Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly⁺ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly⁺ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na⁺-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na⁺-independent systems L and Ly⁺remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (V_max) rather than substrate concentration for half-maximal velocity (K_m). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.     

Potassium transport in normal and transformed mouse 3T3 cells

The components of unidirectional K influx and efflux have been investigated in the 3T3 cell and the SV40 transformed 3T3 cell in exponential and stationary growth phase. Over the cell densities used for transport experiments the 3T3 cell goes from exponential growth to density dependent inhibition of growth (4 × 10⁴ to 4 × 10⁵ cell cm^?2) whereas the SV40 3T3 maintains exponential or near exponential growth (4 × 10⁴ to 1 × 10⁶ cell cm^?2). In agreement with previous observations, volume per cell and mg protein per cell decrease with increasing cell density. Thus, transport measurements have been expressed on a per volume basis. Total unidirectional K influx and efflux in the 3T3 cell is approximately double that of the SV40 3T3 cell at all cell densities investigated. Both cell types have similar volumes initially and show similar decreases with increasing cell density. Thus, in this clone of the 3T3 cell SV40 transformation specifically decreases unidirectional K flux. The magnitude of the total K flux does not change substantially for either cell line during transition from sparse to dense cultures. However, the components of the K transport undergo distinct changes. Both cell lines possess a ouabain sensitive component of K influx, presumably representing the active inward K pump. Both also possess components of K influx and efflux sensitive to furosemide. The data suggest this component represents a one-for-one K exchange mechanism. The fraction of K influx mediated by the ouabain sensitive component is reduced to one half its value when exponential versus density inhibited 3T3 cells are compared (63% versus 31% of total influx). No comparable drop occurs in the SV40 3T3 cell at equivalent cell densities (64% versus 56% of total influx). Thus, the pump mediated component of K influx would appear to be correlated with growth. In contrast, the furosemide sensitive component represents approximately 20% of the total unidirectional K influx and efflux in both cell lines in sparse culture. At high cell densities, where growth inhibition occurs in the 3T3 cell but not the SV40 3T3, the furosemide sensitive component doubles in both cell lines. Thus, the apparent K-K exchange mechanism is density dependent rather than growth dependent.     

Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density.

Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.     

Control by cell interaction of phosphate uptake in 3T3 cells.

Phosphate uptake by monolayers of 3T3 cell decreases when the cultures enter the stationary phase, even when incubated in fresh medium containing 10% serum. However, SV 3T3 cultures retain a high rate of phosphate uptake when the cells reach saturation densities.We have observed that 3T3 cells grown to stationary phase in monolayers and then trypsinized and incubated in suspension, display an increase in phosphate uptake when the cell concentration is decreased from 10⁶ cells/ml to 10⁵ cells/ml. Where the cell concentration is further reduced from 10⁵ cells/ml to 2.5 × 10⁴ cells/ml there is no further increase in the rate of phosphate uptake. We observed, on the contrary, a small decrease.The “concentration effect” (the decrease of phosphate uptake when the cell concentration increases from 10⁵ to 10⁶ cells/ml) is larger when cells originate from a culture in stationary phase than when they originate from a culture in log phase.The “concentration effect” may be observed 10 min after cell incubation but is larger after a lag time of 40 min incubation.Differences in the “concentration effect” may be noted between 3T3 and SV 3T3 cells. In SV 3T3 cells no significant variations of phosphate uptake were observed when the cell concentration was changed. Thus, differences between phosphate uptake in 3T3 and SV 3T3 cells are large when cells are incubated at high concentrations or at high densities and small when they are incubated at low concentrations or at low densities.The “concentration effect” in 3T3 cells supports the assumption that interactions between cells cause the decrease of phosphate metabolism in dense culture. Diffusion of an inhibitor into the medium remains the more plausible explanation of the data.     

Regulation of passive membrane permeability for potassium ions by cell density of 3T3 and SV40-3T3 cells.

The passive K⁺ permeability of 3T3 and SV40-3T3 cells was evaluated from experiments on passive K⁺ efflux and electrical transmembrane potential measurements at different cell growth densities, external calcium concentrations and temperatures. Passive K⁺ permeability was shown to decrease markedly with increasing cell growth density, to increase with the lowering of external calcium concentration, and at low cell densities to be higher at low temperature (25 °C) than at physiological temperature (37 °C). These and further results taken from the literature are fully consistent with the notion of regulation of proliferation being effected by control of intracellular K⁺ concentrations. The phenomenon of high temperature inactivation of passive K⁺ permeabilities observed at low cell densities is discussed in analogy to recent results on model systems from phospholipid/cholesterol doted with channel-forming antibiotics.     

Cell growth and ouabain-sensitive Rb uptake and (Na + K)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts

The uptake of ouabain-sensitive ⁸⁶Rb⁺ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive ⁸⁶Rb⁺ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive ⁸⁶Rb⁺ uptake in both cell types. This increased ouabain-sensitive cation transport was reflected in increased levels of (Na⁺ + K⁺)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same K_rmm as 3T3 cells.These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.     

Pyridine nucleotide levels as a function of growth in normal and transformed 3T3 cells.

The levels of NAD (NAD⁺ + NADH) and NADP (NADP⁺ + NADPH) and their redox states were measured as a function of growth in 3T3 mouse fibroblasts which exhibit density-dependent inhibition of growth and SV40 (simian virus #40)-transformed 3T3 cells (SVT2) which have lost this property. The levels were related to cell numbers, protein content, and rates of DNA synthesis. At corresponding cell densities, 3T3 cells contain approximately twice as much total protein as SVT2 cells. The levels of NAD relative to total cellular protein are density dependent in both 3T3 and SVT2, increasing with increasing cell density. Over a 30-fold range of cell densities, the NAD levels in 3T3 increase 2.4-fold, while the levels in SVT2 increase 1.6-fold. The levels of NAD are very similar in dividing 3T3 and SVT2 cells at corresponding cell densities; however, a marked increase in the levels of NAD is observed in 3T3 cells, but not in SVT2 cells, at cell densities just prior to where 3T3 cells enter density-dependent inhibition of growth. This increase in NAD levels is correlated with the cessation of DNA synthesis. The NAD pools are 15–25% NADH for 3T3 and 5–15% NADH for SVT2. NADP levels relative to protein in 3T3 and SVT2 are less density dependent, with overall increases of 1.3- and 1.2-fold, respectively, observed over the range of cell densities examined. NADP levels relative to protein are nearly twice as high in SVT2 cells as in 3T3 cells of corresponding cell densities. The NADP pools are approximately 70–80% NADPH in both cell types.     

Inhibition of DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes

3T3 plasma membranes were added to subconfluent cultures of SV3T3 cells in the presence of fusogens. If this protocol results in the introduction into the SV3T3 cell membrane of 3T3 plasma membrane components responsible for density-dependent inhibition of growth, then the SV3T3 cell cultures would be expected to show decreased rates of DNA synthesis as they approach confluence. Results of these experiments indicate that rates of DNA synthesis in SV3T3 cultures so treated were as much as 63% less than in untreated controls. This effect could not be attributed to the fusogens or to the 3T3 plasma membranes alone. This growth-inhibitory effect is specific for 3T3 membranes and is not observed when SV3T3 plasma membranes are fused with SV3T3 cell cultures. These data support the hypothesis that one aspect of the loss of density-dependent inhibition of growth in SV3T3 cells is a deletion or alteration in plasma membrane components and, further, that density- dependent inhibition of growth can be in part restored to SV3T3 cell cultures by fusing the cells with 3T3 plasma membranes.     

Isolation and characterization of revertant cell lines. 3. Isolation of density-revertants of SV40-transformed 3T3 cells using colchicine

Treatment of the SV40 transformed 3T3 cell line SV101 with colchicine permits the isolation of polyploid revertant sublines Which have lower saturation densities than SV101. These low saturation density lines have also reverted to a high serum requirement for growth, and are unable to form colonies in methocel. Normal SV40 has been recovered from these revertants. 3T3 cells are more resistant to colchicine than SV3T3 cells at all cell densities. Colchicine revertants do not display a 3T3-like resistance to colchicine at low density, but do survive colchicine at confluent cell densities, presumably due to their increased contact inhibition.     

Cell surface changes correlated with density-dependent growth inhibition. Glycosaminoglycan metabolism in 3T3, SV3T3, and con A selected revertant cells.

A 35SO4-labeling/chromatography technique has been developed which facilitates quantitation of sulfated glycosaminoglycan (GAG) synthesis in mammalian cell cultures. The technique has been used to compare sulfated GAG biosynthesis, degradation, and turnover in three related cell lines with differing degrees of density-dependent inhibition of growth in vitro (Balb/c 3T3, SV3T3, and SV3T3 revertant cells). Viral transformation of Balb 3T3 cells is accompanied by a 2-5-fold decrease in cell associated sulfated GAG. SV3T3 revertant cells, which show partial reversion to low saturation density in vitro, show a 2.5-8-fold increase in cell-associated sulfated GAG compared to the parental SV3T3 cells from which they were selected. In addition, the distribution of 35SO4 and [3H]glucosamine among the different GAG species produced by SV3T3 revertant cells reverts so that it is similar to the distribution characteristic of untransformed 3T3 cells rather than SV3T3 cells. Mild trypsin treatment of 35SO4-labeled cells removed 68-84% of the cellular sulfated GAG, suggesting that at least this proportion of the total cellular sulfated GAG was located at the cell periphery. Removal of 35SO4-labeled cells from the Petri dish with a Ca2+ selective chelating agent revealed a fraction of the sulfated GAG that remained tightly bound to the Petri dish. A higher proportion of the total cell-associated sulfated GAG remained attached to the Petri dish in cultures of untransformed and revertant cells compared to that present in cultures of transformed cells. A role for sulfated GAG in density-dependent growth inhibition of fibroblast cultures is proposed and discussed in the light of the data obtained.     

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells was studied using ¹²⁵I-labeled canine plasminogen. Throughout a 3-day period, ¹²⁵I-plasminogen in the incubation medium bound to the cells and was degraded, first to intermediate-sized macromolecules that were the same size as the large (74,600-dalton) and small (25,000-dalton) chains of active plasmin, and to smaller fragments including 3-iodo-L-tyrosine. Binding to SV3T3 cells was independent of the protease-dependent morphological change (PDMC)¹ characteristic of these and many other transformed cells. The SV3T3, and to a somewhat lesser extent, the 3T3 cells, both accumulated and released into the incubation medium 3-iodo-L-tyrosine, a terminal lysozymal digestion product. The results of a sublethal cell-surface trypsinization assay suggest that the cell-associated plasminogen was primarily bound to the surfaces of the 3T3 and SV3T3 cells while the macromolecular degradation products including active plasmin were inside the cells. The rate of ¹²⁵I plasminogen degradation exhibited by SV3T3 cells was approximately two times greater than that of 3T3 cells, which presumably reflects differences in endocytosis or lysosomal hydrolysis, or both. The rates were unaffected by addition of pancreatic or soybean trypsin inhibitor sufficient to inhibit PDMC. In the incubation medium, plasminogen was activated to plasmin by SV3T3, but not by 3T3 cells. However, 95–100% of plasmin covalently bound to a 47,000-dalton canine serum component, which could be dissociated from plasmin by hydroxylamine: 95–100% of the plasmin was inactive to reaction with DF³²P. Thus the serum component is a plasmin inhibitor. The plasmin-containing complex in the medium had an apparent molecular weight of 212,000. Under denaturing conditions, the complex dissociated into two covalently modified plasmin-containing species of 153,000 and 127,000 daltons. In addition to forming a complex with a serum component, the plasmin is cleaved into two small fragments (～10,000 and 12,000 daltons) by as-yet uncharacterized serum factors.     

Cell cycle dependent modulations of the surface membrane of normal and SV40 virus transformed 3T3 cells

Agglutinability with Concanavalin was studied as function of cell cycle transition in normal and SV40 virus transformed 3T3 cells. In synchronized cultures of normal cells, agglutinbility was high during mitosis and disappeared rapidly. Agglutinability of transformed cells remained high in G₁ phase but diminished gradually upon entering S phase and reached minimum in G₁ phase. Decreased agglutinability a the end of the cell cycle was also observed in synchronous SV3T3 cultures by a combined technique of haemadsorption and density gradient centrifugation. In normal 3T3 cells, similar variations in agglutin ability during interphase could not be observed.     

Quiescent SV40 virus transformed 3T3 cells in culture

Serum counteracts low nutrient concentrations in the culture medium in SV40 virus transformed 3T3 (SV3T3) cells. The transport of [³H]-leucine into TCA soluble material in SV3T3 cells is stimulated by serum and inhibited by But₂-cAMP. When SV3T3 cells are cultured in low leucine concentrations (? 8 × 10^?6 M), the cell's morphology is similar to the one of cells incubated in complete medium in the presence of But₂-cAMP and cells become quiescent. Cells become arrested throughout the cell cycle. The results suggest that the mechanism by which But₂-cAMP inhibits growth of SV3T3 cells is by inhibiting the transport of leucine in SV3T3 cells.     

Amino acid and22Na+ uptake in membrane vesicles from confluent simian virus 40 transformed Balb/c3T3 and Balb/c3T3

Summary The studies reported here were carried out to characterize further previously described changes in membrane localized amino acid transport associated with simian virus 40 transformation of the mammalian cell line, Balb/c3T3. Membrane vesicles were prepared from confluent cultures of both simian virus 40 transformed Balb/c3T3 (SV3T3) and the untransformed parent line, Balb/c3T3 (3T3). An initial, externally imposed out>in, 100mm Na⁺ gradient produces acceleration of early ingress of -aminoisobutyric acid (AIB) in vesicles from both cell lines, but transient, concentrative uptake (overshooting) only in SV3T3 vesicles. Early ingress ofl-leucine is also accelerated in SV3T3 vesicles by a Na⁺ gradient, and overshooting is also demonstrable.Na⁺-gradient independent AIB permeability of SV3T3 and 3T3 membranes was estimated using uptake data, a first order rate equation and measurements of vesicle size derived from quasi-elastic light-scattering studies. AIB permeability of SV3T3 membranes is greater than that of 3T3 membranes (113 Å/min and 43 Å/min, respectively), suggesting that overshooting in 3T3 vesicles is not attenuated by a Na⁺-independent AIB leak. Na⁺ permeability of the two membranes is similar, ruling out the possibility that a slower rate of Na⁺ equilibration across the SV3T3 membrane allows development of the overshoot.In SV3T3 vesicles the height of a Na⁺-gradient dependent overshoot varies with the initial [Na⁺] _o /[Na⁺] _i ratio, and [Na⁺] _o /[Na⁺] _i is linearly related to ln AIB uptake at overshoot peak/AIB uptake at equilibrium, consistent with the possibility that for [Na⁺] _o /[Na⁺] _i ratios in the range studied, AIB overshoot is energized by a constant proportion of the energy available from the initial electrochemical gradient for Na⁺.These results are consistent with the possibility that Na⁺-gradient dependent overshooting in SV3T3 vesicles is produced by Na⁺-amino acid carrier interactions resulting in either an increase in maximum transport velocity or an incrase in carrier affinity for AIB.Abbreviations used 3T3 Balb/c3T3 - SV3T3 simian virus 40 transformed Balb/c3T3 - AIB -aminoisobutyric acid     

The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells

Balb/c 3T3 cells synthesize 5–10 times more ³⁵SO₄^2?-labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular ³⁵SO₄^2?-labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70–80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65–75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular ³⁵SO₄^2?-labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 ³⁵SO₄^2?-labeled proteoglycans and contains chondroitin sulfate exclusively. It is altogether absent in the extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5–10-fold decreased synthesis of ³⁵SO₄^2?-labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.     

Induction of cell proliferation by a migration factor released from a transformed cell line

A protein released by an invasive tumour cell line (SV28) was purified. It then had 20000 times the activity of serum in stimulating the migration of 3T3 cells. At each step in the purification there was a parallel activity that stimulated proliferation of 3T3 cells. The purified material was shown to stimulate proliferation of normal 3T3 cells at low serum concentrations where only transformed 3T3 cells proliferate and to stimulate the growth of 3T3 cultures to above their normal saturation density. The one substance could therefore account for the growth and the invasiveness of the SV28 cells. At limiting dilution of the protein only the cells along the edge of a wounded monolayer incorporate [³H]TdR. The significance of this edge effect to contact inhibition and the possible role of the diffusion boundary layer are discussed.     

Direct Isolation and Characterization of “Flat” SV40-transformed Cells

UNDER standard culture conditions cells of the permanent mouse embryo line BALB/c-3T3 cease to divide when cell to cell contact is made and thus are characterized as sensitive to density dependent inhibition of growth. A loss of this aspect of growth control is commonly used as a selective means for recovering SV40-transformants because such transformed cells continue to divide in conditions in which nontransformed cells remain confined to the monolayer¹. SV40-transformants selected by their ability to overgrow the monolayer commonly attain population densities ten to fifteen times greater than nontransformed cells, although viral transformants of low saturation density termed “flat revertants” may be obtained from transformed clonal lines by negative selection with 5-fluoro-2-deoxyuridine², or by passage on cell layers fixed with glutar aldehyde³.     

Lipids of whole cells and plasma membrane fractions from Balb/c3T3, SV3T3, and concanavalin A-selected revertant cells.

The lipid composition of Balb/c3T3, SV3T3, and the concanavalin A-selected SV3T3 revertant cells has been analyzed at the whole cell and plasma membrane levels. In comparison to untransformed 3T3 whole cells, SV3T3 cells showed an unchanged content of triacylglycerols, free fatty acids, and glycerylether diesters but a lower concentration of total phospholipids, while no significant difference was found in the phospholipid composition. Whole SV3T3 revertant cells exhibited a lipid composition similar to that in untransformed 3T3 cells with the exception of a higher proportion of sphingomyelin. Analysis of isolated plasma membranes did not reveal any significant differences in the cholesterol to phospholipid molar ratio between 3T3 and SV3T3 or SV3T3 revertant cells. The major changes in the acyl chain pattern SV3T3 compared with whole 3T3 cells consisted of an increase of oleic and palmitoleic acids coupled with a decrease of C20 and C22 polyunsaturated acids in phosphatidylethanolamine and phosphatidylcholine; an increase of oleic acid was also evident in SV3T3 phosphatidylinositol plus phosphatidylserine. An increase of palmitoleic and oleic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine of SV3T3 plasma membranes; the only change in SV3T3 plasma membrane phosphatidylcholine was an increase of oleic acid. An increase of monoenoic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol plus phosphatidylserine of SV3T3 revertant cells at the level of both whole cells and plasma membranes.     

Protein synthesis in 3T3 and SV40-transformed 3T3 cells. Activity of ribosomes and cytosol proteins in cell-free protein synthesis.

The activity of specific components involved in protein synthesis in 3T3 cells and its SV40-transformed derivative, SV3T3, were examined in a cell-free protein synthetic system, and the results correlated with previous studies, indicating that a decreasing rate of protein synthesis does not accompany the stationary phase of growth. We found that 3T3 and SV3T3 polysome preparations containing endogenous mRNA were equally efficient in supporting cell-free protein synthesis in this system. Further, the net protein synthesis observed was not altered by an increase in the population density of the cellular polysome source. The activity of the aminoacyl-tRNA synthetase enzymes from 3T3 and SV3T3 cells was examined in vitro after isolation by pH 5 precipitation and by ammonium sulfate fractionation. The activity of these preparations from stationary phase 3T3 and nonexponential phase SV3T3 cells was found to be approximately 3 times higher than the activity of fractions from the homologous exponential phase cell. However, at both growth stages, the SV3T3 preparations were 30 to 40 times more active than the 3T3 preparations. These findings may have implications for the different growth properties observed in the two cell types.     

Correlation of (Na---K)-ATPase activity with growth of normal and transformed cells

The (Na⁺---K⁺)-stimulated Mg²⁺-dependent ATPase activities of 3T3 and SV40 transformed 3T3 cells were compared as a function of cell population density. For normal cells the enzyme activity remained relatively constant during exponential growth, but decreased sharply coincident with contact inhibition of growth at confluence. This decrease in activity could be reversed by stimulating contact-inhibited cultures to undertake renewed short-term growth either by adding fetal calf serum or changing the medium completely. Transformed cells did not experience a decrease in (Na⁺---K⁺)-ATPase activity upon reaching confluence, but this is consistent with the fact that they were still growing exponentially at this stage. However, non-confluent cultures of both normal and transformed cells incurred a marked decrease in levels of the enzyme when growth was inhibited by serum depletion. The results have been interpreted as indicating that levels of (Na⁺---K⁺)-ATPase in both normal and transformed cells are correlated with growth. Hence the different patterns of ATPase activity displayed by malignant cells and their normal counterparts with increase in cell number appear to be a reflection of their dissimilar growth behaviours rather than of any innate difference between them.