monorden and Cell-Transformation--Neoplastic

Trichostatins and trapoxins, structurally unrelated microbial metabolites, are specific inhibitors of histone deacetylases. Radicicol inhibits Src family protein-tyrosine kinases. Recently, these agents were found to induce morphological reversion and enhanced expression of gelsolin, an actin regulatory protein, in a variety of transformed cells. Microinjection of an anti-gelsolin antibody that neutralizes the gelsolin function caused inhibition of the morphological change, suggesting that gelsolin expression is associated with the suppression of transformation.

Topics: Animals; Cell Transformation, Neoplastic; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Lactones; Macrolides; Mice; Neoplasms; Neoplasms, Experimental; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Intrinsic stress response pathways are frequently mobilized within tumor cells. The mediators of these adaptive mechanisms and how they contribute to carcinogenesis remain poorly understood. A striking example is heat shock factor 1 (HSF1), master transcriptional regulator of the heat shock response. Surprisingly, we found that loss of the tumor suppressor gene neurofibromatosis type 1 (Nf1) increased HSF1 levels and triggered its activation in mouse embryonic fibroblasts. As a consequence, Nf1-/- cells acquired tolerance to proteotoxic stress. This activation of HSF1 depended on dysregulated MAPK signaling. HSF1, in turn, supported MAPK signaling. In mice, Hsf1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MAPK signaling. In cell lines from human malignant peripheral nerve sheath tumors (MPNSTs) driven by NF1 loss, HSF1 was overexpressed and activated, which was required for tumor cell viability. In surgical resections of human MPNSTs, HSF1 was overexpressed, translocated to the nucleus, and phosphorylated. These findings reveal a surprising biological consequence of NF1 deficiency: activation of HSF1 and ensuing addiction to this master regulator of the heat shock response. The loss of NF1 function engages an evolutionarily conserved cellular survival mechanism that ultimately impairs survival of the whole organism by facilitating carcinogenesis.

Topics: Active Transport, Cell Nucleus; Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Neurofibromatosis 1; Heat Shock Transcription Factors; Hot Temperature; Humans; Leupeptins; Macrolides; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Nerve Sheath Neoplasms; Neurofibromin 1; NIH 3T3 Cells; Phosphorylation; Protein Processing, Post-Translational; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Transcription Factors; Withanolides

The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Small Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Lung Neoplasms; Models, Chemical; Phosphatidylinositol 3-Kinases; Time Factors

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

The antibiotic radicicol suppresses transformation in a variety of transformed cells. The antineoplastic effects of the drug have been attributed to the degradation of Raf and the inactivation of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. Here we demonstrate that radicicol induces cell spreading, suppresses anchorage-independent cell growth, and increases the expression of the high-molecular weight tropomyosin isoform TM-2 in cells stably expressing a constitutively active form of MEK-1 as well as in ras-transformed cells. Furthermore, the reverting effects of the drug are achieved at concentrations below those required to deplete Raf from the cell or to inhibit the phosphorylation of ERK or its substrates Elk and pp90(RSK). In contrast, low concentrations of radicicol significantly inhibited activator protein (AP-1) and serum response factor (SRF)-mediated transcription. The lack of correlation between the effects of radicicol on cell phenotype and on the signaling activities of the Raf/MEK/ERK pathway indicate that Raf depletion or disruption of proximal signaling events in the mitogen-activated protein kinase pathway are not the predominant mechanisms by which the drug suppresses the transformed phenotype. Our observation that low concentrations of radicicol block transcriptional activities mediated by AP-1 and SRF suggests that interference with signaling upstream of these transcription factors may contribute to the reverting effects of the drug.

Topics: 3T3 Cells; Animals; Cell Adhesion; Cell Division; Cell Line, Transformed; Cell Size; Cell Transformation, Neoplastic; DNA-Binding Proteins; Dose-Response Relationship, Drug; ets-Domain Protein Elk-1; Fibroblasts; Gene Expression Regulation, Neoplastic; Lactones; Macrolides; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mutation, Missense; Oncogene Protein p21(ras); Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Ribosomal Protein S6 Kinases; Transcription Factor AP-1; Transcription Factors; Tropomyosin

Radicicol, a macrocyclic anti-fungal antibiotic, has the ability to suppress transformation by diverse oncogenes such as Src, Ras and Mos. Despite this useful property, the mechanism by which radicicol exerts its anti-transformation effects is currently unknown. To understand the transformation-suppressing effects of radicicol, a biotinylated derivative of radicicol was chemically synthesized and used as a probe in a Western-blot format to visualize cellular proteins that interact with radicicol. In transformed and untransformed mouse fibroblasts, the most prominent cellular protein that bound to radicicol had a molecular weight of approximately 90 kDa. Further analysis revealed that this protein was the mouse homologue of the 90 kDa heat shock protein (HSP90). This was confirmed by demonstrating the ability of radicicol to specifically bind purified human HSP90. Specificity of binding was demonstrated by the inhibition of binding of biotinylated radicicol by the native drug. Taken together with other studies the present observations suggest that the anti-transformation effects of radicicol may be mediated, at least in part, by the association of radicicol with HSP90 and the consequent dissociation of the Raf/HSP90 complex leading to the attenuation of the Ras/MAP kinase signal transduction pathway.

Topics: 3T3 Cells; Animals; Antifungal Agents; Benzoquinones; Cell Line, Transformed; Cell Transformation, Neoplastic; Fibroblasts; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Molecular Chaperones; Quinones

Radicicol, an inhibitor of Src-family protein-tyrosine kinases, causes morphological reversion of v-src- and v-Ha-ras-transformed fibroblasts and arrest of the cell cycle at both the G1 and the G2 phases. Radicicol was found to inhibit the growth of several other oncogene-transformed cell lines and human carcinoma cell lines and to revert their cell morphology to be flat. In the radicicol-treated flat cells, actin stress fiber bundles were reorganized. Since this effect of radicicol on these cell lines was inhibited by cycloheximide, de novo protein synthesis is required for the morphological reversion. Screening of cellular proteins enhanced in response to radicicol by two-dimensional gel electrophoresis suggested that the amount of gelsolin, an actin regulatory protein, was distinctly increased upon radicicol treatment. Western blot and Northern blot analyses showed that radicicol enhanced transcription of the gelsolin gene in human carcinoma cell lines, as a result of which the amount of gelsolin was increased several folds. Injection with an anti-gelsolin antibody into cells and successive treatment with radicicol resulted in approximately 80% reduction of the number of flat cells with stress fibers in comparison with controls treated with an irrelevant antibody. These results show that elevated expression of gelsolin is associated, at least in part, with the suppression of transformation and the restoration of actin stress fibers in human carcinoma cells by radicicol.

Topics: 3T3 Cells; Actin Cytoskeleton; Animals; Cell Transformation, Neoplastic; Cycloheximide; Enzyme Inhibitors; Gelsolin; HeLa Cells; Humans; Lactones; Macrolides; Mice; Microinjections; Oncogenes; Protein-Tyrosine Kinases

Activated versions of ras and mos oncogenes subvert the signal transduction pathway by mimicking transducers at the plasma membrane and cytosol respectively. Radicicol (UCS1006), an antifungal antibiotic, had the ability to suppress transformation by ras and mos oncogenes in a rapid, reversible and dose-dependent manner. UCS1006 inhibited MAP kinase activity (both ERK1 and ERK2) in untransformed as well as ras and mos transformed cells. However, ERK2 but not ERK1 activity was constitutively elevated in ras and mos transformed cells used in this study. In addition, a 62 kDa (kilodalton) phosphoprotein was identified whose tyrosine phosphorylation was inhibited by UCS1006, in both ras and mos transformed cells. This 62 kDa phosphoprotein, which was found to be heavily phosphorylated on tyrosine residues only in the ras and mos transformed cells but not in untransformed NIH3T3 cells, was identical to the previously described GAP-associated tyrosine phosphoprotein, p62, that is the major target for phosphorylation in cells transformed by tyrosine kinase oncogenes. These results suggest that agents such as radicicol can suppress transformation by diverse oncogenes such as src, ras and mos at least in part by inhibiting the function of key signal transduction intermediates such as MAP kinase and GAP-associated p62.

Topics: 3T3 Cells; Animals; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Genes, mos; Genes, ras; GTPase-Activating Proteins; Lactones; Macrolides; Mice; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proteins; ras GTPase-Activating Proteins; Tyrosine

A fungal metabolite, radicicol, with a macrocyclic ring induced the reversal of transformed phenotypes of v-src-transformed fibroblasts (Rous sarcoma virus-transformed 3Y1 rat fibroblast) at a quite low concentration of 0.1 microgram/ml. Actin stress fibers reappeared in the transformed cells after treatment with radicicol. Radicicol reduced the intracellular level of autophosphorylation of p60v-src as well as the level of other tyrosine-phosphorylated proteins in a dose-dependent manner. In vitro kinase assay revealed that radicicol effectively inhibited not only autophosphorylation but also transphosphorylation activities of purified p60v-src with a concentration producing 50% inhibition of 0.1 microgram/ml. However, radicicol showed no inhibitory effect on protein kinase C or protein kinase A. These results suggest that radicicol is a novel and specific protein-tyrosine kinase inhibitor and that the decreased level of tyrosine kinase activity of p60v-src causes reversion of transformed phenotypes of Rous sarcoma virus-transformed 3Y1 rat fibroblast. Furthermore, differentiation of Friend leukemia cells, which is one of the known characteristic phenomena associated with the inhibition of tyrosine kinase, was also induced in the concentration range of 0.05-0.5 microgram/ml, suggesting that the agent is useful for the analysis of differentiation as well as the kinase-mediated signal transduction.

Topics: Animals; Antifungal Agents; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Friend murine leukemia virus; Lactones; Leukemia, Experimental; Macrolides; Oncogene Protein pp60(v-src); Phosphorylation; Rats; Tyrosine