mg/kg body weight; etoposide, 20 and 40 mg/kg body weight) from day 8 through day 12 after tumor transplantation. Melatonin was injected s.c. at a dose of 1 mg/kg body weight/day, from day 8 throughout the experiments and from days 8 through 12 or from day 13 onwards. Melatonin did not influence tumor growth but selectively counteracted bone marrow toxicity when administered together with the cancer chemotherapy compounds without interfering with their anticancer action. In vitro, melatonin proved to counteract apoptosis in bone marrow cells incubated with etoposide. Such protection was reflected by an increased frequency of granulocyte/macrophage-colony forming units but not of the pluripotent spleen-colony forming units. The effect of melatonin was neutralized by anti-granulocyte/macrophage-colony-stimulating factor monoclonal antibodies. When athymic, T-cell-deficient mice were used as bone marrow donors, melatonin did not exert any protective effect. This suggested that melatonin is able to stimulate the endogenous production of granulocyte/macrophage-colony-stimulating factor via bone marrow T-cells. Due to the well known lack of toxic and undesirable side effects of melatonin, these findings might have a straightforward clinical application.