Transforming growth factor-beta (TGFb)1 is thought to be implicated in breast cancer progression. However, data about the influence of TGFb1 on breast cancer development are conflicting. To clarify the clinical relevance of TGFb1, TGFb1 protein level has been measured by enzyme-immoassay in 193 breast tumour samples. We found that 94.3% of patients expressed TGFb1 with a range of 0 -684 pg mg À1 protein.In the overall population, an increase of tumoral TGFb1 was observed in premenopausal patients when compared to postmenopausal subgroup (P ¼ 0.0006). When patients were subdivided according to nodal status, TGFb1 was correlated to type-1 plasminogen activator inhibitor in the node-negative subgroup (P ¼ 0.040). Multivariate analysis revealed that, after lymph node status (P ¼ 0.0002) and urokinase-type plasminogen activator (P ¼ 0.004), TGFb1 was an independent prognostic marker for DFS (P ¼ 0.005) in the overall population. In the node-negative population, TGFb1 was the prominent prognostic factor (P ¼ 0.010). In the same population, Kaplan -Meier curves demonstrated that high TGFb1 level was correlated with a shorter diseasefree survival (P ¼ 0.020). These data suggest that the measurement of tumoral TGFb1 protein level, especially for node-negative patients, might help to identify a high-risk population early in tumour progression.

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.

The characterization of novel prognostic markers in breast cancer is necessary to improve the identification of high-risk populations. In our study, the prognostic significance of VEGF and amphiregulin (AR) was investigated and compared to conventional prognostic factors in primary breast cancers. The analysis was performed using enzyme-linked immunoassay in a series of 193 patients, and univariate and multivariate analysis were performed in the overall population as well as in pre-and post-menopausal patients subdivided in nodenegative (N؊) and node-positive (N؉) subsets. AR (median, 44.8 pg/mg protein) appeared strongly correlated with progesterone receptors (PgR) (p ‫؍‬ 0.0018) in the premenopausal N؉ population, and with uPA (p‫؍‬ 0.020) and VEGF (p‫؍‬ 0.0053) in the postmenopausal/N؉ patients. Despite these attractive data, AR expression was not significant for recurrence or survival outcome. Data revealed strong correlation between VEGF and uPA, and PAI-1, in the N؉ population. Moreover, patients with high VEGF levels displayed poor outcome, with an increased risk for N؉ subset. These data were confirmed by multivariate analysis that presented histologic grade (HR, 10.55, p ‫؍‬ 0.001) and VEGF (HR, 3.89, p ‫؍‬ 0.03) as the prominent prognostic markers for overall survival for the N؉ population. Furthermore, infiltrating ductal carcinomas (IDC) were shown to express higher levels of both uPA (p < 0.0001) and VEGF (p ‫؍‬ 0.002) than intralobular carcinomas. This retrospective study reinforces the pejorative biological role of VEGF in the progression of breast tumors. Our data also suggest that VEGF and uPA might play particular role in the biology and progression of IDC.

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 (330 kDa) 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% (¹KI). 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 (¹TSH) 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.

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.

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.

Key words: amphireguline; transforming growth factor-␤1; urokinase-type plasminogen activator; breast cancerNormal and malignant mammary epithelial cells synthesize locally acting growth factors that function through autocrine, juxtacrine or paracrine pathways. Epidermal growth factor (EGF)-related peptides, in combination with their cognate receptors, are involved in the regulation of mammary-gland development, morphogenesis and lactation, and also play a pivotal role in the pathogenesis of human breast cancer. 1 Among growth factors known to be engaged in tissue growth and differentiation, amphireguline (AR) seems to play a special physio-pathological role in the human mammary gland. Amphireguline is a heparin-binding epidermal growth factor-related peptide presenting 38% sequence homology with EGF. It was initially purified from conditioned medium of breast cancer epithelial MCF-7 cells treated with phorbol 12-myristate 13-acetate. 2 Two mature forms of the protein (78 and 84 amino acids) derived from a 252 amino acid transmembrane precursor by proteolytic cleavage are described currently. 3 Both forms have a Mr ranging from 16 -25 kDa, depending on their degree of glycosylation as well as on their NH2-terminal processing. 4 Whereas AR has been demonstrated to bind and to activate EGF-receptor (c-erb B1), direct interaction of AR with other members of the c-erb B family such as c-erb B 2, 3 or 4, has not been reported. 3,5 AR is expressed in several tissues, such as human ovary, placenta, lung, kidney, stomach and breast 6 -8 and induces variable effects on cell proliferation depending on its concentration and on the nature of target cells. 2,3,6 Nevertheless, increased evidences strongly suggest that AR may function as a potential autocrine growth factor in tumoral as well as in their counterpart normal cells such as, prostatic, 9 colic 7 and mammary epithelial cells. 10 Moreover, overexpression of AR has been detected in several estrogen-responsive and -unresponsive breast cancer cell lines as well as in approximately 80% of human primary breast carcinoma. [11][12][13] These data agree with clinical data demonstrating that AR overexpression in solid tumors such as breast cancer is associated with lymph node infiltration and correlates with poor prognosis. 14,15 Degradation of the extracellular matrix (ECM) is a prerequisite for the formation of new vessels, as well as for invasion and metastasis. The degradation of extracellular matrix proteins is catalyzed by the concerted action of several enzymes, including Type IV collagenase and metalloproteinase (MMP). 16 Plasmin system is a broad spectrum of proteolytic enzymes that degrades several extracellular matrix components, activates other matrix metalloproteinases, and thus exhibits a wide array of physiological and pathological processes, such as tissue growth and remodeling, tumor invasion and metastasis. 17 Plasmin is formed from plasminogen, by the tissue-type plasminogen activator (tPA) or by urokinase-type plasminogen activator (uPA). The activation of...

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