mezerein and Leukemia--Monocytic--Acute

Cells of U937, a human monocytic leukemia cell line, differentiate into macrophages by treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA), whereas cells treated with 1 alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3] continue to grow without undergoing differentiation. When U937 cells were successively treated with TPA and 1,25-(OH)2D3, tartrate-resistant acid phosphatase-positive multinucleated cells appeared at 5 days after the treatment. These osteoclast-like cells released a soluble form of 45Ca from 45Ca-labeled bone particles. These cells were not formed when the order of treatment with TPA and 1,25-(OH)2D3 was reversed. Use of either dexamethasone or interferon-gamma (IFN-gamma) was effective in inhibiting the formation of these osteoclast-like cells. The expression of c-src, c-fms, and macrophage colony stimulating factor (M-CSF) was induced by TPA treatment; however, TPA-induced M-CSF gene transcription was attenuated by the subsequent addition of 1,25-(OH)2D3. Furthermore, both dexamethasone and IFN-gamma impaired the attenuation of M-CSF expression, suggesting that the transient expression of M-CSF may be important for the formation of osteoclast-like cells.

Topics: Acid Phosphatase; Animals; Calcitonin; Calcitriol; Carcinogens; Cell Differentiation; Cell Nucleus; Dexamethasone; Diterpenes; Drug Synergism; Gene Expression Regulation, Leukemic; Genes, fms; Genes, src; Humans; Interferon-gamma; Isoenzymes; Leukemia, Monocytic, Acute; Macrophage Colony-Stimulating Factor; Mice; Osteoclasts; Phenotype; Tartrate-Resistant Acid Phosphatase; Terpenes; Tetradecanoylphorbol Acetate

Cells of the monocytic leukemia line, THP-1, mimic several of the functional characteristics of activated monocytes and macrophages following incubation in the tumor promoting agent, mezerein. The current paper explores the nature of the factors in medium conditioned by THP-1 cells which inhibit cell growth and the effects of interferon-gamma (IFN gamma) on their synthesis. Activity inhibiting the growth of the MDA-MB-415 mammary carcinoma line migrated in a pH range of 6.5 to 7.5 and could be resolved into 2 peaks, one corresponding to interleukin-1 beta (IL-1 beta) and the other with an apparent average molecular weight of 43 Kd. Antisera against tumor necrosis factor alpha and beta (TNF alpha and beta) and monocyte colony stimulating factor (M-CSF) had no effect on the 43 Kd inhibitor. IFN gamma enhanced the secretion of IL-1, but decreased the production of the 43 Kd inhibitor. The L-929 cell cytotoxicity assay and an ELISA were used to show that the amount of TNF alpha present was less in medium from IFN gamma treated cells. THP-1 cells also produced an IFN gamma repressed activity separating at pH 4.8 to 6.1 which inhibited the response of T cells to IL-1 in a thymocyte co-mitogenic assay. The inhibitors expressed by THP-1 cells may be comparable to the cytotoxic and cytostatic activities expressed by activated human monocytes and macrophages.

Topics: Animals; Cell Division; Cell Survival; Chromatography, Gel; Diterpenes; Growth Inhibitors; Humans; Hydrogen-Ion Concentration; Immune Sera; Interferon-gamma; Interleukin-1; Isoelectric Focusing; Leukemia, Monocytic, Acute; Lymphotoxin-alpha; Macrophage Colony-Stimulating Factor; Male; Mice; Mice, Inbred C3H; Terpenes; Thymus Gland; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The acute monocytic leukemia cell line, THP-1, is frequently used as a model to study the mechanism of cell-mediated immune response. The model is justified, in part, because THP-1 cells can be induced to express features of activated peripheral blood monocytes or tissue macrophages. The current investigation, however, demonstrates that THP-1 cells differ from normal monocytes in their secretion of interleukin-1 beta (IL-1 beta) following exposure to reagents that induce synthesis. For example, LPS treatment alone did not result in IL-1 beta secretion and very low concentrations were observed when lipopolysaccharide (LPS)-treated cells were simultaneously incubated with silica or silica in combination with hydroxyurea. Silica-enhanced release of IL-1 was related to changes in cell membrane permeability. Recombinant interferon-gamma (rIFN gamma) alone did not induce IL-1 beta secretion and did not significantly increase secretion by LPS- and silica-stimulated cells. In contrast, mezerein stimulation led to higher extracellular concentrations of IL-1 beta and rIFN gamma augmented secretion by mezerein-treated cells. Isoelectrofocusing of conditioned medium and titration of pooled fractions showed a direct correlation between extracellular levels of biologically active and immunoreactive IL-1. The role of IFN gamma in stimulating IL-1 beta secretion was not related to an enhancement of viability or an increase in the proportion of mezerein-treated cells synthesizing DNA. It was concluded that mezerein's regulation of secretion by THP-1 cells depended on the expression of monocyte features, including cell adherence and responsiveness to IFN gamma.

Topics: Animals; Biological Assay; Cell Division; Cell Survival; Diterpenes; Enzyme-Linked Immunosorbent Assay; Humans; Interferon-gamma; Interleukin-1; Isoelectric Focusing; Leukemia, Monocytic, Acute; Lipopolysaccharides; Mice; Mice, Inbred C3H; Recombinant Proteins; Terpenes; Thymus Gland; Tumor Cells, Cultured

This study examined the secretion of IL-1 alpha and IL-1 beta by THP-1 leukemia cells following activation with mezerein and promotion of synthesis by interferon (IFN-gamma). Interleukin-1 (IL-1) was not detected by co-mitogenic thymocyte assays of crude supernates. Isoelectrofocusing of concentrated medium showed that all biologically active IL-1 migrated at a pH of 6.8-7.2, indicating that the major secreted form was IL-1 beta. Double antibody ELISA confirmed the presence of IL-1 beta, but failed to detect IL-1 alpha in isofocused fractions. Although it appeared that THP-1 cells do not secrete IL-1 alpha; an inhibitor of thymocyte response to IL-1 was present in conditioned medium, migrated in an acidic pH range and masked the expression of biologically active rIL-1 alpha and rIL-1 beta. In contrast, IL-1 alpha was detected using a cell blotting assay. This technique permitted visualization of subpicogram levels of IL-1 when secreted by cells attached to an immunoblotting paper. Cell blotting showed that a greater proportion of attached cells incubated for 24 h in medium containing mezerein and IFN-gamma secreted IL-1 than cells in control medium. In conclusion, the amount of immunoreactive or biologically active IL-1 alpha secreted by stimulated THP-1 cells appeared to be much lower than that reported for human peripheral blood monocytes.

Topics: Diterpenes; Humans; Hydrogen-Ion Concentration; Interferon-gamma; Interleukin-1; Leukemia, Monocytic, Acute; Lipopolysaccharides; Terpenes; Tumor Cells, Cultured

Medium conditioned by mezerein-treated human acute monocytic leukemia cells (THP-1) stimulated human fibroblast replication. Maximum mitogenic activity was elaborated by THP-1 cells with a 24-hr incubation in 10(-7) M mezerein (activator phase) followed by a 36-hr incubation in insulin-supplemented serum-free Roswell Park Memorial Institute (RPMI)-1640 medium (effector phase). Growth stimulation was not due to the presence of residual mezerein. We previously reported that leukemia cells also produced a growth inhibitor. Fibroblast stimulation was resolved by isoelectrofocusing into several active fractions separate from the growth inhibitory activity for malignant mammary cells. Conditioned medium was mitogenic for fibroblasts in the presence of high concentrations of fetal bovine and human whole blood sera. Growth stimulation was observed in plasma-derived serum only when supplemented with exogenous platelet-derived growth factor. Thus, this THP-1 cell product does not fulfill the role of a competence factor.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Division; Cell Line; Diploidy; Diterpenes; Fibroblasts; Growth Substances; Humans; Kinetics; Leukemia, Monocytic, Acute; Lung; Phorbol Esters; Phorbols; Platelet-Derived Growth Factor; Terpenes