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.     

The effects of newborn calf serum and foetal bovine serum on the Na+ + K+-activated adenosine triphosphatase activity in 3T3 and SV3T3 cells grown to confluency

The effects of cell density and growth in 10% foetal bovine serum and 10% newborn calf serum on the activity of the enzyme (Na+ + K+)-ATPase were studied in 3T3 and SV3T3 cells. The enzyme activity decreases in 3T3 cells grown in foetal bovine serum as the cells approach confluency while in those grown in newborn calf serum the enzyme activity increases. The (Na+ + K+)-ATPase activity does not change with increase in cell density in SV3T3 cells grown in foetal bovine serum while the enzyme activity in those grown in newborn calf serum increases with increase in cells density up to about 1.35 x 10(5) cells/sq. cm. and then decreases with further increase in cell number. At confluency it was found that the enzyme activity is higher in the SV3T3 as compared to the 3T3 cells when the cells were grown in 10% foetal bovine serum, whereas in those grown in 10% newborn calf serum the enzyme activity is higher in the 3T3 as compared to the SV3T3 cells.     

2-amino-isobutyric acid and 3-O-methyl-D-glucose transport in 3T3, SV 40-transformed 3T3 and revertant cell lines.

In order to further investigate the connection between transport and growth control, 3T3 cells, SV40 transformed 3T3 cells (SV101), and three revertant cell lines derived from SV101 which have regained certain manifestations of growth control were used. Transport rates of 2-amino-isobutyric acid and 3-O-methyl-D-glucose were measured in sparse, confluent, serum-starved, and serum-stimulated cultures. As shown before, cessation of 3T3 cell growth in G0 under conditions of confluence or serum deprivation was associated with reduced rates of transport for both compounds, whereas the density and serum dependence of growth and transport was largely eliminated in SV101. The density revertant F1SV101, which has regained density regulation of growth similar to 3T3 cells, has also regained density regulation of transport. Neither growth nor transport were serum dependent. The serum revertants AgammaSV7 and LsSV6 have regained both density and serum regulation of growth, but not according to the original mechanism of 3T3 cells of entry into a Go state. Transport was high under conditions of confluence or serum deprivation. Thus for these cells rates of transport were not reduced simply as a consequences of slower cell growth nor were low transport rates responsible for growth arrest. The data are consistent with the possibility that growth arrest specifically in the G0 state could shut off a number of cellular activities, including transport.     

The suppression of cellular proliferation in SV40-transformed 3T3 cells by glucocorticoids

Glucocorticosteroids, when added two hours after cell plating to SV40-transformed, 3T3 mouse fibroblasts in low serum (0.3% v/v), biotin-supplemented medium, suppress cellular proliferation by 24 hours. While some cell death probably occurs, the growth inhibition is not primarily due to cytotoxicity and cytolysis. This conclusion is supported by the following: 1) both dead and viable cell numbers are suppressed, 2) little cell debris is evident in the medium, and 3) very high concentrations of glucocorticoids do not cause an increase in the dead cell count. Furthermore, this growth suppression, which is specific for glucocorticoids since several non-glucocorticoid steroids have no inhibitory effect, is not permanent nor irreversible. Removal of the glucocorticoid and replacement with 10% serum restore rapid proliferation. Although higher concentrations (1% and 10%) of serum afford some protection against glucocorticoid inhibition, this protection is not simply a consequence of faster growth rates. SV3T3 cells can be grown in serum-free medium supplemented with biotin, transferrin, insulin, and epidermal growth factor (EGF). Under these conditions growth rates are comparable to high serum media, yet glucocorticoids are still powerful inhibitors. However, the omission of insulin from serum-free, glucocorticoid cultures does result in observable cell death and lysis. Flow microfluorometry and autoradiographic studies have determined that glucocorticoid-inhibited cells are partially blocked in G1. The proportions of S phase and G2 + M cells are greatly reduced with an accompanying accumulation of G1 cells. These results suggest that glucocorticoids regulate a biochemical step(s) in G1 which is critical for DNA initiation.     

The serum growth and survival requirements of SV40-transformed 3T3 cells

Summary Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that has recently been characterized as containing as its major agent, biotin. To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological concentrations (200 to 500 ng/ml) and transferrin (5×10⁻⁸ M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are nearly as growth enhancing as 10% serum. The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization. Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr population doubling time, 1×10⁶ cells/3.2-cm dish final cell density). Insulin addition further stimulates the growth rate (16 to 20 hr) and the final density (1.5×10⁶ cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME supplemented with biotin, transferrin, insulin, EGF and FGF) yields growth comparable but still inferior to 10% serum supplementation (14-versus 12-hr population doubling time, 1 to 2×10⁶ versus 2 to 3×10⁶ cells final cell density). This work was supported by NIH Grant CA 20040.     

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.     

The effects of biotin and fatty acids on SV3T3 cell growth in the presence of normal calf serum

The growth of SV3T3 cells in medium containing a low concentration (0.20% v/v) of normal calf serum is enhanced by the addition of biotin or certain unsaturated fatty acids. The biotin effect on the final viable cell density is 5- to 10-fold over the control and is extremely potent, exerting a saturating response at a a concentration of approximately 200 pg/ml. The optimal growth response observed with fatty acids in 5-fold over the control and requires the combination of nervonic acid, palmitoleic acid, and arachidonic acid. The fatty acids are probably not replacing the function of biotin since these two substances are additive in their growth effects.     

Identification of a PDGF-like mitoattractant produced by NIH/3T3 cells after transformation with SV40

It has previously been shown that fibroblastic cells transformed by SV40 exhibit a reduced requirement for PDGF for growth. In addition, NIH/3T3 cells lose both their chemotactic response to PDGF and specific cell surface binding of PDGF after transformation with SV40. We have now examined whether the SV40 transformed NIH/3T3 cells are producing a factor which acts similarly to PDGF. Our studies indicate that NIH/3T3 cells transformed with SV 40 produce a factor which shares many biological properties with PDGF. We were unable to detect this activity in conditioned media from nontransformed NIH/3T3 cells. The SV40/NIH/3T3 derived factor appears to possess both chemotactic and mitogenic activity for connective tissue cells but not endothelial or epithelial cells. Furthermore, in preliminary studies, this activity competes with 125I-PDGF for binding to smooth muscle cells. The biochemical properties of the SV40/NIH/3T3 derived factor are different from those of PDGF. The SV40 activity appears to reside in a heat labile acidic protein (pI less than 7.0) of MW less than 30,000 whereas PDGF is a heat stable basic protein (pI9.8) of 30,000 MW. Production of this factor may play a role in the decreased serum requirement for cell replication exhibited by SV40-transformed NIH/3T3 cells by supplying the cells with their own PDGF-like growth factor.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

The growth stimulation of SV3T3 cells by transferrin and its dependence on biotin

A critical nutrient for the growth of SV3T3 cells is iron. Iron must be added in the ferrous form or, if in the ferric state, with a suitable complexing agent. Both transferrin and hemoglobin, as iron complexes, will stimulate cell growth in biotin supplemented medium either with low serum (0.15% v/v) or serum-free. The growth stimulation by iron (free or in complexed form) is dependent on the presence of biotin in the medium. These results indicate the importance of transferrin as a serum growth factor.     

The growth requirements of SV40 virus transformed Balb/c-3T3 cells in serum-free monolayer culture

The growth requirements of SV40 transformed Balb/c-3T3 cells have been studied in the absence of serum. For growth in serum-free medium, the cells require (i) insulin, (ii) transferrin, and (iii) cis-unsaturated fatty acids added in combination with fatty acid free bovine serum albumin. The growth rate, saturation density, and morphology of cells grown in this serum-free medium are the same as those of cells grown in serum supplemented medium. This mixture also supports the growth of SV40 transformed Swiss-3T3 cells and SV40 transformed primary mouse embryo cells, but does not support the growth of untransformed Balb/c-3T3 cells. The addition of fibronectin to this mixture allows routine subculture, repeated passage, and indefinite propagation of SV40 transformed Balb/c-3T3 cells. Cells grown in this medium for a period of two months retain their ability to induce tumors when injected into athymic nude mice.     

Lipid composition of Balb/c3T3, SV3T3, and Concanavalin A-selected revertant cells grown in media containing lipid-depleted serum

The effects of growth in media supplemented with lipid-depleted fetal calf serum (LDS-media) on morphology, saturation density, and lipid composition were studied in Balb/c3T3, SV3T3, and Concanavalin A selected SV3T3 revertant cells (SV3T3 Rev cells). Cells grown in media containing complete fetal calf serum (FCS-medium) or reconstituted FCS (RS-medium) were used as controls. Growth in LDS-media reduced saturation densities of both SV3T3 and SV3T3 Rev cells while it affected only slightly the saturation density of normal parental cells. Similar inhibitory effects on growth were also induced by exposure of RS-medium. Growth in LDS-medium did not change the typical morphology of the three cell lines. 3T3, SV3T3, and SV3T3 Rev cells grown in LDS-medium showed an accumulation of triacylglycerols and free fatty acids together with a reduction of free cholesterol. All these changes were also present, however, in cells grown in these changes were also present, however, in cells grown in RS-Medium. Growth in LDS-medium induced an increase of 16:1 and 18:1, a decrease of 20:4, and an accumulation of 20:3 (n-9) in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol + phosphatidylserine of 3T3 cells. By contrast, only a slight accumulation of 20:3 (n-9) accompanied by a moderate increase of monoenoic acids was found in the phospholipids of SV3T3 cells grown in LDS-medium. SV3T3 Rev cells grown in LDS-medium showed changes in phospholipid fatty acids composition similar to those found in SV3T3 cells grown under the same conditions.     

Regulation of uridine kinase activity in BALB/C-3T3 cells by serum components

After the stimulation of quiescent density-inhibited BALB/c-3T3 cells with fresh bovine calf serum, uridine kinase activity measured in cellular extracts increased between hours 3 and 6 of incubation and remained elevated through 12 h after stimulation. The addition of either partially purified platelet-derived growth factor (PDGF) or platelet-poor plasma (PPP) also caused increased uridine kinase activity by 6 h, but the increased activity was not maintained and the activity returned to the prestimulated level by 12 h. However, when PDGF and PPP were added in combination an increased level of uridine kinase activity was maintained in a manner similar to that seen after the addition of serum. The components of PPP eluted in the void volume from Sephadex G-50 chromatography did not induce uridine kinase activity when present alone, although they did act synergistically with PDGF to allow the maintenance of elevated levels or uridine kinase activity over the period from 6 to 12 h after stimulation. Thymidine kinase activity was not induced by the addition of either PDGF or PPP alone, although either serum or the combination of PDGF and PPP did produce and induction of thymidine kinase activity in late G1.     

The identification of a serum viability factor for SV3T3 cells as biotin and its possible relationship to the maintenance of Krebs cycle activity

A low-molecular weight-factor (“Peak III”) from calf serum, which enhances the viability (and hence growth) of simian virus 40-transformed 3T3 (SV3T3), but not 3T3, cells when grown in low-serum (0.15–0.30%, $\text{v}\text{v}$) Dulbecco's modified Eagle's medium, has been identified as the vitamin biotin. The extraction procedure involved acidification of the serum to pH 4.5, boiling, ultrafiltration of the supernatant through a Pellicon membrane, and Sephadex G-25 chromatography. Peak III was identified as biotin for the following reasons. (i) The viability/growth activity was completely retained on an avidin-Sepharose but not a Sepharose column. (ii) Peak III preparations contained a compound which reacted with the cyclic ureide-specific reagent, p-dimethyl-aminocinnamaldehyde, and which migrated on thin-layer chromatography with the same R_f as biotin. (iii) Peak III and biotin had the same biological effects on SV3T3 cells, including a reduction in the number of dead cells, a lowering of the amount of lactic acid accumulated, and a synergism with iron in stimulating growth. (iv) They were not additive in their effects at saturating doses. To test the hypothesis that at least part of the biotin viability/growth effect may be due to the maintenance of Krebs cycle intermediate levels through the activation of pyruvate carboxylase, Krebs cycle intermediates were added singly to cells in low-serum medium without biotin. Malate, citrate, isocitrate, and fumarate (but not oxaloacetate, α-ketoglutarate, and succinate) were growth stimulatory for SV3T3 but not 3T3 cells. When added in combinations they were no more effective than alone.     

Transformed SV3T3 cells have a reduced lysosomal compartment and lower levels of enzyme activity than 3T3 cells.

The secreted and intracellular activities of a number of lysosomal hydrolases were higher in 3T3 cells than in SV40-transformed cells. The number of lysosomes and their total volume were also much larger in 3T3 cells and the surface area of their lysosomal membranes was almost twice that of SV3T3 cells. These differences alone were not sufficiently large, however, to account for the disparity seen in activity of some enzymes. Gel electrophoresis showed that a number of protein components present in lysosomal membranes purified from 3T3 cells were absent from SV3T3 membrane preparations. The absence of these components may be correlated with the reduced enzyme activity of SV3T3 cells particularly with respect to beta-glucosidase and acid phosphatase, both of which are normally found associated with lysosomal membranes.     

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.     

Control of the Balb/c-3T3 cell cycle by nutrients and serum factors: analysis using platelet-derived growth factor and platelet-poor plasma.

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractionated into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.     

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.     

Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells.

Components of the renin-angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus-transformed SV3T3 cells. With time after infection, renin decreased in Simian virus 40 transformed cells, while it increased steadily in mock-infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells. Angiotensin II stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose-related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2-8) heptapeptide and angiotensin (4-8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment. Our results indicate (1) that angiotensin II may be involved in cell growth regulation, (2) that a negative feedback exist between angiotensin II added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non-specific angiotensinase activity is increased under this condition.