herbimycin and geldanamycin

Until recently, Hsp90 was one of the least well understood of the molecular chaperones, but considerable progress is now being made in unravelling its biochemistry. Hsp90 has now been shown to possess an inherent ATPase that is essential for the activation of authentic 'client' proteins in vivo and in vitro. The molecular detail of Hsp90's interactions with co-chaperones is also becoming clearer and the identification of key roles in assembling regulatory and signalling pathways has made it a target for anticancer drug development. Despite this, a clear understanding of how Hsp90 contributes to the folding and/or activation of its client proteins remains some way off.

Topics: Adenosine Triphosphate; Allosteric Regulation; Animals; Bacterial Proteins; Benzoquinones; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Macromolecular Substances; Models, Biological; Models, Molecular; Protein Binding; Quinones; Rifabutin; Signal Transduction; Structure-Activity Relationship

Cytochrome P4501A (the CYP1A1 and CYP1A2 enzymes) is known to metabolize anthropogenic xenobiotics to carcinogenic and mutagenic compounds. CYP1A1 transcriptional activation is regulated via the aryl hydrocarbon receptor (AhR)-dependent signal transduction pathway. CYP1A2 activation may occur through the AhR-dependent or AhR-independent signal transduction pathways. We used male Wistar rats to explore possible mechanisms of CYP1A activation induced by exposure to cold and the effects of the protein-tyrosine kinase inhibitors genistein, herbimycin A, and geldanamycin on the properties of hepatic CYP1A1 and CYP1A2 proteins following exposure to cold and to classic CYP1A inducers. The molecular mechanisms of cold-induced CYP1A1 and CYP1A2 activation are different. The CYP1A2 activation apparently occurs at the post-transcriptional level. The CYP1A1 activation, whether caused by exposure to cold or by classic CYP1A inducers, is AhR-dependent and occurs at the transcriptional level. Protein tyrosine kinase inhibitors have no effect on benzo(a)pyrene-induced CYP1A expression but alter cold-induced CYP1A1 activity and the CYP1A1 mRNA level. Thus, treatment with herbimycin A or geldanamycin leads to an increase in CYP1A1 activity, while treatment with genistein increases CYP1A1 mRNA expression and decreases CYP1A2 activity. These data elucidate the molecular mechanisms of cold-induced CYP1A activation and the role of protein kinases in the regulation of CYP1A during exposure to cold. Our results can also help identify the differences between the molecular mechanisms underlying the effects of the classic CYP1A inducers and the effects of cooling.

Topics: Animals; Benzo(a)pyrene; Benzoquinones; Cold Temperature; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Enzyme Activation; Genistein; Lactams, Macrocyclic; Male; Microsomes, Liver; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Receptors, Aryl Hydrocarbon; Rifabutin; RNA, Messenger; Transcriptional Activation

In resident mouse peritoneal macrophages, group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) mediates arachidonic acid (AA) release and eicosanoid production in response to diverse agonists such as A23187, phorbol myristate acetate, zymosan, and the enterotoxin, okadaic acid (OA). cPLA(2)alpha is regulated by phosphorylation and by calcium that binds to the C2 domain and induces translocation from the cytosol to membranes. In contrast, OA activates cPLA(2)alpha-induced AA release and translocation to the Golgi in macrophages without an apparent increase in calcium. Inhibitors of heat shock protein 90 (hsp90), geldanamycin, and herbimycin blocked AA release in response to OA but not to A23187, PMA, or zymosan. OA, but not the other agonists, induced activation of a cytosolic serine/threonine 54-kDa kinase (p54), which phosphorylated cPLA(2)alpha in in-gel kinase assays and was associated with cPLA(2)alpha in immunoprecipitates. Activation of the p54 kinase was inhibited by geldanamycin. The kinase coimmunoprecipitated with hsp90 in unstimulated macrophages, and OA induced its loss from hsp90, concomitant with its association with cPLA(2)alpha. The results demonstrate a role for hsp90 in regulating cPLA(2)alpha-mediated AA release that involves association of a p54 kinase with cPLA(2)alpha upon OA stimulation.

Topics: Animals; Arachidonic Acid; Benzoquinones; Calcimycin; Calcium; Carcinogens; Cytosol; Enzyme Activation; Enzyme Inhibitors; Female; Golgi Apparatus; Green Fluorescent Proteins; Group IV Phospholipases A2; HSP90 Heat-Shock Proteins; Immunoblotting; Immunoprecipitation; Ionophores; Lactams, Macrocyclic; Macrophages, Peritoneal; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinase 10; NIH 3T3 Cells; Okadaic Acid; Phosphoamino Acids; Phosphorylation; Protein Transport; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Zymosan

[structure: see text] Hsp90 has recently emerged as a promising biological target for treatment of cancer. Herbimycin A and other members of the benzoquinoid ansamycin class of natural products are known to inhibit Hsp90 activity. The total synthesis of herbimycin A was achieved from the commercially available Roche ester 1 by using allylmetals to control the stereogenic centers at C6, C7, C10, C11, and C12 and a ring-closing metathesis to control the (Z)-double bond of the (E,Z)-dienic moiety.

Topics: Benzoquinones; Carbon; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Molecular Structure; Rifabutin

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

[reaction: see text] A general and convergent route to the C(5)-C(15) subunits of the benzoquinone ansamycin antibiotics macbecin I, geldanamycin, and herbimycin A is described. Each subunit is prepared by the stepwise coupling of differentially functionalized aldehydes with a pentenyl dianion equivalent derived from diastereoselective pentynylation and regioselective reductive coupling.

Topics: Anti-Bacterial Agents; Benzoquinones; Lactams, Macrocyclic; Molecular Structure; Rifabutin; Stereoisomerism

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.

Topics: Aminobenzoates; Antibiotics, Antineoplastic; Bacterial Proteins; Benzoquinones; Cloning, Molecular; Gene Deletion; Hydroxybenzoates; Lactams, Macrocyclic; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Multigene Family; Polyketide Synthases; Quinones; Rifabutin; Sequence Analysis, DNA; Streptomyces

The induction of the heat shock response as well as its termination is autoregulated by heat shock protein activities. In this study we have investigated whether Hsp90 functional protein levels influence the characteristics and duration of the heat shock response. Treatment of cells with several benzoquinone ansamycin inhibitors of Hsp90 (geldanamycin, herbimycin A) activated a heat shock response in the absence of heat shock, as reported previously. Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock. Concurrently, the rate of Hsp synthesis and accumulation was substantially increased and prolonged. The cessation of heat shock protein synthesis did not occur until the levels of Hsp70 were substantially elevated relative to its standard threshold for autoregulation. The elevated levels of HSPS 22-28 (the small HSPS) and Hsp70 are not able to promote thermotolerance when Hsp90 activity is repressed by ansamycins; rather a suppression of thermotolerance is observed. These results suggest that a multicomponent protein chaperone complex involving both Hsp90 and Hsp70 signals the cessation of heat shock protein synthesis, the restoration of normal translation, and likely the establishment of thermotolerance. Impaired function of either component is sufficient to alter the heat shock response.

Topics: Animals; Benzoquinones; Cell Line; Cell Proliferation; DNA-Binding Proteins; Drosophila; Drosophila Proteins; Epithelial Cells; Gene Expression; Heat Shock Transcription Factors; Heat-Shock Proteins; Heat-Shock Response; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Phosphorylation; Quinones; Rifabutin; Transcription Factors

We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.

Topics: Antibiotics, Antineoplastic; Antigens, CD34; Apoptosis; Benzoquinones; Blotting, Western; Bone Marrow Cells; Cell Separation; Chlorambucil; Down-Regulation; Enzyme Inhibitors; Flow Cytometry; HSP70 Heat-Shock Proteins; Humans; Inhibitory Concentration 50; Lactams, Macrocyclic; Leukemia, Lymphocytic, Chronic, B-Cell; Polymerase Chain Reaction; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; T-Lymphocytes; Time Factors; Tumor Suppressor Protein p53; Up-Regulation; Vidarabine; ZAP-70 Protein-Tyrosine Kinase

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

Inhibition of the 90 kDa heat shock proteins (Hsp90) represents a promising new chemotherapeutic approach for the treatment of several cancers. Hsp90 is essential to the survival of cancer cells and is inhibited by members of the ansamycin family of antibiotics. In particular, the quinone-containing antibiotics geldanamycin (GDA) and herbimycin A inhibit Hsp90 function in vitro at low micromolar concentrations via interaction with an ATP binding domain. Many proteins bind ATP, and the discovery of selective Hsp90 inhibitors requires the identification of other proteins that bind GDA and may cause undesired effects. Biotinylated analogues of GDA with varying tether lengths have been synthesized to elucidate other proteins that competitively bind GDA. Analogues containing a photolabile tether have also been prepared as a complementary method for the removal of GDA-bound proteins from neutravidin-containing resin. Preliminary studies indicate several proteins other than Hsp90 are isolated with biotinylated GDA.

Topics: Adenosine Triphosphate; Benzoquinones; Biotinylation; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Photochemistry; Protein Binding; Quinones; Rifabutin

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

Src tyrosine kinase belongs to a non-receptor tyrosine kinase family and has been shown to be involved in G protein-coupled receptor desensitization and internalization. Stimulation of ovarian thecal cells with lutein-izing hormone (LH) activates adenylyl cyclase via a G protein-coupled LH receptor leading to an increase in cAMP. Subsequently, cAMP activates protein kinase A (PKA) that increases steroidogenesis. In order to evaluate the role of Src in thecal cell steroidogenesis, a pharmacological approach was utilized by treating a population of mouse ovarian theca-enriched cells (TEC) in vitro with two Src inhibitors, geldanamycin (GA) and herbimycin A (HA). Treatment of TEC with either GA or HA increased basal androstenedione secretion without alteration of cAMP. In the presence of forskolin, GA and HA treatment further increased androstenedione secretion. RT-PCR analysis of RNA from cells treated with GA for 8, 24, and 48 h revealed that GA increased cytochrome P450 17alpha-hydroxylase/lyase (CYP17) mRNA at 48 h. CYP17 promoter activity also increased after treatment of cells with GA and after co-transfection with a Src dominant negative plasmid. Inhibition of PKA using H89 blocked the effect GA and HA on androstenedione secretion. These results indicate that the pharmacological inhibitors of Src, GA and HA, tested in vitro increased thecal CYP17 promoter activity, CYP17 mRNA, and androstenedione secretion. In addition, GA and HA induced thecal androstenedione secretion may be cAMP independent but possibly requires PKA.

Topics: 3-Hydroxysteroid Dehydrogenases; Androstenedione; Animals; Benzoquinones; Colforsin; Cyclic AMP; Female; Isoquinolines; Lactams, Macrocyclic; Mice; Mice, Inbred C57BL; Ovary; Progesterone; Promoter Regions, Genetic; Protein Kinase Inhibitors; Quinones; Reverse Transcriptase Polymerase Chain Reaction; Rifabutin; RNA, Messenger; src-Family Kinases; Steroid 17-alpha-Hydroxylase; Sulfonamides; Theca Cells

Treatment (>/=6 h) of cultured bovine adrenal chromaffin cells with geldanamycin (GA) or herbimycin A (HA), an inhibitor of the 90-kDa heat-shock protein (Hsp90) family, decreased cell surface (125)I-insulin binding. The effect of GA was concentration (EC(50) = 84 nM)- and time (t(1/2) = 8.5 h)-dependent; GA (1 microM for 24 h) lowered the B(max) value of (125)I-insulin binding by 80%, without changing the K(d) value. Western blot analysis showed that GA (>/=3 h) lowered insulin receptor (IR) level by 83% (t(1/2) = 7.4 h; EC(50) = 74 nM), while raising IR precursor level by 100% (t(1/2) = 7.9 h; EC(50) = 300 nM). Pulse-label followed by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that monomeric IR precursor (~190 kDa) developed into the homodimeric IR precursor (approximately 380 kDa) and the mature alpha(2)beta(2) IR (~410 kDa) in nontreated cells, but not in GA-treated cells; in GA-treated cells, the homodimerization-incompetent form of monomeric IR precursor was degraded via endoplasmic reticulum (ER)-associated protein degradation. Immunoprecipitation followed by immunoblot analysis showed that IR precursor was associated with calnexin (CNX) to a greater extent in GA-treated cells, compared with nontreated cells. GA had no effect on IR mRNA levels and internalization rate of cell surface IRs. In GA-treated cells, insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated by 77%, with no change in IRS-1 level. Thus, inhibition of the Hsp90 family by GA or HA interrupts homodimerization of monomeric IR precursor in the ER and increases retention of monomeric IR precursor with CNX; this event retards cell surface expression of IR and attenuates insulin-induced activation of IRS-1.

Topics: Adrenal Glands; Animals; Benzoquinones; Calcium-Binding Proteins; Calnexin; Cattle; Cells, Cultured; Chromaffin Cells; Cysteine; Down-Regulation; Endoplasmic Reticulum; Enzyme Inhibitors; HSP90 Heat-Shock Proteins; Insulin Receptor Substrate Proteins; Iodine Radioisotopes; Lactams, Macrocyclic; Methionine; Phosphoproteins; Phosphorylation; Protein Conformation; Quinones; Receptor, Insulin; Receptors, Cell Surface; Rifabutin; RNA, Messenger; Sulfur Radioisotopes

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

Epidermal growth factor (EGF) stimulates the growth of various types of cells via its cell surface tyrosine kinase receptor. The EGF receptor (EGF-R) has an oncogenic potential when overexpressed in a wide range of tumor cells. Geldanamycin (GA) and herbimycin (HA), specific inhibitors of the cytosolic chaperone HSP 90 and its endoplasmic reticulum homologue GRP 94, were shown to accelerate degradation of the EGF-R and of its homologue p185(c-)(erbB-2). Here we compared the effects of GA and HA on intracellular degradation and maturation of EGF-R. By using an inhibitor of proteasomal degradation, we learned that GA, but not HA, blocks processing of newly synthesized EGF-R. The effects of GA and HA on receptor degradation are mediated by the cytosolic portion of EGF-R and could be conferred to the erythropoietin receptor (EPO-R), by employing the respective chimera. Neither HA nor GA affected stability of newly synthesized EGF-R lacking the cytosolic domain (Ex EGF-R), but GA caused intracellular retention of this mutant. Taken together, our results imply that GA has two distinct targets of action on the EGF-R, one for promoting its degradation and another for mediating its intracellular retention. Apparently, degradation of the EGF-R mediated by GA or HA requires the presence of the EGF-R cytosolic domain, whereas intracellular retention in the presence of GA is coupled to the extracellular domain of the EGF-R.

Topics: Animals; Benzoquinones; COS Cells; Enzyme Inhibitors; ErbB Receptors; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Quinones; Rifabutin; Signal Transduction

Benzoquinoid ansamycins, such as herbimycin A (HA) and geldanamycin (GA), are antibiotics that exhibit anti-tumor effects. These compounds have been shown to result in the intracellular depletion of important growth signaling molecules. Recently, GA has been shown to bind tightly to Hsp90, thereby implicating Hsp90 as a possible chaperone for those signaling molecules adversely affected by the benzoquinoid ansamycins. Here we have investigated the effects of HA and GA on the synthesis, maturation and stability of different protein tyrosine kinases. Exposing cells to either compound blocked normal maturation of the epidermal growth factor (EGF) receptor, platelet-derived growth factor (PDGF) receptor, and pp60(v-src). We show that only the nascent forms of the EGF and PDGF receptors are degraded under these conditions. Once the newly synthesized receptors had been translocated into the endoplasmic reticulum membrane, addition of the drugs no longer affected their stability. For the cytoplasmic tyrosine kinase, pp60(v-src), both the nascent as well as the mature forms of the protein were degraded in cells treated with the drugs. We discuss these observations as they pertain to the possible role of Hsp90 as a substrate-specific molecular chaperone, perhaps involved in the maturation and/or stability of proteins important for growth control.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Cell Division; Cell Line; Enzyme Stability; ErbB Receptors; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Molecular Chaperones; Oncogene Protein pp60(v-src); Protein Processing, Post-Translational; Quinones; Receptor Protein-Tyrosine Kinases; Receptors, Platelet-Derived Growth Factor; Rifabutin

It is important to understand the mechanisms by which phosphorylation-dependent events play a role in regulation of apoptosis in toxicant-metabolizing organs such as the kidney. Our previous work demonstrated that the toxicant and phosphatase inhibitor okadaic acid induces apoptosis of renal epithelial cells via a mechanism that appears to involve the modulation of c-raf-1, p38 kinase, and extracellular regulatory kinase (ERK) cascades. Using the benzoquinone ansamycins and tyrosine kinase inhibitors geldanamycin and herbimycin A, we examined the contribution of tyrosine phosphorylation and c-raf-1 activities to okadaic acid-induced apoptosis. In this report we show that both geldanamycin and herbimycin A protected NRK-52E cells from okadaic acid-induced apoptosis, abrogated the overall okadaic acid-induced kinase activation, and specifically inhibited activation of p38 kinase by okadaic acid. Herbimycin A and geldanamycin also abrogated okadaic-acid induced morphologic changes such as cell rounding and cell membrane blebbing. Herbimycin A and geldanamycin caused pronounced cell spreading, cell flattening, and a decrease in okadaic acid-induced loss of actin filaments. Interestingly, herbimycin A showed more potent inhibitory effect than geldanamycin, and herbimycin A alone inhibited okadaic acid-induced movement of p38 kinase into the cytosol. These results imply that decreased p38 activity and its cytosolic translocation together with cellular resistance to cytoskeletal disorganization may play a significant role in resistance to phosphorylation-dependent apoptosis. Furthermore, the results imply that changes in cell shape may partially modulate the observed alterations in signal transduction induced by okadaic acid.

Topics: Actins; Activating Transcription Factor 2; Animals; Apoptosis; Benzoquinones; Cell Line; Cell Size; Chromatin; Cyclic AMP Response Element-Binding Protein; Cytosol; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Imidazoles; Kidney; Lactams, Macrocyclic; Mitogen-Activated Protein Kinases; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-raf; Pyridines; Quinones; Rats; Rifabutin; Signal Transduction; Transcription Factors; Transfection

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2+ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC50 2 nM) and MCF7 (IC50 20 nM), while OVCAR3 was only moderately sensitive (IC50 2 microM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced.

Topics: Adenocarcinoma; Benzoquinones; Breast Neoplasms; Cell Division; Enzyme Activation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Lactams, Macrocyclic; Ligands; Neoplasm Proteins; Ovarian Neoplasms; Protein-Tyrosine Kinases; Quinones; Receptor, ErbB-2; Rifabutin; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Previous studies have shown that in rodent myogenic cells and in the hearts of transgenic mice in which heat shock protein expression is increased there is a marked tolerance to ischemic/reperfusion injury. Furthermore, a recent study has shown that the benzoquinoid ansamycin antibiotic and tyrosine kinase inhibitor, herbimycin A, is capable of inducing the expression of heat shock proteins in fibroblasts. Our intention, in the present study, was to investigate if exposure of rat cardiomyocytes and the myogenic cell line H9c2 to herbimycin A would induce these proteins and, thus, confer protection against ischemic stress. For this purpose, we exposed both rat neonatal cardiomyocytes and H9c2 cells to herbimycin A and another related benzoquinoid ansamycin antibiotic, geldanamycin. We found that cells exposed to these compounds overexpressed heat shock proteins and are also rendered more tolerant to simulated ischemia as measured by the release of cytoplasmic enzymes. In addition, we found that the mechanism of induction of heat shock proteins by these compounds is similar, if not identical, to that of a heat shock (42 degrees C, 60 min). These results suggest that these benzoquinoid ansamycin antibiotics, or closely related analogues, may offer a pharmacological means of increasing the level of heat shock proteins in cardiac tissue and thus protect the heart against ischemic/reperfusion injury.

Topics: Animals; Animals, Newborn; Benzoquinones; Cell Line; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation; Heat Shock Transcription Factors; Heat-Shock Proteins; Hot Temperature; Lactams, Macrocyclic; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Staurosporine; Transcription Factors; Transcription, Genetic; Vanadates

Renaturation of thermally denatured firefly luciferase in rabbit reticulocyte lysate (RRL) requires hsp90, hsc70, and other as yet unidentified RRL components [Schumacher, R.J., et al. (1994) J. Biol. Chem. 269, 9493-9499]. Benzoquinonoid ansamycins (BAs) have recently been shown to specifically bind hsp90 and inhibit its function. In this report, we present data that indicate BAs are specific inhibitors of hsp90 function. The effects of the BA geldanamycin (GA) on the kinetics of the luciferase renaturation in RRL were examined to gain insight into the mechanism by which GA inhibits the function of the hsp90 chaperone machinery. Chaperone-mediated renaturation of luciferase obeyed Michaelis-Menten kinetics. The GA inhibited luciferase renaturation uncompetitively with respect to ATP concentration and noncompetitively with respect to luciferase concentration, indicating that GA binds after the binding of ATP and that it binds to both the hsp90 chaperone machine/ATP complex and the hsp90 chaperone machine/ATP/luciferase complex. GA markedly decreased the Kapp of the hsp90 chaperone machine for ATP, suggesting that GA increases the binding affinity of the hsp90 chaperone machinery for ATP or it slows the rate of ATP hydrolysis. Consistent with the notion that GA specifically binds hsp90 and inhibits its function, addition of hsp90, but not hsc70, p60, or p23, reversed GA-induced inhibition of luciferase renaturation in RRL. Hsp90, hsc70, and the hsp cohorts p60, p48, and p23 were coimmunoprecipitated with luciferase from RRL. GA increased the steady-state levels of luciferase associated with hsp90/hsp70 chaperone machine complexes that contain p60 and blocked the association of the hsp90 cohort p23 with chaperone-bound luciferase. The data suggest that the function of the hsp90 chaperone machinery is not specific to its previously described interaction with steroid hormone receptors, and that it carries out some more generalized function in vivo.

Topics: Animals; Benzoquinones; Cell-Free System; Coleoptera; Enzyme Inhibitors; HSP90 Heat-Shock Proteins; In Vitro Techniques; Kinetics; Lactams, Macrocyclic; Luciferases; Molecular Chaperones; Protein Conformation; Protein Denaturation; Protein-Tyrosine Kinases; Quinones; Rabbits; Reticulocytes; Rifabutin

Nitric oxide (NO) modulates the activity of a number of cell types, but little is known about its possible role in bone metabolism. In the present study we demonstrate that freshly isolated murine osteoblasts and an osteoblastic cell line express NO-synthase mRNA and release NO when stimulated with IL-1 or LPS, thus confirming the results of some recent reports using human and rat osteoblast-like cells. Synergistic effects were found between IL-1 and LPS or TNF. Enzyme induction was blocked by dexamethasone and IL-4. 1,25-dihydroxyvitamin D3 did not modify basal NO synthesis, but it markedly increased the cytokine-induced NO release. M-CSF, GM-CSF, IL-3, LIF, PTH, estradiol and calcitonin did not show significant effects on NO synthesis. NOS induction was blocked by various tyrosine-kinase inhibitors, geldanamycin and herbimycin A being the most potent. These results suggest that endogenous NO might participate in the regulation of bone remodeling at the local level, and may mediate some effects of vitamin D on bone. NO has recently been reported to inhibit osteoclastic bone resorption. The release of NO induced by bone-stimulating factors such as IL-1 may represent a protective mechanism helping to avoid excess resorption and preserve bone integrity in inflammatory conditions.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Benzoquinones; Bone Remodeling; Cell Line; Cytokines; Dexamethasone; Enzyme Induction; Estradiol; Gene Expression Regulation; Hematopoietic Cell Growth Factors; Lactams, Macrocyclic; Lipopolysaccharides; Mice; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Osteoblasts; Parathyroid Hormone; Peptide Fragments; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Sodium Nitrite; Teriparatide; Vitamin D

The erbB-2 oncogene encodes a transmembrane protein tyrosine kinase which plays a pivotal role in signal transduction and has been implicated when overexpressed in breast, ovarian, and gastric cancers. Naturally occurring benzoquinoid ansamycin antibiotics herbimycin A, geldanamycin (GDM), and dihydrogeldanamycin were found to potently deplete p185, the erbB-2 oncoprotein, in human breast cancer SKBR-3 cells in culture. Chemistry efforts to modify selectively the quinoid moiety of GDM afforded derivatives with greater potency in vitro and in vivo. Analogs demonstrated inhibition of p185 phosphotyrosine in cell culture and in vivo after systemic drug administration to nu/nu nude mice bearing Fisher rat embryo cells transfected with human erbB-2 (FRE/erbB-2). Specifically, dosed intraperitoneally at 100 mg/kg, 17-(allylamino)-17-demethoxygeldanamycin and other 17-amino analogs were effective at reducing p185 phosphotyrosine in subcutaneous flank FRE/erbB-2 tumors. Modifications to the 17-19-positions of the quinone ring revealed a broad structure-activity relationship in vitro.

Topics: Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Benzoquinones; Breast Neoplasms; Dose-Response Relationship, Drug; Female; Humans; Lactams, Macrocyclic; Mice; Mice, Nude; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Receptor, ErbB-2; Rifabutin; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured

Geldanamycin is an antibiotic that preferentially inhibits G1/S transition and causes G2/M arrest in human leukemia HL-60 cells. With it, we selectively inhibited recombinant Src tyrosine kinase without significantly inhibiting protein kinase A. The perturbation of cell cycling by geldanamycin was accompanied by marked suppression of c-MYC expression. In contrast to this, pRB expression was remarkably enhanced by geldanamycin. In the untreated HL-60 cells, c-MYC was apparently enriched in nuclear matrix preparation, and significant amounts of hyperphosphorylated pRB, p70 and p40 proteins were observed to associated with the nuclear matrix. The amounts of these proteins associated with the nuclear matrix, however, were markedly decreased by treatment with geldanamycin. This finding suggests that the association of c-MYC, hyperphosphorylated pRB, p70 and p40 proteins with the nuclear matrix is essential in cell cycling, especially in G1/S and G2/M progressions, and that this association is a part of signal transduction pathway in Src kinase activation.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Cell Cycle; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation; Gene Products, tax; Genes, myc; HL-60 Cells; Humans; Lactams, Macrocyclic; Nuclear Matrix; Nuclear Proteins; Quinones; Retinoblastoma Protein; Rifabutin; src-Family Kinases; Tumor Cells, Cultured

Herbimycin A, a benzoquinoid ansamycin, is widely used as an inhibitor of tyrosine kinases. We have examined the effects of herbimycin A and several analogues on p185, the tyrosine kinase encoded by the erbB2 gene in human breast cancer cells. Exposure to 0.35 microM herbimycin A reduced tyrosine phosphorylation of p185 in SKBr3 cells by 80% after 2 h, and the p185 protein level was reduced by 90% after 6 h. The reduction of p185 resulted primarily from increased degradation of p185; cellular protein synthesis was reduced only 16% in SKBr3 cells treated with herbimycin A, RNA synthesis was inhibited only 10%, and erbB2 mRNA levels were unchanged. Examination of the major cellular glycoproteins indicated that most glycoproteins were unaffected under conditions that substantially depleted p185. Studies with cell lines transfected with erbB2 containing defined deletions indicated that susceptibility to the depletion of p185 by herbimycin and its analogues required the domain encoded by amino acids 751-971. The benzoquinoid ansamycins therefore initiate a process of specific degradation of tyrosine kinases by a mechanism that remains unknown.

Topics: Anti-Bacterial Agents; Benzoquinones; Breast Neoplasms; ErbB Receptors; Female; Humans; Lactams, Macrocyclic; Phosphorylation; Proto-Oncogene Proteins; Quinones; Receptor, ErbB-2; Rifabutin; Transfection; Tumor Cells, Cultured

Several benzoquinoid ansamycins, e.g., herbimycin A and geldanamycin, have been widely used as inhibitors of tyrosine kinases. We recently reported that exposure to herbimycin A and several analogs depletes the erbB2 gene product p185 in human breast cancer cells. In order to explore the mechanism of this specific degradation of p185, a biologically active ansamycin incorporating a photoaffinity label was synthesized. This compound, CP202509, specifically bound to a 100 kD protein (p100) in intact SKBr3 cells and in fibroblasts transfected with the c-erbB2 or v-src oncogenes. Binding of other ansamycin analogs to p100, as measured indirectly by their ability to inhibit CP202509 binding, correlated with their ability to lower p185 protein and phosphotyrosine in SKBr3 cells. These results suggest that the ansamycins may deplete tyrosine kinases through binding to this protein.

Topics: Affinity Labels; Anti-Bacterial Agents; Benzoquinones; ErbB Receptors; Humans; In Vitro Techniques; Lactams, Macrocyclic; Macromolecular Substances; Molecular Weight; Protein Binding; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Quinones; Receptor, ErbB-2; Rifabutin; Tumor Cells, Cultured

We recently reported that multidrug-resistant, P-170 glycoprotein-positive, Adriamycin-selected, human breast tumor (MCF7/ADRR) cells were resistant to the benzoquinonoid ansamycin antibiotics geldanamycin (GL) and herbimycin A (HA) and that significantly fewer hydroxyl radicals were formed in resistant cells. We have carried out additional studies to define the mechanisms of cytotoxicity of and resistance to GL and HA, by directly examining the interactions of these drugs with P-170 glycoprotein using photoaffinity labeling. We found that both GL and HA inhibited binding of azidopine to P-170 glycoprotein in a dose-dependent manner. We have developed a 10-fold GL-resistant cell line (MCF7/GLR) by continuous drug exposure. Our studies indicated no significant differences in free radical formation between wild-type MCF7 cells and MCF7/GLR cells. Uptake and efflux studies indicated a small decrease in the GL accumulation but no difference in the efflux of GL in these cells. Verapamil had no effect on cellular accumulation of GL in wild-type MCF7 cells or MCF7/GLR cells. Verapamil significantly increased the accumulation of GL in MCF7/ADRR cells and enhanced GL cytotoxicity 12-fold, suggesting that GL interacted with the P-170 glycoprotein. Using reverse transcription-polymerase chain reaction, we found no expression of the mdr1 gene; however, expression of the multidrug resistance-associated protein was about 2-fold higher in MCF7/GLR cells. Taken together, these studies indicate that the mechanisms of GL resistance are multifactorial. Although decreased free radical formation may not play a significant role in low levels of GL resistance, e.g., in MCF7/GLR cells, both overexpression of mdr1 and decreased free radical formation contribute to GL resistance in highly resistant cells such as MCF7/ADRR cells.

Topics: Affinity Labels; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzoquinones; Biological Transport; Breast Neoplasms; Drug Resistance, Multiple; Free Radicals; Glutathione; Glutathione Peroxidase; Glutathione Transferase; Humans; Lactams, Macrocyclic; Polymerase Chain Reaction; Quinones; Rifabutin; Tumor Cells, Cultured

The benzoquinonoid ansamycin antibiotics, geldanamycin and herbimycin A, are potent cytotoxins against tumor cells in vitro. We have examined the mechanism of their in vitro cytotoxicity against human breast adenocarcinoma (MCF-7) cells and we have found that multidrug-resistant MCF-7/ADRR cells that exhibit the MDR phenotype and the overexpression of P-170-glycoprotein, were cross-resistant to geldanamycin and herbimycin A. Verapamil, which binds competitively with P-170-glycoprotein, enhanced geldanamycin cytotoxicity 12-fold only in resistant cells, suggesting that geldanamycin may interact with the drug efflux protein. Geldanamycin and herbimycin A, like adriamycin, were reductively activated by the NADPH-cytochrome P450-reductase and formed reactive .OH. The formation of .OH was significantly lower in resistant cells. In contrast to adriamycin, the formation of .OH was unaffected by the addition of DNA, indicating that a DNA-complexed drug was redoxactive and may, therefore, may be more effective in killing tumor cells at the DNA level. These observations indicate that both the decreased free radical formation and interactions with P170 glycoprotein may be important in geldanamycin and herbimycin A resistance in multidrug resistant human breast tumor cells.

Topics: Adenocarcinoma; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzoquinones; Breast Neoplasms; Doxorubicin; Drug Resistance, Multiple; Free Radicals; Humans; Hydroxyl Radical; Lactams, Macrocyclic; NADPH-Ferrihemoprotein Reductase; Quinones; Rifabutin; Tumor Cells, Cultured; Verapamil

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

Several protein kinase inhibitors (PKIs) were investigated for their effects on IL-1 beta, TNF alpha and PMA-induced IL-8 production from human umbilical vein endothelial cells (HUVEC). IL-1 beta (ED50 0.07 ng/ml), TNF alpha (ED50 100 ng/ml) and PMA (ED50 20 ng/ml) induced IL-8 production that could be detected as early as 2 h following stimulation. Staurosporine, a potent but non-specific inhibitor of protein kinases, inhibited PMA-induced (IC50 2 nM) but not IL-1 beta or TNF alpha (IC50 > 200 nM) induced IL-8 production. Neither the cAMP-dependent PKI, KT5720, nor the tyrosine PKIs, genistein, tyrphostin (1-100 microM) or lavendustin A (0.0001-1 microM), inhibited IL-8 production elicited by IL-1 beta. However, the macrolide protein kinase inhibitor geldanamycin (IC50 = 30 nM), but not the closely related analog herbimycin A (5-500 nM), inhibited IL-8 production by 60%. Northern blot analysis of IL-8 mRNA revealed that staurosporine suppressed mRNA increase following stimulation by PMA but not by IL-1. It is proposed that a novel protein kinase susceptible to geldanamycin inhibition may be involved in IL-1-mediated signal transduction.

Topics: Alkaloids; Benzoquinones; Carbazoles; Cells, Cultured; Endothelium, Vascular; Humans; Indoles; Interleukin-1; Interleukin-8; Lactams, Macrocyclic; Protein Kinase Inhibitors; Pyrroles; Quinones; Recombinant Proteins; Rifabutin; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Umbilical Veins

The benzoquinonoid ansamycin antibiotics herbimycin A and geldanamycin have been shown to reverse the oncogenic phenotype of pp60v-src transformed cells as well as induce differentiation in a number of in vitro model systems, reportedly due to their inhibition of src family protein tyrosine kinases. We now report that these agents are potent cytotoxins in vitro against a panel of highly malignant human tumor cell lines possessing primitive neural features. Proliferation and/or survival of fibroblasts, primary neuronal cultures, and several leukemia cell lines are unaffected at concentrations resulting in greater than 99% cell loss in sensitive lines. The tumorigenicity in nude mice of sensitive cell lines can also be markedly reduced by either systemic or topical administration of these agents without apparent toxicity to the whole animal. The cytocidal action of these ansamycins is initiated very rapidly, is irreversible, and is clearly distinct from the delayed inhibition of src family kinases that has been reported previously. Due to their potency, relative selectivity, and novel mechanism(s) of action, these drugs could prove clinically useful in the therapy of a number of human cancers of neural derivation.

Topics: 3T3 Cells; Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Division; Cell Line; Cell Line, Transformed; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Kinetics; Lactams, Macrocyclic; Mice; Oncogene Protein pp60(v-src); Protein-Tyrosine Kinases; Quinones; Rifabutin; Transfection

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