[Proteoglycans and breast cancer]. / Protéoglycannes et cancer du sein.

Proteoglycans (PG) are complex sulphated macromolecules composed of linear polysaccharide chains of glycosaminoglycans (GAG) covalently attached to a core protein. These GAG chains contain sulphate groups at various positions, giving them a high density of negative charges, and allowing them to interact with extracellular matrix molecules, including various growth factors. In the developing mammary gland, sulphated proteoglycans participate in morphogenesis and interact with extracellular matrix components in order to constitute a functional matrix. In breast pathogenesis, qualitative or quantitative changes in PG may have important consequences on cell proliferation and/or differentiation. Thus, several studies showed large variations in the nature and distribution of PG/GAG in breast cancer. Accumulation of chondroitin sulfate proteoglycans was described in the stromal compartment of mammary biopsy sections, and content in heparan sulfate proteoglycans, which were more specifically distributed in the epithelial compartment, increased with the level of malignancy and invasiveness of breast cancer tissues. Furthermore, heparan sulfate proteoglycans seem to be involved in control of the growth-promoting activity of numerous growth factors such as fibroblast growth factors also named Heparin-Binding Growth Factors (HBGF). The implication of PG in growth factor activity suggest that PG may have prognostic value in breast cancer. In future, structural studies into the specific HS-sequences involvement in growth factors binding could allow the development of new antiproliferative strategies.

Production of sulfated proteoglycans by human breast cancer cell lines: binding to fibroblast growth factor-2.

The cellular distribution and nature of proteoglycans synthesised by human breast cancer cells in culture were studied. Proteoglycans were labelled with [35S] sulfate, purified, and characterised after ion-exchange chromatography followed by gel-filtration chromatography and treatment with glycosaminoglycan degrading enzymes. Proteoglycans were isolated from the culture medium and from cell layers of the hormono-dependent well-differentiated MCF-7 cell line, the hormono-independent poorly-differentiated MDA-MB-231 and the HBL-100 cell line which is derived from non malignant breast epithelium. HBL-100 and MDA-MB-231 cells produced larger amounts of proteoglycans which had a lower degree of sulfation than MCF-7 cells. Gel-filtration chromatography on Sepharose CL-6B indicated that HBL-100 and MDA-MB-231 cells accumulated cell surface heparan sulfate proteoglycans (HSPG), with a high apparent molecular weight (Kav 0.1). In contrast, the MCF-7 cell monolayers synthesised small sulfated macromolecules (Kav 0.4) which possessed mostly chondroitin sulfate chains. Moreover, considerable differences in the nature of the sulfated proteoglycans released into the culture medium of these breast epithelial cell lines were observed. MCF-7 cells released into the culture medium HSPG as the main proteoglycan component while MDA-MB-231 and HBL-100 cells released mainly chondroitin sulfate proteoglycans. In these three cell lines, medium-released sulfated macromolecules have a higher hydrodynamic size than cell-associated ones. Proteoglycans purified by ion-exchange chromatography were tested for their ability to bind 125I FGF-2. We demonstrated that HBL-100 and MDA-MB-231 cells bind more FGF-2 to their heparan sulfate proteoglycans than MCF-7 cells. Taken together, these results suggest that differences in proteoglycan synthesis of human breast epithelial cells could be responsible for differences in their proliferative and/or invasive properties.

Heparan sulfate proteoglycans play a dual role in regulating fibroblast growth factor-2 mitogenic activity in human breast cancer cells.

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of 125I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10-30 mM; higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.

[Involvement of sulfated proteoglycans in the control of proliferation of MCF-7 breast cancer cells]. / Implication des protéoglycanes sulfatés dans le contrôle de la prolifération des cellules cancéreuses mammaires MCF-7.

The MCF-7 breast cancer cells exhibit remarkable growth enhancement in response to basic fibroblast growth factor (FGF-2) stimulation in a dose dependent manner. To investigate the involvement of proteoglycans on control of FGF-2 induced proliferation, polysaccharide chains were degraded by specific enzymes. Our results showed that MCF-7 cells were unsensitive to FGF-2 after enzymatic degradation of heparin sulfate proteoglycans (HSPG) by heparinase. After metabolic inhibition of sulphation by sodium chloride, radiolabelled proteoglycans were purified and quantified by ion exchange chromatography. Sodium chlorate treatment reduced by 70% sulfation of proteoglycans. This decrease of sulphation totally inhibited FGF-2-mediated proliferation. The sulphated glycosaminoglycans which were critical in FGF-2-induced proliferation were strictly HSPG, as an addition of heparin in cell culture medium can restore FGF-2 mitogenic activity. In contrast, other glycosaminoglycans (chondroitin sulfate/hyaluronic acid) did not show any effect. These results provide clear evidence for the critical role of HSPG in FGF-2-induced proliferation on MCF-7 breast cancer cells.

Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles waltl.

Denervation of the amputated limb of newts stops the regeneration process by decreasing blastema cell proliferation. We investigated the effect of the denervation on each of the two compartments (epidermal cap, mesenchyme) in mid-bud blastemas on the level of sulphated glycosaminoglycans (GAGS). Denervation resulted in an increase of about threefold in the incorporation of [35S] sulphate into mesenchyme GAGs but had no effect on the epidermal cap. The increase of GAG synthesis in the mesenchymal part of the blastema involved both heparan sulphates and chondroitin-dermatan sulphates. Gel filtration showed no change in GAGs size after denervation. These results confirm that the mesenchymal part of the mid-bud blastema is the main target of nerves and, as heparan sulphates are known to store acidic fibroblast growth factor (aFGF), a polypeptide found in the blastema (Boilly et al.. 1991), this suggest that the nerves' effect on glycosaminoglycans turnover could be implicated in the control of bioavailability of this growth factor in the blastema.

Identification of decorin proteoglycan in bovine tracheal serous cells in culture and localization of decorin mRNA in situ.

Bovine tracheal submucosal gland serous cells in culture synthesize and secrete proteoglycans and not mucin glycoconjugates. We are interested in the characterization and role of these proteoglycans in airway secretions. The major [35S]methionine-labeled proteoglycan present is identified as the small chondroitin/dermatan sulfate proteoglycan decorin (PG II. PG40). Consistent with its identity as decorin this proteoglycan showed average apparent molecular weights of 75,000 to 130,000 with a core protein of an average, M(r) of about 40,000 and with glycosaminoglycan chains sensitive to chondroitinase ABC lyase of an average M(r) of about 25,000. These data were obtained from gel chromatographic and SDS-PAGE analyses. Northern blot analysis and partial amino acid sequencing of the purified protein further confirmed its identity as decorin. In situ hybridization studies using a decorin riboprobe revealed no expression of decorin in the surface epithelium and only low levels of expression in submucosal gland epithelial cells of bovine tracheal tissue. However, high levels of expression were localized to cells which are peripheral to tracheal submucosal gland epithelial cells and which contact with the extracellular matrix.

Embryonic brain-derived heparan sulfate inhibits cellular membrane binding and biological activity of basic fibroblast growth factor.

We have investigated the ability of glycosaminoglycans from embryonic chick brain (15 days old) to interact with basic fibroblast growth factor (bFGF). 35SO4 metabolically labeled glycosaminoglycans were purified and separated on DEAE-cellulose chromatography. Material which eluted between 0.20 and 0.35 M NaCl displaced the binding of [125I]bFGF to brain membrane. This activity was dose-dependent and on the basis to its heparinase sensitivity and chondroitinase insensitivity, has been attributed to heparan sulfate. CL-6B-Sepharose chromatography of this material revealed two glycosaminoglycans of molecular masses of about 15,000 and 65,000. Incubation with [125I]bFGF followed or not by heparinase and chondroitinase treatment of electrotransfert from SDS-PAGE revealed that both of these forms correspond to heparan sulfate chains and bind bFGF. In vitro, embryonic brain-derived heparan sulfate inhibited both bFGF induced [3H]thymidine incorporation in CCL39 cells and neurite outgrowth in PC12 cells. These results suggest that heparan sulfate play an important function in the control of the biological activity of bFGF during brain development.

Growth-related production of proteoglycans and hyaluronic acid in synchronous arterial smooth muscle cells.

1. The growth-stimulating effect of serum on the proteoglycan and hyaluronic acid production in arterial smooth muscle cells was investigated, using cells synchronized by serum deprivation. 2. After stimulation, synthesis of [35S]sulfated proteoglycans and [14C]hyaluronic acid increased during G1 and G2 phases (about 2- and 5-fold, respectively, in the culture medium), in comparison with quiescent cells. 3. Neither the size, nor the charge, nor the relative proportions of [35S]glycosaminoglycans of the proteoglycans were modified. 4. However, when the cells were stimulated to divide, increased synthesis of large [14C]hyaluronic acid was observed concomitantly with the production of higher hydrodynamic size [35S]proteoglycans, which aggregated with hyaluronic acid (20%).

Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell-conditioned medium.

We have previously shown (Berrou et al., J. Cell. Phys., 137:430-438, 1988) that porcine endothelial cell-conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. 1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]-sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. 2) Whereas ECCM stimulation of [35S]-methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine-labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. 3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine-labeled proteoglycans 7 protein cores of high apparent molecular mass (550-200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]-serine incorporation in response to ECCM (51%, as measured by Sepharose CL-6B chromatography). 4) Finally, incorporation of [35S]-sulfate into chondroitinase-generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL-6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that 1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and 2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.

Changes in proteoglycans of cultured pig aortic smooth muscle cells during subculture.

Smooth muscle cells were cultured from pig aorta. Changes in both the growth and the properties of sulfated proteoglycans were observed during passage. The population doubling time during log phase growth was 34 h from Passages 3 to 7-8 but 20 h at the Passage 11, and the cell density at the stationary phase, was 86,000 and 136,000 cells/cm2 at Passages 3 and 11, respectively. Structural characteristics of sulfated proteoglycans secreted into the medium were investigated after metabolic labeling with [35S]-sulfate. Significant differences were observed with age in vitro: a) [35S]proteoglycan complexes were in a greater amount at Passage 10 than at Passage 3; b) the hydrodynamic size of at least 45% of subunits and about 90% of monomers decreased with in vitro aging; c) this decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains; d) an increase of 15% in the proportion of dermatan sulfate was observed when cells were subjected to 10 passages.

Effect of endothelial-cell-conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta.

The effect of porcine endothelial-cell-conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum-free conditions. Maximal stimulation of [35S]-sulfate incorporation (50%) into medium-secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium-secreted [35S]-labeled proteoglycans. Those [35S]-proteoglycans secreted (for 24 h) in the presence of endothelial cell-conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]-sulfate into glycanic chains (50.1%) and [14C]-serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]-serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para-nitrophenyl-beta-D-xyloside. Fractionation of medium-secreted [35S]-proteoglycans and xyloside-initiated glycosaminoglycans revealed that stimulation of [35S]-glycosaminoglycan protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.

Effect of insulin on sulfated proteoglycan synthesis in cultured smooth muscle cells from pig aorta.

The effect of insulin upon proteoglycan synthesis was studied in cultured smooth muscle cells from pig aorta blocked in the G0 phase by serum deprivation. Insulin enhanced [35S]sulfate incorporation into cell layer and medium-secreted proteoglycans. The increase in incorporation of the precursor was not due to a mitogenic response by smooth muscle cells to the hormone and the specific radioactivity of proteoglycans showed that the stimulation reflected a real increase in sulfated proteoglycan synthesis. Maximal stimulation was observed, for the cell layer as well as for the medium, 40 h after the addition of 1.7 x 10(-7) M insulin and reached respectively 65 and 53%. This stimulation was about 80 and 60% of the level achieved with 10% fetal calf serum for cell layer and medium-secreted proteoglycans, respectively. The half-maximal effect was attained, for both the cell layer and the medium, in the presence of 2.1 x 10(-9) M insulin. Proteoglycans secreted into the medium, in the presence of 1.7 x 10(-8) M insulin for 40 h, showed a higher proportion of complexes (24%) than those synthesized in control medium (11%) and at least 95% of the monomers from culture treated with insulin were characterized by a smaller hydrodynamic size than those synthesized by cells maintained in control medium. This decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains.

Role of proteoglycan in the binding of low-density lipoprotein to cultured arterial smooth muscle cells.

The role of proteoglycans in the binding of 125I-labeled low-density lipoproteins (LDL) to cultured arterial smooth muscle cells was examined. About 60% of cell bound 125I-labeled LDL could be released by unlabeled LDL, heparin, dextran sulfate or proteoglycan. Binding of 125I-labeled LDL decreased by about 50% when incubated in the presence of exogenous arterial proteoglycans. Exposure of cell cultures to rho-nitrophenyl-beta-D xyloside resulted in a 40% decrease in both the amount of 35S-labeled proteoglycan in the cell layer and the 125I-labeled LDL binding, without modifying significantly the cell number and amount of cell layer protein. These data suggest that cell surface and/or cell matrix proteoglycans may influence binding of LDL to either specific receptor or non-receptor sites and thereby play a role in the intracellular deposition of lipid in the arterial wall.

Characterization and macromolecular association of proteoglycans produced by pig arterial smooth muscle cells in culture.

1. Medium and cell-layer proteoglycans from pig aorta smooth muscle cells in culture were compared. In both compartments, the main proteoglycans contained chondroitin sulfate-dermatan sulfate chains of 40 kDalton. 2. However, cell-layer proteoglycans differed from those of the medium by the presence of: (a) some small-size proteoglycans; (b) a greater amount of heparan sulfate; (c) chondroitin sulfate-dermatan sulfate enriched in iduronate and in 4 sulfate- (instead of 6 sulfate-) residues. 3. During dissociation-reassociation assays of arterial proteoglycans with exogenous hyaluronate or "aggregate" proteoglycans, the in vitro formation of complexes appeared to involve inter-associations between proteoglycans molecules, in addition to aggregation with hyaluronate.

Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta.

Cultured smooth muscle cells from pig aorta arrested in G0 phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in G1 phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30-32 h; 75% of these cells multiplied after 30-32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [35S]sulfate. Smooth muscle cells in G1 or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [35S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during G1 phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CL-6B columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied: [35S]Proteoglycan complexes represented at the end of G1 phase and of the cell cycle respectively 19 and 16% of the total [35S]proteoglycans secreted into the medium. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar kav after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (kav 0.68 at the end of G1 phase and 0.65 at the end of full cell cycle). About 95% of monomers synthesized at the two stages of the cell cycle were eluted at kav 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the medium during the G1 and G2 phases.

The structural characterization of proteoglycans of cultured aortic smooth muscle cells and arterial wall of the pig.

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (rho av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (rho av = 1.60 g/ml) fraction and three fifths (rho av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83-90%) with lower proportions of heparan sulfate (5-11%) and hyaluronic acid (3-6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11-13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with Kav 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column Vo and the rest at a Kav of 0.34. The majority of subunit macromolecules eluted at the Vo fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4-7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.

[The role of arterial wall proteoglycan in arteriosclerosis (author's transl)]. / Le role des protéoglycanes de la paroi artérielle dans l'athérosclérose.

Arteriosclerosis is the consequence of numerous factors which may be studied by inspecting; the composition of blood (cholesterol) and haemostasis, as well as, modifications in the composition and structure of aging arterial-wall tissue. Arterial-tunica cells synthesise in particular, elastin and proteoglycan (mucopolysaccharide-protein complexes) which account for the artery's mechanical and elastic properties. We have shown by biochemical separation and characterization that proteoglycan synthesis is considerably modified in aging arterial tissue. The metabolism of these macromolecules declines. Large aggregates of proteoglycan form. Alterations are observed in the distribution of the different protein-associated long chain polysaccharides, in fatty acids and in calcium bound to these proteoglycans favorising the formation of arteriosclerotic plaques. Studies on the aging of arterial cells in culture also demonstrate these same alterations in proteoglycans.