Endoplasmic reticulum vacuolization and valosin-containing protein relocalization result from simultaneous hsp90 inhibition by geldanamycin and proteasome inhibition by velcade.

Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER--associated protein degradation, a sequential process requiring valosin-containing protein (VCP)-dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis.

Measuring ubiquitin conjugation in cells.

Protein ubiquitination is crucial to many diverse and critical functions of cells. Although it has been long known that conjugation of ubiquitin to proteins results in their destruction by the proteasome, recently it has become apparent that reversible protein ubiquitination, particularly monoubiquitination, performs regulatory functions in cells, analogous to protein phosphorylation. The most powerful and sensitive technique for measuring specific protein ubiquitination is antiubiquitin immunoblotting of the immunoprecipitated protein after gel electrophoresis.

Surface charge and hydrophobicity determine ErbB2 binding to the Hsp90 chaperone complex.

The molecular chaperone Hsp90 modulates the function of specific cell signaling proteins. Although targeting Hsp90 with the antibiotic inhibitor geldanamycin (GA) may be a promising approach for cancer treatment, little is known about the determinants of Hsp90 interaction with its client proteins. Here we identify a loop within the N lobe of the kinase domain of ErbB2 that determines Hsp90 binding.

Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vacuolization, and enhances antitumor activity.

The ansamycin antibiotic, geldanamycin, targets the hsp 90 protein chaperone and promotes ubiquitin-dependent proteasomal degradation of its numerous client proteins. Bortezomib is a specific and potent proteasome inhibitor. Both bortezomib and the geldanamycin analogue, 17-N-allylamino-17-demethoxy geldanamycin, are in separate clinical trials as new anticancer drugs.

17AAG: low target binding affinity and potent cell activity--finding an explanation.

The ansamycin geldanamycin (GM) and its derivative, 17AAG, now in early clinical trials in cancer patients, have potent activity against several cancer cells at low nanomolar concentrations. The main target of these drugs is the molecular chaperone heat shock protein 90. Contrary to the high antitumor potency, the affinity of these drugs for the chaperone was determined to be approximately 1 microM.

Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu.

Overexpression of the transmembrane receptor tyrosine kinase ErbB2 is common in multiple malignancies, including breast and ovarian cancer. ErbB2 is resistant to degradation mediated by c-Cbl, the E3 ubiquitin ligase responsible for ligand-induced ubiquitination of ErbB1 (epidermal growth factor receptor). Because of its resistance to degradation, ErbB2 is the preferred dimerization partner for other members of the ErbB family, and its overexpression in vivo is associated with poor prognosis.

Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1 alpha-degradative pathway.

HIF-1 alpha is a normally labile proangiogenic transcription factor that is stabilized and activated in hypoxia. Although the von Hippel Lindau (VHL) gene product, the ubiquitin ligase responsible for regulating HIF-1 alpha protein levels, efficiently targets HIF-1 alpha for rapid proteasome-dependent degradation under normoxia, HIF-1 alpha is resistant to the destabilizing effects of VHL under hypoxia. HIF-1 alpha also associates with the molecular chaperone Hsp90.

Lactacystin enhances cisplatin sensitivity in resistant human ovarian cancer cell lines via inhibition of DNA repair and ERCC-1 expression.

Cisplatin is among the most effective chemotherapeutic agents in the treatment of human ovarian cancer. The cytotoxicity of cisplatin results primarily from its ability to bind covalently to DNA and prevent DNA replication and transcription. The ubiquitin-proteasome pathway plays important roles in a broad array of basic cellular processes.

Hsp90, not Grp94, regulates the intracellular trafficking and stability of nascent ErbB2.

The benzoquinone ansamycin geldanamycin (GA) stimulates proteasome-mediated degradation of plasma membrane-associated ErbB2, a receptor tyrosine kinase. Drug sensitivity is mediated by ErbB2's kinase domain and occurs subsequent to the disruption of Hsp90 interaction with this domain. Full-length ErbB2 is efficiently processed via the endoplasmic reticulum (ER) and Golgi network, so that at steady state most of the detectable protein is plasma membrane associated.

Caspase-dependent deubiquitination of monoubiquitinated nucleosomal histone H2A induced by diverse apoptogenic stimuli.

Enzymatic deubiquitination of mono-ubiquitinated nucleosomal histone H2A (uH2A) and H2B (uH2B) is closely associated with mitotic chromatin condensation, although the function of this histone modification in cell division remains ambiguous. Here we show that rapid and extensive deubiquitination of nucleosomal uH2A occurs in Jurkat cells undergoing apoptosis initiated by anti-Fas activating antibody, staurosporine, etoposide, doxorubicin and the proteasome inhibitor, N-acetyl-leucyl-leucyl-norlucinal. These diverse apoptosis inducers also promoted the accumulation of slowly migrating, high molecular weight ubiquitinated proteins and depleted the cellular pool of unconjugated ubiquitin.

Effect of the proteasome inhibitor ALLnL on cisplatin sensitivity in human ovarian tumor cells.

Small molecules suppressing proteasome function inhibit the post-translational ubiquitination of selected proteins. Ubiquitin H2A is an example of an abundant chromatin-associated protein that is known to be ubiquitinated, which is important for several proteins involved in the repair of DNA damage. We therefore studied the effect of the proteasome inhibitor, N-acetyl leucyl-leucyl norlucinal (ALLnL), on cisplatin sensitivity in three human ovarian tumor cell lines.

Hsp90 as an anti-cancer target.

Heat shock protein 90 is one of the most abundant cellular proteins. Although its functions are still being characterized, it appears to serve as a chaperone for a growing list of cell signaling proteins, including many tyrosine and serine/threonine kinases, involved in cell proliferation and/or survival. The recent discovery of natural products which are able to inhibit Hsp90 function have allowed for both identification of its client proteins and for a better understanding of its role in their activity.

Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90.

ErbB receptors are a family of ligand-activated tyrosine kinases that play a central role in proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed in >25% of breast and ovarian cancers and is correlated with poor prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond quite differently to geldanamycin (GA), an antibiotic that is a specific inhibitor of the chaperone protein Hsp90.

Prevention of cisplatin-DNA adduct repair and potentiation of cisplatin-induced apoptosis in ovarian carcinoma cells by proteasome inhibitors.

Histones H2A and H2B are known to be reversibly post-translationally modified by ubiquitination. We previously observed in cultured tumor cells that proteasome inhibition stabilizes polyubiquitinated proteins, depletes unconjugated ubiquitin, and thereby promotes the deubiquitination of nucleosomal histones in chromatin. Provocative indirect evidence suggests that histone ubiquitination/deubiquitination cycles alter chromatin structure, which may limit accessibility of DNA repair proteins to damaged sites.

Geldanamycin as a potential anti-cancer agent: its molecular target and biochemical activity.

Heat shock protein 90 is one of the most abundant cellular proteins. Although its functions are still being characterized, it appears to serve as a chaperone for a growing list of cell signaling proteins, including many tyrosine and serine/threonine kinases, involved in proliferation and/or survival. The benzoquinone ansamycin geldanamycin has been shown to bind to Hsp90 and to specifically inhibit this chaperone's function, resulting in client protein destabilization.

The measurement of ubiquitin and ubiquitinated proteins.

Ubiquitination of key cellular proteins involved in signal transduction, gene transcription and cell-cycle regulation usually condemns those proteins to proteasomal or lysosomal degradation. Additionally, cycles of reversible ubiquitination regulate the function of certain proteins in a manner analogous to phosphorylation. In this short review we describe the current methodology for measuring ubiquitin and ubiquitination, provide examples which illustrate how various techniques have been used to study protein ubiquination, alert the readers of pitfalls to avoid, and offer guidelines to investigators newly interested in this novel post-translational protein modification.

Rapid deubiquitination of nucleosomal histones in human tumor cells caused by proteasome inhibitors and stress response inducers: effects on replication, transcription, translation, and the cellular stress response.

The proteasome inhibitors, lactacystin and N-acetyl-leucyl-leucyl-norlucinal, caused a rapid and near-complete loss of approximately 22-23-kDa ubiquitinated nucleoproteins, which we have identified as monoubiquitinated nucleosomal histones H2A and H2B by immunological and two-dimensional electrophoretic techniques. In human SKBr3 breast tumor cells, depletion of monoubiquitinated histones by the proteasome inhibitors coincided with the accumulation of high molecular weight ubiquitinated proteins in both nucleoprotein and cytosolic fractions and decreased unconjugated ubiquitin in the cytosol, without changes in the nonubiquitinated core histones. Unconjugated ubiquitin was not detected in isolated tumor cell nuclei.

The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.

Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain.

Polyubiquitination and proteasomal degradation of the p185c-erbB-2 receptor protein-tyrosine kinase induced by geldanamycin.

Treatment of SKBr3 human breast carcinoma cells with the benzoquinoid ansamycin, geldanamycin, rapidly depletes p185c-erbB-2 protein-tyrosine kinase. Loss of p185c-erbB-2 is initiated by disruption of a heteromeric complex between p185c-erbB-2 and the 94-kDa glucose-regulated protein, GRP94, to which geldanamycin binds avidly. Here we report that within minutes of exposure to geldanamycin, mature p185c-erbB-2 becomes polyubiquitinated.

p185erbB2 binds to GRP94 in vivo. Dissociation of the p185erbB2/GRP94 heterocomplex by benzoquinone ansamycins precedes depletion of p185erbB2.

Treatment of SKBr3 cells with benzoquinone ansamycins, such as geldanamycin (GA), depletes p185erbB2, the receptor tyrosine kinase encoded by the erbB2 gene. In the same cells, a biologically active benzoquinone photoaffinity label specifically binds a protein of about 100 kDa, and the ability of various GA derivatives to reduce the intracellular level of p185erbB2 correlates with their ability to compete with the photoaffinity label for binding to this protein. In this report, we present evidence that the approximately 100-kDa ansamycin-binding protein is GRP94.

Possible role for serine/threonine phosphorylation in the regulation of the heteroprotein complex between the hsp90 stress protein and the pp60v-src tyrosine kinase.

The abundant, cytoplasmic 90-kDa heat-shock protein associates transiently with the Rous sarcoma virus oncogenic protein tyrosine kinase, pp60v-src, directs its cellular trafficking and negatively regulates its kinase activity. Here we report that the serine/threonine phosphatase inhibitor, okadaic acid, destabilized the heat-shock protein 90-pp60v-src chaperone complex in v-src-transfected cells. Concomitant with complex destabilization by okadaic acid, phosphoserine was doubled and phosphothreonine was increased 20-fold in the heat-shock protein 90.

Taxol induction of p21WAF1 and p53 requires c-raf-1.

Taxol stabilizes microtubules, prevents tubulin depolymerization, and promotes tubulin bundling and is one of the most effective drugs for the treatment of metastatic breast and ovarian cancer. Although its interaction with tubulin has been well characterized, the mechanism by which taxol induces growth arrest and cytotoxicity is not well understood. Herein, we show that taxol induced dose- and time-dependent accumulation of the cyclin inhibitor p21WAF1 in both p53 wild-type and p53-null cells, although the degree of induction was greater in cells expressing wild-type p53.

Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation.

The molecular mechanisms by which oncogenic tyrosine kinases induce cellular transformation are unclear. Herbimycin A, geldanamycin, and certain other benzoquinone ansamycins display an unusual capacity to revert tyrosine kinase-induced oncogenic transformation. As an approach to the study of v-src-mediated transformation, we examined ansamycin action in transformed cells and found that drug-induced reversion could be achieved without direct inhibition of src phosphorylating activity.

Synergistic antiproliferative effects of interleukin-1 alpha and doxorubicin against the human ovarian carcinoma cell line (NIH:OVCAR-3).

Interleukin-1 alpha (IL-1 alpha) exerts antiproliferative effects on a human ovarian carcinoma cell line, NIH:OVCAR-3, which is resistant to clinically relevant concentrations of doxorubicin (DOX) and other chemotherapeutic agents. This action of IL-1 alpha depends on the presence of type I (80 kDa) receptors, although no quantitative relationship has been established between receptor occupancy and inhibition of cell growth. When NIH:OVCAR-3 cells were exposed to IL-1 alpha and DOX in combination, a mutual potentiation of the antiproliferative effects of the two agents was observed.