Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17b-hydroxysteroid dehydrogenases (17b-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17b-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17b-HSD, 17b-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17b-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17b-HSD1, using a novel murine model. 17b-HSD1 and 17b-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17b-HSD12 was highly expressed in breast cancer cell lines, only 17b-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 lg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17b-HSD, 17b-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17b-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer.

The proteoglycans aggrecan, versican, neurocan, and brevican bind hyaluronan through their N-terminal G1 domains, and other extracellular matrix proteins through the C-type lectin repeat in their C-terminal G3 domains. Here we identify tenascin-C as a ligand for the lectins of all these proteoglycans and map the binding site on the tenascin molecule to fibronectin type III repeats, which corresponds to the proteoglycan lectinbinding site on tenascin-R. In the G3 domain, the C-type lectin is flanked by epidermal growth factor (EGF) repeats and a complement regulatory protein-like motif. In aggrecan, these are subject to alternative splicing. To investigate if these flanking modules affect the C-type lectin ligand interactions, we produced recombinant proteins corresponding to aggrecan G3 splice variants. The G3 variant proteins containing the C-type lectin showed different affinities for various ligands, including tenascin-C, tenascin-R, fibulin-1, and fibulin-2. The presence of an EGF motif enhanced the affinity of interaction, and in particular the splice variant containing both EGF motifs had significantly higher affinity for ligands, such as tenascin-R and fibulin-2. The mRNA for this splice variant was shown by reverse transcriptase-PCR to be expressed in human chondrocytes. Our findings suggest that alternative splicing in the aggrecan G3 domain may be a mechanism for modulating interactions and extracellular matrix assembly.The aggregating proteoglycans aggrecan, versican, neurocan, and brevican form the lectican (1) or hyalectan (2) family and are major components of the extracellular matrix (ECM) 1 with important functions in many tissues. The core proteins of these proteoglycans have extended central glycosaminoglycan attachment regions of varying length that are flanked by globular domains (3-6). In the cartilage proteoglycan aggrecan, the large extent of glycosaminoglycan side chain substitution and the resulting fixed charge density attracts counter-ions and water through osmotic processes. The resulting swelling pressure is crucial for the biomechanical properties of this tissue (7). The conserved N-terminal globular G1 domains anchor these proteoglycans to hyaluronan in an interaction stabilized by the link protein (8 -12). Aggrecan contains an additional globular G2 domain of unknown function between the G1 domain and the glycosaminoglycan attachment region (13). The C-terminal G3 domain is highly conserved and found in all four of these proteoglycans.We have shown previously that the G3 domain mediates binding to other ECM molecules, e.g. tenascin-R (14, 15), fibulin-1 (16), fibulin-2 (17), and fibrillin-1 (18). The G3 domain also binds sulfated glycolipids on the cell surface (19). In addition, neurocan has been reported to bind to tenascin-C (20). The ECM protein ligands for the G3 domains are all dimeric or multimeric proteins, and we have shown that they can crosslink proteoglycans from different hyaluronan/proteoglycan aggregates (17). This may well be of functional importance for the organi...

The mechanism, by which transhydrogenase couples transfer of H-equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichiu coli using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP' or NADPH. This indicates that the rates of release of NADP' and NADPH from their binary complexes with the enzyme are slow. The dependences on pH and salt concentration suggest that (a) release of both NADP' and NADPH are accompanied by the release of H' from the enzyme and (b) increased ionic strength decreases the value of the pK, of the group responsible for H' release. Modification of the enzyme with N,Wdicyclohexylcarbodiimide led to inhibition of the rate of release of NADP' and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD', NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates.It is considered that the binding and release of H' , which accompany the binding and release of NADP+/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane-bound state.