Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation.

Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB.

RhoB promotes γH2AX dephosphorylation and DNA double-strand break repair.

Unlike other Rho GTPases, RhoB is rapidly induced by DNA damage, and its expression level decreases during cancer progression. Because inefficient repair of DNA double-strand breaks (DSBs) can lead to cancer, we investigated whether camptothecin, an anticancer drug that produces DSBs, induces RhoB expression and examined its role in the camptothecin-induced DNA damage response. We show that in camptothecin-treated cells, DSBs induce RhoB expression by a mechanism that depends notably on Chk2 and its substrate HuR, which binds to RhoB mRNA and protects it against degradation.

Use of phage display for the identification of molecular sensors specific for activated Rho.

We describe a phage display approach to select active Rho-specific scFv sensors. This in vitro technique allows preserving the antigen conformation stability all along the selection process. We used the GTP locked RhoBQ63L mutant as antigen against the Griffin.

Detection of label-free cancer biomarkers using nickel nanoislands and quartz crystal microbalance.

We present a technique for the label-free detection and recognition of cancer biomarkers using metal nanoislands intended to be integrated in a novel type of nanobiosensor. His-tagged (scFv)-F7N1N2 is the antibody fragment which is directly immobilized, by coordinative bonds, onto ~5 nm nickel islands, then deposited on the surface of a quartz crystal of a quartz crystal microbalance (QCM) to validate the technique. Biomarker GTPase RhoA was investigated because it has been found to be overexpressed in various tumors and because we have recently isolated and characterized a new conformational scFv which selectively recognizes the active form of RhoA.

Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis.

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM).

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection.

Background: The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation.

Synthesis of estradiol-pheophorbide a conjugates: evidence of nuclear targeting, DNA damage and improved photodynamic activity in human breast cancer and vascular endothelial cells.

The synthesis, physico-chemical properties, cellular localization and photocytotoxicity of estradiol-pheophorbide a conjugates in estrogen-dependent cancer and vascular endothelial cells are described with the aim of increasing the photodynamic activity by targeting the nucleus of both tumor and blood vessel cells.

Syntheses of functionalized biotin N-1' derivatives: new tools for the control of gene expression with small molecules.

The exceptionally high affinity of biotin toward avidin and streptavidin is at the basis of (strept)avidin-biotin biotechnology, which has numerous applications in life sciences. Recent biotin developments for in vivo and in vitro acylation of selective targeted protein and intein-mediated site specific protein biotinylation require the free biotin carboxyl function to covalently bind with the targeted protein. However, recently this carboxylic function has been used to substitute biotin with numerous ligands and flags.

Farnesyl-transferase inhibitor R115,777 enhances tamoxifen inhibition of MCF-7 cell growth through estrogen receptor dependent and independent pathways.

Introduction: We have previously shown that FTI-277, a farnesyl transferase inhibitor (FTI), enhances the efficacy of tamoxifen (Tam) in inhibiting the proliferation of the estrogen dependent MCF-7 cell line. As the cellular response to Tam is the result of an inhibition of both estrogen receptor-dependent and -independent pathways, we have used the estrogen receptor selective anti-estrogen ICI182,780 and N-pyrrolidine(-phenylmethyl-phenoxy)-ethanamine-HCl (PBPE), a selective ligand of anti-estrogen binding site (AEBS), to dissect out the mechanism(s) associated with the observed additivity resulting from combination treatment with FTI-277 and Tam. Moreover, for these studies, FTI-277 has been replaced by R115,777, a FTI currently in phase III clinical trials.

Prenylation inhibitors stimulate both estrogen receptor alpha transcriptional activity through AF-1 and AF-2 and estrogen receptor beta transcriptional activity.

Introduction: We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) alpha to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERalpha and by ERbeta.

A recombinant cell bioassay for measurement of overall estrogenic activity of serum: preliminary results in women with breast cancer.

The estrogenic status of patients with breast cancer may influence the prognosis and the response to treatment and is currently assessed by immunological measurement of serum estradiol. This does not account for estrogenic or anti-estrogenic activity related to growth factors able to activate the estrogen receptor, to anti-estrogenic drugs or to exogenous supply of estrogen-like compounds. We developed a recombinant bioassay based on a mammary cell line expressing luciferase in an estrogen receptor-dependent way.

Pharmacological-based translational induction of transgene expression in mammalian cells.

In the quest for the development of pharmacological switches that control gene expression, no system has been reported that regulates at the translational level. To permit small-molecule control of transgene translation, we have constructed a farnesyl transferase inhibitor-responsive translation initiation factor. This artificial protein is a three-component chimaera consisting of the ribosome recruitment core of the eIF4G1 eukaryotic translation initiation factor, the RNA-binding domain of the R17 bacteriophage coat protein and the plasma membrane localization CAAX motif of farnesylated H-Ras.

Molecular characterization of the microsomal tamoxifen binding site.

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 ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands.

Induction of T-cell antitumor immunity and protection against tumor growth by secretion of soluble human CD70 molecules.

One of the strategies to promote an antitumor response is the genetic modification of tumor cells to induce expression of costimulatory molecules. We have tested the capacity of a soluble form of CD70 molecule (sCD70). After construction of a vector carrying the sCD70, we obtained stable sCD70-secreting TS/A tumor cells and allogenic MC57 fibroblasts.

Tamoxifen is a potent inhibitor of cholesterol esterification and prevents the formation of foam cells.

Tamoxifen is a selective estrogen receptor modulator (SERM) used for the treatment and prevention of breast cancer. Tamoxifen has been reported to protect against the progression of coronary artery diseases in human and different atherosclerosis animal models by blocking the appearance of the atheromatous plaque. However, the molecular mechanism of this effect remains unknown.

Additive effects of tamoxifen and the farnesyl transferase inhibitor FTI-277 on inhibition of MCF-7 breast cancer cell-cycle progression.

The efficacy of tamoxifen in the hormonal therapy of breast cancer is well established, but therapeutic resistance is inevitable. FTIs are a new class of anticancer drugs that are in phase III clinical evaluation. Since the mechanisms of action of these 2 classes of drugs are different, we tested the combination of tamoxifen and FTI-277 on inhibiting proliferation of hormone-dependent MCF-7 human breast cancer cells.

Cloning of the human RHOB gene promoter: characterization of a VNTR sequence that affects transcriptional activity.

The RHOB gene is an immediate-early gene implicated in cell growth control, cytoskeletal organization, and neoplastic transformation. Although the mouse RHOB gene (Arhb) promoter has been described, the human promoter is unknown. We cloned the human RHOB gene (ARHB) 5'-flanking region from the human genome and characterized its promoter region.

Contrasting effects of prenyltransferase inhibitors on estrogen-dependent cell cycle progression and estrogen receptor-mediated transcriptional activity in MCF-7 cells.

Activation of estrogen receptors (ERs) by estrogens triggers both ER nuclear transcriptional activity and Src/Ras/Erks pathway-dependent mitogenic activity. The present study implicates prenylated proteins in both estrogenic actions. The farnesyltransferase and geranylgeranyltransferase I inhibitors (FTI-277 and GGTI-298, respectively) antagonize estradiol-stimulated cell cycle progression, progesterone receptor, cyclin D1, and c-Myc expression.

Insulin and estrogen receptor ligand influence the FGF-2 activities in MCF-7 breast cancer cells.

From the MCF-7 cell line we have developed, a human mammary cancer cell subline with the same karyotype as the mother strain and named MCF-7(SF), able to grow in serum-free chemically defined medium. This cell subline was firstly used to analyze the effect of basic fibroblast growth factor (FGF-2) in estrogen-receptor-positive human breast cancer cells. FGF-2 like estradiol is able to increase cell proliferation and pS2 expression but was also found to inhibit progesterone receptor (PR) expression.

Identification of two tamoxifen target proteins by photolabeling with 4-(2-morpholinoethoxy)benzophenone.

Our quest to identify target proteins involved in the activity of tamoxifen led to the design of photoaffinity ligand analogues of tamoxifen able to cross-link such proteins. A new tritiated photoprobe, 4-(2-morpholinoethoxy)benzophenone (MBoPE), was synthesized and used to identify proteins involved in tamoxifen binding in rat liver. MBoPE, which has structural features in common with the potential antagonist of the intracellular histamine receptor (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl: DPPE) is unable to bind the estrogen receptor although it does compete with tamoxifen for an antiestrogen binding site (AEBS).