herbimycin and Leukemia--Monocytic--Acute

Intracellular cross-talk between LFA-1 and its counter receptor, intercellular adhesion molecule-1 (ICAM-1), on human monocytic cell line THP-1 was analyzed. Stimulation with mAb YH384 specific for LFA-1 alpha (CD11a) up-regulated ICAM-1 expression on THP-1 cells. Cell surface expression of ICAM-1 on THP-1 cells was dose-dependently up-regulated and reached the maximal level 24 hr after stimulation with mAb YH384. Up-regulation of ICAM-1 by mAb YH384 was further confirmed by Northern blotting analysis at the mRNA level, and the maximal message of ICAM-1 was observed 4 hr after stimulation. mAb YH384-induced upregulation of cell surface expression was ICAM-1-specific, and the expression of the other nine molecules tested was not augmented. Up-regulation of ICAM-1 was also observed following stimulation with other mAb specific for CD11a (S6F1), CD11b (JML-H11), and CD18 (IB4); all of these mAb recognized members of the beta 2-integrin family, but not with isotype-matched control mAb. The mAb 4B4 (specific for beta 1-integrin) similarly, but more weakly, augmented ICAM-1 expression. The effect of mAb YH384 on expression of ICAM-1 was dose-dependently suppressed by treatment with herbimycin A or genistein, both inhibitors of tyrosine kinase. These results suggest that beta 2-integrin-mediated up-regulation of ICAM-1 is mediated via a tyrosine phosphorylation pathway.

Topics: Animals; Antibodies, Monoclonal; Benzoquinones; CD18 Antigens; Cell Line; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; Genistein; Humans; Intercellular Adhesion Molecule-1; Isoflavones; Lactams, Macrocyclic; Leukemia, Monocytic, Acute; Lymphocyte Function-Associated Antigen-1; Mice; Mice, Inbred BALB C; Monocytes; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Up-Regulation

Exogenous dolichyl phosphate (Dol-P) induced apoptosis in the human monoblastic leukemia cell line U937 within 4 hours. Phosphorylation of p42 mitogen-activated protein kinase (MAP kinase) increased prior to DNA fragmentation. MAP kinase activation occurred within 5 min, and the maximum response was observed at 30 min. Inhibition of tyrosine phosphorylation of MAP kinase by herbimycin A resulted in complete inhibition of DNA fragmentation and partial inhibition of cell death. These results suggested that Dol-P-induced apoptosis is mediated by the MAP kinase cascade.

Topics: Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Survival; DNA Damage; DNA, Neoplasm; Dolichol Phosphates; Enzyme Activation; Humans; Kinetics; Lactams, Macrocyclic; Leukemia, Monocytic, Acute; Quinones; Rifabutin; Time Factors; Tumor Cells, Cultured

Activation of cytoplasmic tyrosine kinases is an important aspect of signal transduction mediated by integrins. In the human monocytic cell line THP-1, either integrin-dependent cell adhesion to fibronectin or ligation of beta 1 integrins with antibodies causes a rapid and intense tyrosine phosphorylation of two sets of proteins of about 65-75 and 120-125 kDa. In addition, integrin ligation leads to nuclear translocation of the p50 and p65 subunits of the NF-kappa B transcription factor, to activation of a reporter gene driven by a promoter containing NF-kappa B sites, and to increased levels of mRNAs for immediate-early genes, including the cytokine interleukin (IL)-1 beta. The tyrosine kinase inhibitors genistein and herbimycin A block both integrin-mediated tyrosine phosphorylation and increases in IL-1 beta message levels, indicating a causal relationship between the two events. The components tyrosine phosphorylated subsequent to cell adhesion include paxillin, pp125FAK, and the SH2 domain containing tyrosine kinase Syk. In contrast, integrin ligation with antibodies induces tyrosine phosphorylation of Syk but not of FAK or paxillin. In adhering cells, pre-treatment with cytochalasin D suppresses tyrosine phosphorylation of FAK and paxillin but not of Syk, while IL-1 beta message induction is unaffected. These observations indicate that the Syk tyrosine kinase may be an important component of an integrin signaling pathway in monocytic cells, leading to activation of NF-kappa B and to increased levels of cytokine messages.

Topics: Benzoquinones; Cell Adhesion; Cell Adhesion Molecules; Enzyme Activation; Enzyme Precursors; Extracellular Matrix Proteins; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genistein; Humans; Inflammation; Integrin beta1; Integrins; Interleukin-1; Intracellular Signaling Peptides and Proteins; Isoflavones; Lactams, Macrocyclic; Leukemia, Monocytic, Acute; Monocytes; NF-kappa B; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Signal Transduction; Syk Kinase; Tumor Cells, Cultured; Tyrosine

Herbimycin A, a benzoquinonoid ansamycin antibiotic, reduces intracellular phosphorylation by some protein tyrosine kinases and inhibits the proliferation of malignant cells which express high tyrosine kinase activity. Herbimycin A inhibited the proliferation of human monoblastic leukemia U937 cells, but this inhibition was abrogated by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). On the other hand, a derivative of herbimycin A, 19-allylaminoherbimycin A, inhibited the proliferation of such cells without interference by the addition of GM-CSF. Phosphorylation of MAP kinase and c-myc expression induced by GM-CSF in U937 cells were inhibited by both herbimycin A and 19-allylaminoherbimycin A. The time courses of growth inhibition showed that the growth-inhibitory activity of herbimycin A in U937 cells was initially potent, but gradually decreased in the presence of GM-CSF. Thiol compounds, glutathione (GSH) and 2-mercaptoethanol, abrogated the inhibition of the growth of U937 cells by herbimycin A, but not by 19-allylaminoherbimycin A, like GM-CSF. Intracellular GSH content in U937 cells was increased by treatment with GM-CSF, and decreased with herbimycin A, but returned to the control level with the addition of GM-CSF to herbimycin A. In thin-layer chromatography, after in vitro incubation with herbimycin A and GSH, nothing could be detected at the position of intact herbimycin A, while 19-allylaminoherbimycin A was stably detected. These findings suggest that changes in the intracellular concentration of GSH play a role in the abrogation of the inhibition of U937 cell growth by herbimycin A. In the presence of GSH, 19-allylaminoherbimycin A inhibited the proliferation of U937 cells and Philadelphia chromosome-positive K562 cells more effectively than herbimycin A. Since GSH plays a role in detoxicating several anticancer drugs, 19-allylaminoherbimycin A may have therapeutic advantages over herbimycin A against some types of leukemia.

Topics: Antibiotics, Antineoplastic; Base Sequence; Benzoquinones; Cell Division; DNA Probes; Gene Expression; Genes, myc; Glutathione; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lactams, Macrocyclic; Leukemia, Monocytic, Acute; Mercaptoethanol; Molecular Sequence Data; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Sulfhydryl Compounds; Tumor Cells, Cultured

Mouse leukemia Mm-A and Mm-S2 cells are subclones of mouse monocytic leukemia Mm cells, Mm-A cells having much higher leukemogenicity than Mm-S2 cells. The growth-inhibitory effects of several protein kinase inhibitors on leukemogenic Mm-A and non-leukemogenic Mm-S2 cells were examined. Most inhibitors of protein serine/threonine kinases inhibited the growth of Mm-A and Mm-S2 cells similarly, but some protein tyrosine kinase inhibitors exhibited differential inhibitory effects on Mm-A and Mm-S2 cells. Genistein inhibited growth of Mm-A cells more effectively than that of Mm-S2 cells, but another inhibitor of tyrosine kinase, herbimycin A, preferentially inhibited growth of non-leukemogenic Mm-S2 cells. Genistein induced or enhanced several differentiation markers of Mm-S2 cells, such as cell spreading, immunophagocytosis, nitroblue tetrazolium (NBT) reduction and lysozyme activity in a dose-dependent manner, but herbimycin A did not. Genistein was cytotoxic to Mm-A cells rather than inducing cell differentiation. Genistein has effects on several other cellular events as well as inhibition of tyrosine kinases. However, it effectively inhibited protein tyrosine phosphorylation in Mm-A cells and its decrease of tyrosine phosphorylation was closely associated with its inhibition of cell growth. Thus, a genistein-sensitive tyrosine kinase(s) may play an important role in the growth and/or survival of leukemogenic Mm-A cells.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Benzoquinones; Cell Differentiation; Cell Division; Drug Screening Assays, Antitumor; Genistein; Isoflavones; Lactams, Macrocyclic; Leukemia, Monocytic, Acute; Mice; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured; Tyrosine