formazans and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Gelonin is a single chain ribosome-inactivating protein (RIP) with potential applications as a bullet of immunoconjugate for the treatment of cancer and AIDS. Using truncated forms of gelonin, we now report the relationship between its conformation and function. Circular dichroism (CD) and fluorescence spectra show that the N-terminus forms beta-sheets whereas the C-terminus contains alpha-helices of secondary structures. Biological experiments indicate that all gelonin truncation mutants lose partial toxicity compared to intact gelonin, an effect most strongly seen with C-terminally truncated gelonin. Similar evidence is also provided using a DNase-like activity assay. In addition, the intact gelonin exhibits the highest cytotoxicity to cancer cells. These results suggest that truncations of the terminal region of gelonin negatively regulate its function dominantly and that, due to its toxicity, intact gelonin is an important potential immunoconjugate.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Survival; Circular Dichroism; DNA, Neoplasm; Drug Screening Assays, Antitumor; Formazans; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Peptide Fragments; Plant Proteins; Recombinant Fusion Proteins; Ribosome Inactivating Proteins, Type 1; Sequence Deletion; Structure-Activity Relationship; Tetrazolium Salts

Fulvic acid (FA) was extracted and purified from Canadian Sphagnum peat (CP-FA) and characterized by using an element analysis meter, Fourier transform infrared (FT-IR) spectroscopy, electron spin resonance (ESR) spectroscopy, and (13)C-nuclear magnetic resonance ((13)C-NMR) spectroscopy. To investigate the antiallergic effect of CP-FA, we incubated rat basophilic leukemia (RBL-2H3) cells with 0.001-10.0 microg/ml of CP-FA and determined the beta-hexosaminidase release inhibition at different response stages. The intracellular calcium [Ca(2+)](i) level was also determined by using Fluo 3-AM, a calcium-specific fluorescent probe, and the cytotoxicity of CP-FA was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The results revealed that RBL-2H3 cells incubated for 48 h with 0.001-10.0 microg/ml of CP-FA did not show any decreased viability. CP-FA inhibited the beta-hexosaminidase release by IgE-sensitized, antigen-stimulated RBL-2H3 cells at the antigen-antibody binding stage and the antibody-receptor binding stage. CP-FA also inhibited histamine release from A23187 plus PMA- or compound 48/80-stimulated KU812 cells. Furthermore, there was a decrease in the intracellular [Ca(2+)](i) level in IgE-sensitized cells incubated with CP-FA and stimulated with antigen. Our results show that CP-FA may be useful for the treatment or prevention of allergic diseases.

Topics: Animals; Anti-Allergic Agents; Antigen-Antibody Complex; Antigens; Basophils; Benzopyrans; beta-N-Acetylhexosaminidases; Calcimycin; Calcium; Cell Line, Tumor; Dose-Response Relationship, Drug; Fluorescent Dyes; Formazans; Histamine Release; Humans; Immunoglobulin E; Ionophores; Leukemia, Basophilic, Acute; Leukemia, Experimental; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nuclear Magnetic Resonance, Biomolecular; p-Methoxy-N-methylphenethylamine; Plant Extracts; Rats; Spectroscopy, Fourier Transform Infrared; Sphagnopsida; Tetradecanoylphorbol Acetate; Tetrazolium Salts

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is the only non-steroidal anti-inflammatory drug so far which has been approved by the FDA for adjuvant treatment of patients with familial adenomatous polyposis. The molecular mechanism responsible for the anti-cancer effects of celecoxib is not fully understood. There is little data on the potential role of COX-2 in lymphoma pathogenesis. In view of the reported induction of apoptosis in cancer cells by cyclooxygenase-2 inhibitors, the present study is undertaken to test the effect of celecoxib on human chronic myeloid leukemia cell line, K562 and other hematopoietic cancer cell lines like Jurkat (human T lymphocytes), HL60 (human promyelocytic leukemia) and U937 (human macrophage). Treatment of these cells with celecoxib (10-100 microM) dose-dependently, reduced cell growth with arrest of the cell cycle at G0/G1 phase and induction of apoptosis. Further mechanism of apoptosis induction was elucidated in detail in K562 cell line. Apoptosis was mediated by release of cytochrome c into the cytoplasm and cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). This was followed by DNA fragmentation. The level of anti-apoptotic protein Bcl-2 was decreased without any change in the pro-apoptotic Bax. Celecoxib also inhibited NF-kB activation. Celecoxib thus potentiates apoptosis as shown by MTT assay, cytochrome c leakage, PARP cleavage, DNA fragmentation, Bcl-2 downregulation and possibly by inhibiting NF-kB activation.

Topics: Apoptosis; Celecoxib; Cell Proliferation; Cyclooxygenase Inhibitors; DNA Damage; Down-Regulation; Formazans; HL-60 Cells; Humans; Jurkat Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophages; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Sulfonamides; Tetrazolium Salts; Tumor Cells, Cultured

The hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) is commonly utilized to estimate cellular viability in drug screening protocols. The present investigation was prompted, in part, by observations that reduction of MTT to its colored reaction product, MTT formazan, varied between cell lines and with culture age. A correlation was established between the D-glucose concentration of the culture medium at the time of assay and the production of MTT formazan for cell lines representing seven tumor histologies. A decrease in the concentration of D-glucose from culture medium was accompanied by a decrease in MTT specific activity (MTT formazan/microgram cell protein) for a number of cell lines. Cells which extensively metabolized D-glucose exhibited the greatest reduction in MTT specific activity. Further evidence that the D-glucose concentration of the culture medium played an important role in MTT reduction was provided by experiments which demonstrated that transfer of cells to a glucose-free medium (L-15) was accompanied by an immediate decrease in MTT reduction which was pH independent. These studies suggested that cellular transport and constant metabolism of glucose were required for maximum MTT reduction. Decreases in the cellular concentration of the reduced pyridine nucleotides NADH and NADPH were accompanied by concomitant decreases in MTT formazan production. MTT formazan varied significantly among cell lines in both the kinetics of its formation and the degree of saturability exhibited. Apparent IC50 values for Adriamycin varied, in a cell line-specific manner, with MTT exposure time. These results indicate that MTT specific activity is significantly influenced by a number of parameters and suggest that assay conditions should be established which minimize their effects.

Topics: Carcinoma, Renal Cell; Cell Division; Cell Line; Cell Survival; Coloring Agents; Drug Screening Assays, Antitumor; Formazans; Glucose; Humans; Kidney Neoplasms; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; NAD; NADP; Tetrazolium Salts; Thiazoles

Based on the granulocyte-macrophage colony-stimulating factor (GM-CSF) dependency of a newly established human myeloid cell line GM/SO, we developed a highly specific and sensitive bioassay for human GM-CSF. The presence of bioactive GM-CSF could be determined by measuring the formazan concentration produced from MTT by the cells that survived and proliferated in the presence of either natural or recombinant human GM-CSF. With this assay we were able to quantify the level of GM-CSF in two human sera as well as in conditioned media from human bladder cell carcinoma cell line 5637, a human fibroblast line, and phytohemagglutinin-stimulated peripheral blood mononuclear cells. The sensitivity of the assay allows measurement of concentrations of GM-CSF as low as 0.1 U/ml.

Topics: Biological Assay; Cell Division; Cell Survival; Culture Media; Cytokines; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Formazans; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocytes, Mononuclear; Phytohemagglutinins; Recombinant Proteins; Tumor Cells, Cultured; Urinary Bladder Neoplasms