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. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. RESULTS: After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. CONCLUSION: We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.

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. METHODS: The quantitative sulphorhodamine B (SRB) colorimetric assay was used to determine the growth inhibitory effect of agents on MCF-7 cells. Dose response interactions between R115,777-Tam, R115,777-ICI182,780 and R115,777-PBPE were evaluated, at the IC50 point, using the isobologram method. Apoptotic cell death (DNA fragmentation, nucleus condensation and cytokeratin 18 cleavage) and inhibition of the mevalonate pathway (western blot) were also determined. RESULTS: Combinations of the specific FTI R115,777 with either ICI182,780 or PBPE exhibit a synergistic effect on MCF-7 cell growth inhibition, while its combination with Tam is additive, as previously reported for FTI-277. Apoptosis is detected after treatment with combinations of R115,777 with either Tam or PBPE but not with ICI182,780, suggesting that each combination inhibits cell proliferation by different mechanisms. Even though the ER pathway has not yet been deciphered, it is shown here that the AEBS pathway is able to interfere with the mevalonate pathway at the level of protein farnesylation. CONCLUSION: Overall, this work reveals that combinations of R115,777 with either selective ER ligands or a selective AEBS ligand are able to induce large increases in their anti-proliferative activities on MCF-7 cells. Moreover, these results suggest that it may be of definite interest to evaluate combinations of R115,777 with different anti-estrogens in the treatment of ER positive breast tumours. Based on these experimental data, such combinations may prove beneficial in different clinical scenarios or when used in specific sequences; studying the combination of R115,777 with ICI182,780 for early treatment and reserving combinations with either Tam or a selective AEBS ligand, such as BMS-217380-01, for more resistant disease.

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. METHODS: The ERE-beta-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha. The presence of ERalpha was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity. RESULTS: FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERalpha-mediated and ERbeta-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERalpha is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERalpha is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established. CONCLUSIONS: Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

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. In contrast, the inhibitors markedly stimulate transcription from two genes containing estrogen response elements, both in the absence and presence of estradiol. The pure antiestrogen ICI 182,780 inhibits by more than 85% these effects on transcription. We demonstrate that both FTI-277 and GGTI-298 increase the association of steroid receptor coactivator-1 with ER alpha and FTI-277 decreases the association of ER alpha with the histone deacetylase 1, a known transcriptional repressor. In addition, FTI-277 has no marked effect on the association of the two corepressors, nuclear receptor corepressor and silencing mediator of retinoid and thyroid receptor with ER alpha, whereas GGTI-298, similar to tamoxifen, clearly increased these associations. Together, these results demonstrate that prenylated proteins play a role in estradiol stimulation of proliferation and progesterone receptor expression. However, they antagonize the ability of ER alpha to stimulate estrogen response element-dependent transcriptional activity, acting presumably through coregulator complex formation.

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. In all, 45% of wild-type (wt) tumors were rejected in immunocompetent mice when transfected sCD70-secreting cells were injected three times in the periphery of the wt tumors. Furthermore, the sCD70-secreting TS/A cells induced a protective memory against wt TS/A tumor growth: 70% of the wt tumors used for the challenge were rejected by mice, which had rejected tumors 45 days before in the presence of sCD70-secreting TS/A cells. It was also shown that in vitro mock TS/A tumor cell proliferation was inhibited by splenocytes harvested from mice injected with TS/A cells expressing CD70. Growth kinetics of wt TS/A tumors in immunocompetent versus nude mice suggested that T lymphocytes were implicated in the antitumor response, which was confirmed by membrane expression of specific markers. The data suggest that injection of genetically transfected cells secreting sCD70 in the periphery of wt TS/A tumors induces T-cell-mediated inhibition of tumor growth and builds up a protective antitumor memory.

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. The anti-estrogen tamoxifen partly counteracts the effects of FGF-2 and to discriminate between its two main mediators (estrogen receptor vs. anti-estrogen binding site, AEBS) we compare the efficacies of pure anti-estrogen (ICI 182,780) and AEBS ligand (PBPE). It appears that pure anti-estrogen counteracts cell growth and pS2 effects of FGF-2 since AEBS ligand inhibits the cell growth but has no activity on pS2 expression. Secondly, adding insulin (10(-6)M) in the culture medium induces a strong increase in cell proliferation, which then elicits an inhibitory effect of FGF-2 and addition of anti-estrogens, are less efficient to further decrease growth, since the effects of FGF-2 and anti-estrogens on pS2 expression are conserved.

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. RhoB-deficient cells fail to dephosphorylate γH2AX following camptothecin removal and show reduced efficiency of DSB repair by homologous recombination. These cells also show decreased activity of protein phosphatase 2A (PP2A), a phosphatase for γH2AX and other DNA damage and repair proteins. Thus, we propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of γH2AX and DSB repair. Finally, we show that RhoB-deficient cells accumulate endogenous γH2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression.

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. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation.

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.1 library composed of a human synthetic V(H) + V(L) scFv cloned in the pHEN2 phagemid vector. The method described here has permitted to identify an scFv that discriminates between the activated and the inactivated form of the Rho subfamily.

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. We implemented a surface chemistry involving an antibiofouling coating of polyethylene glycol silane (PEG-silane) (<2 nm thick) and Ni nanoislands to reach a label-free detection of the active antigen conformation of RhoA, at various concentrations. The methodology proposed here proves the viability of the concept by using Ni nanoislands as an anchoring surface layer enabling the detection of a specific conformation of a protein, identified as a potential cancer biomarker. Hence, this novel methodology can be transferred to a nanobiosensor to detect, at lower time consumption and with high sensitivity, specific biomolecules.

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). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

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. In the present work, we propose the N-1' labeling possibilities of biotin, keeping the valeric chain free. We describe liquid and solid-phase syntheses of functionalized biotin N-1' derivatives. Although the N-1' modification involves a two-log decrease in affinity, in vitro these molecules kept their high avidin affinity (around 10(-12) M) and the in vivo acylation ability of new biotin derivatives.

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.

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. Unlike its mouse counterpart, the human gene shows a variable number of tandem repeats (VNTR) sequence with a 34-bp repetitive unit between positions -1124 and -821. We demonstrated that this VNTR sequence significantly decreases the transcriptional activity of ARHB and simian virus-40 (SV40) promoters. PCR amplification of the VNTR sequence using genomic DNA from many cell lines revealed the existence of at least four alleles containing a different number of the repetitive unit. Our data suggest a potential regulatory role for the VNTR sequence in ARHB expression.

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. An additive effect on cell proliferation was demonstrated, accompanied by an additive G(0)/G(1) arrest. The major effect of the combination of the 2 drugs was to maintain p21(waf/cip1) at an intermediate level, higher than that observed in the presence of tamoxifen alone. This was associated with an additive effect on inactivation of cyclin E-Cdk2 complexes and decreased phosphorylation of pRb and p130 pocket proteins. These effects were accompanied by increased association of 2 CDIs, p27(kip1) and p21(waf/cip1), with cyclin E-Cdk2 complexes. These data demonstrate that the additive effect is likely predominantly due to the recruitment of p27(kip1) and, to a lesser extent, p21(waf/cip1) into the cyclin E-Cdk2 complexes. Together, these results suggest that the combination of FTI and tamoxifen may increase the antitumor effect of either drug alone in breast cancer.

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). This tritiated benzophenone derivative was obtained by metal-catalyzed halogen-tritium replacement reaction. Because of its high specific activity, four target proteins could be photolabeled, three of which were identified with M(r) of 60,000, 49,500, and 14,000, while the fourth at 27,500 was in too low an amount and could not be sequenced. The 49.5 kDa protein corresponded by mass spectrometry to the microsomal epoxide hydrolase already identified with an aryl azide photoprobe [Mesange, F., et al. (1998) Biochem. J. 334, 107-112]. The 60 and 14 kDa proteins were identified as the carboxylesterase (ES10) and the liver fatty acid binding protein (L-FABP), respectively. The inhibitory effect of tamoxifen on carboxylesterase activity and the competitive efficacy of oleic acid on [(3)H]tamoxifen binding suggest that both proteins are AEBS subunits. Moreover, treatment of hepatocytes with antisense mRNA directed against ES10 or L-FABP abolished both tamoxifen and MBoPE binding. On the basis of previous pharmacological arguments, the 27.5 kDa protein might correspond to the sigma I receptor. Altogether, these results confirm that the microsomal epoxide hydrolase is a target for tamoxifen and provide evidence of two new target proteins implicated in cell lipid metabolism.

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. In a human serum matrix the MELN system was able to detect the transcriptional activity of estradiol, growth factors (epidermal growth factor (EGF), insulin at insulin-like growth factor 1 (IGF-1)-like concentrations), xeno-estrogens (diethylstilbestrol, phytoestrogens) and tamoxifen in a dose-dependent manner. The intra- and inter-assay variations were < 6% and < or = 15%, respectively, whatever the estradiol concentration; the functional sensitivity was < 10 pmol/l equivalents of estradiol. We assessed the overall estrogenic activity of serum (OEAS) in 16 healthy women and in 24 women with advanced breast cancer. The correlation between OEAS and serum log10 17-beta-estradiol (E2) was good for healthy women (r2=0.8568) but poor for patients (r2=0.0563). Assessment of the OEAS/E2 ratio as a prognostic and predictive factor would be of interest in clinical prospective trials involving ER+ breast cancer patients.

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. This membrane-delocalized translation factor is inactive unless liberated in the cytosol. Farnesyl transferase inhibitor FTI-277 prevents the membrane association of the CAAX motif and thus increases the cytoplasmic levels of the eIF4G fusion protein, which is then capable of inducing translation of the second cistron of a bicistronic messenger RNA containing an R17-binding site in its intercistronic space. Such direct translational control by farnesyl transferase inhibitors provides a system for fast, graded and reversible regulation of transgene expression.

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. Acyl-CoA:cholesterol acyl transferase (ACAT) catalyzes the biosynthesis of cholesteryl esters, which are the major lipids found in the atheromatous plaque. In this paper we have tested whether ACAT might be inhibited by tamoxifen. We show, using molecular modeling, that tamoxifen displays three-dimensional structural homology with Sah 58-035 (3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]-propanamide), a prototypical inhibitor of ACAT. We report that tamoxifen inhibits ACAT in a concentration-dependent manner on rat liver microsomal extract. We show that the presence on estrogen receptor ligands of a backbone isosteric to the diphenyl ethane backbone of Sah 58-035 constitutes a pharmacophore for ACAT inhibition. More importantly, tamoxifen was able to inhibit ACAT on intact macrophages stimulated with acetylated low-density lipoproteins and blocked the formation of foam cells, a step that precedes the formation of the atheromatous plaque. This work constitutes the first evidence that tamoxifen is an inhibitor of ACAT and foam cell formation at therapeutic doses and that this may account for its atheroprotective action.

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. 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 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.