herbimycin and Cell-Transformation--Viral

Chronic hepatitis B virus infection is strongly associated with the development of hepatocellular carcinoma (HCC). Epithelial tumors are frequently characterized by loss of cadherin expression or function. Cadherin-dependent adhesion prevents the acquisition of a migratory and invasive phenotype, and loss of its function is itself enough for the progression from adenoma to carcinoma. The HBx protein of hepatitis B virus is thought to contribute to the development of the carcinoma, however, its role in the oncogenic and metastatic processes is far from being fully understood. We report herein the ability of HBx to disrupt intercellular adhesion in three different cell lines stably transfected with an inducible HBx expression vector. The linkage between the actin cytoskeleton and cadherin complex, which is essential for its function, is disrupted in the presence of HBx, as indicated by detergent solubility and immunoprecipitation experiments. In addition, beta-catenin was tyrosine phosphorylated in HBx-expressing cells. Inhibition of the src family of tyrosine kinases resulted in the prevention of the disruption of adherens junctions. These results suggest that HBx is able to disrupt intercellular adhesion in a src-dependent manner, and provide a novel mechanism by which HBx may contribute to the development of HCC.

Topics: Adherens Junctions; Animals; Benzoquinones; beta Catenin; Cadherins; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cell Transformation, Viral; Cocarcinogenesis; Cytoskeletal Proteins; Enzyme Inhibitors; HeLa Cells; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; Lactams, Macrocyclic; Liver Neoplasms; Mice; Neoplasm Metastasis; Phosphorylation; Protein Processing, Post-Translational; Quinones; Recombinant Fusion Proteins; Rifabutin; src-Family Kinases; Trans-Activators; Transfection; Viral Regulatory and Accessory Proteins

We have previously reported the activation of Src by mercuric chloride based on the sulfhydryl modification. To evaluate the significance of cysteine residues in v-Src, we replaced each cysteine to alanine by oligonucleotide-directed mutagenesis and examined its effect on cell transformation. Of ten cysteine residues scattered over v-Src, four cysteines clustered in kinase domain, Cys483, Cys487, Cys496 and Cys498, were important for protein stability and cell transformation, whereas those in SH2 domain were dispensable. A single mutation in Cys498 yielded suppression of kinase activity and a temperature-sensitivity in anchorage independent growth. Double mutation either in Cys483/Cys487 or in Cys496/Cys498 yielded clear temperature-sensitivity in cell transformation and in stability of Src protein. Instability of Src protein was magnified by quadruple mutation in the cysteines, which decreased the half-life of Src to be less than one quarter of that of wild-type. In addition, both Cys483/Cyr487 and Cys496/Cys498 kinases became resistant to in vitro inactivation by herbimycin A, which directly inactivates v-Src in addition to its effect on HSP90. Taken together, our results strongly suggest that the cysteine clustered motif of v-Src are critical for protein stability, cell transformation and in vitro inactivation by herbimycin A.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Benzoquinones; Cell Line; Cell Transformation, Viral; COS Cells; Cysteine; Drug Resistance; Enzyme Stability; Herbicides; Lactams, Macrocyclic; Molecular Sequence Data; Mutagenesis, Site-Directed; Oncogene Protein pp60(v-src); Quinones; Rats; Rifabutin; src Homology Domains; src-Family Kinases; Temperature

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.

Topics: 3T3 Cells; Acylation; Animals; Antigens, Differentiation; Avian Sarcoma Viruses; Benzoquinones; Cell Adhesion; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation, Viral; Genes, src; Half-Life; Lactams, Macrocyclic; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Membrane Glycoproteins; Mice; Myristic Acid; Neural Cell Adhesion Molecule L1; Neural Cell Adhesion Molecules; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Quinones; ras Proteins; Rats; Receptors, Immunologic; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Rifabutin; RNA, Messenger; src-Family Kinases; Transfection

Intracellular cyclic AMP, determined in part by cyclic nucleotide phosphodiesterases (PDEs), regulates proliferation and immune functions in lymphoid cells. Total PDE, PDE3, and PDE4 activities were measured in phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC-PHA), normal natural killer (NK) cells, Jurkat and Kit225-K6 leukemic T-cells, T-cell lines transformed with human T-lymphotropic virus (HTLV)-I (a retrovirus that causes adult T-cell leukemia/lymphoma) and HTLV-II (a nonpathogenic retrovirus), normal B-cells, and B-cells transformed with Epstein-Barr virus (EBV). All cells exhibited PDE3 and PDE4 activities but in different proportions. In EBV-transformed B cells, PDE4 was much higher than PDE3. HTLV-I+ T-cells differed significantly from other T-lymphocyte-derived cells in also having a higher proportion of PDE4 activities, which apparently were not related to selective induction of any one PDE4 mRNA (judged by reverse transcription-polymerase chain reaction) or expression of the HTLV-I regulatory protein Tax. In MJ cells (an HTLV-I+ T-cell line), Jurkat cells, and PBMC-PHA cells, the tyrosine kinase inhibitor herbimycin A strongly inhibited PDE activity. Growth of MJ cells was inhibited by herbimycin A and a protein kinase C (PKC) inhibitor, and was arrested in G1 by rolipram, a specific PDE4 inhibitor. Proliferation of several HTLV-I+ T-cell lines, PBMC-PHA, and Jurkat cells was inhibited differentially by forskolin (which activates adenylyl cyclase), the selective PDE inhibitors cilostamide and rolipram, and the nonselective PDE inhibitors pentoxifylline and isobutyl methylxanthine. These results suggest that PDE4 isoforms may be functionally up-regulated in HTLV-I+ T-cells and may contribute to the virus-induced proliferation, and that PDEs could be therapeutic targets in immune/inflammatory and neoplastic diseases.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adult; B-Lymphocytes; Benzoquinones; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Colforsin; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Enzyme Inhibitors; Gene Products, tax; Human T-lymphotropic virus 1; Humans; Interleukin-2; Jurkat Cells; Killer Cells, Natural; Lactams, Macrocyclic; Leukocytes, Mononuclear; Lymphocytes; Protein Kinase Inhibitors; Quinones; Reverse Transcriptase Polymerase Chain Reaction; Rifabutin; RNA, Messenger; T-Lymphocytes

Quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) proliferate at 35.5 degrees C, a permissive temperature for RSV, but differentiate at 41 degrees C, a nonpermissive temperature, with the formation of multinucleated myotubes and the synthesis of muscle-specific proteins. Tyrosine kinase activity of the src gene product derived from RSV is closely related to regulation of this temperature-dependent differentiation, and the cells obtain commitment to differentiation by incubation for about 12 h at 41 degrees C with dephosphorylation of tyrosine-phosphorylated protein(s). It was examined how myogenin, a member of myogenic regulatory factors, participates in commitment to differentiation and tyrosine dephosphorylation of QM-RSV cells. Myogenin was expressed within 8 h and reached a plateau within 10 h at 41 degrees C. Each cell clone whose differentiation proceeded faster or slower than the parental QM-RSV cells was reflected by a faster or slower myogenin expression, corresponding to the time that is required for commitment to differentiation. It was suggested that there is a lag time between myogenin expression and the acquisition of commitment in QM-RSV cells. On the other hand, at 35.5 degrees C, a condition which suppresses differentiation, myogenin expression was not detected. However, herbimycin A, an inhibitor of protein tyrosine kinase, induced myogenin expression even at 35.5 degrees C. On the contrary, myogenin expression was inhibited at 41 degrees C by sodium orthovanadate, an inhibitor of tyrosine-phosphorylated protein phosphatase. Furthermore, forced induction of myogenin into the cells cultured at 35.5 degrees C resulted in the formation of multinucleated myotubes and the synthesis of muscle-specific proteins. These results suggest that myogenin expression is one of the indispensable conditions for the acquisition of commitment to differentiation and is regulated by tyrosine phosphorylation and dephosphorylation of some protein(s) in QM-RSV cells.

Topics: Animals; Avian Sarcoma Viruses; Benzoquinones; Cell Differentiation; Cell Transformation, Viral; Clone Cells; Enzyme Inhibitors; Gene Expression; Lactams, Macrocyclic; Muscles; Myogenin; Phosphorylation; Protein Tyrosine Phosphatases; Quail; Quinones; Rifabutin; src-Family Kinases; Tyrosine; Vanadates

Myogenic differentiation of quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) depends on the temperature: at 35.5 degrees C, the permissive temperature for the virus, the transformed myoblasts proliferate, without fusion, but at 41 degrees C, the nonpermissive temperature, they become committed to myogenic differentiation until about 10 h and then myoblast fusion occurs within 24 h. This temperature dependency of the differentiation reaction is derived from protein kinase activity of src gene product. Thus, at 41 degrees C, the differentiation proceeds with dephosphorylation of the proteins. For further clarification of relationship between the protein phosphorylation and the control of differentiation, the events during differentiation were examined using inhibitors of tyrosine kinase and phosphatase, respectively. To examine the role of phosphotyrosyl protein phosphatases in skeletal muscle differentiation, these cells were treated with sodium orthovanadate, a potent inhibitor of the enzyme. The treatment with the drug inhibited myoblast fusion and creatine kinase activity of the cells at 41 degrees C, with inhibition of tyrosine dephosphorylation. Moreover, the treatment of the cells with vanadate for 12 h at 41 degrees C, followed by the removal of the drug resulted in myoblast fusion after the lag time about 12 h. On the other hand, herbimycin A was needed to acquire the fusion commitment at 35.5 degrees C. These results are suggestive evidence to reflect that tyrosine dephosphorylation is a key step in commitment of QM-RSV cells to myogenic differentiation. Examination of the tyrosine phosphorylated proteins indicated that vanadate mainly inhibited the dephosphorylations of 70-, 58-, and 38-kDa proteins, suggesting that these proteins may be closely associated with the steps involved in commitment to myogenic differentiation.

Topics: Animals; Avian Sarcoma Viruses; Benzoquinones; Cell Differentiation; Cell Fusion; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Creatine Kinase; Enzyme Inhibitors; Lactams, Macrocyclic; Muscle Fibers, Skeletal; Oncogene Protein pp60(v-src); Phosphoproteins; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Quail; Quinones; Rifabutin; Temperature; Vanadates

We studied the effectiveness of herbimycin A, an inhibitor of the function of the temperature-sensitive src oncogene, to reverse the morphologies of chicken and mammalian cells transformed by various oncogenes. It was found that the antibiotic was effective against the cells transformed by tyrosine kinase oncogenes src, yes, fps, ros, abl, erbB, but did not reverse the transformed morphologies induced by oncogenes raf, ras, and myc. Moreover, decreases in phosphotyrosine content of the total cellular proteins and in 36K protein phosphorylation by herbimycin treatment supported the selective inhibition of the antibiotic on the transforming activity of tyrosine kinase oncogenes.

Topics: Animals; Arvicolinae; Benzoquinones; Cell Line; Cell Transformation, Viral; Chick Embryo; Fibroblasts; Lactams, Macrocyclic; Mice; Oncogene Proteins, Viral; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tyrosine

We have characterized a host range mutant of Rous sarcoma virus in order to identify host cell factors involved in transformation. This mutant, tsLA33-1, which was isolated from a stock of the temperature-sensitive mutant tsLA33, is not temperature-sensitive for transformation of chicken embryo fibroblasts, as judged by its ability to induce morphological changes and agar colony formation at both 36 and 41.5 degrees. In Rat-3 cells, however, this mutant induced a temperature-dependent transformation: infected Rat-3 cells were transformed at 34 degrees but not at 39.5 degrees. Retransformants were isolated from tsLA33-1-infected Rat-3 cells by growth in agar suspension at 39.5 degrees. Virus rescued from these retransformants induced a temperature-dependent transformation when reintroduced into rat cells. The level of expression of pp60v-src at 39.5 degrees was unchanged in the retransformants. When the retransformants were treated with herbimycin, an antibiotic which induces turnover of certain protein-tyrosine kinases, they reverted to a normal phenotype, indicating that the transformed phenotype of the retransformants was dependent on continued expression of pp60v-src. The retransformants are therefore pseudorevertants in which a cellular alteration has occurred that allows transformation at 39.5 degrees by the mutant pp60v-src. Thus the temperature-dependence of transformation by tsLA33-1 is affected by the cellular environment, and is suppressed or complemented both in chicken cells and in the rat cell pseudorevertants. No clear correlation between levels of phosphorylation at tyrosine and transformation was observed. In Rat-3 cells the pp60v-src encoded by tsLA33-1 may be defective in its interaction with low abundance substrates that are critical for transformation; alternatively the nonpermissive cells may require a higher threshold dose of pp60v-src for transformation.

Topics: Animals; Avian Sarcoma Viruses; Benzoquinones; Cell Line; Cell Transformation, Viral; Chick Embryo; Lactams, Macrocyclic; Mutation; Oncogene Protein pp60(v-src); Oncogene Proteins, Viral; Oncogenes; Phosphoproteins; Protein-Tyrosine Kinases; Quinones; Rats; Retroviridae Proteins; Rifabutin; RNA, Viral; Temperature

Herbimycin A specifically increased the level of fibronectin mRNA in Rous sarcoma virus-infected rat kidney cells, and the time course of fibronectin expression was found to be closely related to that of morphological change induced by herbimycin A.

Topics: Animals; Anti-Bacterial Agents; Avian Sarcoma Viruses; Benzoquinones; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Fibronectins; Gene Expression Regulation; Kidney; Lactams, Macrocyclic; Quinones; Rats; Rifabutin; RNA, Messenger

During the course of screening of agents active in converting the transformed morphology of Rous sarcoma virus-infected rat kidney cells to the normal morphology, we identified an active substance produced by Streptomyces sp. MH237-CF8 as herbimycin. Herbimycin converted almost all cells into the normal morphology. The antibiotic was found to be an inhibitor of p60src-associated protein kinase in the cells.

Topics: Animals; Benzoquinones; Cell Transformation, Viral; Cells, Cultured; Kidney; Lactams, Macrocyclic; Oncogene Protein pp60(v-src); Oncogene Proteins, Viral; Phenylglyoxal; Protein-Tyrosine Kinases; Quinones; Rats; Retroviridae Proteins; Rifabutin