Glutaraldehyde-fixed transformed and non-transformed cells induce contact-dependent inhibition of growth in non-transformed C3H/10T1/2 mouse fibroblasts, but not in 3-methylcholanthrene-transformed cells

C3H/10T1/2 mouse fibroblasts showed a pronounced inhibition of growth when reaching a critical cell density. The situation of high cell density could be mimicked by the addition of glutaraldehyde-fixed cells to sparsely seeded proliferating cells. Treatment of the C3H/10T1/2 cells with 3-methylcholanthrene led to a high frequency of piled up foci (118 type II and type III foci in 78 cultures). Cells of a type III focus of a treated culture were cloned. These cells grew in soft-agar and reached 10 times higher cell densities when grown in culture dishes, than did their non-transformed counterparts. Glutaraldehyde-fixed transformed cells did not differ from fixed non-transformed cells in the ability to inhibit the growth of sparsely seeded non-transformed cells. On the other hand, both the addition of fixed normal or transformed C3H/10T1/2 cells did not affect the growth rate of transformed cells. In a concept explaining the density-dependent inhibition of growth of non-transformed cells by a specific interaction of plasma membrane-localized effectors with plasma membrane-localized receptors, the present findings would indicate that the transformed cells used express active effectors but are functionally defective in the receptors or in the signal transmission.     

ATP analogues induce membrane permeabilization in transformed mouse fibroblasts

The mechanism underlying ATP-induced permeabilization of transformed mouse fibroblasts was studied by using nonhydrolyzable analogues of ATP. Incubation of 3T6 cells with 0.6 mM of either ATP, 5'-adenylyl imidodiphosphate (p[NH]ppA) or adenosine 5'-[beta, gamma-methylene]triphosphate (p[CH2]ppA) resulted in an increase of 17-, 8- or 5-times, respectively, in the cell membrane permeability, measured by the efflux of normally impermeant metabolites from the cells. The induced cell permeabilization was preceded by a reduction in the membrane potential (delta psi), determined according to the distribution of the cation tetraphenylphosphonium (TPP+) between the cells and the medium. Reduction of 26, 18 and 13 mV in delta psi was exerted by 0.6 mM of either ATP, p[NH]ppA or p[CH2]ppA, respectively. In 3T3 cells the untransformed counterparts of 3T6 cells, neither reduction of delta psi, nor alterations in membrane permeability were exerted by either ATP or by its analogues. The data indicate that the dissociation of the beta, gamma-phosphate bond is not essential for membrane permeabilization by external ATP, implying that the binding of ATP to the cell surface of transformed cells is sufficient to initiate the permeabilization process. The data also suggest that delta psi is involved in the control of membrane permeability.     

Effect of serum on the growth of Balb oT3 A31 mouse fibroblasts and an SV40-transformed derivative.

The effect of serum on the growth properties of non-transformed Balb 3T3 A31 and SV40-transformed Balb 3T3 A31 was studied. The concentration of serum in the growth medium of non-transformed cells had little effect on the initial population doubling time, but did regulate the cell density at which the population became quiescent in G1. The doubling time of transformed cells, however, was increased significantly as the concentration of serum was decreased below 4%. This effect on the growth of transformed cells was seen at serum concentrations so low that non-transformed cells did not complete one population doubling. Flow microfluorometric analysis of these populations indicated that the primary effect of different serum concentrations on the non-transformed cells was to modulate the average residence time in G1, whereas, all the cell cycle phases of the transformed cells were affected by serum. At saturation densities, the non-transformed cells became quiescent in G1, but the transformed cells still traversed the cell cycle and their saturation density appeared to be a balance between cell production and cell death occurring primarily in the G1 phase of the cell cycle.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

ATP analogues induce membrane permeabilization in transformed mouse fibroblasts

The mechanism underlying ATP-induced permeabilization of transformed mouse fibroblasts was studied by using nonhydrolyzable analogues of ATP. Incubation of 3T6 cells with 0.6 mM of either ATP, 5′-adenylyl imidodiphosphate (p[NH]ppA) or adenosine 5′-[β,γ-methylene]triphosphate (p[CH₂]ppA) resulted in an increase of 17-, 8- or 5-times, respectively, in the cell membrane permeability, measured by the efflux of normally impermeant metabolites from the cells. The induced cell permeabilization was preceded by a reduction in the membrane potential (Δψ), determined according to the distribution of the cation tetraphenylphosphonium (TPP⁺) between the cells and the medium. Reduction of 26, 18 and 13 mV in Δψ was exerted by 0.6 mM of either ATP, p[NH]ppA or p[CH₂]ppA, respectively. In 3T3 cells the untransformed counterparts of 3T6 cells, neither reduction of Δψ, nor alterations in membrane permeability were exerted by either ATP or by its analogues. The data indicate that the dissociation of the β,γ-phosphate bond is not essential for membrane permeabilization by external ATP, implying that the binding of ATP to the cell surface of transformed cells is sufficient to initiate the permeabilization process. The data also suggest that Δψ is involved in the control of membrane permeability.     

Growth inhibition of breast cancer cells induced by exogenous ATP

Addition of ATP (>0.1 mM) to cultures of human breast cancer T47D cells resulted in an inhibition of cell proliferation. The inhibition was found to be specific for ATP, and dependent on its concentration. Growth inhibition continued for at least three days, although ATP and its hydrolysis products were metabolized within one day. Conditioned medium from ATP-treated cultures (CM+) was found to inhibit the growth of cells that were not exposed to ATP. This is an indication that extracellular factors, besides ATP, are involved in the inhibition process. The inhibition was maintained after dialysis of the CM+, using an 8 kDa cut-off membrane. Conditioned medium from untreated cultures (CM-), however, only slightly affected cell growth. The data suggest that the CM+ -induced cell growth inhibition is mediated by an ATP-activated growth inhibiting factor. Flow microfluorometry and thymidine incorporation experiments have shown that the growth arrest is mainly due to the elongation of the S-phase of the cell cycle. © 1993 Wiley-Liss, Inc.     

A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.

Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.     

External ATP-induced passive permeability change and cell lysis of cultured transformed cells: action in serum-containing growth media

External ATP causes a marked increase in the passive permeability to phosphorylated metabolites in several types of transformed cells in alkaline medium containing low concentrations of Ca2+, but not in untransformed cells. Such increased membrane permeability with external ATP was also observed in B16 melanoma cells at pH 7.4-7.5 in both Tris-buffered saline and a growth medium containing 10% calf serum and divalent ions at normal concentrations, although a higher concentration of ATP was required. The permeability change in the growth medium was significantly enhanced by calmodulin-interacting drugs, such as trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and chlorpromazine (CPZ). As expected, prolonged exposure of the cells to ATP in the serum-containing medium led to cell lysis. This ATP-dependent cell lysis was observed only in several transformed cell lines, and not in untransformed mouse fibroblasts. These results indicate that the effect of ATP on the membrane permeability in transformed cells is elicited under the physiological conditions and this would be useful in some limited way for cancer chemotherapy management.     

Type-beta transforming growth factor inhibits proliferation and expression of alkaline phosphatase in murine osteoblast-like cells

TGF-beta modulates growth and differentiation in many cell types. MC3T3E1 is a clonal non-transformed murine bone cell line which differentiates in culture. We tested the effect of porcine TGF-beta on the proliferation and differentiation of MC3T3E1 cells in monolayer cultures by following cell number, and alkaline phosphatase activity. TGF-beta treatment (2 ng/ml) altered the shape of MC3T3E1 cells from cuboidal to elongated/spindle-shape. TGF-beta inhibited the growth of MC3T3E1 by up to 40% (P less than 0.02) in a dose-dependent manner with half maximal inhibition at 1 ng/ml. Growth inhibition depended on serum concentration, maximal inhibition occurring at 2% serum. Expression of alkaline phosphatase, which peaks in vitro when the cells reach confluence, was strongly inhibited by TGF-beta, in a dose-dependent manner with half maximal inhibition at around 0.05 ng/ml and complete inhibition at 2 ng/ml. Alkaline phosphatase inhibition was irreversible after 24 hours exposure to TGF-beta.     

Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts.

Fructose 2,6-bisphosphate (F-2,6-P2) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P2 than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F1) or inorganic phosphate (Pi) further stimulated lactate production in both cell lines. F-2,6-P2 stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P2. In the absence of F-2,6-P2 the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mM, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mM ATP. F-2,6-P2 reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 microM (in the presence of 2 mM ATP and 1 microM F-2,6-P2) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 microM F-2,6-P2) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P2 was observed after transfection of rat-1 fibroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.     

Passive membrane permeability to small molecules and ions in transformed mammalian cells: probable role of surface phosphorylation

Addition of ATP to medium surrounding intact, transformed 3T3 cells causes the formation of aqueous channels in the plasma membrane. This effect of extracellular ATP is sharply dependent on the pH and temperature of the incubation medium, and is inhibited by low levels of La3+ or ruthenium red; inhibition is also obtained with concentrations of Mg2+ ions that exceed a ratio of Mg/ATP of one. The effect of ATP on membrane channel formation is unaffected by chelators of metal ions or by prior modification of the cell surface with various surface-active enzymes or sulfhydryl reagents. Under conditions which favor aqueous channel formation, incubation of intact 3T6 cells with ATP (gamma-32P) leads to phosphorylation of two membrane components with apparent molecular weight of 40,000 (40K) and 110,000 (110K) daltons; the 110K component which is unaffected by trypsin under normal conditions is rendered trypsin-sensitive by the phosphorylation reaction, probably as a result of a conformational change. Conditions which inhibit aqueous channel formation also inhibit phosphorylation of the 110K protein and decrease the labeling of the 40K component. These results indicate the probable role of cell surface phosphorylation, involving one or both of these components, in the formation of aqueous channels in transformed 3T3 cells. Aqueous channel formation by extracellular ATP is not associated with gross unfolding of the cell surface as revealed by lactoperoxidase-catalyzed iodination of the 3T6 cell surface.     

BALB/C mouse 3T3 fibroblasts expressing human estrogen receptor: effect of estradiol on cell growth

Mouse 3T3 fibroblasts (clone A31) were stably transfected with human estrogen receptor (hER). Among the four sublines expressing functional hER at approximately 10(4) estrogen binding sites/cell, three retained a non-transformed morphology and growth characteristics while the fourth displayed a transformed phenotype (criss-cross growth, lack of density arrest, reduced dependence on exogenous growth factors). Estradiol (E2) had no effect on the growth of the three non-transformed hER expressing sublines. In contrast, low concentrations (1 to 20 nM) of E2 strongly inhibited the proliferation of the subline with transformed phenotype and high (100 nM) concentrations were toxic in these cells.     

Insulin receptors in 3T3 fibroblasts : Relationship to growth phase, transformation and differentiation into new cell types

The amount of [¹²⁵I]insulin binding per 2 × 10⁶ cells is measured in three lines of mouse embryonic 3T3 fibroblast at different growth stages. Insulin binding is found to be lowest in growing cells of all three types, increasing as cells reach stationary phase. Binding in 3T3-M cells approaches a plateau as cells become stationary. Insulin binding in 3T3-L cells, many of which differentiate into adipocytes following cessation of growth, show further increase in insulin binding post-confluence, in parallel with their differentiation into adipocytes. Binding of insulin in spontaneously transformed cells is higher at all phases of growth than the other two lines, rising to a much higher eventual plateau at approx. 17 days post-confluence. Scatchard plots of insulin binding tend to reflect the same degree of relative insulin binding in these three cell lines. Previously starved cells of all three types exhibit a drop in insulin binding following their first feeding, which corresponds with a second growth spurt in response to nutrients in fresh serum. These results suggest that insulin, as reflected by binding per cell, may play only a minor role in actively growing adequately fed cells of all three types, its major role developing as these cells approach confluence. It is also suggested that higher insulin binding in transformed vs non-transformed cells may indicate a special role for insulin in the loss of contact inhibition, by preserving transport of limiting nutrients in dense, nutrient-depleted transformed cultures.     

Transformation and phosphorylation of purified molybdate-stabilized chicken oviduct progesterone receptor

The non-transformed, molybdate-stabilized chick oviduct cytosol progesterone receptor was purified approx. 7000-fold using biospecific affinity resin (NADAC-Sepharose), DEAE-Sephacel chromatography and gel filtration on Bio-Gel A-0.5m agarose. The purified preparation contained progesterone receptor which sedimented as a 7.9S molecule, had a Stokes' radius of 7.5 nm, was composed of three major peptides corresponding to Mr 108,000, 90,000 and 79,000. Upon removal of molybdate, the purified [3H]progesterone-receptor complex could be transformed from the 8S form to a 4S form by exposure to 23 degrees C or by an incubation with 10 mM ATP at 0 degrees C. The purified thermally transformed receptor could be adsorbed to columns of ATP-Sepharose. No cytosol factor(s) appeared to be required for the 8S to 4S transformation of purified receptor or for its subsequent binding to ATP-Sepharose. Incubation of purified non-transformed receptor preparation with [gamma-32P]ATP and cAMP-dependent protein kinase led to incorporation of radioactivity in all the three major peptides at serine residues. The results of this study show for the first time that purified 8S progesterone receptor can be phosphorylated in vitro by a cAMP-dependent protein kinase, and that it can be transformed to a 4S form by 0 degrees C incubation with 10 mM ATP.     

ATP concentrations in multicellular tumor spheroids assessed by single photon imaging and quantitative bioluminescence

To evaluate the interrelationship among the cellular energy status and the development of necrosis in tumor microregions, local ATP concentrations and the extent of necrosis were determined in multicellular tumor spheroids, i.e., in spherical tumor cell aggregates. The spheroids were grown in rotated suspension cultures using EMT6 cells that were derived from a murine mammary sarcoma. The distribution of viable and necrotic cell areas was assessed by histological investigations. The regional distribution of ATP concentrations was measured with a novel technique using quantitative bioluminescence and single photon imaging. This method makes it possible to determine ATP concentrations in absolute terms with a spatial resolution at the level of a single cell. The results show that ATP concentrations in the center of EMT6 spheroids decrease from values of 1.0 to 1.5 mM in small spheroids with 300 microns in diameter to values close to or at the background level in 750 microns spheroids. Necrosis was detectable in spheroids larger than 300 microns, and virtually no spheroid without necrosis was found at sizes larger than 600 microns. Since the emergence of central necrosis precedes the drop in ATP to undetectably low values, the data suggest that energy metabolism is not or not directly involved in the development of necrosis in tumor spheroids under the growth conditions investigated.     

Regulation of calcium transport in bovine spermatozoa

Calcium uptake into bovine epididymal spermatozoa is enhanced by introducing phosphate in the suspending medium (Babcock et al. (1975) J. Biol. Chem. 250, 6488-6495). This effect of phosphate is found even at a low extracellular Ca2+ concentrations (i.e., 5 microM) suggesting that phosphate is involved in calcium transport via the plasma membrane. Bicarbonate (2 mM) cannot substitute for phosphate, and a relatively high bicarbonate concentration (20 mM) causes partial inhibition of calcium uptake in absence of Pi. In the presence of 1-2 mM phosphate, 20 mM bicarbonate enhances Ca2+ uptake. The data indicate that the plasma membrane of bovine spermatozoa contains two carriers for Ca2+ transport: a phosphate-independent Ca2+ carrier that is stimulated by bicarbonate and a phosphate-dependent Ca2+ carrier that is inhibited by bicarbonate. Higher phosphate concentrations (i.e., 10 mM) inhibit Ca2+ uptake into intact cells (compared to 1.0 mM phosphate) and this inhibition can be relieved partially by 20 mM bicarbonate. This effect of bicarbonate is inhibited by mersalyl. Calcium uptake into the cells is enhanced by adding exogenous substrates to the medium. There is no correlation between ATP levels in the cells and Ca2+ transport into the cell. ATP levels are high even without added exogenous substrate and this ATP level is almost completely reduced by oligomycin, suggesting that ATP can be synthesized in the mitochondria in the absence of exogenous substrate. Calcium transport into the sperm mitochondria (washed filipin-treated cells) is absolutely dependent upon the presence of phosphate and mitochondrial substrate. Bicarbonate cannot support Ca2+ transport into sperm mitochondria. There is good correlation between Ca2+ uptake into intact epididymal sperm and into sperm mitochondria with the various substrates used. This indicates that the rate of calcium transport into the cells is determined by the rate of mitochondrial Ca2+ uptake and respiration with the various substrates.     

Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.     

Hyperoxia-induced clonogenic killing of HeLa cells associated with respiratory failure and selective inactivation of Krebs cycle enzymes

Cellular intoxication by elevated concentrations of O2 may be considered as a model for accelerated cellular aging processes resulting from excessive free radical production by normal metabolic pathways. We describe here that exposure of HeLa cell cultures to 80% O2 for 2 days causes progressive growth inhibition and loss of reproductive capacity. This intoxication was correlated with inhibition of cellular O2 consumption and inactivation of 3 mitochondrial flavoproteins, i.e., partial inactivation of NADH and succinate dehydrogenases and total inactivation of alpha-ketoglutarate dehydrogenase. As alpha-ketoglutarate dehydrogenase controls the influx of glutamine/glutamate into the Krebs cycle, which is the major pathway for oxidative ATP generation in HeLa cells, the inactivation of alpha-ketoglutarate dehydrogenase was expectedly correlated with a net fall in glutamine/glutamate utilization. Furthermore, a simultaneous increase in glucose consumption and lactate production was observed, indicating that the cellular response to respiratory failure is to generate more ATP from glycolysis. In spite of this response, extensive depletion of ATP was observed. Thus, hyperoxia-induced growth inhibition and loss of clonogenicity seem to be due primarily to an impairment of mitochondrial energy metabolism resulting from inactivation of SH-group-containing flavoprotein enzymes localized at or near the inner mitochondrial membrane. These observations may be relevant for theories implicating loss of mitochondrial function as a prime factor in the aging process.     

Biochemical characteristics of normal and virally transformed mouse cell lines

Normal and virally transformed mouse cells respond differently in vitro to high concentrations of potassium. A higher potassium concentration is required to inhibit multiplication of SV40 transformed 3T3 cells to the same extent as that of normal 3T3 cells. This potassium effect correlates to specific activity of Na-K dependent ATPase in membranous fraction, normal 3T3 cells having the higher enzyme activity being more sensitive to potassium than SV40-3T3 cells which have the lower specific activity of the enzyme. Contact inhibition of growth and changes of concanavalin A binding sites which are characteristics of viral transformation influence specific activities of Na-K dependent ATPase and of adenyl cyclase. Incubation with trypsinized concanavalin A causes SV40-3T3 to show contact inhibition of growth and at the same time, higher specific activities of both enzymes than the observed in untreated cells. Cellular content of cyclic 3′,5′-AMP of contact inhibited 3T3 is higher than that in transformed SV40-3T3, but no difference is detectable in ATP content.     

Induction of permeability change and restoration of membrane permeability barrier in transformed cell cultures

Transformed 3T3 cells incubated with ATP at an alkaline pH become permeable to phosphorylated compounds. The increase in membrane permeability can be induced by incubation with ATP at a neutral pH but only if sodium fluoride is present. Fluoride is not necessary for activation of the permeability change in these cultures at the alkaline pH. The effect of fluoride is very rapid, and sodium fluoride by itself does not alter membrane permeability. The alteration of membrane permeability by ATP in 3T6 cells is reversible; the permeability barrier is restored by switching to neutral buffer in the presence or absence of divalent cations. The restoration of the membrane permeability barrier is prevented by fluoride, and by ATP itself; this action of ATP is specific and no other nucleoside triphosphates or chelating agents produce this effect. Untransformed 3T3 cells do not exhibit any appreciable change in permeability as a result of ATP treatment either in the presence or absence of fluoride. These results are consistent with the presence on the transformed cell surface of an ATP-requiring protein kinase and a fluoride-inhibitable protein phosphatase, which would be involved in the control of membrane permeability.