Lack of correlation between the decreased expression of cell surface LETS protein and tumorigenicity in human cell hybrids

An analysis of the correlation between tumorigenicity and the loss of expression of the large external transformation-sensitive glycoprotein (LETS) was performed on human cell hybrids and their respective normal and tumorigenic parental cell lines. The distribution of cell surface LETS protein in a series of cell lines was examined by both specific immunofluorescent staining and by gel electrophoresis of lactoperoxidase-catalyzed, iodinated cell surface proteins. The tumorigenicity of these cell lines was assayed in nude mice. Although the series of cell lines studied provided a broad spectrum of LETS protein expression, both quantitatively and qualitatively, there does not appear to be a correlation between tumorigenicity and decreased expression of the LETS protein.In a series of transformed, nontumorigenic hybrids, the LETS protein expression was found to be altered with respect to both decreased organizational complexity and decreased content. These hybrids continue to express a number of other transformed phenotypes. Conversely, a number of tumorigenic hybrids continue to express relatively high levels of LETS protein when compared with nontumorigenic hybrids. Thus an alteration in LETS protein expression by itself, or in concert with a spectrum of other transformation properties, does not appear to be a sufficient requirement for tumorigenicity and lends further support to an apparent separate control of the transformed versus tumorigenic phenotype.     

Effects of LETS glycoprotein on cell motility

Addition of LETS glycoprotein to normal or transformed cells produces increased migration of the cells, as determined by formation of phagokinetic tracks on gold particle-coated coverslips. These tracks arise by a combination of phagocytosis of the gold particles and cellular migration. Increased motility is also evident on plastic in the absence of gold particles. The added LETS protein attaches to the cells in a fibrillar network, and binding is greater to normal than to transformed cells. The effects of LETS protein on migration are consistent with its effects on cell adhesion, morphology and cytoskeleton, and have potential implications for the determination of cellular migration in vivo.     

Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein.

Transformed cells lack a large, external, transformation-sensitive (LETS) glycoprotein which is a major surface component of their normal counterparts. Addition of LETS glycoprotein isolated from normal cells to transfomed cells restores certain morphological features and adhesive properties characteristic of normal cells. LETS protein is detected on the cell surface both by iodination using lactoperoxidase and by immunofluorescent staining. The surface distribution pattern detected by immunofluorescence is strikingly similar to that of normal cells. After addition of LETS protein, transformed cells also exhibit well defined actin cables which are not seen in untreated, transformed cells. All these alterations can be blocked by treating LETS protein with specific antisera or by subjecting it to mild trypsinization prior to addition to transformed cells. The effects are rapidly reversible by mild trypsinization, which removes the added LETS protein. The high rate of uptake of 2-deoxyglucose, characteristic of transformed cells, is not affected by LETS protein. These results suggest that LETS protein may have a role in cell attachment and spreading, and affect the organization of cytoskeleton.     

Cell surface protein decreases microvilli and ruffles on transformed mouse and chick cells.

Transformation of cultured fibroblasts usually results in a decrease in a high molecular weight cell surface glycoprotein (LETS protein) and often in increased numbers of surface microvilli and ruffles. We have isolated such a major cell surface glycoprotein from chick embryo fibroblasts; this protein, CSP, is decreased after transformation. Treatment of a mouse tumor cell line (SV1), L929 cells, and transformed chick fibroblasts with CSP results in a decrease in the number of microvilli and marginal ruffles, accompanied by restoration of a more normal morphology.     

Effects of cytochalasin B and colchicine on attachment of a major surface protein of fibroblasts

We have investigated the effects of the drugs cytochalasin B and colchicine on the surface levels of the large, external, transformation-sensitive (LETS) glycoprotein. Colchicine neither removed LETS protein from the surface, nor inhibited its regeneration after removal by mild trypsinization. Cells treated with cytochalasin B, however, showed both a 2–3-fold increase in the turnover rate of their surface LETS protein and a marked inhibition in its regeneration. Inhibition of regeneration was not due to inhibition of synthesis or transport to the surface. In fact, in the presence of cytochalasin B, increased quantities of LETS protein were released into the medium. The results are consistent with the idea of an association of LETS protein with the actin-containing microfilaments. However, other possible explanations, such as effects on cellular morphology or on transport of sugar precursors cannot yet be excluded.     

Role of disulfide bonds in the attachment and function of large, external, transformation-sensitive glycoprotein at the cell surface

Reduction of disulfide linkages by dithiothreitol removes LETS (large, external, transformation-sensitive) protein from the cell surface. This process is dependent upon the concentration of dithiothreitol and the time and temperature of reaction. At 0 degrees C the release of LETS protein by dithiothreitol is completely blocked, but this is apparently not due to a requirement for metabolic energy. At this temperature, reduction of LETS protein is incomplete. These results suggest that intact disulfide bonds are involved in the retention of this protein on the cell surface. Furthermore, reduction of purified LETS protein interferes with its ability to confer flattened morphology and increased adhesivity when added to transformed cells. It appears, therefore, that disulfide bonds are functionally important at the cell surface.     

Alteration in cell surface LETS protein during myogenesis.

Cell surface alterations during myogenesis have been investigated in Yaffe's myogenic cell line L8, using indirect immunofluorescence with an antibody against the large external transformation-sensitive (LETS) protein. The immunofluorescent technique reveals a susbstantial alteration in the distribution of this surface antigen. With the prefused myoblasts, LETS protein is dispersed all over the cell surface; following myoblast fusion, this pattern is markedly changed. All of the fibril-like surface LETS protein disappears, and in some myotubes, discrete clusters of LETS protein become conspicuous. By use of radioimmunological assay, the total LETS protein is quantitatively reduced upon myoblast fusion.     

Density and cell cycle dependence of cell surface proteins in hamster fibroblasts

The large, external, transformation-sensitive (LETS) glycoprotein of hamster fibroblasts, detected by lactoperoxidase-catalyzed iodination, is shown to vary in its accessibility at the cell surface, depending upon the growth state and position in the cell cycle. High levels correlate with arrest in early G1, and these fall after growth stimulation by serum. Very low levels are detectable on mitotic cells.     

Two functionally distinct pools of glycosaminoglycan in the substrate adhesion site of murine cells

Footpad adhesion sites pinch off from the rest of the cell surface during EGTA-mediated detachment of normal or virus-transformed murine cells from their tissue culture substrates. In these studies, highly purified trypsin and testicullar hyaluronidase were used to investigate the selective destruction or solubilization of proteins and polysaccharides in this substrate-attached material (SAM). Trypsin-mediated detachment of cells or trypsinization of SAM after EGTA-mediated detachment of cells resulted in the following changes in SAM composition: (a) solubilization of 50-70% of the glycosaminoglycan polysaccharide with loss of only a small fraction of the protein, (b) selective loss of one species of glycosaminoglycan-associated protein in longterm radiolabeled preparations, (c) no selective loss of the LETS glycoprotein or cytoskeletal proteins in longterm radiolabeled preparations, and (d) selective loss of one species of glycosaminoglycan-associated protein, a protion of the LETS glycoprotein, and proteins Cd (mol wt 47,000 and Ce' (mol wt 39,000) in short term radiolabeled preparations. Digestion of SAM with testicular hyaluronidase resulted in: (a) almost complete solubilization of the hyaluronate and chondroitin sulfate moieties from long term radiolabeled SAM with minimal loss of heparan sulfate, (b) solubilization of a small portion of the LETS glycoprotein and the cytoskeletal proteins from longterm radiolabeled SAM, (c) resistance to solubilization of protein and polysaccharide in reattaching cell SAM which contains principally heparan sulfate, and (d) complete solubilization of the LETS glycoprotein in short term radiolabeled preparations with no loss of cytoskeletal proteins. Thus, there appear to be two distinct pools of LETS in SAM, one associated in some unknown fashion with hyaluronate-chondroitin sulfate complexes, and a second associated with some other component in SAM, perhaps heparan sulfate. These data, together with other results, suggest that the cell-substrate adhesion process may be mediated principally by a heparan sulfate--LETS complex and that hyaluronate-chondroitin sulfate complexes may be important in the detachability of cells from the serum-coated substrate by destabilizing LETS matrices at posterior footpad adhesion sites.     

Analyses of cell surface and secreted proteins of primary cultures of mouse extraembryonic membranes

Procedures have been developed for primary culture of 13th day mouse parietal and visceral endoderm, yolk sac mesoderm, and amnion cells. We have analyzed cell surface and secreted proteins of these cultures by labeling the cells with radioactive iodine, glucosamine, or amino acids, and/or by immunofluorescence. Cell surface and secreted proteins of visceral endoderm, yolk sac mesoderm, and amnion cells resemble each other closely, whereas parietal endoderm cells are strikingly different. Unlike the other cell types, parietal endoderm cells synthesize and secrete substantial quantities of a protein tentatively identified as procollagen. These cells also secrete a number of other glycoproteins not observed in the media from the other cultures. It is proposed that the procollagen and one or more of the other unique, secreted glycoproteins are normally constituents of Reichert's membrane. Compared to the other cultures, parietal endoderm cells appear to be deficient in production of LETS protein. However, parietal endoderm—Reichert's membrane complexes analyzed by immunofluorescence directly after dissection from the uterus show an abundant association with LETS protein. It is not clear whether this LETS protein is actually synthesized by the parietal endoderm cells themselves. If so, it is possible that this protein is rapidly degraded after its secretion in parietal endoderm primary cultures. The studies reported here represent a first step in the characterization of cell surface properties of embryonic and extraembryonic cell types. The information already accumulated should be useful in investigations aimed at identification of cells derived from blastocysts and teratocarcinomas in vitro.     

Identification of a high molecular weight nervous system specific cell surface glycoprotein on murine neuroblastoma cells

An antiserum to N18 neuroblastoma cells has been used to identify a glycoprotein of apparent molecular weight greater than 200 000 D in SDS-polyacrylamide gels. This glycoprotein (Band 1) is found in culture medium of N18 cells. An immunologically similar component can be immunoprecipitated from detergent extracts of enzymatically iodinated or biosynthetically labelled viable cells. Anti-band 1 activity can be adsorbed from the antiserum by intact N18 cells but not four other cultured murine cell lines. Normal adult murine brain also adsorbs anti-band 1 activity but adult murine adrenal, heart, kidney, liver, lung, and spleen do not. Several experiments indicate that band 1 is not myosin heavy chain or the fibroblast LETS protein. Thus band 1 is a newly identified high molecular weight nervous system specific glycoprotein.     

Mechanism of the decrease in the major cell surface protein of chick embryo fibroblasts after transformation.

The major cell surface glycoprotein of cultured chick embryo fibroblasts (CSP, a LETS protein) is substantially decreased after neoplastic transformation. We investigated the regulation of this glycoprotein by determining the kinetics of CSP biosynthesis, transit to the cell surface, and degradation before and after transformation by Rous sarcoma virus. CSP synthesis, as measured by immunoprecipitation after pulse-labeling with ¹⁴C-leucine, is decreased 3–6 fold after transformation by the Bryan high titer, Schmidt-Ruppin and temperature-sensitive ts68 and T5 strains of Rous sarcoma virus. Steady state quantities of CSP in intracellular pools are also decreased 4–5 fold after transformation. However, the rate at which newly synthesized CSP is processed and exported to the cell surface is similar before and after transformation.Degradation and release of CSP from cells were measured after labeling for 24 hr. The half-life of CSP on normal cells is 36 hr and is decreased to 16–26 hr after transformation. The absolute amount of intact CSP released into the culture medium is decreased 3 fold after transformation; these amounts, however, represent losses of approximately 20 and 40% of the total CSP synthesized by normal and transformed cells, respectively. These results indicate that the major mechanism for the decrease in CSP after transformation is reduction in its biosynthesis, although increased degradation and loss from the cell surface also contribute significantly. These changes can account for the observed 5–6 fold decreases in cell-associated CSP after transformation of chick embryo fibroblasts.     

Investigations of the possible role of proteases in altering surface proteins of virally transformed hamster fibroblasts

Virally transformed fibroblasts have on their surfaces zero or reduced amounts of a large external transformation-sensitive (LETS) glycoprotein. This protein is extremely sensitive to proteolysis. When prelabeled normal fibroblasts are cocultivated with transformed cells, the LETS glycoprotein of the normal cells shows an increased rate of turnover. Experiments are described which investigate the possibility that this phenomenon and the absence of LETS glycoprotein are due to proteolysis by the transformed cells. In particular, the role of plasminogen activation is examined by the use of protease inhibitors and plasminogen-depleted serum. It is concluded that activation of plasminogen is not required for the disappearance of the LETS glycoprotein although the involvement of other proteases cannot be ruled out. The role of proteases in affecting cell growth and behavior is discussed.     

Loss of LETS protein is not a marker for salivary gland or bladder epithelial cell transformation

LETS protein was demonstrated by indirect immunofluorescence techniques in adult mouse submandibular gland and bladder epithelial cells at various stages during neoplastic transformation, after the application of a chemical carcinogen in vitro. There was no clear relationship between transformation and the loss of LETS protein; most normal and preneoplastic epithelium lacked the distinctive fibrillar distribution of fluorescent staining. In contrast, some carcinoma-producing cell lines did possess appreciable amounts of LETS protein.     

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.     

Molecular composition and origin of substrate-attached material from normal and virus-transformed cells

The proteins and polysaccharides which are left adherent to the tissue culture substrate after EGTA-mediated removal of normal, virus-transformed, and revertant mouse cells (so-called SAM, or substrate-attached material), and which have been implicated in the cell-substrate adhesion process, have been characterized by SDS-PAGE and other types of analyses under various conditions of cell growth and attachment. The following components have been identified in SAM: 3 size classes of hyaluronate proteoglycans; glycoprotein C_o (the LETS glycoprotein); protein C_a (a myosin-like protein); protein C_b (MW 85,000); protein C₁ (MW 56,000, which is apparently not tubulin); protein C₂ (actin); proteins C₃–C₅ (histones) which are artifactually bound to the substrate as a result of EGTA-mediated leaching from the cell; and proteins C_c, C_d, C_e, and C_f. The LETS glycoprotein (C_o) and C_d appear in newly-synthesized SAM (which is probably enriched in “footpad” material – “footpads” being focal areas of subsurface membranous contact with the substrate) in greater relative quantities than in the SAM accumulated over a long period of time (which is probably enriched in “footprint” material – remnants of footpads left behind as cells move across the substrate). C_o and C_d turn over very rapidly following short radiolabeling periods during chase analysis. The SAM's deposited during a wide variety of cellular attachment and growth conditions contained the same components in similar relative proportions. This may indicate well-controlled and coordinate deposition of a cell “surface” complex involving the hyaluronate proteoglycans, the LETS glycoprotein, actin-containing microfilaments with associated proteins, and a limited number of additional proteins in the substrate adhesion site. Evidence indicates that SAM is the remnant of “footpad” vesicles by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell. Three different models of cell-substrate adhesion are presented and discussed.     

Surface proteins of young and senescent cultured avian fibroblasts

Proliferation of senescent cultured chick fibroblasts is arrested at densities that are 3-4 fold lower than densities inhibiting growth of young cells. The effects of density and growth rate of young and aged cultures on the accessibility of their surface proteins to external iodination were studied. LETS glycoprotein and a protein of 110,000 daltons are the major iodinated proteins of resting, highly dense and of sparse young cells, respectively. By contrast, LETS is minimally exposed on undividing, relatively disperse old cells. Therefore, exposure of LETS is correlated with cell density rather than with growth rate.     

In vitro traits of adenovirus-transformed cell lines and their relevance to tumorigenicity in nude mice

Six independently isolated adenovirus 2-transformed rat cell lines and one adenovirus 5-transformed human cell line have been examined in vitro for serum growth requirements, saturation density, anchorage-independent growth, proteolytic enzyme activity and the presence of LETS glycoprotein and T antigen. This series of adenovirus-transformed cell lines exhibits an oncogenic spectrum ranging from being tumorigenic in immunocompetent rats through to nontumorigenic in adult nude mice. The relevance of the in vitro findings to growth potential in vivo is discussed.     

Response of cultured rat liver epithelial cell lines to tumour-promoting phorbol esters

The membrane effects of a potent tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), were studied in a series of cultured rat liver epithelial cell lines. Treatment with TPA resulted in the formation of strand-like aggregates (ridges) of viable cells over the monolayer of IAR 6-1 cells, but not of three other cell lines tested (IAR 20, IAR 6, IAR 6-7). The morphological response of IAR 6-1 to TPA was investigated by determination of phorbol ester receptors, analysis of cellular fucoproteins, surface galactoproteins and iodinatable surface proteins, and specific immunofluorescence for components of the extracellular matrix (fibronectin, laminin-entactin, procollagen type III). A class of specific, saturable, high-affinity receptors for phorbol esters was demonstrated in all four cell lines employing a conventional [20(-3)H]phorbol-12,13-dibutyrate ([3H]PDBu)-binding assay. The dissociation constants were similar in four lines, but the number of receptors per cell in IAR 6-1 cells was about twice that in other lines. Down-regulation of receptors was demonstrated in IAR 20 and IAR 6-1 cells with similar characteristics. Iodinatable surface proteins and galactose-containing surface glycoproteins did not respond to TPA. The distribution of fibronectin, laminin-entactin and procollagen type III was not affected by TPA. A TPA-responsive cell line, IAR 6-1, contained considerably less laminin-entactin than did the other lines. TPA had no influence on metabolic labelling of [3H]fucose-containing cellular glycoprotein in IAR 6-1 cells. One specific protein, with molecular mass of 78 kD, was more heavily labelled with [3H]fucose in IAR 6-1 cells than in the other cell lines. Taken together, the results of this study show that the responsive cells (IAR 6-1) differed from non-responsive ones in having more phorbol ester receptors, increased fucosylation of a specific glycoprotein and decreased deposition of laminin-entactin in the extracellular matrix. These surface properties of IAR 6-1 cells may contribute to their ability to respond to TPA.     

Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape

The major cell surface glycoprotein of chick embryo fibroblasts, cellular fibronectin (formerly known as CSP or LETS protein), was purified and used to produce monospecific antisera. After affinity purification, the anti-fibronectin was used to investigate fibronectin's localization, its transfer from intracellular to extracellular pools, its antibody-induced redistribution on the cell surface, and its role in cell shape. Anti-fibronectin localizes to extracellular fibrils located under and between sparse cells, and to a dense matrix that surrounds confluent cells. Cellular fibronectin is also present in granular intracytoplasmic structures containing newly synthesized fibronectin before secretion. This intracellular staining disappears 2 h after treatment with cycloheximide or puromycin, and returns after removal of these protein synthesis inhibitors. In pulse-chase experiments using cycloheximide, fibronectin was sequentially transferred from the intracellular to the fibrillar extracellular forms. Transformation of chick fibroblasts results in decreases in both extracellular and intracellular fibronectin, and in altered cell shape. Treatment of untransformed chick fibroblasts with anti-fibronectin results in rapid (30 min) alteration to a rounder cell shape resembling that of many transformed cells. These rapid shape changes are followed by a slow, antibody-induced redistribution of fibronectin to supranuclear caplike structures. This "capping" is inhibited by metabolic inhibitors. Reconstitution of cell surface fibronectin onto transformed cells restores a more normal fibroblastic phenotype. The reconstituted fibronectin on these cells organizes into fibrillar patterns similar to those of untransformed cells. As with untransformed cells, treatment of these reconstituted cells with anti-fibronectin also results in cell rounding and "capping" of fibronectin.