Matrix-degrading proteinases are shed in membrane vesicles by ovarian cancer cells in vivo and in vitro.

The in vitro release of matrix-degrading proteinases from breast cancer cells is associated in part with shed membrane vesicles. To determine whether shed vesicles might play a similar role in ovarian cancer cells, we analyzed the shedding phenomenon in vivo and in vitro as well as the enzymatic content of their vesicles. This is the first time that an immunoelectron microscopical analysis revealed membrane vesicles carrying tumor-associated antigen alpha-Folate Receptor (alpha-FR), circulating in biological fluids (ascites and serum) of an ovarian carcinoma patient. These vesicles were trapped in a fiber network with characteristic fibrin periodicity. An ovarian cancer cell line (CABA I) established from ascitic fluid cells of this patient, grew in Matrigel and formed tubular structures suggesting invasive capability. Immunofluorescence analysis demonstrated strong cytoplasmic staining of CABA I cells with anti-matrix metalloproteinase-9 (MMP-9) and anti-urokinase-type plasminogen activator (uPA) antibodies. CABA I cells shed membrane vesicles, which were morphologically similar to those identified in vivo, as determined by electron microscopy. Gelatin zymography of vesicles isolated both in vivo and in vitro revealed major gelatinolytic bands of the MMP family, identified as the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2). By casein-plasminogen zymography we observed high-molecular weight (HMW)-uPA and plasmin bands. Incubation of purified vesicles from CABA I cells with Matrigel led to cleavage of Matrigel components. Taken together, our results point to a possible role of shed vesicles, both in vivo and in vitro, in proteolysis that mediates invasion and spread of ovarian epithelial carcinoma cells.

Membrane vesicles in ovarian cancer fluids: a new potential marker.

The rate of membrane vesicle shedding by tumor cells is probably related to their invasive capability. In order to verify whether the vesicle amount could be utilized as a marker of different pathologies, we analyzed biological fluids obtained from 33 patients with gynecological diseases. In fluids of benign serous cysts, vesicle content was extremely low; in cystoadenomas and fibromas generally it was low. On the contrary, large amounts of vesicles were found in malignant tumor fluids. Gelatin zymographies showed the presence of MMP-2 and MMP-9 in all vesicles except in those recovered from fluids of some serous cysts. A positive correlation between tumor malignancy and both vesicle-amount and vesicle-associated MMP-2 activity was noticed. We also analyzed vesicle content in ascitic fluids recovered from two carcinomas at different times during clinical treatment. In both cases, tumor progression, not monitored by Ca 125 levels, was pointed out by an increased amount of vesicles in ascites. These findings suggest that vesicle content in biological fluids could represent a new useful marker of tumor aggressiveness and tumor progression.

The amount and proteolytic content of vesicles shed by human cancer cell lines correlates with their in vitro invasiveness.

Cancer cells are known to shed extracellular membrane vesicles both in vitro and in vivo. To analyse their possible involvement in the metastatic behaviour of tumours, we measured the Matrigel invasion capability and amounts of vesicles shed by four human tumour cell lines (8701-BC, MCF-7, MDA-MB-231 and HT-1080), and by MCF-10A, an immortalised human breast cell line. The proteolytic activity content of vesicles was analysed by gelatin and casein zymographies. While MCF-10A cells do not release a measurable amount of vesicles, all tumour lines analysed, when cultured in presence of serum, shed vesicles rich in MMP-9. Other vesicle-associated proteinases include MMP-2 and uPA. Amounts and proteolytic activities of shed vesicles correlate with the in vitro invasiveness of cells. Since vesicles appear to promote the proteolytic cascade required for the localised degradation of the extracellular matrix, their shedding from cancer cells might represent an important feature of tumour progression.

Selective localization of matrix metalloproteinase 9, beta1 integrins, and human lymphocyte antigen class I molecules on membrane vesicles shed by 8701-BC breast carcinoma cells.

The shedding of membrane vesicles from the cell surface is a vital process considered to be involved in cell-cell and cell-matrix interactions and in tumor progression. By immunoelectron microscopic analysis of surface replicas of 8701-BC human breast carcinoma cells, we observed that membrane vesicles shed from plasma membranes contained densely clustered gelatinase B [matrix metalloproteinase 9 (MMP-9)], beta1 integrins, and human lymphocyte antigen class I molecules. By contrast, alpha-folate receptor was uniformly distributed on the smooth cell membrane and shedding areas. Both cell surface clustering of selected molecules and membrane vesicle release were evident only when cells were cultured in the presence of serum. Vesicle shedding occurred preferentially at the edge or along narrow protrusions of the cell. Specific accumulation of proMMP-9 and active forms of MMP-9 in shed vesicles was also demonstrated by gelatin zymography. In addition, Western blotting analysis showed the presence of a large amount of proMMP-9/tissue inhibitor of metalloproteinase 1 complex. The release of selected areas of plasma membranes enriched with MMP-9 and beta1 integrins indicates that membrane vesicle shedding from tumor cells plays an important role in the directional proteolysis of the extracellular matrix during cellular migration. The presence of human lymphocyte antigen class I antigens suggests a mechanism for tumor cells to escape from immune surveillance.

Modulation of vesicle shedding in 8701 BC human breast carcinoma cells.

Vesicles, shed in the extracellular medium by several kinds of normal and tumoral cells, are known to play important roles in cell-cell and cell-matrix interactions and to participate in mechanisms by which tumoral cells acquire metastatic capability and evade immune surveillance. Regulation of the shedding phenomenon and molecular mechanisms involved in extracellular vesicle production are not known and are the subject of this investigation. Fetal calf serum stimulated shedding short after its addition and its stimulatory effect was dose dependent. This effect was reduced after gelatin-Sepharose adsorption indicating a possible involvement of gelatinases on its stimulatory effect. This conclusion was confirmed by the inhibitory effect of bathophenanthroline. Shedding of membrane vesicles decreased after treatment with all trans retinoic acid, a molecule known for its capability to induce cell differentiation. Brefeldin A, an inhibitor of intracellular vesicle movements, and methylamine, an inhibitor of exocytosis, did not abolish shedding. Quercetin, an inhibitor of phosphatidyl inositol 4 kinase and 1,4 phosphatidyl inositol 5 kinase, and 8-Cl-cAMP, a site selective cAMP analogous which induces growth inhibition and differentiation, significantly decreased the amount of shed vesicles.

Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells.

Membrane vesicles are shed by tumor cells both in vivo and in vitro. Although their functions are not well understood, it has been proposed that they may play multiple roles in tumor progression. We characterized membrane vesicles from human HT1080 fibrosarcoma cell cultures for the presence of proteinases involved in tumor invasion. By gelatin zymography and Western blotting, these vesicles showed major bands corresponding to the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2) and to the MMP-9. tissue inhibitor of metalloproteinase 1 complex. Both gelatinases appeared to be associated with the vesicle membrane. HT1080 cell vesicles also showed a strong, plasminogen-dependent fibrinolytic activity in 125I fibrin assays; this activity was associated with urokinase plasminogen activator, as shown by casein zymography and Western blotting. Urokinase was bound to its high affinity receptor on the vesicle membrane. Addition of plasminogen resulted in activation of the progelatinases associated with the vesicles, indicating a role of the urokinase-plasmin system in MMP-2 and MMP-9 activation. We propose that vesicles shed by tumor cells may provide a large membrane surface for the activation of membrane-associated proteinases involved in extracellular matrix degradation and tissue invasion.

Ultrastructural and phenotypic characterization of CABA I, a new human ovarian cancer cell line.

We have established an ovarian cancer cell line (CABA I) from ascitic fluid obtained from a patient with papillary adenocarcinoma of the ovary prior to drug treatment. The epithelial origin of the cell line was confirmed by morphology and by immunofluorescence analysis using anticytokeratin antibodies. Ultrastructural analysis revealed a very irregular membrane surface and a clear cytoplasm rich in electron-lucent vesicles. CABA I cells grow rapidly in culture (doubling time 18 h) in an anchorage-independent manner. Exogenously added beta-estradiol and epidermal growth factor (EGF) treatments did not influence cell growth rate. FACS analysis to determine the phenotypic profile of tumor-associated antigen, membrane receptor, and adhesion molecule expression indicated that the cell line was positive for different members of the c-erbB family, for alpha 6 and beta 1 integrin receptors, and intensively positive for HLA class I antigens and the folate receptor. Molecular characterization revealed no mutations for c-myc and c-k-ras genes, but did detect an exon 5 mutation in the p53 gene. CABA I cells grew poorly as heterotransplants in nude mice, and tumors showed long latency periods. Because early (15-20) and late (55-60) passage cells maintain the same growth and phenotypic characteristics, the CABA I cell line might provide a good in vitro model system to investigate the cellular and molecular events involved in ovarian carcinogenesis.

Cell adhesion-dependent regulation of cell growth during sea urchin development.

In the sea urchin embryo there are at least two cell adhesion molecules related to mammalian cadherins, one of them, similar to E-cadherin, is expressed in embryos at very early developmental stages, the second appears at the blastula stage (G. Ghersi et al. Mech. Dev. 41, 47-55 (1993)). We show here that when sea urchin embryos are treated with monovalent fragments of antibodies directed against the extracellular domain of these molecules, the decompaction of the embryo is accompanied by a sharp reduction of the rate of cell division. Treatment of the embryos with Fab fragments inhibits thymidine incorporation, but does not affect thymidine uptake or amino acid incorporation. After the first day of development treated embryos have 10 times less blastomeres than normal; later, however, they resume development and give eventually rise to normal-looking plutei. Analysis of putative second messengers shows that treatment of the embryos with anti-cadherin Fabs leads to a decreased tyrosine phosphorylation of the two cadherins and of two cadherin-associated proteins and to a doubling of the intracellular concentration of cAMP. These results are discussed in view of the importance of cell adhesion signals for cell growth control.

Inhibitory effects of vesicles shed by human breast carcinoma cells on lymphocyte 3H-thymidine incorporation, are neutralised by anti TGF-beta antibodies.

When membrane vesicles shed in vitro by 8701-BC, a human breast carcinoma cell line, are added to peripheral blood lymphocytes, a strong, dose dependent inhibition of the lymphocyte capability to incorporate 3H-thymidine is observed. Inhibition is evident on both PhA stimulated and non stimulated lymphocytes, it is not specie-specific and occurs after three days of culture. Vesicles shed by the human breast carcinoma cell line MCF-7 have inhibitory effects similar to those observed with 8701-BC vesicles, but vesicles shed by HT-1080, a human fibrosarcoma cell line, do not inhibit, but rather stimulate 3H-thymidine incorporation by peripheral blood lymphocytes. The inhibitory effect of vesicles shed by human breast carcinoma cells is recovered in their acid soluble components, and it is completely neutralised by anti TGF-beta 1 antibodies. These findings suggest a role for shed vesicles, in the escape of breast carcinoma cells from immunological surveillance. The immune suppressing cytokine TGF-beta, which is produced by breast carcinoma cells, could be specifically delivered to lymphocytes reacting with vesicles, which are HLA positive, tumour-associated antigen-rich, membrane structures.

Membrane vesicles shed into the extracellular medium by human breast carcinoma cells carry tumor-associated surface antigens.

We have compared the pattern of surface antigen expression, as detected by monoclonal antibodies (mAbs), in plasma membranes vs shed membrane vesicles of two human breast carcinoma cell lines, MCF-7 and 8701-BC. Antigen expression was detected on cells by immunofluorescence (IF) analysis, whilst, due to their small dimensions, the same technique was not applicable to vesicles. For these structures dot-blot analysis and immunoelectron microscopy (IEM) were employed. When applicable, both cell membranes and membrane vesicles were immunoprecipitated and the precipitate (IP) was analyzed by SDS-PAGE. Cells of both lines expressed HLA class I antigens, epithelial cytokeratins, beta 1 integrins, CEA and the glycoprotein detected by mAb 19.9, but only MCF-7 cells expressed Lewis Y, episialin and globo-H antigens and only 8701-BC cells expressed folate receptor. Membrane vesicles of both cell lines appeared to be rich in beta 1, alpha 3 and alpha 5 integrin chains, expressed HLA class I antigens and carried most of the plasma membrane antigens found in the cell membranes. Overall we have analyzed 17 antigens on the two cell lines and on their vesicles. The results obtained for cells (IF and IP) and those for vesicles (dot-blot and IP) were generally concordantly positive or concordantly negative. We obtained a total of 26 clearly concordant combinations on 34 analyses. In three cases we found discordant results, whereas in the remaining combinations we observed slight reactivity and we found difficulties in determining concordance. Discordant results concerned the expression of the following antigens: folate receptors, which were clearly expressed in 8701-BC cells but not detected by dot-blot analysis or IEM on their shed membrane vesicles; neu (c-erb-B2) receptor found in MCF-7 cell membranes but not in their vesicles; and the globo-H antigen recognized by mAb MBr1, detected at low levels on 8701-BC plasma membranes but undetectable on their membrane vesicles. Like vesicles shed in vitro by cultured cells, the vesicles shed in vivo by human breast carcinoma cells could be tagged with several antibodies against tumor-associated antigens. The vesicles shed in vivo were found in association with a fiber network. Some of the fibers had the characteristic fibrin periodicity. These data suggest that tumor markers detected in the circulation of carcinoma patients, at least in part, are carried by shed membrane vesicles. Moreover the observation that membrane vesicles carry both tumor-associated antigens and HLA class I molecules indicate that these structures could in principle present antigens to the immune system.(ABSTRACT TRUNCATED AT 400 WORDS)

Human breast carcinoma cells cultured in the presence of serum shed membrane vesicles rich in gelatinolytic activities.

Human breast carcinoma cell lines, 8701-BC and MCF-7, in culture shed membrane vesicles with similar morphology. Vesicles shed in the presence of serum were rich in gelatinolytic activities, but not those obtained in the absence of serum. Zymographic analyses of the vesicles from 8701-BC and MCF-7, using gelatin as substrate, showed three predominant activities at 68-kDa, 97-kDa, and above 200-kDa. The ratio of the three activities was similar in the vesicles recovered from the two cell lines, but the vesicles from 8701-BC cells contained greater amounts of activities than those from MCF-7 cells. Optimal pH and sensitivity to protease inhibitors suggest that all gelatinolytic activities detected in vesicles are metalloproteinases. Treatment of the vesicles extracts with 4-aminophenylmercuric acetate and comparison with the purified enzyme indicate that 97-kDa gelatinase is the precursor of matrix metalloproteinase-9 (gelatinase B). These results support the early hypothesis that vesicle shedding from the plasma membrane may participate in metastatic cascade of cancer cells.

Differential expression and function of cadherin-like proteins in the sea urchin embryo.

Cadherins are Ca(+2)-dependent cell surface proteins involved in the specification of the adhesive properties of cells. They are supposed to play a critical role in morphogenesis and pattern formation. In this paper we show that in the sea urchin embryo there are at least two different cadherins of relative molecular masses 140 and 125 kDa. The 140 kDa cadherin is already present in the fertilized egg and is the sea urchin equivalent of E-cadherin. The 125 kDa cadherin, which can be detected using a broad-spectrum anti-cadherin antibody, appears only at later stages of development. In later embryos these two molecules are distributed differently: E-cadherin is present predominantly in the invaginating endoderm of the gastrula while the 125 kDa protein is present on the cell surface of most epithelia. Consistently with the observed differences in expression and in distribution, antibodies directed against these two cadherins differently perturb sea urchin development. For example, when these antibodies are added to early gastrulas only the antibodies against the 125 kDa component can induce a complete disaggregation of the ectoderm, while anti E-cadherin antibodies induce an abnormal development of the endoderm while the embryo maintains its basic integrity. These results are discussed in view of the need for multiple adhesion receptors during pattern formation and embryogenesis.

An acid extract from dissociation medium of sea urchin embryos, induces mesenchyme differentiation.

When material extracted by 1 M acetic acid from the dissociation medium of sea urchin embryos is added at low concentrations to isolated primary mesenchyme cells, it induces skeletogenesis. The same material added to dissociated blastula cells, or to embryos at the blastula stage, stimulates skeleton formation and pigment cell differentiation. On dissociated cells, it also increases cell reaggregation, thymidine incorporation and survival. On embryos, it induces exogastrulation and appearance of extraembryonic pigment cells. The activity of the extract is resistant to raised temperatures and partially to tryptic digestion but is abolished by trypsin treatment followed by heating. The active fraction does not readily filter through Amicon XM-50 and is retarded by column chromatography on Bio-Gel P-60.

Immunological evidence for the presence in sea urchin embryos of a cell adhesion protein similar to mouse uvomorulin (E-cadherin).

A tryptic fragment (88 kDa), obtained from external digestion of sea urchin embryos carried out in the presence of calcium, shows immunological cross-reactivity with polyclonal and monoclonal antibodies (DECMA-1) against mouse teratocarcinoma uvomorulin. Fab fragments obtained from anti-mouse terato-carcinoma uvomorulin mono- and polyclonal antibodies, and from polyclonal antibodies against the partially purified 88-kDa tryptic fragment, decompact early sea urchin embryos and block reaggregation of dissociated sea urchin blastula cells. These data indicate the presence of an uvomorulin-like protein in sea urchin embryos and suggest an important role for this protein in embryonic development.

Different aggregation properties of sea urchin embryonic cells at different developmental stages. I. Stage specificity of aggregation factors solubilized by butanol.

Surface proteins solubilized with butanol from purified plasma membranes of sea urchin embryos at different developmental stages were tested for their aggregation promoting activity on dissociated cells. Cells used for the assays were obtained either from blastulae or from embryos at the 16 cell stage. Results show that a strong enhancement of cell aggregation was produced only when extracted proteins and dissociated cells were obtained from embryos at the same developmental stage.

Different aggregation properties of sea urchin embryonic cells at different developmental stages. II. Stage specific response to fibronectin and collagen.

Fibronectin and collagen were added to cells dissociated from embryos at the blastula and at the 16 cell stages. Both molecules stimulate aggregation of cells dissociated from blastula but they do not affect aggregation of blastomeres dissociated from the 16 cell stage. The stage-specific response to fibronectin and collagen appears to be related to the onset of new functional role(s) of the two molecules at the blastula stage.

Trypsin treatment which elicits DNA synthesis, removes a high molecular weight glycoprotein from the plasma membrane of sea urchin embryonic cells.

SDS-polyacrylamide gel electrophoresis of plasma membrane proteins from sea urchin embryos at the swimming blastula stage allows the identification of fifteen-seventeen components, five of which are glycoproteins. One of these (molecular weight about 145,000) is almost completely absent in the plasma membrane of trypsin treated cells.

Isolation of the plasma membrane from sea urchin embryos.

A method for isolation of sea urchin embryos plasma membranes is described. Purification of the obtained fraction was assayed by several enzymatic markers and electron microscopy. The isolated plasma membranes appear to be pure from contamination of other cell membranes (endoplasmic reticulum and mitochondria), and they can therefore be used for analytical studies on the composition and structure of plasma membrane.