vitamin-k-semiquinone-radical and Leukemia--Erythroblastic--Acute

The intracellular molecular oxygen concentration in control and menadione-treated K562 (an erythroleukemic cell line that grows in suspension) and A431 (an epidermal carcinoma that grows in monolayer) cells was measured directly by using the new electron paramagnetic resonance (EPR) probe fusinite. Because the oxidizing agent menadione is known to damage mitochondria and the cytoplasmic membrane in other cell systems, before conducting measurements of oxygen concentration in K562 and A431 cells, it was necessary to establish injury in these systems as well. Consequently, morphological and flow cytometric analyses were conducted after menadione treatment. The data presented here show that the two cell lines are heavily damaged by menadione. Once this menadione-induced injury was demonstrated, measurements of oxygen concentration were carried out in both K562 and A431 cells. Treatment with this quinone induces a sharp increase in intracytoplasmic molecular oxygen in both cell lines (from about 1% to about 10 and 15% in K562 and A431 cells, respectively). In addition, to gain a more complete understanding of the effects of menadione on cells, the extracellular molecular oxygen concentration and the oxygen consumption rate were also measured in control and menadione-treated K562 cells. These measurements demonstrate that menadione treatment results in an increase in the extracellular oxygen concentration (from about 5% in controls to 15% in treated cells) as well as a decrease in the oxygen consumption rate (from about 10 ng O/min/10(6) cells in controls to 3 ng O/min/10(6) cells after menadione exposure). The importance of the new EPR probe fusinite in monitoring directly cellular functions in which oxygen is involved and the effects of menadione on cellular oxygen balance are discussed.

Topics: Calibration; Carbon; Electron Spin Resonance Spectroscopy; Flow Cytometry; Leukemia, Erythroblastic, Acute; Molecular Probes; Oxidants; Oxygen; Oxygen Consumption; Skin Neoplasms; Tumor Cells, Cultured; Vitamin K

Carbonyl reductase (CBR) catalyzes the reduction of daunorubicin (DN) to its corresponding alcohol, daunorubicinol (DNOL), and changes the pharmacological properties of this cancer chemotherapeutic drug. The DN reductase associated with CBR reduces the C13 methyl ketone group and does not metabolize the quinone ring of DN. Reports comparing DN and DNOL toxicity have resulted in various conclusions depending on the cells tested. Differences in toxicity could be due to variations in several enzymes involved in DN metabolism. In this report, the effects of CBR expression on DN metabolism and cell toxicity were determined by cloning and expressing a human CBR cDNA in DN reductase-deficient myeloid erythroleukemia K562 cells. CBR activity increased 83-fold in the K562-transfected cells and was associated with a 2-3-fold reduction in DN toxicity. Maximum protection occurred at 30 nM DN where 94% of the intracellular DN was converted to DNOL within 2 h. The reduced toxicity was specific for DN. Other CBR substrates such as menadione, phenanthrenequinone, and doxorubicin were equally toxic to both the CBR expresser cells and the control cells under the conditions tested. Our results suggest that high levels of CBR in tumor cells could contribute to drug resistance. The results also suggest that reduction of DN to DNOL protects against DN toxicity by altering interaction of the drug at one or more of the many target sites.

Topics: Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductases; Antibiotics, Antineoplastic; Base Sequence; Cells, Cultured; Cloning, Molecular; Daunorubicin; DNA Primers; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression; Humans; In Vitro Techniques; Leukemia, Erythroblastic, Acute; Mitomycin; Molecular Sequence Data; Oxidation-Reduction; Phenanthrenes; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Vitamin K

Topics: Cell Line; DNA Damage; DNA, Neoplasm; Humans; Leukemia, Erythroblastic, Acute; NAD; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Tumor Cells, Cultured; Vitamin K

It has been demonstrated that perturbation of oxidative balance plays an important role in numerous pathological states as well as in physiological modifications leading to aging. In order to evaluate the role of the oxidative state in cells, biochemical and ultrastructural studies were carried out on K562 and HL-60 cell cultures. Particular attention was given to the transferrin receptor, which plays an important role in cellular iron metabolism. In order to evaluate if oxidative stress influences the transferrin receptor regulation process, the free-radical inducer menadione was used. The results obtained seem to indicate that oxidative stress is capable of inducing a rapid and specific down-modulation of the membrane transferrin receptor due to a block of receptor recycling on the cell surface, without affecting ligand-binding affinity. These effects were observed in the early stages of menadione treatment and before any typical signs of subcellular damage, including surface blebbing, a well-known cytopathological marker of menadione-induced injury. The mechanisms underlying such phenomena appear to be related to cytoskeletal protein thiol group oxidation as well as to the perturbation of calcium homeostasis, both induced by menadione. It is thus hypothesized that the data reported here represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be influenced by oxidative balance.

Topics: Actin Cytoskeleton; Calcimycin; Calcium Channel Blockers; Cytochalasin B; Edetic Acid; Endocytosis; Humans; Iron; Leukemia, Erythroblastic, Acute; Leukemia, Promyelocytic, Acute; Microscopy, Electron; Microtubules; Oxidation-Reduction; Phalloidine; Reactive Oxygen Species; Receptors, Transferrin; Stress, Physiological; Tumor Cells, Cultured; Vitamin K

Topics: Animals; Antimetabolites; Cell Count; Cell Division; Cell Line; Deoxyuridine; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Interactions; Fluorouracil; Folic Acid; Leucovorin; Leukemia, Erythroblastic, Acute; Mice; Vitamin E; Vitamin K

Topics: Adrenal Cortex Hormones; Humans; Leukemia, Erythroblastic, Acute; Vitamin A; Vitamin K; Vitamins