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Biomedical subjects

S Barlati

Publications and source records attributed to S Barlati.

At least 73 records · Page 4Linked to original sources
PubMed1986

A family of fibronectin mRNAs in human normal and transformed cells.

Previously, two fibronectin mRNAs, generated by alternative splicing of the extra domain (ED) and type III connecting segments (IIICS) sequences, have been described in a human transformed cell line and in human liver, respectively. We now report on a family of fibronectin mRNAs identified by Northern blotting analysis in two normal human fibroblast strains (HEL 299 and Flow 7000) and five transformed cell lines (8387 and HT-1080, fibrosarcomas; G-361, melanoma; JEG-3, choriocarcinoma; and RD, rhabdomyosarcoma). Seven different fibronectin mRNA forms with electrophoretic mobilities ranging between 8.6 and 7.7 kb were identified. Each cell line contains three (HEL 299, Flow 7000 and 8387) or two (HT-1080, G-361, JEG-3 and RD) fibronectin mRNAs species with characteristic size. In all cell lines we detected one fibronectin mRNA form which lacks the ED sequence (ED- fibronectin mRNA) and one or two fibronectin mRNAs containing it (ED+ fibronectin mRNA). These data show that the presence of ED+ and ED- fibronectin mRNAs is a general feature of all cells tested. Moreover, the fibronectin mRNA pattern is characteristic of the cell type analyzed, suggesting the occurrence of specifically programmed splicing mechanisms in each cell line.

Cell Line↗
PubMed1986

Relationship between circulating plasminogen activators and tumor development in mice.

Plasminogen activators (PAs) present in the plasma of BALB/c mice and produced in vitro by murine tumor cell cultures (B77-3T3, SR-BALB, AA6) have been characterized using electrophoretic-zymographic techniques. BALB/c mouse plasma contains a main PA activity with an approximate molecular weight of 88,000 and pI 6.3, inhibited by anti-human tissue-type plasminogen activator (t-PA) serum, here defined as murine t-PA. On the contrary, all tumor cells tested release a PA activity with a molecular weight of 44,500 and pI 9.2 characteristic of urokinase-type activator (murine u-PA). The injection s.c. of the different tumorigenic cells into BALB/c mice leads to tumor development and to a rapid increase of t-PA from the first day following the injection. This early enhancement of t-PA activity is not detectable in mice given injections of lethally irradiated B77-3T3 cells. Moreover the development of the tumor in the animals is related to the appearance of increasing levels of u-PA in the blood. This activity is detectable in the plasma of treated mice almost 2 wk before detection of a tumor 1 mm in diameter. During tumor development, the molecular weight of the u-PA and t-PA forms present in the plasma does not change, while there is a decrease of the isoelectric point of the u-PA leading to the appearance of distinct PA activities with pI 7.6 and 8.9. Syngenic and allogenic lymphocytes, injected in BALB/c mice, do not induce any modification in the pattern of the plasma PA. The injection of highly metastatic cells, as opposed to nonmetastatic or low-metastatic cells, does not give rise to detectable levels of u-PA in the plasma of treated mice. These data suggest that the lack of plasma u-PA activity facilitates the formation of metastates, while the increase of this activity is important in tumor development and is independent of the metastatic potential of the injected cells.

Animals↗
PubMed1985

Fibronectin, laminin in hybrids of Rous sarcoma virus transformed and normal mouse fibroblasts.

Hybrid clones derived from the fusion of normal and Rous sarcoma virus-transformed 3T3 fibroblasts were analyzed for fibronectin and laminin pattern of expression in order to find a possible correlation with tumorigenicity. Both organization in the pericellular matrix and secretion into the culture media were investigated by immunofluorescence and ELISA techniques. No significant difference in fibronectin or laminin release was found among hybrid clones exhibiting different levels of tumorigenicity. In contrast, distinctive immunofluorescence patterns of concomitant presence of low levels of fibronectin and high levels of laminin were constantly observed in all the tumorigenic clones.

Animals↗
PubMed1984

A rapid and highly sensitive solid-phase enzyme immunoassay specific for human fibronectin using a characterized monoclonal antibody.

A solid-phase enzyme immunoassay (EIA) was developed for the quantitation of human fibronectin in body fluids and cell culture media. In the assay a human fibronectin-specific murine monoclonal IgG1 (f-33) was used as capture antibody and polyclonal rabbit anti-fibronectin as detector antibody. The antibody showed no reactivity to purified monkey, dog, rabbit, horse, sheep, mouse, bovine or chicken fibronectins. The determinant of the monoclonal antibody was mapped to the cell-binding region of the fibronectin molecule. This localization was based on the use of purified fragments of fibronectin, immunoblotting, EIA and inhibition of fibroblast adhesion and spreading by the antibody. The detection limit of the fibronectin assay was 2 ng/ml. The assay was used for the quantitation of fibronectin in human plasma, urine and cerebrospinal fluid specimens and culture media of human cells.

Animals↗
PubMed1984

Tissue type plasminogen activator, but not urokinase, exerts transformation-enhancing activity.

Previous studies have established that plasma cryoprecipitates of tumor patients, culture media of transformed cells and defined proteolytic fragments of fibronectin enhance the morphological cell transformation ( TEF activity) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We now report that purified human tissue type plasminogen activator (t-PA), but not urokinase (u-PA), has a similar TEF activity, at doses as low as 2 ng/ml (30 pM). Specific antibodies effectively neutralized the activity. No significant contamination (less than or equal to 1%) between the preparations of t-PA and fibronectin (FN) or its fragments ( FNdp ) was detected. The results suggest that t-PA may have a direct role in the process of morphological cell transformation in vitro.

Cell Transformation, Neoplastic↗
PubMed1984

Plasminogen activators, activation inhibitors and alpha 2-macroglobulin produced by cultured normal and malignant human cells.

In the present paper we have characterized the plasminogen activators (PA) synthesized by 25 different human cell lines. Technically easy methods were adopted for concentration and immunological characterization of the activators even in the presence of PA inhibitors. Most cell lines produced u-PA (mol. wt 55,000), melanoma and HeLa cells t-PA (mol. wt 66,000) and two carcinoma cell lines and normal skin fibroblasts produced no detectable PA. The classical 125I-fibrin method was compared to a caseinolytic assay and some of the discrepancies between results obtained with the two methods were shown to be due to cell-derived NaDodSO4-sensitive proteinase inhibitors in culture media. Additionally, synthesis and uptake by the cells of the wide-spectrum proteinase inhibitor alpha-2-macroglobulin ( alpha 2M ) were studied by radioimmunoassay and immunofluorescence. No production of alpha 2M could be measured in any of the malignant cell lines. In normal cells no correlation existed between the production of alpha 2M and the observed inhibition of PA activity, which indicates that other proteinase inhibitors are produced by the cells.

Cells, Cultured↗
PubMed1984

Relationship between multiple forms of plasminogen activator in human breast tumors and plasma and the presence of metastases in lymph nodes.

Plasminogen activators (PAs), a family of proteases active in blood coagulation, may play an important role in cancer. Indeed, blood coagulation disorders, such as altered fibrinogen and fibrin metabolism and increased incidence of vascular thrombosis, are common in patients with advanced malignant disease. Different types of human tumors are known to contain high levels of PA. The isoelectric focusing patterns of the PAs present in tumors and plasma from patients with breast cancer were compared with those of purified human urokinase and melanoma tissue PA. The pattern of isoelectric molecular forms of PA active at pH 8 showed two groups of several bands: in plasma from tumor-bearing patients and controls, these groups were in the pl ranges of 6.6 to 6.8 and 8.0 to 8.5; in mammary adenocarcinoma tissue, the ranges were 6.8 to 7.9 and 9.0 to 9.4. These patterns were different from those obtained with purified markers; the latter were 5.8 to 9.4 and 5.9 to 7.6 for purified human urokinase and melanoma plasminogen tissue activator, respectively. PA activity in tumor-bearing patients was very high in malignant tissue and, on the contrary, very decreased in plasma; this latter decrease was correlated with the presence of metastases in the axillary lymph nodes. These results suggest that the high PA activity in the tumor tissue might participate in the destruction of the peritumoral tissue, thus allowing its invasion by tumor cells, whereas the low activity of PA in the plasma might increase plasma fibrin, reflecting thus an early disorder in blood coagulation which would enhance the formation of metastases.

Adenocarcinoma↗
PubMed1983

Monoclonal antibody against the N-terminal end of human plasma fibronectin.

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.

Antibodies, Monoclonal↗
PubMed1983

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them.

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.

Antibodies, Monoclonal↗
PubMed1983

Studies on transformation markers and tumorigenicity in segregant clones from a human hybrid line.

Thirty segregant clones were back-selected in 8AG or 5BUdR media from a non-tumorigenic human intraspecific hybrid line (HeLa TK- X fibroblasts HPRT-) displaying a high plasminogen activator (PA) level, a disorganized fibronectin (FN) matrix and anchorage-independence. These clones exhibited a widely modulated expression of the above markers concomitantly with different degrees of chromosome loss. Out of six representative segregant clones tested in nude mice, two were found to re-express tumorigenicity. No significant correlation was observed between PA or FN levels and anchorage-independence, as well as between these markers and tumorigenicity.

Animals↗
PubMed1983

Human fibroblasts X mouse cell hybrids, containing a human 11/X translocation, do not express human fibronectin.

Using a monoclonal antibody specific for human fibronectin (FN), we screened hybrid clones derived from the fusion of FN+ human fibroblasts, carrying a 11/X translocation, and FN-, HPRT- mouse cells for the production of this glycoprotein. Since no hybrid clone retaining the human der 11 chromosome was found to produce any human fibronectin, the segment of chromosome 11 included in the rearranged chromosome (11qter leads to 11p13) probably does not carry the structural locus for fibronectin.

Animals↗
PubMed1983

Fibronectin and its proteolytic fragments. Potential as cancer markers.

Since the discovery of fibronectin as a transformation-sensitive 'cell surface' protein, it has been the focus of intensive studies. Fibronectin has multiple interactions and functions and is composed of distinguishing properties of malignant cells, properties. Invasiveness and metastasis, distinguishing properties of malignant cells, involve penetration through components of the extracellular matrix. Enzymatic degradation of matrix components is involved in these phenomena. Proteolytic targets of the pericellular matrices of cells in culture include fibronectin that has been found to be even selectively susceptible to proteinases. Defined fragments of fibronectin have transformation-promoting activity in experimental conditions and such fragments, detected in tumor patient body fluids, may serve as markers for tumor progression.

Amino Acids↗
PubMed1982

Expression of transformation markers and suppression of tumorigenicity in human cell hybrids.

Somatic human cell hybrids produced by fusion of HeLa cells and diploid fibroblasts were analysed in a study designed to test the coordinate expression of transformation markers and tumorigenicity. The great majority of these hybrids displayed a finite lifespan in culture, but some of them inherited from the HeLa parent the capability to grow as permanent cell lines. Hybrids from both groups all had a plasminogen activator activity 20 to 100-fold higher and a cloning efficiency in semisolid medium 2 to 10-fold lower than the HeLa parent. Cell surface fibronectin was expressed at variable levels, albeit in a disorganized form. No correlation between the level of plasminogen activator or fibronectin content and cloning efficiency in agar was observed. Two hybrid lines, assayed for tumorigenicity in nude mice, did not produce tumors, even at inocula 20-fold greater than those at which the HeLa cells formed tumors.

Animals↗
PubMed1982

Enhancement of plasminogen activator activity by the culture medium of Rous sarcoma virus-transformed cells.

We have tested the effect of the culture medium of chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) on plasminogen activator (PA) activity of normal and RSV-infected cells. The results obtained showed that fibrin digestion by cultures of normal uninfected and RSV-infected cells directly attached to a fibrin substrate was increased when the cells were exposed to the supernatant of an 18 h-culture of CEF transformed by RSV. Addition of cycloheximide to the supernatant completely abolished this effect. Similarly, exposure of both normal and RSV-infected cells to the culture medium of RSV-transformed CEF resulted in a marked increase of the soluble PA activity present in the supernatant. This effect, however, was not abolished by inhibition of protein synthesis. For both the cell-bound and soluble PA activities of normal and RSV-infected cells the stimulatory effect was transient, being maximal between 8 and 12 h of exposure to the transformed cell-conditioned medium and disappearing after 24 h. A possible correlation with transformation-enhancing factor(s), previously reported to be present in the culture medium of transformed cells, is discussed.

Animals↗