A crosstalk between the Wnt and the adhesion-dependent signaling pathways governs the chemosensitivity of acute myeloid leukemia.

Relapses following chemotherapy are a major hindrance to patients' survival in acute myeloid leukemia (AML). To investigate the role of the hematopoietic niche in the chemoresistance of leukemic cells, we examined two pathways: one mediated by adhesion molecules/integrins, and the other by soluble factors of the morphogen Wnt pathway. In our study, both the adhesion of leukemic blasts to fibronectin and the addition of Wnt antagonists induced, independently, resistance of AML cells to daunorubicin in a cell survival assay. Using pharmacological inhibitors and siRNA, we showed that both resistance pathways required the activity of the glycogen synthase kinase 3beta (GSK3beta). Moreover, the AML cell protection downstream of GSK3beta was mediated by NF-kappaB. A link between the adhesion and the Wnt pathway was found, as adhesion of U937 on human osteoblasts, a component of the hematopoietic niche, triggered the secretion of the Wnt antagonist sFRP-1 and supported resistance to daunorubicin. The osteoblast-conditioned medium could also confer chemoresistance to U937 cells cultured in suspension, and this cell protective effect was abrogated after depletion of sFRP-1. In the context of this potential double in vivo resistance, modulators of the common signal GSK3beta and of its target NF-kappaB could represent important novel therapeutic tools.

Modifications of benzylphenoxy ethanamine antiestrogen molecules: influence affinity for antiestrogen binding site (AEBS) and cell cytotoxicity.

The antiestrogen binding site (AEBS) is a membranous protein complex that has been shown to be intimately linked with the antiproliferative and antiretroviral effects of certain antiestrogenic compounds such as tamoxifen (Tx). Various specific ligands of AEBS derived from benzylphenoxy ethanamine and a new benzoyl structure were synthesized either by modification of the aminoether side chain or by halogen substitution at the meta-, ortho-, and para position on the benzoyl group. Using the MCF-7 cellular strain and its RTx6 variant (a clone selected for its antigrowth resistance to tamoxifen), it was shown that under high drug concentrations the cytotoxicity of the ligands was directly correlated with their affinity for AEBS. In agreement with previous observations made on triphenylethylenic ligands, modification of the basic ethanamine side chain modulated the ligand affinities. Chloride in meta increased ligand efficacy, whereas chloride substitution in ortho and para decreased it. Effects on AEBS-positive MCF-7 cells were drug concentration- and time-dependent, whereas they were unspecific on the AEBS-negative RTx6 cell line. These cytotoxic effects were confirmed in the absence of estrogen receptor on human AEBS-positive uterine cervix cell carcinoma HeLa cells, but were non-specific on rat fibroblastic AEBS-negative (low concentration) NRK cells. The cytotoxicities of these ligands are related to their affinities for AEBS.

Microsomal epoxide hydrolase of rat liver is a subunit of theanti-oestrogen-binding site.

A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039-17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.

Ligands of the antiestrogen-binding site are able to inhibit virion production of human immunodeficiency virus 1-infected lymphocytes.

Since the discovery of human immunodeficiency retrovirus, the drug arsenal against retrovirus has rapidly increased. Concomitantly, new challenges in the therapy of acquired immune deficiency syndrome have arisen, including drug toxicities, drug resistance, and the development of various cancers as effective therapies prolong survival. Tamoxifen, a nonsteroidal antiestrogen with a low incidence of side effects, is widely used in cancer therapy; it is known to exert pleiotropic activities by binding essentially to the estrogen receptor and other unidentified proteins. In the present work, quantification of the p24 core protein of human immunodeficiency virus 1 produced by infected lymphocytes shows an inhibitory effect of tamoxifen on virion production. Moreover, we assume that this effect is not mediated by the estrogen receptor because antiestrogen ligands interacting with the antiestrogen-binding site exhibit efficacy related to their affinity for this site, although specific antiestrogens of the estrogen receptor are ineffective.

Characterization of the membranous antiestrogen binding protein: I. Partial purification of the protein in its active state.

We previously demonstrated that, in addition to the estrogen receptor, the Antiestrogen Binding Site (ABS) is also a potent mediator of the antitumorous activity of the clinical drug tamoxifen. Because of report discrepancies in the binding parameters of rat liver ABS we first attempted to improve binding study conditions. In this way buffer, protein concentration, methodology for bound/free ligand separation and phospholipidic ratio were determined. This work was used to evaluate the Stoke radius (4.4 S) and isoelectric point (pH = 6.6) of the protein in its native state. These studies constituted the obligatory transition from rat liver to pure ABS protein.

Characterization of the membranous antiestrogen binding protein: II. Purification to homogeneity.

Our knowledge of the biological role of the antiestrogen binding site ABS in the antitumoral activity of tamoxifen, will be increased with the determination of its coding gene sequence. To this end our team has for some time attempted to purify this membranous protein. In this work we report the purification to homogeneity of ABS from rat liver in a six step succession. Specific photolabeling with a tritiated photoprobe, solubilization of rat liver microsomes, chromatofocusing of the labeled proteins, preparative electrophoresis on polyacrylamide gel, and two consecutive high performance liquid chromatography separations on C4 hydrophobic resin produced 2.5 micrograms of pure ABS by silver stain analysis of SDS-PAGE. The NH2-terminal residue of the protein appears to be blocked, which hinders the Edman degradation method for obtention of the whole protein sequence.

Antiestrogens inhibit the replication of the retroviral Moloney murine leukemia virus in vitro.

Widely used in breast cancer therapy, tamoxifen exhibits in vitro and in vivo pleiotropic activities that are generally attributed to its binding to the estrogen receptor. However, several reports have shown that the antiestrogen binding site (ABS) is also an intracellular target of the drug. This dual affinity determines at least two modes of action for the triphenylethylenic antiestrogens; one would be estrogen reversible and the other irreversible. Here, tamoxifen is shown to inhibit the production of Moloney murine leukemia virus virions by fibroblastic A9 cells, in which estrogen receptor is not detectable either by binding or by radioimmunoassay. Moreover, a specific ligand of the ABS induces effects equivalent to those of tamoxifen, suggesting that tamoxifen inhibits Moloney murine leukemia virus replication through an estrogen-independent pathway involving the ABS.

Ligands of the antiestrogen binding site block endothelial cell proliferation reversibly.

Benzylphenoxyethanamine derivatives are known to display antiproliferative activities on tumor cell lines consistently with their binding affinity to the microsomal antiestrogen binding site. In the present study we show that pyrrolidinobenzylphenoxyethanamine, a new efficient compound of this series, exhibits reversible effects on exponentially growing adult bovine aortic endothelial cells inducing (1) lamellated cytoplasmic inclusions, (2) cell proliferation inhibition, (3) dose-dependent transition delay of cells in the G0-G1 phase of the cell cycle. Complete reversal of these effects is achieved only by withdrawing the drug from the medium. The ultrastructural cellular modifications disappeared, and flow cytometry and thymidine incorporation analysis showed the effect on degree of synchronization of this one-step methodology.