Lack of responsiveness to TGF-beta1 in a thyroid carcinoma cell line with functional type I and type II TGF-beta receptors and Smad proteins, suggests a novel mechanism for TGF-beta insensitivity in carcinoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine. In the present study we have investigated the expression of TGF-beta receptors (TbetaR's) and SMAD proteins in non-neoplastic and neoplastic thyroid follicle cells. We found expression of all TbetaR's (type I, II and III) and SMAD proteins analysed (Smad2, Smad3, Smad4, Smad6 and Smad7). Five out of six human anaplastic thyroid carcinoma cell lines were growth inhibited by addition of TGF-beta1, and therefore considered to be TGF-responsive. One cell line however, HTh 7, did not respond to TGF-beta1 with growth inhibition, induction of the extracellular matrix protein fibronectin or immediate early genes junB, Smad6 and Smad7 mRNA. Analysis of the TGF-beta intracellular signalling pathway in HTh 7 cells showed that receptors were capable of signalling, e.g. Smad2 phosphorylation and SMAD nuclear translocation. In summary, our data shows abundant expression of TGF-beta signalling components in thyroid follicle cells, and the escape from TGF-beta sensitivity in one anaplastic thyroid carcinoma despite an apparently functional TGF-beta/SMAD-signalling pathway, indicating a novel mechanism for TGF-beta insensitivity.

Structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase.

The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated. Optimal incubation conditions for assay of the microsomal (S)-adenosyl-L-methionine:sterol delta 24-methyl transferase (SMT) have been established for the first time. The microsomal preparation was found to catalyze the formation of a delta 24(28)-sterol and to be free of contaminating methyl transferase enzymes, e.g. those which form delta 23-24 methyl sterols (cyclosadol) and delta 25-24 beta-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate. From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT. In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates. Of the chemical groups which distinguishes cycloartenol, a free 3 beta-OH,9 beta,19-cyclopropyl group, trimethylated saturated nucleus, and delta 24-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation. Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W. D., Benson, M., Lundin, R. E., and Le, P. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5759-5763).

Regulation of sterol biosynthesis in sunflower by 24(R,S),25-epiminolanosterol, a novel C-24 methyl transferase inhibitor.

Whereas sitosterol and 24(28)-methylene cycloartanol were competitive inhibitors (with Ki = 26 microM and 14 microM, respectively), 24(R,S)-25-epiminolanosterol was found to be a potent non-competitive inhibitor (Ki = 3.0 nM) of the S-adenosyl-L-methionine-C-24 methyl transferase from sunflower embryos. Because the ground state analog, 24(R,S)-oxidolanosterol, failed to inhibit the catalysis and 25-azalanosterol inhibited the catalysis with a Ki of 30 nM we conclude that the aziridine functions in a manner similar to the azasteriod (Rahier, A., et al., J. Biol. Chem. (1984) 259, 15215) as a transition state analog mimicking the carbonium intermediate found in the normal transmethylation reaction. Additionally, we observed that the aziridine inhibited cycloartenol metabolism (the preferred substrate for transmethylation) in cultured sunflower cells and cell growth.

Purification and Some Properties of a Daucus carota Lectin which Enhances the Activation of Prophenoloxidase by CaCl(2).

Carrot (Daucus carota) cell cultures were found to secrete a protein, with the molecular weight of 58,000, which could strongly enhance the activation of carrot prophenoloxidase by CaCl(2). The protein was isolated from the culture filtrate and purified to electrophoretic homogeneity. The purified protein did also show hemagglutinating activity toward rat as well as rabbit erythrocytes and this activity was inhibitied by N-acetylglucosamine or fetuin.

Purification of a trypsin inhibitor secreted by embryogenic carrot cells.

A protease inhibitor with a molecular weight of about 12,800 was purified to electrophoretic homogeneity from Daucus carota cells. The protease inhibitor was heat stable and inhibited trypsin but had no activity toward chymotrypsin or subtilisin. Nonembryogenic as well as embryogenic strains contained the inhibitor in similar amounts, but in the embryogenic strains the trypsin inhibitor was released from the cells and as a result accumulated in high concentrations in the culture medium, whereas no release of the trypsin inhibitor was found during cultivation of the nonembryogenic strains. Very low amounts of acid phosphatase or alpha-mannosidase activity were found in the culture filtrate of both embryogenic and nonembryogenic strains, which suggest that the release of the inhibitor from embryogenic strains was not due to cell lysis.