In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.DOI: http://dx.doi.org/10.7554/eLife.16228.001

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

Purpose: RhoB is a low molecular weight GTPase belonging to the Ras protein superfamily. Whereas most Rho proteins have been shown to have a positive role in proliferation and malignant transformation, the specific role of RhoB appears more divergent. We reported previously that RhoB inhibits cell proliferation in various human cancer cells. Here, we studied the specific role played by RhoB in human lung cancer.Experimental Design: We analyzed the expression of RhoB protein by immunostaining in human lung tissues ranging from normal to invasive carcinoma from different histological types in two large independent studies of, respectively, 94 and 45 samples. We then studied the cellular effect of RhoB overexpression in a model of lung cancer (A549, adenocarcinoma) and tumorigenicity in nude mice.Results: We showed in both studies that RhoB protein was expressed in normal lung and decreased dramatically through lung cancer progression (P < 0.01). Interestingly, RhoB expression was lost in 96% of invasive tumors and reduced by 86% in poorly differentiated tumors compared with the nonneoplastic epithelium. Moreover, the loss of expression of RhoB correlated significantly with tumor stage and proliferative index, whereas no correlation was found between RhoB and p53 or Bcl-2 expression. We then showed that ectopic expression of RhoB in lung cancer cell line A549 suppressed cell proliferation, anchorage-independent growth, and xenograft tumor growth in nude mice.Conclusions: RhoB loss of expression occurs very frequently in lung carcinogenesis, reinforcing its putative tumor suppressive activity, and raising the value of its potential use in cancer therapy.

Whereas the GTPase RhoA has been shown to promote proliferation and malignant transformation, the involvement of RhoB in these processes is not well understood. In this manuscript RhoB is shown to be a potent suppressor of transformation and human tumor growth in nude mice. In several human cancer cell lines, RhoA promotes focus formation whereas RhoB is as potent as the tumor suppressor p53 at inhibiting transformation in this assay. RhoB is both farnesylated (F) and geranylgeranylated (GG), and RhoB-F has been suggested as a target for the antitumor activity of farnesyltransferase inhibitors. Here we demonstrate that both RhoB-F and RhoB-GG inhibit anchorage-dependent and -independent growth, induce apoptosis, inhibit constitutive activation of Erk and insulin-like growth factor-1 stimulation of Akt, and suppress tumor growth in nude mice. The data demonstrate that RhoB is a potent suppressor of human tumor growth and that RhoB-F is not a target for farnesyltransferase inhibitors.

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoidinduced apoptosis was preceded by an arrest in G 0 /G 1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.

Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ERindependent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration-and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5␣-cholest-8-en-3␤-ol for tamoxifen treatment and 5␣-cholest-8-en-3␤-ol and cholesta-5,7-dien-3␤-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3␤-hydroxysterol-⌬ Tamoxifen is a selective estrogen receptor modulator (SERM) widely used for the treatment and the prevention of breast cancer (1). More than 20 years ago, Sutherland et al. (2) discovered that tamoxifen bound to a high affinity binding site that was different from the estrogen receptor (2). This site has been named the microsomal antiestrogen binding site (AEBS) 1 because it is localized in the microsomes of cells; it binds principally aryl aminoethoxy antiestrogens and has no affinity for estrogens (3). Two classes of selective ligands have been developed so far to selectively target the AEBS. The first class includes diphenylmethane derivatives of tamoxifen (DPPE) and N-pyrrolidino-2-[4-(benzyl)-phenoxy-ethanamine⅐HCl (PBPE) (4 -6), while the second class includes oxygenated derivatives of cholesterol such as 7-ketocholestanol (7-9). We and others (5, 10 -14) have shown that AEBS ligands inhibit the growth of tumor cell lines in vitro and in vivo, demonstrating that the AEBS was involved in the mediation of the effects of these structural classes of its cognate ligands. These compounds represent not only specific tools to study AEBS function but are also anticancer drug candidates because the selective AEBS ligand DPPE (Tesmilifene) was brought up to phase II and III clinical trials for the treatment of breast and prostate cancer in association with doxorubicin (15-17). Two principal points remained unsolved: the first is the precise molecular nature of the AEBS and the second the explanation of the difference observed between the nanomolar affinity of AEBS ligands and their micromolar effectiveness for growth control and cytotoxicity.We have been involved in the identification of the AEBS for several years. The AEBS can be found in most tissues in mammals and is abundant in microsomes of liver that contained 20 -30ϫ the amount found in tumor cell lines (18). For this reason, the liver have been chosen for the purification of the AEBS but the pharmacological profiles of the AEBS found in the ...

BackgroundThe microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours.Methodology/Principal FindingsWe isolated an original type of stromal cells, referred to as “Hospicells” from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance.Conclusions/SignificanceThis is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient's tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.

Integrins are extracellular matrix receptors involved in tumour invasion and angiogenesis. Although there is evidence that inhibiting integrins might enhance the efficiency of radiotherapy, little is known about the exact mechanisms involved in the integrin-dependent modulation of tumor radiosensitivity. The purpose of this study was to investigate the role of avb3 and avb5 integrins in glioblastoma cell radioresistance and overall to decipher the downstream biological pathways. We first demonstrated that silencing avb3 and avb5 integrins with specific siRNAs significantly reduced the survival after irradiation of 2 glioblastoma cell lines: U87 and SF763. We then showed that integrin activity and integrin signalling pathways controlled the glioma cell radiosensitivity. This regulation of glioma cell response to ionising radiation was mediated through the integrin-linked kinase, ILK, and the small GTPase, RhoB, by two mechanisms. The first one, independent of ILK, consists in the regulation of the intracellular level of RhoB by avb3 or avb5 integrin. The second pathway involved in cell radiosensitivity consists in RhoB activation by ionising radiation through ILK. Furthermore, we demonstrated that the avb3/avb5 integrins/ILK/ RhoB pathway controlled the glioma cells radiosensitivity by regulating radiation-induced mitotic cell death. This work identifies a new biological pathway controlling glioblastoma cells radioresistance, activated from the membrane through avb3 and/or avb5 integrins via ILK and RhoB. Our results are clues that downstream effectors of avb3 and avb5 integrins as ILK and RhoB might also be promising candidate targets for improving the efficiency of radiotherapy and thus the clinical outcome of patients with glioblastoma. ' 2008 Wiley-Liss, Inc.Key words: glioblastoma; radiosensitivity; integrin; ILK; RhoB Despite recent advances in diagnostic imaging, neurosurgical techniques, radiation therapy and chemotherapy, 1 glioblastoma, one of the most common primary brain tumours, is still associated with a dismal prognosis. Almost all of the patients will die of a relapse in the radiation field as a result of low tumour sensitivity to ionising radiation. The low tumour sensitivity to radiotherapy is not only due to the modulation of different biological signal transduction pathways in tumour cells but also due to a cross-talk between the tumour cells and their microenvironment. Our previous results have shown that factors controlling the microenvironment, such as basic fibroblast factor (FGF-2), modulate the tumour cell radioresistance via the small GTPase RhoB which itself also regulates angiogenesis via metalloproteinase 2 (MMP2) and hypoxia in the glioblastoma U87 cell model. Cell adhesion to extracellular matrix (ECM) is known to confer a cell-adhesion-mediated chemotherapeutic drug resistance for hematopoietic and solid tumours.9 Among integrins, the b1 integrin has been largely involved in cell survival after a genotoxic injury by modulating DNA repair 10 or protecting cells from apoptosis. 11Fe...