Heat shock protein 90α (Hsp90α) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90α has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90α ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90α coated magnetic beads can be used to "fish" from complex chemical and biological mixtures for new lead drug candidates and client proteins.Heat shock protein 90 (Hsp90) is a family of cellular proteins (Hsp90α, Hsp90β, Grp94, Trap1) that act as molecular chaperones, which guide the normal folding, intracellular disposition, and proteolytic turnover of many key regulators of cell growth and survival. 1 It is one of the most abundant molecular chaperones in eukaryotes 2 and is essential in the activation of over 100 client proteins, including receptors, protein kinases, and transcription factors. 1,3 Increased expression and activity of Hsp90 have been observed in human cancers, and this protein has become a therapeutic target for the development of new anticancer agents. 4 The intrinsic ATPase activity of Hsp90 is also a target in drug discovery as ATPase activity is a key component in the chaperone function. 5Although a large number of Hsp90 client proteins have been identified, it is likely that there are many as of yet unidentified client proteins whose function could help elucidate novel oncogenic signaling pathways. At the present time, co-immunoprecipitation (IP) is the primary method used to identify client proteins. 6 However, in this approach an antibody raised against the coprecipitating protein is required for identification by Western blot analysis. Thus, it is difficult to find new proteins without first knowing what you are looking for. Additional problems with this technique include antibody contamination, nonspecific binding interactions, and leaching contamination that can result from protein A or protein G immobilization on agarose.We now report an alternative approach to the isolation of Hsp90 client proteins based upon the immobilization of Hsp90α on the surface of magnetic beads. In a previous study, we demonstrated that Hsp90 could be immobilized on a silica-based stationary phase through

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The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRα and ERRγ proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC50 and IC50 values were consistent with previous reported values determined in other types of cancer cell lines. Induction of luciferase expression under the control of ERR isoform-specific promoters was also observed in these cells. These results indicate that ERRα and ERRγ are differentially expressed in these tumor cell lines and likely contribute to agonist-dependent ERR transcriptional activity.

Abstract. Urokinase-type plasminogen activator (uPA) is implicated in various pathophysiological processes, including extracellular matrix turnover, cell migration and invasion. Our study aimed to determine the role of uPA in both proliferation and mitogen-activated protein kinase (MAPK) pathway. Hence, we analyzed the effects induced by exogeneous addition of domain-specific uPA antibodies and uPAinteracting molecules on proliferation of uPA-suppressed MDA-MB-231 breast cancer cells. uPA expression was reduced to 53% by stable transfection with an antisense/ vector construct and to 65% by siRNA transfection. Immunocytochemical Ki67 staining and flow cytometry (S-phase) analysis indicated a strong decrease of cellular proliferation activity (35% and 38%, respectively). Exogenous addition of high molecular weight-uPA (HMW-uPA) or incubation with the amino terminal fragment (ATF), which lacks the enzymatic activity of uPA, lead to increased cell proliferation. A strong increase of proliferation was absent when the monoclonal antiuPAR antibody IIIF10 (blocking uPA binding site), soluble uPAR (scavenger effect) and phosphatidyl-inositol-specific phopholipase C (PI-PLC, degrading uPAR) was added prior to the addition of HMW-uPA. In conclusion, HMW-uPA and ATF induce proliferation of breast cancer cells by binding to uPAR. Thereby, integrins situated adjacent to uPAR carry the signals into the cell, thus stimulating proliferation that is mediated via the MAPK pathway.

Estrogen related receptors, ERRα, ERRβ, and ERRγ, form a subfamily of orphan nuclear receptors that have significant amino acid homology with the estrogen receptors ERα and ERβ. ERRs have been identified in breast cancer tumors and ERRα has been associated with a negative response to treatment while ERRγ is considered an indicator of a positive response. We have recently demonstrated that ERRα and ERRγ are expressed in cell lines derived from astrocytoma and glioblastoma tumors. We have also demonstrated that there are different levels of expression of ERRα and ERRγ in the cell lines and this difference may be the source of the variation of these tumors to anticancer agents. MTT assays were performed to measure the metabolic activity of T98G cells treated with the ERRγ antagonists. While ERRs share significant Ligand binding domain (LBD) homology with ERs, they have different responses to characterized ER agonists and antagonists. For example, the ER agonist estradiol is inactive in ERR expressing cell lines, the ER agonist diethyl stilbestrol is an antagonist of all three ERR subtypes, 4-hydroxytamoxifen does not affect ERRα and acts instead as an ERRγ antagonist. In addition, we have recently demonstrated that ERRα and ERRγ antagonists can effectively inhibit tumor growth. Based on this observation, we have created liquid chromatographic columns containing immobilized LBDs of the ERRα and ERRγ. In the initial study, the LBD of ERRγ was covalently immobilized onto the surface of an aminopropyl silica liquid chromatography stationary phase. The protein was immobilized via the N-terminus to create the ERRγ-column. The column was characterized using frontal chromatographic techniques and a series of tamoxifen derivatives were studied. Additionally, we have identified a second site to which diethylstilbestrol (DES) binds. Acknowledgement This research was supported entirely or in part by the Intramural Research Program of the NIH, National Institute on Aging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5500.

Aim: Desmoplastic infantile ganglioglioma (DIG), is a rare tumor arising mainly during the first 2 years of life. Molecular characterization of these benign yet rapidly proliferating tumors has been limited to evaluating a few mutations in few genes. Our aim was to establish a live cell culture to enable the understanding of the cellular processes driving the non-malignant growth of these tumors.Methods: Tumor tissue from a rare non-infantile 8-year-old female DIG patient was dissociated and digested using collagenase to establish live cultures. Both 2D monolayer and 3D neurospheres were successfully cultured and characterized for proliferative potential, intrinsic plasticity, presence of cancer stem cells and the expression of stem cell markers. Cells cultured as 3D were embedded as tissue blocks. Immunohistochemistry was performed in both tissue and 3D sections for markers including synaptophysin, vimentin, neurofilament and MIB-1. Mutation analysis by NGS was performed using a-100 gene panel.Results: Using immunohistochemistry, the 3D cultures were shown to express markers as in the original DIG tumor tissue indicating that the spheroid cultures were able to maintain the heterogeneity found in the original tumor. Cells continued proliferating past passage 10 indicative of immortalization. Enrichment of cancer stem cells was observed in neurospheres by FACS using CD133 antibody and RT-PCR. Mutation analysis indicated the presence of germline mutations in three genes and somatic mutations in two other genes.Conclusion: A spontaneous cell line-like cell culture with high percentage of stem cells has been established from a DIG tumor for the first time.

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