Heparan sulfates of mouse cells. Analysis of parent and transformed 3T3 cell lines.

Heparan sulfate from the surface of a variety of mouse cells at different cell densities was examined by ion-exchange chromatography. The results of this analysis show that: (1) The heparan sulfate from new isolates of Swiss 3T3 cells transformed by SV40 virus (a DNA tumor virus) elutes from DEAE-cellulose at a lower ionic strength than that from the parent cell type. This finding confirms our earlier observation with an established SV40-transformed cell line (Underhill and Keller, '75) and eliminates the possibility that this change is caused by extended passage in culture. (2) For both parent and transformed 3T3 cells, the heparan sulfates from low and high density cultures were the same as judged by chromatography on DEAE-cellulose. This result demonstrates that the transformation-dependent change which we have observed is independent of cell density. (3) The heparan sulfate from Balb/c 3T3 cells transformed with Kirsten murine sarcoma virus (an RNA tumor virus) elutes from DEAE-cellulose prior to that from parent Balb/c 3T3 cells. This result extends the transformation dependent change in heparan sulfate to the Balb/c 3T3 cell line and to cells transformed with an RNA virus.     

Loss of density-dependent regulation of multiplication of BALB-3T3 cells chemically transformed in vitro

Balb/3T3 cells show density-dependent regulation of multiplication with the final cell density depending on serum concentration in the media. Chemically transformed Balb/3T3 cells (Balb/3T3-D) pile up on each other, multiply to a high cell density, but have decreased DNA synthesis at very high cell densities. Balb/3T3-D cells require less serum for multiplication compared with original Balb/3T3 cells. A rat serum fraction and a bovine β-globulin fraction stimulate the multiplication of Balb/3T3 cells but only slightly stimulate Balb/3T3-D cells indicating different serum factors stimulate growth of these two cell types. The multiplication properties of Balb/3T3-D cells are very similar to those of SV-40 transformed 3T3 cells, however, these properties were brought about by a single treatment by a chemical carcinogen, without an exogenous virus. The transformation altered the contact of cells to one another, indicating a permanent chemical change in the membrane structure.     

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

CELL CONTACT DEPENDENCE OF SURFACE GALACTOSYLTRANSFERASE ACTIVITY AS A FUNCTION OF THE CELL CYCLE

Mitotic, nonmalignant Balb/c 3T3 cells exhibit endogenous, surface galactosyltransferase activity that does not require intercellular contact throughout the assay period. In this respect, mitotic 3T3 cells resemble malignant Balb/c 3T12 cells which similarly show no contact requirement for optimum transferase activity in any phase of their cell cycle. Previously, it was shown that randomly growing populations of 3T3 cells have lower galactosyltransferase activity when assayed under conditions which decreased cell contact. This led to the conclusion that these normal (3T3) and malignant (3T12) cells differed in that intercellular contact is required for optimum activity of surface galactosyltransferases on the normal cell type. The present data indicate that mitotic 3T3 cells may be capable of expressing enzyme activities exhibited at all times by malignant cells. That is, mitotic 3T3 cells and randomly growing 3T12 cells may readily catalyze galactosyltransferase reactions between enzymes and acceptors on the same cell. Interphase 3T3 cells, on the other hand, might require that enzymes glycosylate acceptors on adjacent cells. A model is proposed that suggests that changes in the spatial arrangement of surface enzymes and acceptors or variations in the fluidity of the cell membrane can account for this contact-related glycosylation.     

Glucocorticoids modulate transformation by v-Src in a cell-specific manner

In Balb 3T3 murine fibroblasts infected with retroviruses carrying the v-src oncogene, treatment with the glucocorticoid hormone dexamethasone induces a 10-fold increase in the number of transformed foci and of anchorage-independent colonies. In contrast, in NIH-3T3-infected cells the number of foci and of colonies growing in soft agar is considerably reduced by the addition of the hormone. The effect of dexamethasone on both Balb 3T3 and NIH 3T3 cells is dose-dependent and mediated by specific receptors. The expression of glucocorticoid receptors as well as transactivation of a mouse mammary tumor virus promoter in the presence of dexamethasone is comparable in the two cell lines. Dexamethasone does not change the expression and kinase activity of v-Src proteins either in freshly infected Balb 3T3 and NIH 3T3 cells or in morphologically normal clones or in transformed foci derived from infected Balb 3T3 cells stably expressing v-Src. However, in cocultivation assays of phenotypically normal clones of v-Src expressing Balb 3T3 cells mixed with a large excess of parental Balb 3T3 cells, the hormone is able to rescue the ability to form transformed foci of these otherwise normal cells. The present data point out a new role of glucocorticoid hormones in controlling transformation in a cell-specific manner through epigenetic mechanisms.     

Amino acid transport by membrane vesicles of virally transformed and nontransformed cells: effects of sodium gradient and cell density.]

Mixed membrane vesicle populations composed of plasma membrane and endoplasmic reticulum were prepared from Balb/c 3T3 and simian virus 40-transformed Balb/c 3T3 mouse fibroblasts. The initial rates of uptake of L-leucine and alpha-aminoisobutyric acid by these vesicles were stimulated by a NaCl gradient (external greater than internal). Cation specificity for stimulation of L-leucine uptake was Na+ greater than Li+ greater than K+. NaSCN was as effective as NaCl. Stimulation of uptake of both amino acids by a NaCl gradient was twice as great in vesicles from transformed as compared to non-transformed cells. The NaCl gradient produced transient accumulation of both L-leucine and alpha-aminoisobutyric acid to twice the equilibrium level in vesicles from transformed cells. No such "overshoot" was observed in vesicles from nontransformed cells. In vesicles from the contact-inhibitable Balb/c 3T3 cells, transport of alpha-aminoisobutyric acid, but non L-leucine, exhibited a density-dependent decrease in Na+ gradient induced stimulation, from 248% for sub-confluent to 109% with confluent cells. No density-related changes in uptake were noted with vesicles from the transformed cells. These studies suggest that variation in amino acid uptake associated with viral transformation may be related, at least in part, to alterations in Na+ permeability of the surface membrane.     

The expression of ceruloplasmin, an angiogenic glycoprotein, by mouse embryonic fibroblasts

Balb/c 3T3, Swiss 3T3 and Rous sarcoma virus transformed Balb/c 3T3 mouse embryonic fibroblasts produced ceruloplasmin in vitro, whereas primary cultures prepared from the Balb/c mouse embryos did not produce ceruloplasmin. The amount of ceruloplasmin synthesis by the Balb/c 3T3 cell line is enhanced by Rous sarcoma virus-transformation (1.5-3 fold) and by treatment with dexamethasone (about 2.4 fold). The protein was identified as ceruloplasmin by immunoprecipitation with ceruloplasmin-specific polyclonal antibody, and by similarity of peptide maps, and subunit molecular weight (135,000 dalton) to that of authentic ceruloplasmin from primary cultures of mouse hepatocytes.     

Selection of Revertants of Kirsten Sarcoma Virus Transformed Nonproducer BALB/3T3 Cells

Revertants of Kirsten sarcoma virus transformed nonproducer BALB/3T3 cells (KA31 cells) were isolated after exposing the transformed cells to 5-fluorodeoxyuridine at high cell density, or when suspended in methylcellulose. Revertants were also isolated by treating KA31 cells with the lectin, concanavalin A, which is manyfold more toxic to transformed cells than for normal cells. The revertants resemble BALB/3T3 cells in their morphology and growth characteristics in that they have a low saturation density, fail to grow in 1% calf serum or when suspended in methylcellulose, and cease to synthesize DNA after reaching their saturation density. Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus. The concanavalin A revertants also become transformed after infection with murine leukemia virus. All the revertants can be transformed by Kirsten sarcoma virus but not by simian virus 40.     

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.     

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.     

Membrane glycopeptides from virus-transformed hamster fibroblasts and the normal counterpart.

Comparisons of membrane glycopeptides from baby hamster kidney fibroblasts (BHK21/C13) and a clone transformed by Rous sarcoma virus (C13/B4) were made by using cells metabolically labeled with radioactive D-glucose and L-fucose. Most of the glycopeptides were metabolically labeled with both the general and the specific glycoprotein precursors. The glycopeptides obtained from the cell surface by controlled trypsinization were representative of the surface membrane as shown by comparing them with those of purified membrane preparations. The trypsin-removable glycopeptides from both cell types were further processed and examined by successive chromatography on Sephadex G-50 and DEAE-cellulose. The chromatographic distribution patterns showed that each cell type had glycopeptides of similar characteristics, although the proportions of the glycopeptides differed dramatically between the two cell types. After transformation there was an increase in the larger, more highly charged glycopeptides. This was verified by the increased sialic acid content in these glycopeptides. Some of the glycopeptides were homogeneous after the size and charge separations, since a variety of procedures did not separate them further. The apparent homogeneity and reasonably few species obtained may be due to the methods of isolation, with the procedures selecting particular glycopeptides from the external portion of the membrane. These results corroborate the concept and show for the first time that virus transformation is accompanied by an increase in certain species of glycopeptides rather than de novo synthesis.     

Insulin receptors in normal and transformed fibroblasts: relationship to growth and transformation

The insulin receptors in normal and transformed lines of mouse Balb/3T3 fibroblasts have been studied. In the normal fibroblasts, the binding of insulin was low in growing cells and increased 2–9 fold in confluent stationary cells. Insulin binding was increased whether growth arrest was due to contact inhibition of growth or serum starvation. When serum-starved cells were stimulated to grow by the addition of fresh serum, insulin binding declined. In cells transformed by simian virus 40, Kirsten, Moloney, and Harvey sarcoma viruses, methylcholanthrene, X rays, or spontaneously, the binding was low, in the same range as growing normal cells. In simian virus 40-transformed cells, insulin binding increased 4 fold as the cells reached higher densities in culture. No relationship to changes in cell size was found. The differences in binding were due to changes in the concentration of the receptors, without changes in their affinity for the hormone.     

Activation of the Murine Sarcoma Virus Genome After Infection with the Murine Leukemia Virus as Determined by Cell Agglutination

Non-virus-producing NIH/3T3 cells transformed by the murine sarcoma virus are agglutinated by conconavalin A to the same low level as normal NIH/3T3 cells. Infection with the murine leukemia virus greatly increases the agglutination of transformed cells but not that of normal cells. These data suggest that the morphological expression of cell transformation and the surface alterations associated with increased cell agglutination are controlled by the expressions of different sarcoma virus genes.     

Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.     

Surface Membrane Glycopeptides Which Coincide with Virus Transformation and Tumorigenesis

Glycopeptides from the surface of clones of hamster embryo cells were examined at various intervals after infection with polyoma virus. Two types of transformed cells were examined: (i) clones that showed delayed transformation or an initially low tumorigenicity, and (ii) clones that were rapidly transformed showing an initially high tumorigenicity. The glycopeptides were removed from the cell surface by trypsin and, after Pronase digestion, were examined by filtration through Sephadex G-50. With delayed transformation, a specific group of glycopeptides was increasingly evident over an 85-day period as the cells showed phenotypic properties of transformation and the ability to form tumors. In the other series, all but one clone of hamster embryo cells showed rapid transformation after infection with polyoma virus. This clone was less tumorigenic and showed little of the specific glycopeptides. In all cases of delayed or rapid transformation examined, the specific group of glycopeptides increased proportionately to the ability of the cells to form tumors. All of the cells derived from progressively growing tumors formed by injection of these transformed hamster cells into adult animals showed an abundance of this group of glycopeptides. These results suggest that specific surface membrane glycopeptides accompany viral transformation and tumorigenesis.     

Enucleation of normal and transformed cells

A quantitative analysis based on centrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3-K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3-K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well-formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2-deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation. It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.     

Sulfated mucopolysaccharides from normal and virus transformed rodent fibroblasts.

The sulfated mucopolysaccharide composition of normal and virus transformed Balb 3T3 and BHK21 cell lines is reported. It is shown that normal 3T3 cells contain mainly chondroitin sulfate B and heparitin sulfate. Relatively higher amounts of chondroitin sulface AC were observed in polyoma virus transformed 3T3 cells, besides an absolute increase of all the three sulfated mucopolysaccharides in the polyoma and SV 40 transformed cells. It is shown also that the three sulfated mucopolysaccharides are at least in part at the cell surface. Similar differences in sulfated mucopolysaccharide composition of normal and virus transformed BHK cell lines were also observed.     

The chick chorioallantoic membrane as a model system for the study of tissue invasion by viral transformed cells.

The chick chorioallantoic membrane (CAM) was used as an assay system to investigate the the invasive properties of viral transformed NIH/3Y3 cells. Scanning electron microscopy demonstrated that single Kirsten sarcoma virus (KiSV)-transformed cells passed between the epithelial cells of the CAM ectoderm within 6 hr of application, while viable NIH/3T3 cells did not penetrate the ectoderm within 24 hr. The transformed cells entered the mesoderm of the CAM and formed tumors of proliferating cells. The application of 5 X 10(5) KiSV-transformed cells resulted in the formation of donor cells resulted in the formation of the donor cell tumors within 5 days in 43% of the membranes. No tumors were formed when as many as 5 X 10(6) NIH/3T3 cells were applied to the membrane. NIH/3T3 cells transformed by the Abelson leukemia virus or the Moloney sarcoma virus also ivaded the CAM and formed tumors of proliferating cells within the mesoderm, while cells infected with the Moloney leukemia virus did not. NIH/3T3 cells inoculated onto the CAM 8 days after infection and transformation with KiSV formed tumors with a frequency similar to that of KiSV transformed cells that have been passaged in culture for many generations. Cells that formed invasive tumors within the mesoderm also attracted loops of host blood vessels.     

Glucose-regulated membrane properties of untransformed and virus-transformed BHK21 cells.

BHK21 fibroblasts transformed by hamster sarcoma virus have a higher rate of uptake of hexoses than their untransformed counterparts, and therefore rapidly exhaust glucose from the culture medium. The effects of culturing normal and transformed BHK cells, both in limiting and in excess glucose, on several membrane properties related to malignant transformation have been studied. The increase in the rate of hexose uptake in transformed cells is partially but not entirely dependent on extracellular glucose concentration. Two transformation-increased membrane proteins of molecular weights 95 000 and 78 000 are shown to be regulated by extracellular glucose concentration in both normal and transformed cells. The loss of LETS-protein, the high density of intramembranous particles, the increase in the amount of a 177K integral plasma membrane protein and the increase in the amount of high molecular weight surface glycopeptides in transformed cells, are not related to glucose depletion of the medium. Beside LETS, another iodinated protein, of molecular weight 160 000, is decreased in transformed cells. The exposure of this protein increased in both normal and transformed cells when arrested in G1 by asparagine deprivation.     

D-galactosyltransferase and its endogenous substrates in chick embryo fibroblasts transformed by Rous sarcoma virus

UDP-D-galactose: 2-acetamido-2-deoxy-beta-D-glucopyranosyl 4-beta-D-galactosyltransferase (GalTase) activity was purified, from primary chick embryo fibroblast (CEF) transformed by a temperature-sensitive, Rous sarcoma virus mutant (CEF-RSV), by chromatography on an affinity resin prepared with monoclonal antibodies to GalTase. Cellular glycopeptides from CEF, as well as CEF-RSV, maintained at permissive (37 degrees) [CEF-RSF (37 degrees)] and nonpermissive temperatures (41 degrees) [CEF-RSV (41 degrees)], were solubilized and galactosylated in vitro by incubation with purified GalTase substrates, composed of at least six discrete complex glycopeptides having bi- to tetra-antennary structures. The glycopeptides isolated from transformed cells, CEF-RSV (37 degrees), included the six types observed in nontransformed cells, but demonstrated alterations in their relative amounts, including an increase in the content of a glycopeptide containing 3 mannose and 4 glucosamine residues. Furthermore, two additional complex-type glycopeptides were isolated from CEF- but demonstrated alterations in their relative amounts, including an increase in the content of a glycopeptide containing 3 mannose and 4 glucosamine residues. Furthermore, two additional complex type glycopeptides were isolated from CEF-RSV (37 degrees). These malignant transformation-related glycopeptides were partially characterized and found to represent tri- and tetra-antennary complex glycopeptides. Endogenous galactosylation appeared to have occurred in a branched, nonspecific manner in these transformed cell-derived glycopeptides. These findings indicate that transformed cells may contain a greater preponderance of more highly branched, complex oligosaccharides which are randomly galactosylated at nonreducing termini by cellular GalTase.