mezerein and Leukemia--Myeloid

Bryostatin 1 is a macrocyclic lactone protein kinase C (PK-C) activator which has demonstrated promising antileukemic activity in preclinical studies. We have examined the effect of this agent on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in both log phase and high-density human promyelocytic leukemia cells (HL-60). Exposure of low-density cells to 12.5 nM bryostatin 1 for 24 hr prior to a 4-hr incubation with 1 or 10 microM ara-C resulted in nearly a 2-fold increase in ara-CTP formation. When cells were maintained under high-cell density conditions (e.g. 5 x 10(6) cells/mL) for 24 hr prior to ara-C exposure, a 90% reduction in ara-CTP formation and ara-C DNA incorporation was observed. However, coincubation of high-density cells with bryostatin 1 for 24 hr increased ara-CTP formation 6- to 8-fold, yielding levels essentially equivalent to those achieved in low-density cells. Smaller (but still significant) increases in ara-C DNA incorporation were also noted. Enhancement of ara-CTP formation by bryostatin 1 occurred over a broad ara-C concentration range (0.1 to 100 microM), involved a temperature-dependent process, could not be mimicked by addition of hematopoietic growth factors, and was not related to neutralization of toxic or inhibitory substances in high-density medium. Exposure of cells to bryostatin 1 did not lead to morphologic or functional evidence of HL-60 cell maturation or an increase in cell viability, but did produce a decline in cellular proliferative activity as determined by thymidine and bromodeoxyuridine incorporation and cytofluorometric analysis. Bryostatin 1 did not exert its effects in high-density cells by inhibiting ara-C deamination or by interfering with ara-CTP dephosphorylation, but instead appeared to act by enhancing ara-C phosphorylation. Although cell-free extracts obtained from high-density cells exposed to bryostatin 1 exhibited levels of deoxycytidine kinase activity compared to controls, treated cells did display a significant decline in intracellular dCTP levels (e.g. 0.7 vs 1.3 pmol/10(6)), and nearly a 2-fold increase in ATP and UTP concentrations. Ara-CTP formation was also increased substantially by other PK-C activators including phorbol dibutyrate and mezerein (10-100 nM); this process was inhibited more than 70% by the PK-C inhibitor H-7 (50 microM), but not by the PK-C inhibitors staurosporine, tamoxifen, and HA1004. Finally, coadministration of ara-C and bryostatin 1 resul

Topics: Antineoplastic Agents; Arabinofuranosylcytosine Triphosphate; Bryostatins; Cell Count; Cytarabine; Diterpenes; DNA, Neoplasm; Humans; Kinetics; Lactones; Leukemia, Experimental; Leukemia, Myeloid; Macrolides; Phorbol 12,13-Dibutyrate; Phosphorylation; Terpenes; Time Factors; Tumor Cells, Cultured

Growth inhibitors and interleukin-1 (IL-1) are two biological response modifiers produced by mezerein-treated THP-1 cells maintained in serum-free medium. The activities comigrated with isoelectrofocusing in a pH range of 6.7 to 7.3. Subsequent molecular sieving on an AcA-54 column revealed that a portion of the growth-inhibitory activity for the mammary cell line MCF-7 remained associated with IL-1. IL-1-containing fractions were further analyzed by chromatography with DEAE-Sephacel, phenyl:Sepharose, and concanavalin A:Sepharose. In each instance, IL-1 coeluted with growth-inhibitory activity. IL-1 and growth-inhibitory activities partially purified by sequential isofocusing, AcA-54 chromatography, and DEAE-Sephacel were located in a single region following preparative polyacrylamide gel electrophoresis. Elution, concentration, and analytical polyacrylamide gel electrophoresis of this region resulted in a single band with an apparent molecular weight of 17,000. Stability studies revealed similarities between the IL-1 activity and growth-inhibitory activity in their sensitivity to a variety of physical and chemical treatments. A commercial source of human IL-1 also inhibited the growth of MCF-7 cells. DEAE-purified IL-1 derived from THP-1 cells inhibited the growth of 7 of 11 cell types tested, and all inhibited cell lines were established from malignant sources. Prostaglandin synthesis by MCF-7 cells in response to IL-1 was not responsible for growth inhibition.

Topics: Cell Differentiation; Cell Division; Culture Media; Diterpenes; Growth Inhibitors; Humans; Indomethacin; Interleukin-1; Isoelectric Point; Leukemia, Myeloid; Molecular Weight; Prostaglandins; Protein Denaturation; Terpenes

After mezerein treatment of suspension cultures of the acute monocytic human leukemia cell line THP-1, cells became adherent to plastic culture surfaces, lost division potential, acquired Fc receptors, displayed phagocytic activity, and expressed increased nonspecific esterase staining. Serum-free RPMI 1640 medium conditioned by adherent THP-1 cells was examined for the presence of biological response modifiers. Preparative isoelectrofocusing of concentrated medium in the presence of various pH gradients of Ampholine ampholytes resulted in the separation of the following activities: fibroblast growth-stimulating activities in pH ranges of 4.10-4.55 and 5.30-5.45; colony-stimulating factor (CSF) for mouse bone marrow cells at pH 4.10-4.55; and a malignant cell growth inhibitor comigrating with a lymphocyte-activating factor (interleukin-1) at pH 6.70-6.95. Both CSF and the fibroblast growth stimulator isofocused at pH 4.10-4.55 coeluted following molecular-sieve chromatography through P-100. CSF-induced colonies were composed of nongranulocytic mononuclear cells. Chromatography through an ACA-54 column separated interleukin-1 from most of the growth-inhibitory activity.

Topics: Antineoplastic Agents, Phytogenic; Biological Assay; Cell Adhesion; Cell Line; Colony-Stimulating Factors; Diterpenes; Growth Substances; Humans; Interferons; Interleukin-1; Leukemia, Myeloid; Macrophages; Terpenes

Serum-free medium conditioned by activated cells of the acute monocytic leukemia line, THP-1, was examined for growth-inhibitory activity with several established human cell lines. Free-floating clusters of THP-1 cells were activated into adherent nonproliferating cells by a 24-hr exposure to 10(-7) M mezerein in Roswell Park Memorial Institute 1640 medium containing 1% fetal bovine serum. Adherent cells were incubated for an additional 24 hr in serum-free medium containing insulin (5 micrograms/ml). Dose-response studies revealed that a cervical carcinoma (HeLa), a melanoma (A375Ag5), and several mammary carcinoma cell lines (MCF-7, BT474, MDA-MB415, and T47D) were growth inhibited by this conditioned medium. We concluded, from the results of thymidine release assays and from experiments on reversibility, that inhibition was a cytostatic and not a cytolytic response. In contrast, THP-1 conditioned medium stimulated the growth of two mammary lines (ZR75-1 and HBL-100), a lung type II carcinoma (549), and a colon adenocarcinoma (SW48). Preliminary characterization showed that the inhibitory activity was stable to acid and urea treatment but was destroyed by trypsin and sodium dodecyl sulfate. Molecular sieve chromatography of acetic acid-extracted material separated the inhibitory and stimulatory components.

Topics: Animals; Cell Division; Cell Line; Culture Media; Diterpenes; Drug Synergism; Growth Inhibitors; Humans; Insulin; Leukemia, Myeloid; Lipopolysaccharides; Mammary Neoplasms, Experimental; Phorbol Esters; Terpenes