Loss Of Cell Surface Fibronectin Research Articles

NEGATIVE REGULATION OF FIBRONECTIN GENE-TRANSCRIPTION BY EJ-HA-RAS AND C-SIS ONCOGENES IN BALB/C 3T3 CELLS

Down-regulation of fibronectin gene expression at cell surfaces by various oncogenes is an important phenotypic change of many transformed cells. Mechanisms effecting this loss of cell surface fibronectin remain poorly understood. An isogenic mouse fibroblast system, Balb/c 3T3, was used to investigate the effect of oncogene transformation on fibronectin gene transcription. Fibronectin mRNA levels were shown to decrease by >80% when 3T3 cells became stably transformed by EJ-Ha-ras or c-sis oncogenes. The mouse fibronectin gene promoter was subsequently cloned from a mouse genomic library and inserted into reporter gene constructs. Transient transfection assays with the 1.2 kb fibronectin promoter demonstrated that its activity was also decreased in the two transformed cell lines to a similar extent as fibronectin mRNA levels. Co-transfections into 3T3 cells with both the promoter construct and either Ha-ras or c-sis expression constructs provided further support for negative regulation of fibronectin gene transcription by oncogene expression. Potential mechanisms for such transcriptional regulation are proposed based on deletion analysis of the fibronectin promoter. These data suggest that down-regulation of fibronectin gene transcription can contribute directly to the loss of cell surface fibronectin in transformed murine fibroblasts.

A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event.

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event.

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

Stress relaxation of contracted collagen gels: Disruption of actin filament bundles, release of cell surface fibronectin, and down-regulation of DNA and protein synthesis

Relaxation of stressed collagen gels provides a model system uniquely suited to studying the regulation of cell morphology and biosynthetic function by tissue organization. Stress relaxation results in rapid, synchronous changes in cell morphology without enzymatic or other drug treatments, and makes possible an analysis of the initial cellular events associated with changes in tissue organization. During the first hour after stress relaxation, we observed transient hypercontraction of collagen gels and loss of collagen fibril organization as stress in the system dissipated. Morphological changes in the fibroblasts included retraction of pseudopodia, collapse of cytoplasmic actin filament bundles, and loss of cell surface fibronectin. Accompanying these morphological changes, we observed marked decreases in DNA and protein synthesis, especially of fibronectin and type I procollagens. These results show that changes in tissue organization can exert rapid and profound effects on the morphology and biosynthetic function of cells Within the tissue.

Malignant progression of an SV40-transformed human epidermal keratinocyte cell line

Human foetal keratinocytes were transfected with a recombinant plasmid (pSV6-1) which contained an origin defective SV40 genome. The resulting transformed cell line had many properties in common with previously described SV40-transformed keratinocytes, including expression of simple epithelial-type keratins. It was non-tumourigenic in nude mice at early passages, forming small benign cysts, however, after approximately 46 in vitro passages, these transformed keratinocytes formed invasive squamous cell carcinomas in athymic nude mice. Several in vitro changes were associated with this acquisition of tumourigenicity (a) an alteration in cellular morphology, (b) development of a cytogenetically marked clone and (c) loss of cell surface fibronectin. The loss of fibronectin was also observed in vivo; cysts formed by SV6-1 Bam/HFK produced human fibronectin whereas tumours did not, although both tumours and cysts were laminin- and keratin-positive. These results indicate that the spontaneous development of secondary events in immortalised human cells may lead to the acquisition of a malignant phenotype.

Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin.

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.

Restoration of anchorage regulation in transformed cells by retinoic acid (RA) is independent of the presence of cytoplasmic RA-binding proteins

In an attempt to analyse the cause-effect relationship between anchorage-independent growth (a property which correlates best with in vivo tumorigenicity) and a set of other common transformation-related properties, the effect of retinoic acid (RA) treatment on six unrelated transformed cell lines (including DNA tumor virus, retrovirus, and spontaneously transformed cells) were studied. The data show that the changes in morphology and cellular orientation in culture, loss of cell surface fibronectin, disruption of actin microfilaments, increased hexose uptake, loss of density-dependent growth, and decreased binding of EGF, properties which are often associated with oncogenic transformation of cells, are dissociable from one another and from anchorage-independent growth. RA appears to interfere with anchorage-independent growth of all the retrovirus and spontaneously transformed cell lines (responsive cells) that we examined; however, such treatment failed to inhibit achorage-independent growth in both of the DNA tumor virus-transformed cell lines (non-responsive cells) that we used in the present study. The presence of RA-binding proteins in both responsive and non-responsive cells suggests that the mechanism of RA action for the inhibition of anchorage-independent growth in transformed cells may be independent of the presence of such cytoplasmic proteins. Finally, the present study clearly indicates that the use of RA treatment, like partial transformation mutants of oncogenic viruses, can be a novel approach in analysing the general mechanism by which transformed cells grow without anchorage.

Fibronectin and type III collagen in epithelial neoplasms of gastrointestinal tract and salivary gland.

The distribution of fibronectin (FN) and collagen type III (IIIC) have been compared in a series of epithelial neoplasms from the gastrointestinal tract and salivary gland. The difficulty of distinguishing between FN of epithelial and fibroblastic origin is emphasised and evidence is presented for the validity of this distinction. In carcinomas FN was sometimes, but not invariably, lost from epithelial cell surfaces. Both FN and IIIC were increased in reactive connective tissue stroma. It is concluded that loss of cell surface FN is unlikely to be a useful diagnostic marker for malignancy, but that the occurrence of this phenomenon in vivo as in vitro indicates that it is biologically significant.

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.

Transformation parameters of chick embryo fibroblasts transformed by fujinami, PRCII, PRCII-p, and Y73 avian sarcoma viruses

The transformation parameters of chicken embryo fibroblasts transformed by replication defective avian sarcoma viruses were compared with those of cells transformed by Rous sarcoma virus (RSV). Two classes of avian sarcoma virus with transforming genes distinct from the src gene of RSV were examined: Fujinami sarcoma virus (FSV), PRCII, and PRCII-p were used as representatives of one class, and Y73 as a representative of the other class. The transformation parameters examined included morphological markers: cell surface morphology, the disappearance of microfilament bundles, and the loss of cell surface fibronectin; alterations in growth properties: growth to high densities and growth in suspension; and metabolic changes: increased uptake of hexose, release of plasminogen activator, and increased synthesis of hyaluronic acid. In general, all of the viruses induced a phenotype which was qualitatively similar to that of RSV-transformed cells, although the extent of each change was less than that observed with RSV. One strain of FSV was found to be coordinately temperature sensitive for the induction of all phenotypic markers examined, whereas another strain of FSV and the other viruses examined were temperature resistant. PRCII-transformed cells had a lower efficiency of colony formation in agar suspension and showed only partial disruption of microfilament bundles; this partial transformation phenotype may be correlated with the low oncogenicity of this virus. These findings in conjunction with previous studies on protein phosphorylation in ASV-transformed cells are consistent with the idea that the replication-defective avian sarcoma viruses transform cells by a mechanism similar to that involved in transformation by RSV.

A possible role for cold insoluble globulin in chemotactic factor mediated polymorphonuclear leukocyte adherence to plastic

We examined the potential role of fibronectin in chemotactic factor stimulation of neutrophil adherence to plastic. Monoclonal antibody to human fibronectin significantly reduced chemotactic peptide stimulation of adherence but did not reduce adherence stimulated by phorbol myristate acetate or aggregation stimulated by either agent. Stimulation of neutrophils by chemotactic peptide was also associated with loss of cell surface fibronectin detected by immunofluorescence or binding of radiolabeled collagen. These data suggest that chemotactic peptides stimulate neutrophils to release Fn and that Fn mediates the attachment of neutrophils to plastic surfaces.

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells.

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.

Cellular tumorigenicity in nude mice. Test of associations among loss of cell-surface fibronectin, anchorage independence, and tumor-forming ability.

Fibronectin (FN; also called large external transformation-sensitive [LETS] protein or cell-surface protein [CSP]) is a large cell-surface glycoprotein that is frequently observed to be either absent or greatly reduced on the surfaces of malignant cells grown in vitro. Because FN may be a useful molecular marker of cellular malignancy, we have carried out an extensive screening to test the specific association among the degree of expression of FN, anchorage-independent growth, and tumorigenicity in the athymic nude mouse. A variety of diploid cell strains and established cell lines were tested for the expression of surface FN by indirect immunofluorescence using rabbit antisera against human cold insoluble globulin, rodent plasma FN, or chicken cell-surface FN. Concomitantly, the cells were assayed for tumor formation in nude mice and for the ability to form colonies in methylcellulose. Tumorigenic cells often showed very low surface fluorescence, confirming earlier reports. However, many highly tumorigenic fibroblast lines from several species stained strongly with all three antisera. In contrast, the anchorage-independent phenotype was nearly always associated with tumorigenicity in approximately 35 cell lines examined in this study. In another series of experiments, FN-positive but anchorage-independent cells were grown as tumors in nude mice and then reintroduced into culture. In five of the six tumor-derived cell lines, cell-surface FN was not significantly reduced; one such cell line showed very little surface FN. Our data thus indicate that the loss of cell-surface FN is not a necessary step in the process of malignant transformation and that the growth of FN-positive cells as tumors does not require a prior selection in vivo for FN-negative subpopulations.

ORGANIZATION OF EXTRACELLULAR PROTEINS ON THE CONNECTIVE TISSUE CELL SURFACE: RELEVANCE TO CELL‐MATRIX INTERACTIONS IN VITRO AND IN VIVO*

A model has been developed that proposes a cell surface-associated protein meshwork, composed in part of fibronectin and collagen, for a connective tissue cell attached to a substratum. In support of this model are the observations that collagen and fibronectin interact and that these proteins are similarly distributed on the fibroblast cell surface. We suggest that this external meshwork interacts directly or indirectly with the internal cytoskeleton and with the extracellular matrix and thereby mediates several cellular properties, including adhesion, shape, and motility. Loss of cell surface fibronectin as a result of viral transformation, or due to treatment of normal cells with tunicamycin, an inhibitor of protein glycosylation, may contribute to the reduced adhesion and altered morphology observed in these circumstances. We therefore predict that the changes in these properties observed with virally transformed cells, mitotic cells, and cells treated with proteolytic enzymes are related to alterations in the external protein meshwork.