Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells.

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B3H4 (c) pyridoxal phosphate/B3H4 and (d) periodate/B3H4. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated. The LETS protein was also labelled with (14C) glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with (3H) fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.     

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.     

Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B³H₄ (c) pyridoxal phosphate/B³H₄ and (d) periodate/B³H₄. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated.The LETS protein was also labelled with [¹⁴C]glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with [³H]fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.     

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.     

Effects of cocultivation with transformed cells on surface proteins of normal cells.

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformed cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

Effects of cocultivation with transformed cells on surface proteins of normal cells

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformation cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

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.     

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.     

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.     

Quantitation of a transformation-sensitive, adhesive cell surface glycoprotein. Decrease of several untransformed permanent cell lines

We have quantitated the transformation-sensitive, cell surface LETS glycoprotein on many untransformed cell types. By SDS-polyacrylamide gel electrophoresis, this trypsin-sensitive iodinatable glycoprotein comprises 1-3% of total cellular protein of the seven early passage cell types tested. In contrast, it constitutes less than 0.15% of the protein in four of six continuous cell lines. This decrease is reflected in alterations both in [14C]glucosamine labeling and in the immunofluorescent staining of early passage vs. these four permanent cell lines. These results help to clarify previous experiments in which CSP, a purified LETS protein, partially restored a fibroblastic phenotype to cells transformed by tumor viruses. These findings also indicate that a major decrease in this cell surface glycoprotein can occur in the establishment of a continuous cell line without resulting in cellular transformation.     

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.     

A comparison of membrane components of normal and transformed BALB/c cells.

Membrane glycolipids, glycoproteins, and surface proteins of normal and transformed BALB/c cell lines have been compared. Several virally and spontaneously transformed cell lines showed differences in membrane components compared to normal A31 cells. These differences consisted of increased amounts of simpler gangliosides, absence of the large external transformation sensitive (LETS) protein, and the appearance of a major new glycoprotein band of about 105 000 molecular weight. In contrast, the spontaneously transformed cell line that caused the fastest growing tumors in vivo and the most rapid animal death (3T12T) did not have these changes. A31 and 3T12T glycolipid profiles appear similar as did glycoproteins and cell surface proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When Pronase-generated glycopeptides were analyzed by Sephadex G-50 chromatography, and enrichment in faster-eluting species was seen in two killing tumor lines (c5T and 3T12T) compared to A31. Regressing tumor lines (MSC, c5) did not show this change. Isolated membrane glycoproteins yield glycopeptides of different sized after Pronase digestion. In addition, several 3T12T glycoproteins yield glycopeptides that are larger than those from the corresponding glycoproteins of A31 cells. It appears that glycopeptide alterations associated with transformation occur in several membrane glycoproteins.     

Distribution of cell surface lets protein in co-cultures of normal and transformed cells

When LETS protein positive and negative cells were co-cultured, the positive cells remained as positive and the negative cells remained as negative. Apparently the transformed cells do not secrete factors which are sufficient to influence the distribution of surface LETS protein on normal cells.     

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.     

Ia antigen: a murine B lymphocyte receptor for transformed cell induction of interferon-alpha/beta

A receptor on the surface of nonsensitized mouse spleen cells that recognizes a glycoprotein from transformed mouse L-929 cells is described. The interaction of the receptor and glycoprotein inducer results in the production of MoIFN alpha/beta. An assay was developed to assess certain biologic and physicochemical characteristics of the receptor. The receptor and glycoprotein inducer bound in a concentration-dependent manner, which tends to indicate a direct interaction between the two. The receptor was not ubiquitous; spleen cells but not normal mouse embryo cells appeared to be the source. It was specific for MoIFN alpha/beta inducers from transformed cells, but not from other MoIFN alpha/beta or gamma inducers such as NDV, LPS, PWM, or SEA. The receptor appeared to be a cell surface protein in that its activity was abolished by trypsinization of whole spleen cells. Previous studies indicated that the receptor was probably located on B cells. Gel filtration indicated that the receptor had a m.w. of 30,000 to 60,000. Because the receptor appeared to be: 1) B lymphocyte associated, 2) a surface protein, and 3) 30,000 to 60,000 daltons, a similarity to Ia antigen was suggested. This possibility was confirmed by showing binding of the receptor to an anti-IaK antibody-Sepharose affinity column. PAGE analysis of the affinity-purified receptor revealed a single protein band with a m.w. of approximately 60,000. ELISA of the above gel slices with anti-Ia antibody further confirmed the specificity of the column. A physical association of the receptor and inducer was demonstrated by showing binding of the glycoprotein inducer to a receptor (Ia antigen)-Sepharose affinity column. Furthermore, the receptor (Ia antigen) was highly purified by a glycoprotein inducer-Sepharose affinity column.     

Decreased adherence to the substrate in Rous sarcoma virus-transformed chicken embryo fibroblasts.

Cell-substrate adherence in cultures of chicken embryo fibroblasts was examined by determining the number of cells which could be detached from the culture dish by a stream of medium. Transformed cells were significantly less adherent than their normal counterparts. In cultures infected with a mutant of Rous sarcoma virus which is temperature-conditional for transformation, adherence changed promptly following a temperature shift. This change did not require progression through the cell cycle. The transformation-specific decrease in adherence required new protein synthesis, but the restoration of adherence which occurred following a shift to the restrictive temperature could occur in the absence of new protein synthesis. Inhibitor experiments suggested the importance of microfilaments and perhaps microtubules in the changes in detachability. In addition, there was a positive correlation between levels of surface LETS protein and cell substrate adherence following a temperature shift, although it seems probable that the bulk of the surface LETS is neither necessary nor sufficient for maintenance of normal cell substrate adherence.     

Differential expression of Rous Sarcoma virus-specific transformation parameters in enucleated cells.

Chicken embryo fibroblasts transformed with the Ta and ts68 mutants of Rous Sarcoma virus (RSV) were enucleated and studied for their capacity to express reversibly the transformed phenotype in response to temperature changes. After shift to the permissive temperature (35 degrees C), the cytoplasts acquired a transformed morphology and displayed characteristic ruffles and microvilli at their surface. As detected by immunofluorescence, they also lost their actin filament cables and exhibited characteristic changes in the pattern of cell surface structures containing LETS protein. Expression of all these transformation parameters was reversible after shiftback to the nonpermissive temperature (41 degrees C). These results indicate that a whole set of changes characteristic for the transformed phenotype can be expressed independently of the cell nucleus. In contrast, ts mutant-infected cytoplasts were no longer able to respond to temperature shifts with changes in their hexose transport rate. Cytoplasts prepared from cells grown at 41 degrees C retained their low rate of hexose uptake after shift to 35 degrees C, whereas cytoplasts from cells grown at 35 degrees C exhibited a high rate of hexose transport even after 10 hr of shift to 41 degrees C. These results are in accordance with the hypothesis that the product of the src gene of RSV represents a multifunctional protein which acts independently on nuclear and extranuclear sites.     

Agglutination of Normal Cells by Plant Lectins following Infection with Nononcogenic Viruses

MAMMALIAN cells transformed by oncogenic viruses and chemical carcinogens undergo characteristic changes in their surface properties, some of which affect the control of cell multiplication. Certain plant lectins agglutinate transformed cells but not normal cells^1–6, which, although possessing binding sites, can only be agglutinated following treatment with proteolytic enzymes^3–5. Furthermore, both normal and transformed cells bind equal amounts of lectins, indicating that the increased susceptibility of transformed and trypsinized cells to agglutination is not caused by simple “unmasking” of hidden receptor sites. Nevertheless, the increased susceptibility of normal cells to agglutination following trypsinization may well result from changes occurring in the cell coat material. Since lytic infections with certain nononcogenic viruses¹⁰ and various drug treatments¹¹ are known to cause modification of the coat material in normal cells, we were interested to see whether these treatments increased the susceptibility of cells to agglutination by lectins.     

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.