herbimycin and monorden

Persistent infection with hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Here we report that inhibition of heat shock protein 90 (Hsp90) is highly effective in suppressing HCV genome replication. In HCV replicon cells, HCV replication was reduced by Hsp90 inhibitors and by knockdown of endogenous Hsp90 expression mediated by small-interfering RNA (siRNA). The suppression of HCV replication by an Hsp90 inhibitor was prevented by transfection with Hsp90 expression vector. We also tested the anti-HCV effect of Hsp90 inhibition in HCV-infected chimeric mice with humanized liver. Combined administration of an Hsp90 inhibitor and polyethylene glycol-conjugated interferon (PEG-IFN) was more effective in reducing HCV genome RNA levels in serum than was PEG-IFN monotherapy. These results suggest that inhibition of Hsp90 could provide a new therapeutic approach to HCV infection.

Topics: Animals; Benzoquinones; Blotting, Western; Cell Line, Tumor; Heat-Shock Proteins; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Interferon alpha-2; Interferon-alpha; Lactams, Macrocyclic; Macrolides; Mice; Mice, SCID; Polyethylene Glycols; Recombinant Proteins; Replicon; Rifabutin; RNA, Small Interfering; RNA, Viral; Time Factors; Transfection; Transplantation Chimera; Transplantation, Heterologous; Virus Replication

There are many similarities between the interactions of environmental protozoa with pathogenic bacterial species and those observed in mammalian macrophages. Since single-celled protozoa predate mammalian hosts, it is likely that interactions in environmental biofilms have selected for many of the bacterial virulence mechanisms responsible for human disease. In order to better understand bacterial-phagocyte interactions, we developed a selection for Acanthamoeba castellanii variants that are more resistant to killing by bacterial pathogens. We identified four amoebal clones that display decreased phagocytosis of bacteria but no difference in uptake of latex beads compared to wild-type amoebae. These amoebal variants display differences in cellular morphology, partial resistance to killing by bacteria, more bactericidal activity, and higher frequencies of lysosome fusion with the bacterial vacuole. Three proteins are present at lower levels in these variants than in wild-type amoebae, and matrix-assisted laser desorption ionization-time of flight mass spectrometry allowed identification of two of them as actin and hsp90. We found that specific inhibitors of hsp90 produce a similar phenotypic effect in macrophages. These data suggest that hsp90 plays a role in phagocytic and, possibly, bactericidal pathways that affect interactions of phagocytic cells with bacteria.

Topics: Acanthamoeba castellanii; Animals; Benzoquinones; Enzyme Inhibitors; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Lactones; Legionella pneumophila; Lysosomes; Macrolides; Macrophages; Mitochondria; Novobiocin; Phagocytosis; Protozoan Proteins; Quinones; Rifabutin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Heat-shock protein 90 (HSP90) is known to affect a variety of cellular activities. The present study showed that the HSP90-binding agents, geldanamycin, herbimycin A and radicicol, inhibited the murine thymocyte apoptosis induced by dexamethasone and was accompanied by the inhibition of the reduction of the mitochondrial transmembrane potential (delta psi m). HSP90-binding agents did not inhibit etoposide-induced apoptosis. The inhibition of dexamethasone-induced apoptosis was in part due to the interference of HSP90 with the glucocorticoid receptor, resulting in the inhibition of nuclear translocation of the receptor. The expression of inositol 1,4,5-triphosphate receptors, which were shown to be involved in dexamethasone-induced apoptosis, did not participate in the inhibition of apoptosis.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Benzoquinones; Calcium Channels; Dexamethasone; DNA Fragmentation; Dose-Response Relationship, Drug; Etoposide; Glucocorticoids; HSP90 Heat-Shock Proteins; Inositol 1,4,5-Trisphosphate Receptors; Lactams, Macrocyclic; Lactones; Macrolides; Male; Membrane Glycoproteins; Membrane Potentials; Mice; Mice, Inbred BALB C; Mitochondria; Quinones; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Glucocorticoid; Reverse Transcriptase Polymerase Chain Reaction; Rifabutin; T-Lymphocytes

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

Heat shock proteins (HSPs) are major components of eukaryotic and prokaryotic cells with particularly high levels of expression in neurons. HSPs control protein folding, transport of proteins to and from the nucleus, incorporation of proteins into the cell membrane, and maintenance of the functional activity of several proteins involved in transcriptional control. In this study we demonstrate that inhibitors of HSP90 alter currents mediated by the ligand gated channels, P2X and VR1. P2X and VR1 are membrane receptors activated by ATP and capsaicin, respectively, and are thought to be involved in inflammation-related nociception. The HSP90 inhibitors geldanamycin (GLD), radicicol (RAD) herbimycin A (HERB) potentiated ATP induced currents, whereas only GLD altered capsaicin-induced currents in isolated DRG neurons. At low (< 1 microM) concentrations, GLD potentiated the capsaicin-induced current, while at high concentrations (10-25 microM) it inhibited it. The results suggest a potential involvement of HSPs in nociception.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Benzoquinones; Cannabinoids; Capsaicin; Cells, Cultured; Dose-Response Relationship, Drug; Ganglia, Spinal; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Lactones; Macrolides; Male; Neurons; Quinones; Rats; Rats, Sprague-Dawley; Receptors, Drug; Receptors, Purinergic P2; Rifabutin; Synaptic Transmission

Radicicol (RAD) and the benzoquinone ansamycin geldanamycin (GA) are potential anticancer drugs known to inhibit heat shock protein 90 (hsp90) and, therefore, the activation of proteins dependent on its function such as proto-oncogenic kinases and nuclear receptors. Using the glucocorticoid receptor (GR) as a model system we analysed the effects of RAD and various benzoquinone ansamycins. All compounds efficiently abolished GR-dependent transactivation. Surprisingly, whenever one of the ansamycins was applied in combination with RAD, synergistic inhibition of GR-dependent transcription and of hormone binding of GR was observed. In contrast, combination of two ansamycins showed no synergy. These findings suggest synergism within the hsp90 dimer and may open new ways to explore hsp90 as therapeutic target.

Topics: Anti-Bacterial Agents; Benzoquinones; Drug Synergism; Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Quinones; Receptors, Glucocorticoid; Rifabutin; Saccharomyces cerevisiae Proteins; Tumor Cells, Cultured

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

Chronic myelogenous leukemia (CML) is a clonal disorder of a pluripotent hematopoietic stem cells characterized by a chimeric bcr-abl gene giving rise to a p210(Bcr-Abl) protein with dysregulated tyrosine kinase activity. Radicicol, a macrocyclic antifungal antibiotic, binds to the N-terminal of heat shock protein 90 (Hsp90) and destabilizes Hsp90-associated proteins such as Raf-1. This study investigated the effect of radicicol, novel oxime derivatives of radicicol (KF25706 and KF58333), and herbimycin A (HA), a benzoquinoid ansamycin antibiotic, on the growth and differentiation of human K562 CML cells. Although KF25706 and KF58333 induced the expression of glycophorin A in K562 cells, radicicol and HA caused erythroid differentiation transiently. Cell cycle analysis showed that G(1) phase accumulation was observed in K562 cells treated with KF58333. KF58333 treatment depleted p210(Bcr-Abl), Raf-1, and cellular tyrosine phosphorylated proteins in K562 cells, whereas radicicol and HA showed transient depletion of these proteins. KF58333 also down-regulated the level of cell cycle-dependent kinases 4 and 6 and up-regulated cell cycle-dependent kinase inhibitor p27(Kip1) protein without an effect on the level of Erk and Hsp90 proteins. Immunoprecipitation analysis showed that p210(Bcr-Abl) formed multiple complexes with Hsp90, some containing p23 and others Hsp70; KF58333 treatment dissociated p210(Bcr-Abl) from Hsp90/p23 chaperone complexes. Furthermore, KF58333 induced apoptosis in K562 cells and administration of KF58333 prolonged the survival time of SCID mice inoculated with K562 cells. These results suggest that KF58333 may have therapeutic potential for the treatment of CML that involves abnormal cellular proliferation induced by p210(Bcr-Abl).

Topics: Animals; Antibiotics, Antineoplastic; Antifungal Agents; Benzoquinones; Cell Differentiation; Erythroblasts; Fusion Proteins, bcr-abl; G1 Phase; HSP90 Heat-Shock Proteins; Humans; K562 Cells; Lactams, Macrocyclic; Lactones; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrolides; Mice; Quinones; Rifabutin

Six different biotinylated radicicol derivatives were synthesized as affinity probes for identification of cellular radicicol-binding proteins. Derivatives biotinylated at the C-17 (BR-1) and C-11 (BR-6) positions retained the activity of morphological reversion in v-src-transformed 3Y1 fibroblasts. Two radicicol-binding proteins, 120 and 90-kDa in size, were detected in HeLa cell extracts by employing BR-1 and BR-6, respectively. The 90-kDa protein bound to BR-6 was identified to be Hsp90 by immunoblotting. The 120-kDa protein bound to BR-1 was purified from rabbit reticulocyte lysate, and its internal amino acid sequence was identical to that of human and rat ATP citrate lyase. The identity of the 120-kDa protein as ATP citrate lyase was confirmed by immunoblotting. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. These results suggest that radicicol binds the two proteins through different molecular portions of its structure. BR-1-bound ATP citrate lyase isolated from rabbit reticulocyte lysate showed no enzymatic activity. The activity of rat liver ATP citrate lyase was inhibited by radicicol and BR-1 but not by BR-6. Kinetic analysis demonstrated that radicicol was a non-competitive inhibitor of ATP citrate lyase with K(i) values for citrate and ATP of 13 and 7 microm, respectively.

Topics: Animals; ATP Citrate (pro-S)-Lyase; Benzoquinones; Binding, Competitive; Biotinylation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Immunoblotting; Kinetics; Lactams, Macrocyclic; Lactones; Liver; Macrolides; Protein Binding; Quinones; Rabbits; Rats; Reticulocytes; Rifabutin

Activation of Src, which has an intrinsic protein tyrosine kinase (PTK) activity, has been demonstrated in human solid tumors, such as colorectal and breast cancers. To investigate the role of activated Src in drug resistance, we evaluated the effect of v-src on the resistance to various anti-cancer drugs using v-src-transfected HAG-1 human gallbladder adenocarcinoma cells. Compared with parental or mock-transfected HAG-1 cells, v-src-transfected HAG/src3-1 cells showed a 3.5-fold resistance to cis-diamminedichloroplatinum (II) (CDDP) but not to doxorubicin, etoposide or 5-fluorouracil. By contrast, activated H-ras, which acts downstream of src, failed to induce resistance to either of these drugs. Furthermore, wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, and H7, a protein kinase C (PKC) inhibitor, did not alter CDDP resistance. Evaluation of the kinetics of the removal of DNA interstrand cross-links (ICLs), measured by alkaline elution, showed a significant increase in this removal in HAG/src3-1 cells as compared with mock-transfected cells, though no differences were found in the formation of DNA ICLs between these cell lines. CDDP resistance in v-src-transfected cells was reversed, if not completely, by either herbimycin A or radicicol, specific inhibitors of Src-family PTKs, suggesting that Src tyrosine kinase activity induces CDDP resistance. Moreover, significant reduction in the repair of CDDP-induced DNA ICLs was observed upon treatment with radicicol. The intracellular glutathione content and mRNA expression of topoisomerase II and metallothionein were virtually identical between these cell lines, except for topoisomerase I mRNA. Our data strongly suggest that the ability of activated src, but not ras, to induce CDDP resistance is mediated by augmentation of DNA repair through Src to downstream signal-transduction pathways distinct from either the Ras, PI 3-kinase or PKC pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenocarcinoma; Androstadienes; Benzoquinones; Cell Survival; Cisplatin; DNA Adducts; DNA Damage; DNA Repair; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gallbladder Neoplasms; Genes, src; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Oncogene Protein pp60(v-src); Phosphoinositide-3 Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; Transfection; Tumor Cells, Cultured; Wortmannin

The mitogen-inducible cyclooxygenase (COX-2) is selectively expressed in lipopolysaccharide (LPS)-stimulated macrophages. However, the signaling pathways that lead to the expression of COX-2 in LPS-stimulated macrophages are not well understood. LPS activates members of mitogen-activated protein kinases (MAPKs) and NF-kappaB transcription factor in macrophages. We have shown that protein tyrosine kinase (PTK) inhibitors suppress the LPS-induced expression of COX-2 in macrophages (Chanmugam et al., J Biol Chem 270: 5418-5426, 1995). These PTK inhibitors also inhibit LPS-induced activation of MAPKs. Thus, in the present study, we determined whether the activation of MAPKs and NF-kappaB is necessary for the signaling pathway for the LPS-induced expression of COX-2 in the murine macrophage cell line RAW 264.7. The findings demonstrated that inhibition of extracellular signal-regulated protein kinases 1 and 2 (ERK-1 and -2) by the selective inhibitor PD98059 or inhibition of P38 by the specific inhibitor SB203580 results in partial suppression of COX-2 expression. However, activation of MAPKs by phorbol 12-myristate 13-acetate, H2O2, sorbitol, sodium vanadate, or a combination of these agents failed to induce the expression of COX-2. Inhibitors of NF-kappaB suppressed COX-2 expression without affecting tyrosine phosphorylation of MAPKs. The PTK inhibitors that suppressed the activation of MAPKs and COX-2 expression also inhibited the degradation of IkappaB-alpha. Together, these results indicate that the activation of NF-kappaB is required to induce the expression of COX-2 in LPS-stimulated RAW 264.7 cells. Inhibition of ERK-1 and 2 or P38 results in partial suppression of COX-2 expression. However, the activation of MAPKs alone is not sufficient to induce the expression of COX-2 in these cells.

Topics: Animals; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; DNA-Binding Proteins; Enzyme Inhibitors; Flavonoids; I-kappa B Proteins; Imidazoles; Lactams, Macrocyclic; Lactones; Lipopolysaccharides; Macrolides; Macrophages; Mice; Mitogens; NF-kappa B; NF-KappaB Inhibitor alpha; Prostaglandin-Endoperoxide Synthases; Pyridines; Quinones; Rifabutin; RNA, Messenger; Signal Transduction

We have described a microplate colorimetric assay to quantitate anchorage-independent cell growth, using plates coated with an anti-adhesive polymer poly (2-hydroxyethyl methacrylate) (polyHEMA).We investigated whether this method could be applied to human cancer cells of epithelial origin. HAG-1, a non-tumorigenic human gall-bladder carcinoma cell line, and its pSVneo and c-H-ras transfectants, which are also non-tumorigenic, did not grow on a polyHEMA-surface. In contrast, the v-src transformant which produced tumors in nude mice and formed colonies in soft agar, was able to proliferate on the coated surface, suggesting that tumorigenicity of human cancer cells correlates with the ability to grow on a polyHEMA-coated surface. We report on the feasibility of this method as a screening system for inhibitors of oncogenic transformation. Herbimycin A and radicicol, which have been reported to block Src function, suppressed the growth of v-src-transformed NRK and HAG-1 cells on the non-adhesive polyHEMA-surface at concentrations significantly lower than on plastic. Differences in the inhibitory concentrations were not observed with KB cells, and cytotoxic agents such as adriamycin did not show any selectivity between the 2 surfaces. Growth of ras-transformed cells on the coated surface was selectively blocked by L-739,749, a farnesyltransferase inhibitor. The results demonstrate that compounds causing reversion of transformed cells to normal, hence, selectively inhibiting cell growth in anchorage-independent conditions, can be screened using this microtiter plate assay.

Topics: Adenocarcinoma; Animals; Benzoquinones; Cell Adhesion; Cell Division; Cell Line, Transformed; Drug Screening Assays, Antitumor; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Methacrylates; Mice; Neoplasms; Oligopeptides; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Sam68 (Src-associated in mitosis 68 kDa) is a protein that associates with and is tyrosine phosphorylated by Src in a mitosis-specific manner, thereby raising the possibility of a role for Src in the regulation of the cell cycle. This study examines the effects of radicicol, a Src tyrosine kinase inhibitor, upon both the phosphorylation of Sam68 and mitotic progression in Src-transformed mouse fibroblasts. Radicicol reversibly inhibits the mitosis-specific tyrosine phosphorylation of Sam68 in vivo, as determined by antiphosphotyrosine immunoblotting. Radicicol inhibits the tyrosine phosphorylation of both free and Src-associated Sam68, suggesting the presence of two intracellular pools of tyrosine phosphorylated Sam68 in mitotic cells. In addition, radicicol treatment has no effect on the ability of cells to enter mitosis, indicating that tyrosine phosphorylation of Sam68 is probably not important for cells to enter mitosis. However, radicicol reversibly retards the exit of cells from mitosis, as determined by flow cytometric analyses. Radicicol mediated inhibition of Sam68 tyrosine phosphorylation, and its concurrent ability to block mitotic exit suggests the possibility of a significant role for Src kinase and this unique mitotic substrate, Sam68, in cell cycle regulation.

Topics: 3T3 Cells; Adaptor Proteins, Signal Transducing; Animals; Benzoquinones; Cell Line, Transformed; Enzyme Inhibitors; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Mitosis; Nocodazole; Phosphorylation; Proto-Oncogene Proteins pp60(c-src); Quinones; Rifabutin; RNA-Binding Proteins; src Homology Domains; Time Factors; Tyrosine

Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.

Topics: Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Line, Transformed; Enzyme Inhibitors; Genes, ras; Guanosine Triphosphate; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Point Mutation; Protein Binding; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; Reference Values; Rifabutin; Transformation, Genetic; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Transgenic mice lacking a functional c-src gene have osteopetrosis, a bone disorder characterized by defective osteoclast function. We have investigated the effects of selective protein tyrosine kinase inhibitors that are known to inhibit c-src, on osteoclast activity in the bone slice assay. Geldanamycin, herbimycin A and monorden (0.001-10 microM) all dose-dependently inhibited bone resorption with IC50 values of 8, 70 and 86 nM, respectively. At concentrations of 0.001-1 microM, the compounds were not cytotoxic as judged by osteoclast morphology and survival on bone slices. In order to determine whether c-src plays a role in signal transduction associated with osteoclast activation prior to bone resorption commencing, or in the resorptive process itself, we performed kinetic experiments using human calcitonin as a positive control. Calcitonin inhibited all bone resorption subsequent to its addition at t = 0, 3 or 6 hr (100%, approximately 90% and approximately 50% inhibition, respectively), after the start of the 24 hr bone slice assay. Similar results were obtained with herbimycin A and geldanamycin (1 microM) added at t = 0, 3 or 6 hr, and with monorden (1 microM) added at t = 0 and 6 hr. These results indicate that c-src plays a crucial and continuous role in the process of osteoclastic bone resorption, most likely related to the translocation and/or fusion of exocytic vesicles to the ruffled border membrane.

Topics: Animals; Animals, Newborn; Benzoquinones; Bone Resorption; Calcitonin; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Genes, src; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Osteoclasts; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Signal Transduction