Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.     

Effects of ouabain and osmolarity on bumetanide-sensitive potassium transport in simian virus-transformed 3T3 cells

Unidirectional potassium influx in simian virus-transformed 3T3 cells was dissected into a ouabain-inhibitable "pump' component, a bumetanide-sensitive and chloride-dependent "cotransport' component, and a residual "leak' flux. The bumetanide-sensitive component was stimulated 2-3-fold by a 60-min preincubation with ouabain. Subsequent washing of the cells and incubation in ouabain-free saline reversed both the inhibition of the Na+ pump and the stimulation of bumetanide-sensitive flux. Bumetanide-sensitive potassium influx was also stimulated by hypertonic cell shrinkage (induced by 0.1 M or 0.2 M sorbitol). This latter observation suggests that the bumetanide-sensitive system may play a role in cellular volume regulation.     

Virus-specific phosphoproteins in simian sarcoma virus-transformed primate cells.

Cells transformed by simian sarcoma virus (SSV) express a 115000-dalton protein ( p115 ) that is precipitated by a goat antiserum to disrupted SSV/SSAV-infected and transformed cells but not by antibodies directed against the viral gag protein, p30, or envelope proteins. The protein is detected in productively as well as in nonproductively infected, transformed cells. It is not present in untransformed cells infected with helper virus (SSAV). The protein can be phosphorylated in vivo and in vitro at the tyrosine residue and SSV-transformed cells contain elevated levels of phosphotyrosine.     

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.     

Comparison of oxygen consumption rates in minimally transformed BALB/3T3 and virus-transformed 3T3B-SV40 cells

In the recent years, bioenergetics of tumor cells and particularly cell respiration have been attracting great attention because of the involvement of mitochondria in apoptosis and growing evidence of the possibility to diagnose and treat cancer by affecting the system of oxidative phosphorylation in mitochondria. In the present work, a comparative study of oxygen consumption in 3T3B-SV40 cells transformed with oncovirus SV40 and parental BALB/3T3 cells was conducted. Such fractions of oxygen consumption as “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration observed in the presence of protonophore were determined. Maximal respiration was shown to be only slightly decreased in 3T3B-SV40 cells as compared to BALB/3T3. However, in the case of certain fractions of cellular respiration, the changes were significant. “Phosphorylating” respiration was found to be reduced to 54% and “reserve” respiration, on the contrary, increased up to 160% in virus-transformed 3T3B-SV40 cells. The low rate of “phosphorylating” respiration and high “reserve” respiration indicate that under normal incubation conditions the larger part of mitochondrial respiratory chains of the virus-transformed cells is in the resting state (i.e. there is no electron transfer to oxygen). The high “reserve” respiration is suggested to play an important role in preventing apoptosis of 3T3B-SV40 cells.     

Phosphate and the regulation of DNA replication in normal and virus-transformed 3T3 cells.

3T3 cells were cultured in media with different phosphate concentrations and the effects on DNA synthesis were examined. Even a modest phosphate depletion markedly inhibited DNA synthesis and cell multiplication in proliferating cultures. Furthermore, the decrease in the proportion of DNA-synthesizing cells observed after phosphate starvation followed the same time-course as the decrease seen after serum starvation. Cells starved to quiescence in a medium with a 100-fold decrease in phosphate concentration remained viable but non-proliferating for up to 3 weeks, i.e. they had entered a state of quiescence comparable with that seen after serum starvation. Addition of phosphate to phosphate-depleted cultures restored DNA synthesis within 24h. Furthermore, the kinetics of [3H]thymidine labelling after phosphate addition were nearly identical with the labelling kinetics following addition of serum to serum-depleted cultures. In contrast, phosphate deprivation had no inhibitory effects on DNA synthesis in simian-virus-40-transformed 3T3 cells. Furthermore, the inhibitory effects on DNA synthesis in such cells caused by a complete removal of serum could not be further enhanced by decreasing the phosphate concentration in the culture medium.     

Locomotory behavior, contact inhibition, and pattern formation of 3T3 and polyoma virus-transformed 3T3 cells in culture

The social behavior of 3T3 cells and their polynoma virus-transformed derivative (Py3T3 cells) was examined by time-lapse cinemicrography in order to determine what factors are responsible for the marked differences in the patterns formed by the two cell lines in culture. Contrary to expectations, both cell types have been found to exhibit contact inhibition of cell locomotion. Therefore, the tendency of 3T3 cells to form monolayers and of Py3T3 cells to form crisscrossed multilayers cannot be explained on the basis of the presence versus the absence of contact inhibition. Morevover, with the exception of cell division control, the social behavior of the two cell types is qualitively similar. Both exhibit cell underlapping and, after contact between lamelliopodia, both show inhibition of locomotory activity and adhesion formation. Neither cell type was observed to migrate over the surface of another cell. The two cell types do show quantitative differences in the frequency of underlapping, the frequency with which contact results in inhibition of locomotion, and the proportion of the cell margin that adheres to the substratum. The increased frequency pf Py3T3 underlapping is correlated with the reduced frequency of substratum adhesions, which in turn favors underlapping. On the basis of these observations, it is concluded that the differences in culture patterns are the result of differences in the shapes of the individual cells, such that underlapping, and hence crisscrossing, is favored in Py3T3 cell interactions and discouraged in 3T3 cells.     

Interleukin 1 alpha mRNA in virus-transformed T and B cells

IL-1 alpha cDNA clone was isolated from a T cell line infected by the human T lymphotropic retrovirus type-I (HTLV-I/ATLV). We found significant amounts of mRNA hybridizing to IL-1 alpha cDNA not only in HTLV-I-transformed T cells but also in Epstein-Barr Virus-transformed B cells. A part of IL-2 receptor inducing activity in Adult T cell leukemia (ATL) cell line seems to be due to IL-1 alpha.     

Phosphorylation but not transport of sugars is enhanced in virus-transformed mouse 3T3 cells.

The transport and phosphorylation of 2-deoxy-D-glucose are separate and sequential events in both normal and virus-transformed 3T3 cells. The apparent enhancement of 2-dOG uptake by 3T3 cells accompanying virus transformation is not due to an effect on the transport process but to enhanced phosphorylation by intracellular kinases. Phosphorylation of 3-O-methyl-D-glucose does not occur in these cells. Both the rate and extent of transport of this glucose analog is the same in normal cells, SV40 virus-transformed cells and sarcoma virus-transformed cells. The appropriateness of using 3-O-MeG for studies of the glucose transport system of animal cells is examined and discussed.     

Dibutyryl cyclic AMP treatment of 3T3 and SV40 virus-transformed 3T3 cells in aggregates. Effects on mobility and cell contact ultrastructure

The random cell movement of BALB/c 3T3 and SV40 virus-transformed BALB/c 3T3 cells within homogeneous aggregates was studied by observing the degree of penetration of newly attached [3H]thymidine-labeled cells into the interior of the aggregates. The 3T3 cells penetrated into 3T3 aggregates an average of 0.89 cell diameter in 1.5 days, whereas the SV40-3T3 cells penetrated into SV40-3T3 aggregates an average of 3.20 cell diameters in the same time. Treatment of the aggregates with theophylline, theophylline plus prostaglandin E1, or theophylline plus dibutyryl cyclic AMP all decreased the penetration of the SV40-3T3 cells into SV40-3T3 aggregates (2.36, 1.22, and 0.79 cell diameters, respectively). The same treatments had little effect on 3T3 aggregates. The ultrastructure of 3T3 and SV40-3T3 cells in aggregates was examined by transmission electron microscopy. The 3T3 cells in aggregates were surrounded by microvilli and lamellipodia which were in contact with neighboring cells, whereas SV40-3T3 cells were nearly devoid of microvilli and lamellipodia and made contact at broader, less regular surface undulations. Treatment with theophylline plus dibutyryl cyclic AMP resulted in the appearance of microvilli on SV40-3T3 cells and also appeared to increase the area of intercellular contacts in both 3T3 and SV40-3T3 cells. These observations were supported for the surface cells of the aggregates by scanning electron microscopy.     

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated. Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that in normal NIH 3T3 cells.     

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.     

Hyaluronate binding and degradation by cultured embryonic chick cardiac cushion and myocardial cells

Cultured cells obtained from developing chick heart valvular and septal primordial tissues (cardiac cushions) and myocardium were tested for their capacity to bind, internalize, and degrade hyaluronate. A presumptive lysosomal hyaluronidase capable of hyaluronate degradation has been previously isolated and partially characterized from cultures enriched in either cushion tissue cells or myocardial cells (D. H. Bernanke and R. W. Orkin, 1984, Dev. Biol. 106, 351-359). In this study, both types of cultures were found to bind hyaluronate, but only the myocardial cultures could degrade the hyaluronate substrate. The lack of hyaluronate degradative capacity in the mesenchymal cushion tissue cells appears to result from their inability to internalize the macromolecule, thus failing to make it available to the lysosomal hyaluronidase. The data suggest that hyaluronate clearance from the extracellular matrix of the developing cushion is a complex process, involving more than simple extracellular degradation adjacent to the migrating mesenchymal cushion tissue cells. Instead, a sequence of events may be indicated which includes binding of hyaluronate to the cushion tissue cell surfaces and its transport by these cells across the cushion matrix toward the myocardium. The myocardium may be involved in the ultimate removal of hyaluronate from the cardiac jelly.     

Non-histone chromosomal proteins from virus-transformed and untransformed 3T3 mouse fibroblasts.

A peak in the non-histone chromosomal protein polyacrylamide gel electrophoresis profiles has been detected which is higher in log phase 3T3 and 3T3/SV40 cells than in density-inhibited 3T3 cells. Radioactive incorporation is substantially higher into this peak in log phase 3T3 than in 3T3/SV40 and density-inhibited 3T3 cells. Reversion of 3T3/SV40 cells with dibutyryl cyclic AMP and theophylline produces increased radioactive incorporation into the peak. Electrophoresis of non-histone chromosomal proteins extracted at different stages of the cell cycle in density inhibited 3T3 cells following serum stimulation shows a cyclic variation in the amount of this peak with maximum accumulation in late G1. In contrast the height of an equivalent peak in synchronously growing 3T3/SV40 cells remains constant throughout the cell cycle. It is postulated that the protein(s) of this peak may have a regulatory role in cell growth.     

Reverse transformation of Harvey murine sarcoma virus-transformed NIH/3T3 cells by site-selective cyclic AMP analogs

Eighteen site-selective cAMP analogs modified at either the C-8 position or the C-6 position were tested for their growth regulatory effects on the Harvey murine sarcoma virus-transformed NIH/3T3 clone 13-3B-4 cells grown in a serum-free defined medium. All 18 analogs, when tested individually, exhibited an appreciable growth inhibitory effect at micromolar concentrations. The most potent growth inhibitory analogs contained a thio moiety at the C-8 position. In general, C-6 analogs required 5-10-fold greater concentrations than C-8 analogs to produce the same degree of growth inhibition. The growth inhibition induced by these analogs was accompanied by a change in cell morphology; cells treated with the analogs exhibited the morphology characteristic of untransformed fibroblasts, while untreated cells retained a transformed phenotype. The regulatory subunit of cAMP-dependent protein kinase, the cAMP receptor protein, has two different intrachain cAMP binding sites, and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1, while analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2. Thus, C-8 and C-6 analogs were tested in combination to enhance the growth regulatory effect. Both growth inhibition and morphological change were enhanced synergistically by a combination of the C-6 and C-8 analogs. Two C-6 analogs or two C-8 analogs added together did not cause synergism. For both growth inhibition and phenotypic change, C-8 thio analogs acted far more synergistically than C-8 amino analogs when cells were treated in combination with C-6 analogs, suggesting a response of the RII rather than the RI cAMP receptor protein. DEAE-cellulose chromatography revealed that the growth inhibition, in fact, correlates with an increase of the RII cAMP receptor protein and a decrease of the RI receptor protein. The growth inhibitory effect of the site-selective analogs was not due to the cytotoxic effect of adenosine metabolites as shown by the different behavior of 8-Cl-cAMP compared with 8-Cl-adenosine in 1) cell cycle effects and 2) release from growth inhibition. It is concluded that the observed growth inhibition and phenotypic reversion of 13-3B-4 cells is most likely mediated through the cellular effector, the RII cAMP receptor protein.     

Increased collagen synthesis in Kirsten sarcoma virus-transformed BALB 3T3 cells grown in the presence of dibutyryl cyclic AMP

Growth of Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells in the presence of dibutyryl cyclic AMP (dbcAMP) resulted in alteration of morphology, inhibition of growth, and increased collagen synthesis as measured by incorporation of ¹⁴C-proline into collagenase-digestible protein. There was an increase in incorporation of ¹⁴C-proline into collagen when expressed not only as dpm per μg DNA or protein, but also as the relative rate of collagen synthesis compared to total cellular protein synthesis, which suggests that an alteration in amino acid transport cannot totally account for the increased incorporation into collagen. The three properties studied were all affected over a concentration range of 0.10 to 1.0 mM dbcAMP, but each had a slightly different dose-response curve. At 0.5 mM dbcGMP or sodium butyrate, there was no affect on growth, morphology, or the relative rate of collagen synthesis indicating specificity for the dibutyryl analog of cAMP. Growth of the parent line, BALB 3T3, was inhibited by 0.5 mM dbcAMP, but the relative rate of collagen synthesis did not increase. These results suggest that although growth, morphology, and collagen synthesis are altered in transformed cells so that they more closely resemble those of the parent line, each property may be regulated independently.     

Phosphorylation and proteolytic degradation of nucleolin from 3T3-F442A cells

The effect of phosphorylation on the proteolysis of nucleolin has been investigated. Nucleolin is readily phosphorylated both in vitro and in vivo. Utilizing phosphorylation assays and immunoblotting with anti-nucleolin serum, we have observed that phosphorylation enhances nucleolin as a substrate for a protease. This protease activity cleaves the protein into a highly phosphorylated 30 kDa peptide and a 72 kDa peptide. The involvement of casein kinase II is suggested since this cleavage is promoted by spermine and inhibited by heparin, which are, respectively, a stimulator and an inhibitor of casein kinase II activity. The molecular identity of the protease and the physiologic significance of the proteolytic cleavage of nucleolin remain to be studied.