herbimycin and Lymphoma--Non-Hodgkin

We previously reported that natural killer (NK)-sensitive target cells, K562, kill interleukin-2-stimulated (lymphokine-activated killer [LAK]) but not unstimulated NK cells. We have now investigated the molecular basis of this phenomenon. Soluble monoclonal antibody (MoAb) to CD18 inhibited 75% of K562-induced DNA fragmentation and membrane disruption, whereas blocking MoAb to Fas partially inhibited only the DNA fragmentation. MoAbs to CD2, CD11a, CD11b, B7, or CD16 had limited or no effect on K562-induced death of LAK cells. Receptor ligation with either immobilized MoAb to CD18 or Fas induced membrane disruption and DNA degradation in LAK cells independently of K562, and MoAb to CD18, CD11a, or CD11b enhanced DNA fragmentation induced by anti-Fas. Fas-L-transfected Raji cells also killed LAK cells, but only if Fas-L expression was amplified. K562 cells rapidly triggered protein phosphorylation in LAK cells, and the tyrosine kinase inhibitor, Herbimycin A, inhibited DNA fragmentation and membrane disruption. Protease inhibitors strongly suppressed K562-mediated DNA fragmentation of LAK cells, but not membrane disruption. In conclusion, (1) K562-induced death of LAK cells involves primarily CD18, although other molecules, such as Fas, may also be involved; (2) K562-mediated apoptosis of LAK cells requires tyrosine phosphorylation and protease activity; (3) engagement of Fas by immobilized MoAb or Fas-L on target cells can also kill LAK cells; and (4) Fas-immobilized MoAb synergizes with coimmobilized MoAb to CD11a, CD11b, or CD18 for LAK cell killing. Activation-induced death of NK cells may represent a mechanism for NK cell regulation.

Topics: Antibodies, Monoclonal; Antigens, CD; Apoptosis; Benzoquinones; CD18 Antigens; Cell Membrane; Cysteine Endopeptidases; Cytotoxicity, Immunologic; DNA; Fas Ligand Protein; fas Receptor; Humans; Interleukin-2; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoma, Non-Hodgkin; Melanoma; Membrane Glycoproteins; Protease Inhibitors; Quinones; Recombinant Fusion Proteins; Rifabutin; Serine Endopeptidases; Transfection; Tumor Cells, Cultured

We have shown that geldanamycin (GDM), an antibiotic of benzoquinoid ansamycin group, inhibits DNA replication in cultured mouse lymphoblastoma L5178Y cells. Here we report that GDM selectively inhibited the expression of c-myc gene, proto-oncogene, along with suppression of DNA replication in L5178Y cells, which are consistent with our previous results that c-myc protein promotes cellular DNA replication. The significantly enhanced inhibition by GDM of DNA replication was observed, when the antibiotic was introduced at G1 stage prior to S phase of cell cycle. The results are in favor of the prospects that GDM inhibits DNA replication mainly at time of initiation, and that c-myc protein is essential for the initiation of cellular DNA replication. Furthermore, when c-myc expression was inhibited by GDM, the expression of p53 gene, the product of which may be another DNA replication protein, was stimulated in the tumor cells. Thus, GDM should be useful to investigate the molecular mechanism of DNA replication promoted by c-myc protein and also to distinguish the function of c-myc protein from that of p53 protein in DNA replication.

Topics: Animals; Anti-Bacterial Agents; Benzoquinones; Cell Cycle; DNA Replication; DNA, Neoplasm; Gene Expression Regulation; Lactams, Macrocyclic; Lymphoma, Non-Hodgkin; Mice; Molecular Structure; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogenes; Quinones; Rifabutin; Tumor Cells, Cultured