Proteomic dissection of dome formation in a mammary cell line: role of tropomyosin-5b and maspin.

In this work we extended the study of genes controlling the formation of specific differentiation structures called "domes" formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the beta-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC beta-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC beta-subunit and the rat8 proteins, and is under the negative control of maspin.

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.

["Classic" vs autolubricant catheterization for endovesicular chemotherapy. Preliminary experience]. / Cateterismo "classico" vs autolubrificante per chemioterapia endovescicale. Esperienza preliminare.

AIM: The aim of this study was to evaluate the frequency of urinary tract infections (UTI) after catheterisation for instillation comparing two systems: the "classic" method and the catheterisation using a new autolubricant device: EasiCath Coloplast. METHODS: During the period of endovesical chemotherapy (between 4 and 48 weeks), 22 patients (6 females and 18 males) were studied, aged between 53 and 78 years old. We have performed 139 instillations using Nelaton Ch 14 or 12 type catheters lubricated with gel based on lidocaine, neomicyn and fluocinolone ("classic" method). Instead 135 patients have been treated with autolubricant devices according to the manufacturer's instructions. After 48 hours from instillation, a total of 274 catheterisation have been examined using urine tests and urine culture with antibiogram. We administered a 5-point visual analogic score to the patients weighing the post-instillation dysuria. RESULTS: With "classic" method UTI frequency is 7.19% (10/139). The most common pathogen has been E. coli (7/10). With autolubricant catheters UTI frequency is 2.96 (4/135). Klebsiella, Enterobacter, as well as E. coli (2/4) have been identified as pathogen. All patients with infections have been treated with targeted antibiotics based on the antibiogram. CONCLUSIONS: We have observed the people with autolubricant catheters left more comfort then those undergoing to the "classic" catheterisation. The frequency of post-catheterisation, dysuria was also reduced. Our data show that the new method is safer and easier to handle then the "classic" one. Moreover, common anaesthetic/antibiotic lubricant have important bacteriostatic effects that reduce the BCG viability.

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.

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.

Relationship between dose, mode of administration and effects of triiodothyronine on two hepatic responsive enzymes.

We have examined serum 3,3',5-triiodo-L-thyronine (T3) levels and the activity of two hepatic responsive enzymes [malic enzyme (ME) and alpha-glycerophosphate dehydrogenase (alpha-GPD)] in the livers of hypothyroid rats, under conditions where different doses of T3 (1 and 2.5 micrograms/100 g b.w.) were administered daily for one week either by intraperitoneal injection or by continuous infusions. In infused animals, serum T3 concentrations were constant for the whole period of treatment while in injected groups, widely oscillating diurnal levels were observed. The injection of 2.5 micrograms/100 g b.w. resulted, at the end of the treatment, in serum T3 levels which were higher than in animals receiving the same dose by infusion. No significant differences were observed when the administered dose was 1 microgram/100 g b.w. The basal levels of alpha-GPD and ME, which were markedly reduced in the livers of hypothyroid rats, were returned to normal both in infused rats (both with the dose of 1 microgram and 2.5 micrograms/100 g b.w. of T3) and in rats injected with a dose of 1 microgram/100 g b.w.). On the other hand, the dose of 2.5 micrograms/100 g b.w. when administered by injection, resulted in alpha-GPD and ME activities which were significantly higher even than those found in normal ones. The results indicate that both the diurnal T3 profile and the activity of the two hepatic T3 responsive enzymes are dependent not only on the dose but also on the administration mode.

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.