leupeptins and geldanamycin

In this work we tried to identify mechanisms that could explain how chemical inhibition of heat-shock protein 90 reduces nerve growth factor signaling in rat pheochromocytoma PC12 cells. Geldanamycin is an antibiotic originally discovered based on its ability to bind heat-shock protein 90. This interaction can lead to the disruption of heat-shock protein 90-containing multimolecular complexes. It can also induce the inhibition or even degradation of partner proteins dissociated from the 90 kDa chaperone and, eventually, can cause apoptosis, for instance, in PC12 cells. Before the onset of initial apoptotic events, however, a marked decrease in the activity of extracellular signal-regulated kinases ERK 1/2 and protein kinase B/Akt can be observed together with reduced expression of the high affinity nerve growth factor receptor, tropomyosine-related kinase, TrkA, in this cell type. The proteasome inhibitor MG-132 can effectively counteract the geldanamycin-induced reduction of TrkA expression and it can render TrkA and ERK1/2 phosphorylation but not that of protein kinase B/Akt by nerve growth factor again inducible. We have found altered intracellular distribution of TrkA in geldanamycin-treated and proteasome-inhibited PC12 cells that may, at least from the viewpoint of protein localization explain why nerve growth factor remains without effect on protein kinase B/Akt. The lack of protein kinase B/Akt stimulation by nerve growth factor in turn reveals why nerve growth factor treatment cannot save PC12 cells from geldanamycin-induced programmed cell death. Our observations can help to better understand the mechanism of action of geldanamycin, a compound with strong human therapeutical potential.

Topics: Animals; Benzoquinones; Blotting, Western; DNA Fragmentation; Enzyme Inhibitors; Immunoprecipitation; Lactams, Macrocyclic; Leupeptins; Microscopy, Confocal; Nerve Growth Factor; Neurons; PC12 Cells; Proteasome Inhibitors; Rats; Receptor, trkA; Signal Transduction

Rab7 plays an important role in regulating endocytic traffic. In view of an emerging role of membrane traffic in signaling and diseases, we have examined the possible role of Rab7 in oncogenesis. The role of Rab7 was investigated using shRNA-mediated knockdown in A431 and MCF7 cancer cells. To our surprise, Rab7 knockdown effectively suppressed anchorage-independent growth of cancer cells in soft agar. Anoikis (matrix-detachment triggered apoptosis) was enhanced, while the level of phosphorylated (active) Akt (which is a key survival factor) was significantly reduced. Also intriguing was the observation that EGFR and Her2 levels were significantly reduced when Rab7 was knocked-down. More robust reduction of EGFR and Her2 levels was observed when knocked-down cells were treated with HSP90 inhibitor geldanamycin (GA). Low concentration of GA (50-100 nm)-induced apoptosis of the Rab7 knocked-down cells but not control cells, suggesting that Rab7 and HSP90 together contribute to the optimal stability of EGFR and Her2 as well as to protect cancer cells from apoptosis. Rab7 seems to protect EGFR and Her2 from proteosome-mediated degradation. These results suggest that Rab7 is likely involved in protecting EGFR and Her2 from being degraded by the proteosome and in maintaining optimal Akt survival signal (especially during cell detachment or when HSP90 is inhibited). Rab7 is potentially a novel target for combinatory therapy with Hsp90 inhibitors.

Topics: Animals; Anoikis; Apoptosis; Benzoquinones; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; ErbB Receptors; Genetic Vectors; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lentivirus; Leupeptins; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Stability; Proto-Oncogene Proteins c-akt; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Receptor, ErbB-2; RNA Interference; Signal Transduction; Time Factors; Transduction, Genetic; Tumor Burden

Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein-dependent retrograde trafficking to a juxtanuclear location. We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation. These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.

Topics: Acetylation; Benzoquinones; Cell Extracts; Cell Line; Cell Nucleus; Epithelial Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Lactams, Macrocyclic; Leupeptins; Pigment Epithelium of Eye; Protein Structure, Quaternary; Tubulin; Ubiquitination

The underlying cause of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder, remains unknown. However, there is strong evidence that one pathophysiological mechanism, toxic protein misfolding and/or aggregation, may trigger motor neuron dysfunction and loss. Since the clinical and pathological features of sporadic and familial ALS are indistinguishable, all forms of the disease may be better understood and ultimately treated by studying pathogenesis and therapy in models expressing mutant forms of SOD1. We developed a cellular model in which cell death depended on the expression of G93A-SOD1, a mutant form of superoxide dismutase found in familial ALS patients that produces toxic protein aggregates. This cellular model was optimized for high throughput screening to identify protective compounds from a >50,000 member chemical library. Three novel chemical scaffolds were selected for further study following screen implementation, counter-screening and secondary testing, including studies with purchased analogs. All three scaffolds blocked SOD1 aggregation in high content screening assays and data on the optimization and further characterization of these compounds will be reported separately. These data suggest that optimization of these chemicals scaffolds may produce therapeutic candidates for ALS patients.

Topics: Amyotrophic Lateral Sclerosis; Animals; Benzoquinones; Cell Death; Cytoprotection; Drug Design; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Humans; Lactams, Macrocyclic; Leupeptins; Macrolides; Mutant Proteins; PC12 Cells; Rats; Recombinant Fusion Proteins; Small Molecule Libraries; Superoxide Dismutase

Glucocorticoid (GC) hormones are secreted from the adrenal gland in a characteristic pulsatile pattern. This ultradian secretory activity exhibits remarkable plasticity, with distinct changes in response to both physiological and stressful stimuli in humans and experimental animals. It is therefore important to understand how the pattern of GC exposure regulates intracellular signaling through the GC receptor (GR). We have previously shown that each pulse of ligand initiates rapid, transient GR activation in several physiologically relevant and functionally diverse target cell types. Using chromatin immunoprecipitation assays, we detect cyclical shifts in the net equilibrium position of GR association with regulatory elements of GC-target genes and have investigated in detail the mechanism of pulsatile transcriptional regulation of the GC-induced Period 1 gene. Transient recruitment of the histone acetyl transferase complex cAMP response element-binding protein (CREB) binding protein (CBP)/p300 is found to precisely track the ultradian hormone rhythm, resulting in transient localized net changes in lysine acetylation at GC-regulatory regions after each pulse. Pulsatile changes in histone H4 acetylation and concomitant recruitment of RNA polymerase 2 precede ultradian bursts of Period 1 gene transcription. Finally, we report the crucial underlying role of the intranuclear heat shock protein 90 molecular chaperone complex in pulsatile GR regulation. Pharmacological interference of heat shock protein 90 (HSP90) with geldanamycin during the intranuclear chaperone cycle completely ablated GR's cyclical activity, cyclical cAMP response element-binding protein (CREB) binding protein (CBP)/p300 recruitment, and the associated cyclical acetylation at the promoter region. These data imply a key role for an intact nuclear chaperone cycle in cyclical transcriptional responses, regulated in time by the pattern of pulsatile hormone.

Topics: Acetylation; Activity Cycles; Animals; Benzoquinones; Cell Line; Cell Nucleus; Chromatin Immunoprecipitation; Corticosterone; CREB-Binding Protein; Histones; HSP90 Heat-Shock Proteins; Humans; Hydrocortisone; Lactams, Macrocyclic; Leupeptins; Ligands; Mice; p300-CBP Transcription Factors; Period Circadian Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Rats; Receptors, Glucocorticoid; Regulatory Elements, Transcriptional; RNA Polymerase II; RNA, Messenger; Transcription, Genetic

Heat shock transcription factor A2 (HsfA2) is induced under environmental stress and regulates transcription of various defense-related genes. Thus HsfA2 plays an important role in induction of defenses against different types of environmental stress, but its mode of regulation remains unknown. To clarify the signal transduction pathway involved in the regulation of HsfA2 expression, we investigated the effect of MG132, a 26S proteasome inhibitor, or geldanamycin (GDA), a heat shock protein 90 (Hsp90) inhibitor, on the transcription of HsfA2 and its targets, Hsp18.1-CI and ascorbate peroxidase 2 (Apx2), in Arabidopsis T87 cells. The levels of transcripts were significantly increased by treatment with MG132 or GDA. Overexpression of a dexamethazone-inducible dominant-negative form of Hsp90.2 in Arabidopsis plants caused significant expression of HsfA2 and its target gene on treatment with the compound. Treatment with MG132 or GDA had no effect on intracellular levels of reactive oxygen species (ROS). Interestingly, the levels of polyubiquitinated proteins as well as the levels of HsfA2 transcript were rapidly increased under oxidative stress derived from treatment with H2O2 or methylviologen, while they were completely suppressed by pre-treatment with ascorbate, a scavenger of ROS, under oxidative stress. The present findings suggest that the inhibition of 26S proteasome function and/or Hsp90 activity is involved in the induction of HsfA2 expression in response to oxidative stress.

Topics: Arabidopsis; Arabidopsis Proteins; Ascorbic Acid; Benzoquinones; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Gene Expression Regulation, Plant; Heat Shock Transcription Factors; Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Hydrogen Peroxide; Lactams, Macrocyclic; Leupeptins; Oxidative Stress; Paraquat; Plant Proteins; Proteasome Endopeptidase Complex; Reactive Oxygen Species; Signal Transduction; Transcription Factors; Ubiquitinated Proteins

The heat shock protein HSP90 serves as a chaperone for receptor protein kinases, steroid receptors, and other intracellular signaling molecules. Targeting HSP90 with ansamycin antibiotics disrupts the normal processing of clients of the HSP90 complex. The platelet-derived growth factor receptor alpha (PDGFRalpha) is a tyrosine kinase receptor up-regulated and activated in several malignancies. Here we show that the PDGFRalpha forms a complex with HSP90 and the co-chaperone cdc37 in ovarian, glioblastoma, and lung cancer cells. Treatment of cancer cell lines expressing the PDGFRalpha with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) promotes degradation of the receptor. Likewise, phospho-Akt, a downstream target, is degraded after treatment with 17-AAG. In contrast, PDGFRalpha expression is not affected by 17-AAG in normal human smooth muscle cells or 3T3 fibroblasts. PDGFRalpha degradation by 17-AAG is inhibited by the proteasome inhibitor MG132. High molecular weight, ubiquitinated forms of the receptor are detected in cells treated with 17-AAG and MG132. Degradation of the receptor is also inhibited by a specific neutralizing antibody to the PDGFRalpha but not by a neutralizing antibody to PDGF or by imatinib mesylate (Gleevec). Ultimately, PDGFRalpha-mediated cell proliferation is inhibited by 17-AAG. These results show that 17-AAG promotes PDGFRalpha degradation selectively in transformed cells. Thus, not only mutated tyrosine kinases but also overexpressed receptors in cancer cells can be targeted by 17-AAG.

Topics: 3T3 Cells; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Benzamides; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Humans; Imatinib Mesylate; Lactams, Macrocyclic; Leupeptins; Mice; Piperazines; Platelet-Derived Growth Factor; Pyrimidines; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction

The objective of this study was to investigate the role of heat shock protein 90 (hsp90) and the proteasome in regulating aryl hydrocarbon receptor (AhR) activation and cytochrome P450 1A (Cyp1A) protein expression in rainbow trout (Oncorhynchus mykiss). We exposed trout hepatocytes in primary culture to the AhR agonist beta-napthoflavone (betaNF; 10(-6) M) and examined AhR and Cyp1A expression. betaNF-induced a significant temporal accumulation of AhR and Cyp1A1 mRNA abundance in trout hepatocytes. This transcript response was followed by a significantly higher AhR and Cyp1A protein expression. Exposure to geldanamycin (GA; 1000 ng mL(-1)), a benzoquinone ansamycin antibiotic used to inhibit hsp90 function, significantly reduced ( approximately 70%) betaNF-induced Cyp1A protein expression. Also, exposure to the proteasomal inhibitor MG-132 (50 microM) completely abolished betaNF-induced Cyp1A protein expression in trout hepatocytes. In addition, MG-132 treatment further enhanced the GA-mediated suppression of the Cyp1A response. The effect of MG-132 on Cyp1A response corresponded with a significant inhibition of BNF-mediated AhR mRNA abundance, but not protein content. Altogether our results suggest a betaNF-mediated autoregulation of AhR content in trout hepatocytes. We propose a key role for hsp90 and the proteasome in this ligand-mediated AhR regulation and Cyp1A response.

Topics: Animals; Benzoquinones; beta-Naphthoflavone; Cells, Cultured; Cytochrome P-450 CYP1A1; Gene Expression Regulation, Enzymologic; Hepatocytes; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Leupeptins; Oncorhynchus mykiss; Proteasome Endopeptidase Complex; Receptors, Aryl Hydrocarbon; RNA, Messenger; Signal Transduction

The 90-kDa heat shock protein (HSP90) normally functions as a molecular chaperone participating in folding and stabilizing newly synthesized proteins, and refolding denatured proteins. The HSP90 inhibitor geldanamycin (GA) occupies the ATP/ADP binding pocket of HSP90 so inhibits its chaperone activity and causes subsequent degradation of HSP90 client proteins by proteasomes. Here we show that GA reduces the level of endogenous c-Jun in human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and that this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids in the absence of GA increases the level of endogenous c-Jun protein, but has no obvious affect on c-Jun mRNA levels. We also showed that HSP90 prolongs the half-life of c-Jun by stabilizing the protein; the proteasome inhibitor N-benzoyloxy-carbonyl (Z)-Leu-Leu-leucinal (MG132) blocks the degradation of c-Jun promoted by GA. Transfection of HSP90 plasmids did not obviously alter phosphorylation of c-Jun, and a Jun-2 luciferase activity assay indicated that over-expression of HSP90 elevated the total protein activity of c-Jun in HEK293 cells. All our evidence indicates that HSP90 stabilizes c-Jun protein, and so increases the total activity of c-Jun in HEK293 cells.

Topics: Benzoquinones; Cell Line; Half-Life; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-jun; Thermodynamics

Studies have shown that zebrafish and rodent aryl hydrocarbon receptors (AHRs) are degraded following ligand exposure and that reductions in AHR protein can impact growth and development in vivo. The current study was designed to evaluate the degradation of the AHR in seven human cell lines that were derived from various carcinomas or from normal tissue. Consistent with studies in other species, the results show that the human AHR (hAHR) is degraded in a ligand dependent manner following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin for up to 16h. However, the hAHRs expressed in the various cell lines show differences in the time course and magnitude of degradation. The ligand dependent degradation is completely blocked by treatment with the proteasome inhibitor, MG-132. Ligand-independent degradation of the hAHR following exposure to geldanamycin (GA) is also observed in the different cell lines, although the magnitude of hAHR degradation is also is variable. These findings are significant since they indicate that ligand-dependent and independent degradation of the AHR is a conserved aspect of this signal transduction cascade from fish to human. In addition, the study identifies several cell lines that may provide novel models to further assess the regulation of AHR-mediated signaling and degradation of the human AHR.

Topics: Benzoquinones; Biodegradation, Environmental; Cell Culture Techniques; Cell Line; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Environmental Pollutants; Humans; Lactams, Macrocyclic; Leupeptins; Ligands; Models, Biological; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Signal Transduction; Time Factors

The androgen receptor (AR) is a member of the nuclear receptor superfamily that requires the action of molecular chaperones for folding and hormone binding. C-terminal Hsp-interacting protein (Chip) is a cochaperone that interacts with Hsp70 and Hsp90 molecular chaperones via a tetratricopeptide domain and inhibits chaperone-dependent protein folding in vitro. Chip also stimulates protein degradation by acting as an E3 ubiquitin ligase via a modified ring finger domain called a U box. We analyzed whether Chip affected AR levels using a transient transfection strategy. Chip overexpression led to a large decrease in AR steady state levels and increased levels of AR ubiquitinylation. However, Chip effects were not fully reversed by proteasome inhibitors, suggesting that mechanisms alternative to or in addition to proteasome-mediated degradation were involved. This hypothesis was supported by the finding that Chip overexpression reduced the rate of AR degradation, consistent with an effect on AR folding, perhaps leading to aggregation. The possibility that Chip affected AR folding was further supported by the finding that the effects of exogenous Chip were reproduced by a mutant lacking the U box. These results are discussed in terms of the role played by molecular chaperones in AR biogenesis.

Topics: Acetylcysteine; Benzoquinones; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dihydrotestosterone; Gene Expression; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Ligases; Male; Molecular Chaperones; Multienzyme Complexes; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Quinones; Receptors, Androgen; Receptors, Estrogen; Receptors, Glucocorticoid; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-Protein Ligases

Changes in the concentration or subcellular location of the key proteins involved in signal transduction pathways have been shown to impact gene regulation. Studies were designed to evaluate the relationship between aryl hydrocarbon receptor (AHR) localization, stability, and gene regulation in a defined system where the endogenous AHR protein could be evaluated. The findings indicate that treatment of cells with geldanamycin (GA) or MG-132 (an inhibitor of the 26S proteasome) results in nuclear translocation of the endogenous AHR in both human HepG2 and murine Hepa-1 cells without induction of endogenous CYP1A1 protein. Exposure to GA resulted in the degradation of AHR by >90% in the nucleus via the 26S proteasome. Importantly, the reduced level of AHR resulted in a 50% reduction in the maximal level of CYP1A1 induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In all treatments the concentration of the AHR nuclear translocator (ARNT) protein was unchanged and had no impact on the localization of the AHR. Thus, ligand-independent translocation of the AHR to the nucleus was not sufficient to induce CYP1A1 in the absence of ligand, but reductions in the level of the endogenous AHR protein pool shifted the dose-response curve for TCDD to the right.

Topics: Animals; Antineoplastic Agents; Aryl Hydrocarbon Receptor Nuclear Translocator; Benzoquinones; Cell Nucleus; Cytochrome P-450 CYP1A1; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation; Hepatocytes; Humans; Lactams, Macrocyclic; Leupeptins; Ligands; Mice; Polychlorinated Dibenzodioxins; Quinones; Receptors, Aryl Hydrocarbon; Subcellular Fractions; Teratogens; Transcription Factors; Tumor Cells, Cultured

The caspase 8 homologue FLICE-inhibitory protein (cFLIP) is a potent negative regulator of death receptor-induced apoptosis. We found that cFLIP can be upregulated in some cell lines under critical involvement of the NF-kappaB pathway, but NF-kappaB activation was clearly not sufficient for cFLIP induction in all cell lines. Treatment of SV80 cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) or geldanamycin, a drug interfering with tumor necrosis factor (TNF)-induced NF-kappaB activation, inhibited TNF-induced upregulation of cFLIP. Overexpression of a nondegradable IkappaBalpha mutant (IkappaBalpha-SR) or lack of IkappaB kinase gamma expression completely prevented phorbol myristate acetate-induced upregulation of cFLIP mRNA in Jurkat cells. These data point to an important role for NF-kappaB in the regulation of the cFLIP gene. SV80 cells normally show resistance to TNF-related apoptosis-inducing ligand (TRAIL) and TNF, as apoptosis can be induced only in the presence of low concentrations of cycloheximide (CHX). However, overexpression of IkappaBalpha-SR rendered SV80 cells sensitive to TRAIL-induced apoptosis in the absence of CHX, and cFLIP expression was able to reverse the proapoptotic effect of NF-kappaB inhibition. Western blot analysis further revealed that cFLIP, but not TRAF1, A20, and cIAP2, expression levels rapidly decrease upon CHX treatment. In conclusion, these data suggest a key role for cFLIP in the antiapoptotic response of NF-kappaB activation.

Topics: Antibodies, Monoclonal; Apoptosis; Benzoquinones; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; CD40 Antigens; Cell Line; Cycloheximide; DNA-Binding Proteins; HeLa Cells; Humans; I-kappa B Proteins; Interleukin-1; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Lactams, Macrocyclic; Leupeptins; Mutation; NF-kappa B; NF-KappaB Inhibitor alpha; Quinones; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Signal Transduction; Tumor Necrosis Factor-alpha; Up-Regulation

The death domain kinase, receptor interacting protein (RIP), is one of the major components of the tumor necrosis factor receptor 1 (TNFR1) complex and plays an essential role in tumor necrosis factor (TNF)-mediated nuclear factor kappaB (NF-kappaB) activation. The activation of NF-kappaB protects cells against TNF-induced apoptosis. Heat-shock proteins (Hsps) are chaperone molecules that confer protein stability and help to restore protein native folding following heat shock and other stresses. The most abundant Hsp, Hsp90, is also involved in regulating the stability and function of a number of cell-signaling molecules. Here we report that RIP is a novel Hsp90-associated kinase and that disruption of Hsp90 function by its specific inhibitor, geldanamycin (GA), selectively causes RIP degradation and the subsequent inhibition of TNF-mediated IkappaB kinase and NF-kappaB activation. MG-132, a specific proteasome inhibitor, abrogated GA-induced degradation of RIP but failed to restore the activation of IkappaB kinase by TNF, perhaps because, in the presence of GA and MG-132, RIP accumulated in a detergent-insoluble subcellular fraction. Most importantly, the degradation of RIP sensitizes cells to TNF-induced apoptosis. These data indicate that Hsp90 plays an important role in TNF-mediated NF-kappaB activation by modulating the stability and solubility of RIP. Thus, inhibition of NF-kappaB activation by GA may be a critical component of the anti-tumor activity of this drug.

Topics: Apoptosis; Benzoquinones; Cell Survival; Cycloheximide; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Genes, Reporter; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; I-kappa B Kinase; Kinetics; Lactams, Macrocyclic; Leupeptins; Luciferases; NF-kappa B; Protein Serine-Threonine Kinases; Proteins; Quinones; Receptor-Interacting Protein Serine-Threonine Kinases; Recombinant Proteins; Transfection; Tumor Necrosis Factor-alpha

Mutation of the tumor suppressor gene p53 is the most common genetic abnormality detected in human cancers. Wild type p53 is a short-lived protein with very low basal intracellular levels. Most mutated forms of the protein, however, display markedly increased intracellular levels as an essential feature of their positive transforming activity. In this report, we have used selective inhibitors of the 20S proteasome to demonstrate that processing of p53 by ubiquitination and proteasome-mediated degradation is impaired by commonly occuring mutations of the protein. We found that this impairment of p53 turnover can be reversed by treatment of tumor cells with the benzoquinone ansamycin, geldanamycin, leading to a marked reduction in intracellular p53 levels. Finally, using cells which over-express a mutant p53 protein, we were able to demonstrate that restoration of proteasome-mediated degradation by geldanamycin is accompanied by p53 polyubiquitination. Although much remains to be learned about the mechanisms involved, our data demonstrate that selective de-stabilization of mutant transforming proteins such as p53 can be achieved pharmacologically with agents such as geldanamycin which modify the function of molecular chaperone proteins within tumor cells.

Topics: Acetylcysteine; Animals; Benzoquinones; Cycloheximide; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Detergents; Enzyme Inhibitors; Half-Life; Humans; Lactams, Macrocyclic; Leupeptins; Mice; Multienzyme Complexes; Mutation; Octoxynol; Polyethylene Glycols; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Quinones; Rats; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Ubiquitins