Determination of TGFbeta1 protein level in human primary breast cancers and its relationship with survival.

Transforming growth factor-beta (TGFbeta)1 is thought to be implicated in breast cancer progression. However, data about the influence of TGFbeta1 on breast cancer development are conflicting. To clarify the clinical relevance of TGFbeta1, TGFbeta1 protein level has been measured by enzyme-immunoassay in 193 breast tumour samples. We found that 94.3% of patients expressed TGFbeta1 with a range of 0-684 pg mg(-1) protein. In the overall population, an increase of tumoral TGFbeta1 was observed in premenopausal patients when compared to postmenopausal subgroup (P=0.0006). When patients were subdivided according to nodal status, TGFbeta1 was correlated to type-1 plasminogen activator inhibitor in the node-negative subgroup (P=0.040). Multivariate analysis revealed that, after lymph node status (P=0.0002) and urokinase-type plasminogen activator (P=0.004), TGFbeta1 was an independent prognostic marker for DFS (P=0.005) in the overall population. In the node-negative population, TGFbeta1 was the prominent prognostic factor (P=0.010). In the same population, Kaplan-Meier curves demonstrated that high TGFbeta1 level was correlated with a shorter disease-free survival (P=0.020). These data suggest that the measurement of tumoral TGFbeta1 protein level, especially for node-negative patients, might help to identify a high-risk population early in tumour progression.

Thyrotropin regulates tyrosine sulfation of thyroglobulin.

OBJECTIVE: To study the regulation of thyroglobulin sulfation by thyrotropin (TSH) and iodide. Sulfation, a widespread post-translational modification of proteins, is involved in various biological activities. Thyroglobulin has been reported to be sulfated but, to date, the role of sulfate residues in the metabolism and function of thyroglobulin is not known; moreover, the regulation of thyroglobulin sulfation has not been yet investigated. METHODS: The effect of TSH on thyroglobulin sulfation was studied in porcine thyroid cells cultured on porous collagen-coated filters. Cells cultured with or without TSH and with or without iodide (KI) were incubated for 4 days with radioactive sulfate. The specific radioactivity of thyroglobulin subunit (330kDa) was determined from apical media analyzed by electrophoresis. Enzymatic hydrolysates of the purified thyroglobulin were separated by oligosaccharide affinity chromatography and thin-layer chromatography; alkaline hydrolysates were analyzed only by thin-layer chromatography. RESULTS: Thyroglobulin secreted by TSH-stimulated cells incorporated about twofold less radioactive sulfate. Iodide slightly modified this incorporation. Enzymatic hydrolysates of purified thyroglobulin showed sulfate residues bound essentially to complex oligosaccharide units. Alkaline hydrolysis was necessary to release all sulfated amino acids (tyrosine and serine). In the absence of TSH the proportion of tyrosine sulfate was dramatically increased: 24% compared with 7% (+KI) or 5% (-KI). The ratio of specific radioactivity of thyroglobulin to the specific radioactivity of intracellular inorganic sulfate (determined in each culture condition) gave the number of sulfated residues incorporated: 46 (-TSH) and 31 (+TSH) per mol thyroglobulin. From this distribution, we deduced the number of residues bound to complex oligosaccharide units and to tyrosine. Thus TSH decreased the number of sulfate residues on tyrosine from 11 to 2 per mol thyroglobulin. CONCLUSIONS: TSH regulates the binding of sulfate groups to tyrosine residues. Iodide exerts a slight control over this process.

Thyrotropin controls dolichol-linked sugar pools and oligosaccharyltransferase activity in thyroid cells.

We previously showed that thyroglobulin (Tg) glycosylation is enhanced 1.5-fold under thyrotropin (TSH) stimulation, corresponding to an increased number of oligosaccharide chains per molecule of Tg. Now the steps involving dolichol components and oligosaccharyltransferase activity have been studied. Porcine thyroid cells were cultured on porous bottom filters with or without TSH and incubated with [14C]mevalonate. Under TSH regulation, the level of the whole of dolichol components was increased 1.25-fold without modifying their distribution. Dolichol, and free and monosaccharide-linked dolichyl-phosphate, represented respectively 40% and 45% of total dolichol components while dolichyl-pyrophosphate-oligosaccharide represented 3% only. A marked enhancement (4.2-fold) of oligosaccharyltransferase activity occurred in stimulated cells, which could correspond to the addition of the two TSH effects: stimulation of Tg synthesis (3-fold) and of Tg glycosylation (1.5-fold). The amount of lipid carriers appeared to be insufficiently increased but no component is a limiting step, suggesting that the turnover of dolichol derivatives may be increased under TSH control through their use by higher amounts of Tg.

Divergent effect of TGFbeta1 on growth and proteolytic modulation of human prostatic-cancer cell lines.

Plasminogen activators (PAs) play a key role in malignant transformation. PA secretion by tumoral cells is strongly correlated with their aggressive phenotype. Regulation of invasive potential by growth factors has been also demonstrated. This study was designed to investigate the effects of 5alpha-dihydrotestosterone (DHT), epidermal growth factor (EGF), transforming growth factor beta1 (TGFbeta1), retinoic acid and basic fibroblastic growth factor (bFGF) on cell growth and PA expression and secretion in DU145 and PC3 cells, 2 human prostatic-cancer cell lines. The proliferation of 2 cell lines was significantly increased only by EGF (about 30%), but decreased by TGFbeta1 (40% inhibition). However, EGF-treated cells showed significant enhancement (about 400%) of u-PA secretion. A similar effect was observed when cells were cultured with DHT (200%) and with TGFbeta1 (300%). Nevertheless, u-PA mRNA level in EGF-, TGFbeta1 - or DHT-treated cells was amplified only between 110 and 180% of control, suggesting that growth factors differently controlled the steps of PA expression. Furthermore, our results clearly showed the divergent effect of TGFbeta1, i.e., an inhibition of prostatic-cell-line growth accompanied by an increase in proteolytic activity.

Hormonal regulation of some steps of thyroglobulin synthesis and secretion in bicameral cell culture.

Porcine thyroid cells were cultured for 15 days on porous bottom chambers with or without different mixtures of hormones added to serum-free basal medium. Assays with 10% serum were also performed for comparison with previously published results. The effects of the hormones, particularly insulin, TSH and hydrocortisone, were studied on total RNA content, thyroglobulin mRNA level, the amount of thyroglobulin secreted into the apical medium and on glycosylation. Insulin and TSH similarly increased the total RNA content, and their effects were additive. Thyroglobulin mRNA content was increased twofold by insulin and threefold by TSH. When they were added simultaneously, the maximal level of thyroglobulin mRNA was reached, showing that TSH and insulin effects on thyroglobulin gene expression were additive. Hydrocortisone alone did not modify total RNA or thyroglobulin mRNA content but the hormone amplified total RNA when insulin and TSH were present together. The basal level of thyroglobulin secreted into the apical medium was increased threefold by insulin and fourfold by TSH. The effects of these two hormones added together appeared to be additive. Hydrocortisone had no effect alone or even when combined with insulin or TSH. However, when the three hormones were added together, the hormonal response was amplified. TSH effect and insulin effect on the incorporation of 3H-mannose into thyroglobulin as well as on the anionic residue content of the molecule were additive.

Glycosylation of thyroglobulin secreted by porcine cells cultured in chamber system: thyrotropin controls the number of oligosaccharides and their anionic residues.

Porcine thyroid cells were cultured in porous bottom chambers in the presence or in the absence of TSH added to the basal medium. Radiolabeled-sugar (3H-mannose) was added to the basal medium on day 11 for 4 days and the glycosylation of thyroglobulin (Tg), the major glycoprotein secreted into the apical medium, was studied. The incorporation of 3H-mannose per molecule of Tg was increased 1.5-fold by a 50 microU/ml minimal concentration of TSH. The distribution of glycopeptides (after pronase digestion) on concanavalin A sepharose column was not modified by the presence of TSH. However this distribution was different from that observed for Tg extracted from gland (more multiantennary units than biantennary units and polymannose units). After desialylation and desulfation, the sizes of the oligosaccharide chains analyzed on HPLC appeared similar when cells were cultured under stimulation or not. Thus TSH enhanced sugar incorporation without modifying either the distribution of the different oligosaccharide moieties or their sizes. Consequently the effect of TSH was a 1.5-fold increase in oligosaccharide chains linked to asparagine residues. 3H-Mannose-oligosaccharide chains were then analyzed on ion-exchange HPLC before and after desialylation and desulfation. The number of anionic residues per oligosaccharide unit (particularly sulfate residues) was higher in the absence of TSH than in the presence of TSH. Nevertheless, since TSH increased the number of carbohydrate units per molecule of Tg 1.5-fold, the total content of anionic residues bound to oligosaccharide units per molecule of Tg seems not to be modified by TSH.