herbimycin and Cell-Transformation--Neoplastic

Herbimycin A, a benzoquinonoid anasamycin antibiotic, has been shown to reserve the oncogenic phenotypes of p60v-src transformed cells by the virtue of the inhibition of src protein tyrosine kinase. Furthermore, we previously demonstrated that herbimycin A displayed the antitumor activity on Ph1-positive leukemia cells and bcr/abl oncoprotein-associated transformed murine hematopoietic cells with the transfection of a retroviral vector expressing bcr/abl. Herbimycin A showed preferential inhibition on the in vitro growth of Ph1-positive leukemia cells and bcr/abl oncoprotein-associated murine hematopoietic cells through the inhibition of bcr/abl tyrosine kinase activity and the reduction of subsequent phosphotyrosyl proteins. Recently, from the view of investigating the oncogenic significance or of developing a future clinical application in malignancies, several developing agents targeted against oncoprotein have been tried. We reviewed the present progress in the mechanism of oncoprotein-targeted antitumor effects and focused on herbimycin A-induced antitumor activity on Ph1-positive leukemia cells.

Topics: Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Fusion Proteins, bcr-abl; Humans; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Oligonucleotides, Antisense; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured

Topics: Animals; Benzoquinones; Cell Transformation, Neoplastic; Genes, src; Humans; Lactams, Macrocyclic; Leukemia, Experimental; Lymphocyte Activation; Monophenol Monooxygenase; Quinones; Rifabutin; Signal Transduction

Overexpression and activation of TPM3-ALK tyrosine kinase fusion protein is a causal oncogenic event in the development of Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic ALK-positive tumors. Thus, the development of ALK specific tyrosine kinase inhibitors is a current therapeutic challenge. Animal models are essential to assess, in vivo, the efficiency of ALK-oncogene inhibitors and to identify new and/or additional therapeutic targets in the ALK tumorigenesis pathway. Using the tetracycline system to allow conditional and concomitant TPM3-ALK and luciferase expression, we have developed a unique transplant model for bioluminescent TPM3-ALK-induced fibroblastic tumors in athymic nude mice. The reversible TPM3-ALK expression allowed us to demonstrate that this oncogene is essential for the tumor growth and its maintenance. In addition, we showed that this model could be used to precisely assess tumor growth inhibition upon ALK chemical inactivation. As proof of principle, we used the general tyrosine kinase inhibitor herbimycin A to inhibit ALK oncoprotein activity. As expected, herbimycin A treatment reduced tumor growth as assessed both by tumor volume measurement and bioluminescent imaging. We conclude that this transplant model for TPM3-ALK-induced tumors represents a valuable tool not only to accurately and rapidly evaluate in vivo ALK-targeted therapies but also to gain insight into the mechanism of ALK-positive tumor development.

Topics: Anaplastic Lymphoma Kinase; Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Transformation, Neoplastic; Disease Models, Animal; Drug Screening Assays, Antitumor; Genes, Reporter; Lactams, Macrocyclic; Luciferases; Luminescent Agents; Lymphoma, Large-Cell, Anaplastic; Mice; Mice, Nude; Neoplasm Transplantation; Oncogene Proteins, Fusion; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; Rifabutin; Tropomyosin

An internal tandem duplication of the juxtamembrane (JM) domain of FLT3, a family of ligand-activated receptor tyrosine kinases, has been found in 20% of cases of acute myeloid leukemia (AML), and this mutation is correlated with leukocytosis and a poor prognosis. As a therapeutic approach, we previously reported that herbimycin A (HA) inhibited the growth of tandemly duplicated FLT3 (TDFLT3)-transformed cells (Leukemia 2000; 14: 374). Here, we have investigated the mechanism behind the cytotoxicity of HA, an ansamycin derivative which is now known to target Hsp90. The treatment with HA or another Hsp90 inhibitor, radicicol, induced selective apoptosis in TDFLT3-transformed 32D cells (TDFLT3/32D). The tyrosine-phosphorylation of TDFLT3 was inhibited by HA, whereas FLT3 ligand-induced phosphorylation of wild-type FLT3 (WtFLT3) was not. The downstream signal molecules MAPK, Akt and STAT5a were also dephosphorylated by HA in TDFLT3/32D. Immunoprecipitation analysis showed that TDFLT3 but not WtFLT3 formed a complex with Hsp90, and that the HA treatment dissociated TDFLT3 from the Hsp90 chaperone complex. These findings imply that targeting of Hsp90 will facilitate the development of anti-TDFLT3 therapy, and that Hsp90 is closely involved in the oncogenic activation of FLT3.

Topics: Animals; Apoptosis; Benzoquinones; Cell Transformation, Neoplastic; DNA-Binding Proteins; fms-Like Tyrosine Kinase 3; HSP90 Heat-Shock Proteins; Interleukin-3; Lactams, Macrocyclic; Lactones; Macrolides; Macromolecular Substances; MAP Kinase Signaling System; Mice; Milk Proteins; Myeloid Cells; Neoplasm Proteins; Phosphorylation; Protein Folding; Protein Interaction Mapping; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinones; Receptor Protein-Tyrosine Kinases; Rifabutin; STAT5 Transcription Factor; Tandem Repeat Sequences; Trans-Activators

Src tyrosine kinase plays key roles in signal transduction following growth factor stimulation and integrin-mediated cell-substrate adhesion. Since src-signal transduction defects are implicated in a multitude of human diseases, we have sought to develop new ways to identify small molecule inhibitors using a yeast-based, activated-src over-expression system. In the present study, we describe the identification of a unique src-signal transduction inhibitor, UCS15A. UCS15A was found to inhibit the src specific tyrosine phosphorylation of numerous proteins in v-src-transformed cells. Two of these phosphoproteins were identified as bona-fide src substrates, cortactin and Sam68. UCS15A differed from conventional src-inhibitors in that it did not inhibit the tyrosine kinase activity of src. In addition, UCS15A appeared to differ from src-destabilizing agents such as herbimycin and radicicol that destabilize src by interfering with Hsp90. Our studies suggest that UCS15A exerted its src-inhibitory effects by a novel mechanism that involved disruption of protein-protein interactions mediated by src. One of the biological consequences of src-inhibition by UCS15A was its ability to inhibit the bone resorption activity of osteoclasts in vitro. These data suggest that UCS15A may inhibit the bone resorption activity of osteoclasts, not by inhibiting src tyrosine kinase activity, but by disrupting the interaction of proteins associated with src, thereby modulating downstream events in the src signal transduction pathway.

Topics: 3T3 Cells; Adaptor Proteins, Signal Transducing; Animals; Anti-Bacterial Agents; Benzaldehydes; Benzoquinones; Bone Resorption; Cell Transformation, Neoplastic; Cortactin; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Genes, src; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Male; Mice; Microfilament Proteins; Organ Culture Techniques; Osteoclasts; Phosphorylation; Quinones; Rats; Rifabutin; RNA-Binding Proteins; Signal Transduction; src-Family Kinases; Tyrosine

Somatic mutation of the FLT3 gene, in which the juxtamembrane domain has an internal tandem duplication, is found in 20% of human acute myeloid leukemias and causes constitutive tyrosine phosphorylation of the products. In this study, we observed that the transfection of mutant FLT3 gene into an IL3-dependent murine cell line, 32D, abrogated the IL3-dependency. Subcutaneous injection of the transformed 32D cells caused leukemia in addition to subcutaneous tumors in C3H/HeJ mice. To develop a FLT3-targeted therapy, we examined tyrosine kinase inhibitors for in vitro growth suppression of the transformed 32D cells. A tyrosine kinase inhibitor, herbimycin A, remarkably inhibited the growth of the transformed 32D cells at 0.1 microM, at which concentration it was ineffective in parental 32D cells. Herbimycin A suppressed the constitutive tyrosine phosphorylation of the mutant FLT3 but not the phosphorylation of the ligand-stimulated wild-type FLT3. In mice transplanted with the transformed 32D cells, the administration of herbimycin A prolonged the latency of disease or completely prevented leukemia, depending on the number of cells inoculated and schedule of drug administration. These results suggest that mutant FLT3 is a promising target for tyrosine kinase inhibitors in the treatment of leukemia.

Topics: Animals; Antineoplastic Agents; Benzoquinones; Cell Line, Transformed; Cell Transformation, Neoplastic; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; fms-Like Tyrosine Kinase 3; Genistein; Humans; Hydroquinones; Interleukin-3; Lactams, Macrocyclic; Leukemia, Experimental; Mice; Mice, Inbred C3H; Neoplasm Proteins; Neoplasm Transplantation; Phosphorylation; Phthalimides; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Quinones; Receptor Protein-Tyrosine Kinases; Rifabutin; Signal Transduction; Transfection; Tyrphostins

The roles of activated ras and src oncogene products in the acquisition of fully neoplastic phenotype by human gallbladder adenocarcinoma cells were investigated by co-transfecting non-tumorigenic HAG-I human gallbladder carcinoma cells with the pSV2neo plasmid and a plasmid carrying either activated c-H-ras or v-src oncogene. G418-resistant clones were isolated and assessed for the acquisition of anchorage-independent growth potential. Neither the 10 established clones transfected with pSV2neo alone nor the 17 clones transfected with activated c-H-ras, including 4 clones expressing the mutated p21H-ras protein, could form colonies in soft agar. By contrast, out of 10 clones transfected with v-src, 2 formed colonies in soft agar and produced tumors in athymic nude mice, the resulting progressive neoplasms being poorly differentiated adenocarcinomas. These tumorigenic clones were shown to have v-src DNA and mRNA levels with p60v-src protein, but there were no significant chromosomal alterations following tumorigenic conversion. Moreover, herbimycin A, a selective src-kinase inhibitor, markedly reduced clonogenic growth of these cells in soft agar rather than monolayer growth, suggesting that anchorage-independent growth of the v-src-transformed HAG-I cells might be driven directly by p60v-src kinase activity. Taken together, our data suggest that the fully neoplastic conversion of HAG-I cells depends on src-related tyrosine-kinase activity, but not solely on the function mediated by activated ras, thus providing evidence of an src-related signaling pathway for the acquisition of tumorigenic potential by human gallbladder adenocarcinoma cells.

Topics: Adenocarcinoma; Animals; Benzoquinones; Blotting, Northern; Blotting, Southern; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Disease Progression; Gallbladder Neoplasms; Gene Expression Regulation, Neoplastic; Genes, ras; Genes, src; Humans; Immunoblotting; Karyotyping; Lactams, Macrocyclic; Mice; Mice, Nude; Phenotype; Protein-Tyrosine Kinases; Quinones; Rifabutin; Transfection

We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Hölttä, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60v-src, known to associate also with v-Crk and designated p130CAS. We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphorylation of p130 was markedly reduced, while the level of activity of ODC remained highly elevated. A similar reduction in pp130 phosphorylation and reversion of morphology by herbimycin A were observed in v-src- and c-Ha-ras-transformed cells. In addition, we show that expression of antisense mRNA for p130CAS resulted in reversion of the transformed phenotype of all these cell lines. An increased level of tyrosine kinase activity, not caused by c-Src or c-Abl, was further detected in the cytoplasmic fraction of ODC-transformed cells. Preliminary characteristics of this kinase are shown. These data indicate that p130CAS is involved in cell transformation by ODC, c-ras, and v-src oncogenes, raise the intriguing possibility that p130CAS may be generally required for transformation, and imply that there is at least one protein tyrosine kinase downstream of ODC that is instrumental for cell transformation.

Topics: 3T3 Cells; Actins; Animals; Benzoquinones; Blotting, Western; Cell Line; Cell Transformation, Neoplastic; Crk-Associated Substrate Protein; Enzyme Inhibitors; Genes, ras; Humans; Lactams, Macrocyclic; Mice; Ornithine Decarboxylase; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proteins; Quinones; Rats; Recombinant Proteins; Retinoblastoma Protein; Retinoblastoma-Like Protein p130; Rifabutin; src-Family Kinases; Transfection

We have examined the differentiation-inducing effects of ONO-4007, a new synthetic lipid A derivative with low endotoxic activities, on a rat myelomonocytic cell line, c-WRT-7, in vitro and in vivo. ONO-4007 induced the differentiation of c-WRT-7 cells into macrophage-like cells and inhibited the proliferation of c-WRT-7 cells in vitro. Stimulation with ONO-4007 induced messenger RNA expression of interleukin-1 alpha (IL-1 alpha), IL-6, and tumor necrosis factor-alpha (TNF-alpha), which have been reported to induce differentiation of several leukemia cell lines. However, autocrine production of these cytokines may not be involved in the mechanisms of differentiation induced by ONO-4007, because the treatment with IL-1 alpha, IL-6, or TNF-alpha does not induce the differentiation of c-WRT-7 cells. In vivo treatment by intravenous administration of ONO-4007 resulted in a significant prolongation of survival time of the rats inoculated intravenously with c-WRT-7 cells compared with that of untreated rats. These results suggest that ONO-4007 can be therapeutically useful for the treatment of leukemia.

Topics: Alkaloids; Animals; Base Sequence; Benzoquinones; Calmodulin; Cell Division; Cell Transformation, Neoplastic; In Vitro Techniques; Interleukin-1; Interleukin-6; Lactams, Macrocyclic; Leukemia, Myeloid; Lipid A; Molecular Sequence Data; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; RNA, Messenger; Staurosporine; Sulfonamides; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.

Topics: Animals; Benzoquinones; Blotting, Northern; Bone Marrow; Cell Line; Cell Transformation, Neoplastic; DNA Probes; Fusion Proteins, bcr-abl; Gene Expression; Genes, ras; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Lactams, Macrocyclic; Mice; Oncogenes; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; Rifabutin; RNA; Signal Transduction; Transcription, Genetic

Transformation of chick embryonic fibroblasts with Rous sarcoma virus strongly suppresses N-cadherin-mediated cell-cell adhesion, without inhibiting its expression. This suppression is correlated with tyrosine phosphorylation of N-cadherin and catenins, the cadherin-associated proteins, which are known to regulate cadherin function. Experiments with non-myristylation and temperature-sensitive mutants of RSV and with herbimycin A, a potent inhibitor of tyrosine kinases, suggest that both the suppression of cell adhesion and tyrosine phosphorylation of catenins are highly transformation-specific.

Topics: Animals; Avian Sarcoma Viruses; Benzoquinones; Cadherins; Cell Adhesion; Cell Aggregation; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Cytoskeletal Proteins; Fibroblasts; Lactams, Macrocyclic; Methionine; Oncogene Protein pp60(v-src); Phosphates; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tyrosine

To search for the biochemical prerequisites for morphological transformation by p60v-src, we examined the effect of herbimycin A, a potent inhibitor of cell transformation, on chicken embryonic fibroblasts transformed by Rous sarcoma virus. A small dose of herbimycin (0.1-0.3 micrograms ml-1) was enough to convert the cell morphology to a normal phenotype with a concomitant reassembly of microfilament bundles. In the cells treated with the drug, the majority of the substrates for p60v-src remained phosphorylated and p60v-src was myristylated, membrane associated and fairly active as a protein kinase. Under the same conditions, however, the association of p60v-src with the cytoskeletal structure and with phosphatidylinositol 3' kinase was strongly inhibited, suggesting that the interactions of p60v-src with the cellular structure and the enzyme were indispensable for morphological transformation.

Topics: Animals; Antibiotics, Antineoplastic; Avian Sarcoma Viruses; Benzoquinones; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Cytoskeleton; Lactams, Macrocyclic; Oncogene Protein pp60(v-src); Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases; Quinones; Rifabutin

We developed a new method for evaluating inhibitors of oncogenic signal transduction pathways based on different growth abilities between normal and transformed cells in a defined serum-free medium. The growth rates of src, abl or ras oncogene-transformed cells, activated raf proto-oncogene transformed cells, and normal NIH-3T3 cells were 60-90%, 20-30% and 10% in a serum-free medium, respectively, compared to the growth rates in a serum-containing medium. An addition of a growth factor (PDGF, FGF or TGF-beta) stimulated the growth of normal NIH3T3 cells by 40-80% in a serum-free medium. Herbimycin A, a specific cytoplasmic protein tyrosine kinase inhibitor, selectively inhibited the growth of src or abl transformed cells in the serum-free medium resulting in about 10-fold or fivefold lower IC50 than those in the serum-containing medium. The antibiotic did not show such an effect on ras transformed cells, and the treatment of src transformed cells with other protein kinase inhibitors or cytotoxic drugs showed little IC50 shifts between the two media. Thus, this method of comparing growth inhibition in the serum-free and the serum-containing media may be useful in evaluating specific inhibitors of signaling pathways mediated by growth factors and certain oncogene products.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Benzoquinones; Cell Division; Cell Transformation, Neoplastic; Culture Media, Serum-Free; Doxorubicin; Ethylmaleimide; Lactams, Macrocyclic; Mice; Oncogenes; Protein Kinase Inhibitors; Quinones; Rifabutin; Vinblastine

Herbimycin A, a benzoquinoid ansamycin antibiotic, was demonstrated to decrease intracellular phosphorylation by protein tyrosine kinase (PTK). In Philadelphia chromosome (Ph1)-positive leukemias such as chronic myelogenous leukemia (CML) and Ph1-positive acute lymphoblastic leukemia (ALL), both of which express bcr-abl fused gene products (P210bcr-abl or P190bcr-abl protein kinase) with augmented tyrosine kinase activities, herbimycin A markedly inhibited the in vitro growth of the Ph1-positive ALL cells and the leukemic cells derived from CML blast crisis. However, the same dose of herbimycin A did not inhibit in vitro growth of a broad spectrum of Ph1-negative human leukemia cells, and several other protein kinase antagonists also displayed no preferential inhibition. Furthermore, we demonstrated that herbimycin A has an antagonizing effect on the growth of transformed cells by a transfection of retroviral amphotrophic vector expressing P210bcr/abl into a murine interleukin (IL)-3-dependent myeloid FDC-P2 cell line. This inhibition was abrogated by the addition of sulfhydryl compounds, similar to the reaction previously described for Rous sarcoma virus transformation. The inhibitory effect of herbimycin A on the growth of Ph1-positive cells was associated with decreased bcr/abl tyrosine kinase activity, but no decrease of bcr-abl mRNA and protein, suggesting that the inactivation of bcr-abl tyrosine kinase activity by herbimycin A may be induced by its binding to the bcr-abl protein portion that is rich with sulfhydryl groups. The present study indicates that herbimycin A is a beneficial agent for the investigation of the role of the bcr-abl gene in Ph1-positive leukemias and further suggests that the development of agents inhibiting the bcr-abl gene product may offer a new therapeutic potential for Ph1-positive leukemias.

Topics: Benzoquinones; Cell Division; Cell Transformation, Neoplastic; Fusion Proteins, bcr-abl; Genetic Vectors; Humans; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Protein-Tyrosine Kinases; Quinones; Retroviridae; Rifabutin; Sulfhydryl Compounds; Transfection

Topics: Animals; Antibiotics, Antineoplastic; Avian Sarcoma Viruses; Benzoquinones; Cell Division; Cell Line; Cell Transformation, Neoplastic; Genes, src; Humans; Lactams, Macrocyclic; Oncogene Protein pp60(v-src); Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Streptomyces

Herbimycin A, which has been known to inactivate and degrade p60v-src tyrosine kinase, induced an elevated synthesis of a protein with a molecular size of 70 kDa in A431 human epidermoid carcinoma cells. This protein showed the same migration distance on SDS-polyacrylamide gel electrophoresis as that of the protein induced in the cells by heat shock treatment, and this 70-kDa protein was identified as a member of the heat shock protein 70 family (hsp70) through immunoprecipitation with anti-hsp72/73 antibody and partial digestion with V8 protease. The induced level of the 70-kDa protein was dependent on the length of period and the concentration of herbimycin A treatment. Cellular fractionation and indirect immunofluorescence analyses revealed that the 70-kDa protein induced by herbimycin A was localized in the cytoplasm, in contrast to the nuclear distribution of hsp70 induced by heat treatment. Induction of hsp70 by herbimycin A was also observed in several other cells, including HeLa S3 cells, chicken embryo fibroblasts, NIH3T3 cells, and Rous sarcoma virus-transformed NIH3T3 cells.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Cell Transformation, Neoplastic; Heat-Shock Proteins; Humans; Kinetics; Lactams, Macrocyclic; Oncogenes; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured

Topics: Actinomyces; Animals; Benzoquinones; Cell Transformation, Neoplastic; Lactams, Macrocyclic; Oncogenes; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Signal Transduction; Tumor Cells, Cultured

Herbimycin A is an antiobiotic which reverses transformation caused by src family oncogenes. It inactivates p60v-src in vitro, possibly by binding to reactive SH-group(s) of the kinase. We examined effects of various SH-reagents on p60v-src and observed that N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) or N-(9-acridinyl)maleimide (NAM) were potent inactivators of the kinase, whereas N-ethylmaleimide (NEM) required high concentrations, and iodoacetamide was totally ineffective in reducing the kinase activity. Pretreatment of p60v-src immune-complex with NEM and iodoacetamide, however, protected the kinase from inactivation by herbimycin A, BIPM, and NAM. The results suggest that SH-group(s) to which herbimycin A binds is not essential for the kinase activity, but is positioned in the vicinity of the active center.

Topics: Animals; Antibiotics, Antineoplastic; Avian Sarcoma Viruses; Benzoquinones; Cell Line; Cell Transformation, Neoplastic; Ethylmaleimide; Iodoacetamide; Kinetics; Lactams, Macrocyclic; Mice; Oncogene Protein pp60(v-src); Protein-Tyrosine Kinases; Quinones; Rifabutin; Sulfhydryl Reagents

We studied the mechanism of reversion of Rous sarcoma virus (RSV)-transformation by herbimycin A in a temperature-sensitive RSV-infected NRK cell line. Herbimycin reduced the total cellular phosphotyrosine level to 10% of the control value. Intracellular p60src kinase activity was reduced significantly within 3 h of herbimycin treatment, but recovered to approximately 50% of the control within 24 h, and the kinetics paralleled the decrease of the phosphorylation of a cellular target p36. Partial proteolytic phosphopeptide analysis of p60src revealed decreased phosphorylation of tyrosine 416 in the C-terminal half. Analysis of the steady state level of p60src indicated 30% decrease in src protein. Measurement of the rates of p60src synthesis and degradation showed that the decrease in the level was due to the accelerated degradation of src protein. Cycloheximide blocked this enhanced turnover of p60src, but did not block the herbimycin-induced inactivation of p60src kinase. Subcellular distribution and myristoylation of p60src protein were not altered. These results support the idea that RSV transformation is associated with elevated phosphotyrosine levels and indicate that inhibition of src kinase activity can reverse RSV-transformation by reducing cellular phosphotyrosine content.

Topics: Amino Acids; Animals; Avian Sarcoma Viruses; Benzoquinones; Cell Line; Cell Transformation, Neoplastic; Herbicides; Kinetics; Lactams, Macrocyclic; Oncogene Protein pp60(v-src); Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Retroviridae Proteins; Rifabutin; Thermodynamics; Tyrosine

We studied the effectiveness of herbimycin A, an inhibitor of the function of the src oncogene, to reverse the various transformed phenotypes in normal rat kidney (NRK) cells integrating temperature-sensitive v-src (ts/NRK). Elevated glucose transport in ts/NRK cells at a permissive temperature (33 degrees C) was decreased by herbimycin in 8 h to near the level that was observed either in ts/NRK grown at a nonpermissive temperature (39 degrees C) or in untransformed NRK cells at either temperature. Herbimycin caused no significant decrease in glucose uptake in ts/NRK cells grown at 39 degrees C. The effects of herbimycin on serum- and anchorage-independent growth properties of ts/NRK cells and of NRK cells integrating K-ras (KNRK) were also examined. With ts/NRK cells grown at 33 degrees C, the inhibition of cell growth by herbimycin became more pronounced when the serum concentration in the medium was lowered. With KNRK cells, in contrast, almost the same extent of cell growth inhibition was exerted by herbimycin irrespective of the serum concentration. Furthermore, with ts/NRK cells grown at 33 degrees C, herbimycin inhibited the colony formation in the soft agar medium more strongly than on a solid support. No such differential effects were observed with KNRK cells under similar conditions. These results suggest that herbimycin specifically acts on cells expressing the src oncogene and reverses various transformed characteristics to the normal ones.

Topics: Animals; Benzoquinones; Blood Physiological Phenomena; Cell Transformation, Neoplastic; Deoxyglucose; Fibroblasts; Lactams, Macrocyclic; Oncogenes; Phenotype; Quinones; Rats; Rifabutin

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

Topics: Animals; Benzoquinones; Cell Transformation, Neoplastic; Flavonoids; Genistein; Hydroquinones; Isoflavones; Lactams, Macrocyclic; Oncogenes; Phospholipids; Protein-Tyrosine Kinases; Quinones; Rifabutin