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 3b (GSK3b). Moreover, the AML cell protection downstream of GSK3b was mediated by NF-jB. 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 GSK3b and of its target NF-jB could represent important novel therapeutic tools.

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.

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.