ascorbic-acid and Leukemia--Erythroblastic--Acute

K562 erythroleukaemic cells produced ascorbate when incubated with dehydroascorbic acid. The reduction depended on the number of cells and on the concentration of dehydroascorbic acid. The observed rate consists of a high affinity (apparent Km 7 mu M, Vmax 3 center dot 25 pmol min-1 (10(6) cells)-1 and a low affinity component, which was non-saturable up to 1 mM of DHA (rate increase of 0 center dot 1 pmol min-1 (10(6) cells)-1 (1 mu M of DHA-1). The rate was dependent on temperature and was stimulated by glucose and inhibited by phloretin, N-ethylmaleimide, parachloro-mercuribenzoate and the noyltrifluoroacetone. Although uptake of DHA proceeded at a higher rate than its extracellular reduction, the generation of extracellular ascorbate from DHA cannot be accounted for by intracellular reduction and the release of ascorbate, since the latter was not linear with time and had an initial rate of approximately 3 pmol min-1 (10(6) cells-1). At a concentration of DHA of 100 mu M this is 25 per cent of the observed reduction.

Topics: Ascorbic Acid; Chelating Agents; Chloromercuribenzoates; Dehydroascorbic Acid; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethylmaleimide; Free Radicals; Humans; Leukemia, Erythroblastic, Acute; Leukocytes; p-Chloromercuribenzoic Acid; Thenoyltrifluoroacetone; Tumor Cells, Cultured

Electroporation is a most popular method of cell membrane permeabilization, by pulsed electric fields. It allows foreign molecules to enter the cell and has been used for many biotechnological applications, including transformation of mammalian cells and plant protoplasts by exogenous genetic material. However, the mechanism underlying membrane electropermeabilization is still largely unknown. Evidence is presented here that electroporation under conditions compatible with cell survival induces lipid hydroperoxide formation in the membranes of animal and plant cells. Exposure to electric fields also enhanced up to 5-fold the spontaneous emission of light from both cell types, which paralleled the amount of conjugated hydroperoxides detected in cell membranes. The emitted photons were mainly in the red edge of the spectrum, suggesting the involvement of singlet oxygen. The presence of antioxidants during electroporation did not reduce the formation of hydroperoxides nor the permeability but quenched the luminescence.

Topics: Antioxidants; Ascorbic Acid; Cell Membrane; Chromans; Electroporation; Fabaceae; Humans; Leukemia, Erythroblastic, Acute; Lipid Peroxidation; Luminescent Measurements; Membrane Lipids; Oxygen; Photochemistry; Plants, Medicinal; Protoplasts; Singlet Oxygen; Tumor Cells, Cultured; Vitamin E

Topics: Antioxidants; Ascorbic Acid; Azo Compounds; Cell Line; Cell Nucleus; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Etoposide; Free Radicals; Glutathione; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Molecular Structure; Monophenol Monooxygenase; Phenols; Tumor Cells, Cultured

More than half of the 67Cu recovered from K562 cells following a brief incubation with 67Cu-ceruloplasmin was recovered in particulate fractions of the cell. The fractions in Percoll had densities that ranged between 1.040 and 1.060 g/dl. In as early as 5 min, two fractions, densities of 1.051 and 1.056, respectively, were discernible. Components in the 1.051 fraction tested positive for clathrin and catalase. Those in the 1.056 fraction sedimented near the marker for lysosomes. The 67Cu in both fractions was stable to treatment by EDTA, nitrilotriacetate, alpha,alpha'-dipyridyl, heparinase, and ascorbate, but dissociated when treated with pronase, trypsin, or sodium dodecylsulfate. Continuous incubation with 67Cu-ceruloplasmin intensified the 67Cu activity in the 1.051 and 1.056 fractions. Cells incubated with 125I-transferrin displayed the label primarily in the 1.051 fraction. Continuous incubation intensified the label but unlike 67Cu, it did not shift to lighter or heavier fractions. Electron micrographs of the 1.051 fraction showed fields dominated by membranous structures some of which were enclosed. Micrographs of whole cells showed numerous invaginations resembling coated pits with sealed structures along and beneath the membrane surface suggesting the membrane was engaged in a rather extensive endocytosis. These data provide evidence that a large fraction of Cu from ceruloplasmin enters the K562 cell bound to membranous-like vesicles, part of which are sealed and coated with clathrin. This particulate pathway accounts for most of the copper entering the cell.

Topics: 2,2'-Dipyridyl; Ascorbic Acid; Biological Transport; Cell Fractionation; Cell Line; Centrifugation, Density Gradient; Ceruloplasmin; Copper; Copper Radioisotopes; Edetic Acid; Heparin Lyase; Humans; Leukemia, Erythroblastic, Acute; Nitrilotriacetic Acid; Polysaccharide-Lyases; Subcellular Fractions; Tumor Cells, Cultured

The present study characterizes the transport of nontransferrin (non-Tf) iron by K562 cells. Accumulation of radiolabel by cells incubated with 55Fe-nitrilotriacetate (NTA) is a saturable process that is time and temperature dependent (Ea approximately 20 kcal/mol). Initial rate analysis of iron influx yields values of Vmax = 855 fmol/min/10(6) cells and apparent Km = 0.54 microM. NHCL4 and chloroquine, agents that block cellular acquisition of iron from Tf, do not interfere with assimilation from FeNTA, demonstrating that uptake is truly independent of the Tf-mediated pathway. Furthermore, the inactivation of this transport mechanism by limited proteolytic digestion on ice indicates that specific cell surface proteins are involved. The extent of radiolabel incorporation into heme and ferritin is the same regardless of whether K562 cells acquire iron from 55FeNTA via the cell surface mechanism or from 55Fe-Tf via receptor-mediated endocytosis. Unlike other Tf-independent iron transport pathways that have been described, the K562 cell transport mechanism is not inhibited by divalent cations such as Ni2+, Co2+, or Mn2+. Uptake from 55FeNTA can be blocked by Cu2+ but at concentrations > 1500-fold molar excess. However, Cd2+ is a fairly specific inhibitor of 55Fe uptake by K562 cells (IC50 approximately 50 microM). Additionally, the K562 cell transport mechanism is not Ca2+ dependent and does not appear to be regulated by extracellular iron salts, in contrast to features noted for non-Tf iron uptake by fibroblasts (Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1991) J. Biol. Chem. 265, 3139-3145; Kaplan, J., Jordan, I., and Sturrock, A. (1991) J. Biol. Chem. 266, 2997-3004). These unique characteristics of the K562 cell uptake mechanism suggest that multiple transport systems function in Tf-independent iron assimilation.

Topics: Ascorbic Acid; Biological Transport; Calcium; Ferric Compounds; Humans; Iron; Kinetics; Leukemia, Erythroblastic, Acute; Nitrilotriacetic Acid; Transferrin; Tumor Cells, Cultured

Transmembrane reduction of extracellular oxidants by K562 and U937 leukemic cells was stimulated by catalytic amounts of ascorbate or dehydroascorbate. This stimulation was not due to transport of ascorbate in different redox states in and out of the cells. The membrane redox cycle was strictly dependent on the presence of the cells at every stage, and showed high affinity for ascorbate with simple linear kinetics. Metabolic inhibitors and sulfhydryl reagents inhibited this stimulation. Ascorbate uptake was also dependent on oxidation, but in a very different manner and with much lower affinity for ascorbate. The uptake was non-saturable in the concentration range used. There was some release of ascorbate from the cells, which cannot account for an appreciable part of the reduction of extracellular electron acceptors.

Topics: Ascorbic Acid; Biological Transport; Cell Membrane; Culture Media; Dehydroascorbic Acid; Electron Transport; Ferricyanides; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Lymphoma, Large B-Cell, Diffuse; Oxidation-Reduction; Tumor Cells, Cultured

The antitumour, antimetastatic and antileukemic effect of cyclophosphane and adriamycin in combination with vitamins A. E. C was studied according to the scheme developed by the authors. The preliminary administration of vitamins was established to intensify the effect of cytostatics and to lower considerably their toxic action. Cyclophosphane proved to be more effective relative to the Lewis lung carcinoma than adriamycin.

Topics: Animals; Ascorbic Acid; Cyclophosphamide; Doxorubicin; Drug Therapy, Combination; Leukemia L1210; Leukemia P388; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasms, Experimental; Sarcoma, Experimental; Vitamin A; Vitamin E

Ceruloplasmin binds to the membranes of K562 cells. The binding has been shown to result in a temperature-dependent transfer of ceruloplasmin-bound copper into the cytosol. Ascorbic acid (100 microM) stimulates the transmembrane transfer nearly 10-fold, depending on the initial concentration of 67Cu-ceruloplasmin. The protein moiety of ceruloplasmin does not enter the cells. Bathocuproine disulfonate, a chelator specific for cuprous copper, inhibits the uptake, suggesting copper atoms are reduced concomitant with their removal from ceruloplasmin. Cytosolic 67Cu from ceruloplasmin was found mainly bound to Cu, Zn superoxide dismutase, the major cytosolic copper protein in these cells. Evidence supporting the various phases in the ceruloplasmin-mediated transport mechanism are presented.

Topics: Ascorbic Acid; Biological Transport; Cells, Cultured; Ceruloplasmin; Copper; Copper Radioisotopes; Hemin; Humans; Leukemia, Erythroblastic, Acute; Phenanthrolines

An important property of ascorbic acid is its ability to increase the availability of storage iron to chelators. To examine the mechanism of this effect, K562 cells were incubated with ascorbate, attaining an intracellular level of 1 nmol/10(7) cells. In contrast to the reductive mobilization of iron seen with isolated ferritin, ascorbate stabilized iron preincorporated into cellular ferritin. Biosynthetic labeling with [35S]methionine demonstrated that ascorbate also retarded the degradation of the ferritin protein shell. Ferritin is normally degraded in lysosomes. The lysosomal protease inhibitors leupeptin and chloroquine produced a qualitatively similar stabilization of ferritin. Ascorbate did not act as a general inhibitor of proteolysis, however, since it did not effect hemoglobin degradation in these cells. The stabilization of cellular ferritin by ascorbate was accompanied by an expansion of the pool of chelatable iron.

Topics: Animals; Ascorbic Acid; Cell Line; Deferoxamine; Dose-Response Relationship, Drug; Ferritins; Iron; Leukemia, Erythroblastic, Acute; Leupeptins; Methionine

The suppressive effect of L-ascorbic acid on the growth of bone marrow cells from patients with acute nonlymphocytic leukemia was studied using a modified agar culture method featuring daily feeding to allow the growth of leukemic cell colonies. In seven of 28 patients (25%), the numbers of leukemic cell colonies grown in culture were reduced to 21% of control by the addition of L-ascorbic acid (0.3 mM) to the culture medium. Glutathione did not suppress leukemic cell colonies although it has a similar oxidation-reduction potential to that of L-ascorbic acid. The addition of L-ascorbic acid reduced the pH of the medium. However, a comparable reduction of pH by the addition of HCl did not suppress leukemic cell colonies. In simultaneous cultures for leukemic and normal marrow cells, the suppression of leukemic cell colony was noted with a concentration of L-ascorbic acid as low as 0.1 mM (a concentration achievable in vivo), but normal myeloid colonies were not suppressed until the concentration of L-ascorbic acid reached an extremely high level (1 mM). In conclusion, growth of leukemic cells in culture was suppressed by L-ascorbic acid in a substantial proportion of patients with acute nonlymphocytic leukemia. This suppression was a specific effect of L-ascorbic acid and was not due to its oxidation-reduction potential or pH change. Leukemic cells were selectively affected at an L-ascorbic acid concentration attainable in vivo while normal hemopoietic cells were not suppressed.

Topics: Adult; Aged; Ascorbic Acid; Bone Marrow; Cell Division; Colony-Forming Units Assay; Female; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Leukemia; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid, Acute; Male; Middle Aged