Growing 3T3 Cells Research Articles

Influence of a silicon (Si14)-based coating substrate for biomaterials on fibroblast growth and human C5a

Despite considerable efforts in biomaterial development there is still a lack on substrates for cardiovascular tissue engineering approaches which allow the establishment of a tight a functional endothelial layer on their surface to provide hemocompatibility. The study aimed to test the biocompatibility of a silicon (Si14)-based coating substrate (Supershine Medicare, Permanon) which was designed to resist temperatures from -40°C up to 300°C and which allows the use of established heat-inducing sterilization techniques respectively. By X-ray photoelectron spectroscopy it could be validated that this substrate is able to establish a 40-50 nm thick layer of silica, oxygen and carbon without including any further elements from the substrate on an exemplary selection of materials (silicone, soda-lime-silica glass, stainless steel). Analysis of the LDH-release, the cell activity/proliferation (MTS assay) and the cell phenotype after growing 3T3 cells with extracts of the coated materials did not indicate any signs of cytotoxicity. Additionally by measuring the C5a release after exposure of the coated materials with human serum it could be demonstrated, that the coating had no impact on the activation of the complement system. These results generally suggest the tested substrate as a promising candidate for the coating of materials which are aimed to be used in cardiovascular tissue engineering approaches.

Innate cellular fluorescence reflects alterations in cellular proliferation.

The objective of this study was to examine the question of whether unique spectral patterns were associated with cell proliferation and could be identified by comparing the fluorescence pattern of slow to rapid growing cells. Three in vitro model systems, (A431 cells inhibited by EGF, serum-starved 3T3 fibroblasts, and normal oral epithelial cells exposed to TGF beta), were analyzed using fluorescence spectroscopy. Growth status was monitored by cell number, 3H-thymidine incorporation, and flow cytometry. The excitation spectra (lambda ex 240-430 nm, lambda em 450 nm) effectively distinguished slow and rapid growing cells in all three systems. Statistical analysis of the ratios of the main broad peak (320-350 nm) to a point on the down-slope of the curve at 370 nm was statistically significant. Ratios in the emission scan (lambda ex 340 nm, lambda em 360-660 nm) could separate slow and rapid growing A431 and oral epithelial cells (P = 0.0001 and P = 0.023, respectively), but not slow and fast growing 3T3 cells (P = 0.56). Innate cellular fluorescence has the potential to discriminate proliferating and nonproliferating cell populations.

Inhibition of basic fibroblast growth factor-induced growth promotion by overexpression of syndecan-1.

The expression of syndecan-1, the prototype member of the cell surface proteoglycan family, follows morphogenetic rather than histological boundaries during organ formation. As a heparan sulfate-containing cell surface molecule, syndecan-1 can simultaneously bind various components of the extracellular matrix and members of the heparin-binding growth factors. Indeed, syndecan-1 may act as a co-receptor for basic fibroblast growth factor (bFGF) (Salmivirta, M., Heino, J., and Jalkanen, M. (1992) J. Biol. Chem. 267, 17606-17610), allowing the growth factor to bind the tyrosine kinase bFGF receptor. We have studied the role of syndecan-1 in growth factor response by growing 3T3 cells transfected with syndecan-1 in the presence of bFGF. The enhanced expression of syndecan-1 caused down-regulation of bFGF-induced cell proliferation and, at the same time, enhancement of cell matrix interactions. Thus, the induced expression of the heparan sulfate co-receptor (syndecan-1) may provide a mechanism to restrict FGF action and modulate cell-matrix interactions to maintain co-ordinated growth of cells during organ formation.

Modulation of the synthesis and glycosylation of the glucose transporter protein by transforming growth factor-β1 in Swiss 3T3 fibroblasts

Transforming growth factor-β1 (TGF-β1) stimulated growth and glucose uptake in Swiss mouse fibroblasts. DNA synthesis was increased 2–3-fold after 48 h incubation of growing 3T3 cells with TGF-β1 in calf serum-containing medium. Glucose transport activity in the cells was increased within 3 h after addition of TGF-β1 and this stimulation continued during incubation for 48 h. TGF-β1 also increased the levels of a brain type-glucose transporter (GLUT1) mRNA and the GLUT1 protein (55 kDa) in the membranes, consistent with the increase in glucose uptake. Furthermore, a longer exposure of TGF-β1 for 24–48 h induced a marked increase in the 65 kDa GLUT1 in 3T3 cell membranes. Other growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor-α, and insulin did not elevate glucose uptake and the levels of 55 and 65 kDa GLUT1 proteins. Adding tunicamycin or deoxymannojirimycin to the TGF-β1-treated and untreated cells caused these 55 and 65 kDa glucose transporters to migrate as one band at 40–43 kDa. In addition, treating membrane proteins with glycopeptidase F, which removes N-linked oligosaccharides, also generated a glucose transporter of 40 kDa, suggesting that the 55 and 65 kDa GLUT1 proteins have a similar or identical core polypeptide but with different N-linked oligosaccharides. These results indicate that TGF-β1 modulates the synthesis of GLUT1 protein as well as its glycosylation in Swiss 3T3 cells, and that these changes may contribute to the control of cell proliferation by TGF-β1.

Compartmentation of dCTP pools. Evidence from deoxyliponucleotide synthesis.

The nucleotide fraction of cultured 3T6 and 3T3 mouse fibroblasts contains deoxy-CDP choline and deoxy-CDP ethanolamine as well as the corresponding riboliponucleotides. In permeabilized cells both deoxyliponucleotides were formed from dCTP. In intact cells they could be labeled from [5-3H] deoxycytidine or cytidine via transformation of the nucleosides to dCTP. Their turnover was slow compared to that of dCTP. When rapidly growing 3T3 cells were labeled during 90 min from deoxycytidine the specific activity of dCDP choline was 2.4 times higher than that of dCTP while after labeling from cytidine both nucleotides (and CTP) reached the same specific activity under steady state conditions. Also dCDP ethanolamine was labeled more rapidly from deoxycytidine than from cytidine. Our results suggest that the deoxyliponucleotides were synthesized from a dCTP pool that was labeled preferentially from deoxycytidine. Earlier work (Nicander, B., and Reichard, P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1347-1351) had demonstrated synthesis of DNA from a dCTP pool labeled preferentially from cytidine. Taken together our results suggest that deoxyliponucleotides and DNA are synthesized from separate dCTP pools.

Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor.

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the G1 phase of the cell cycle. When stimulated to proliferate, quiescent (G0) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for G1 transit to those necessary for the transition from the G0 to the S phase. Cycling cells in the G1 phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late G1 step. All cells in the G1 phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-G1 phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new transcription. This result is in contrast to findings that other growth factors induce new transcription. We examined the requirements for G1 transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early- and mid-G1-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.

Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor.

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the G1 phase of the cell cycle. When stimulated to proliferate, quiescent (G0) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for G1 transit to those necessary for the transition from the G0 to the S phase. Cycling cells in the G1 phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late G1 step. All cells in the G1 phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-G1 phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new transcription. This result is in contrast to findings that other growth factors induce new transcription. We examined the requirements for G1 transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early- and mid-G1-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.

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.

Utilization of γ- 32P-GTP by ectoprotein kinase of 3T3 and SV40 3T3 cells

The ectoprotein membrane kinase(s) of 3T3 and SV40 3T3 cells were found to use GTP as a phosphate donor. Phosphorylation with GTP was similar to that found with ATP, previously used to identify the kinase activity in this system ( Mastro and Rozengurt, 1976). Under the same conditions, phosphorylation was greater with ATP than GTP. However, with either nucleotide the phosphorylation was higher in SV40 transformed and growing 3T3 cells than in quiescent ones. Using SDS polyacrylamide gel electrophoresis and radioautography we found that similar substrates were phosphorylated with GTP or ATP.

Transport of 6-deoxy-D-glucose and D-xylose by untransformed and SV40-transformed 3T3 cells.

Transport rates of the nonphosphorylated D-glucose analogs 6-deoxy-D-glucose and D-xylose were measured in quiescent and serum-stimulated cultures of mouse 3T3 cells, in SV40-transformed 3T3 cells (SV101), and in a density revertant cell line derived from SV101 (FI-SV101). Initial rates of both entry and exit of 6-deoxy-D-glucose and D-xylose were more than threefold higher in serum-stimulated 3T3 and in SV101 cells than they were in quiescent 3T3 cells, but transport rates were not higher in the transformed cells (SV101) than they were in serum-stimulated 3T3. Confluent cultures of FI-SV101 showed lower rates of transport than serum-stimulated FI-SV101, but not as low as quiescent 3T3 cells enter the quiescent G0 state, but emphasize that SV40-transformed 3T3 cells do not show higher activity of the D-glucose carrier than do actively growing 3T3 cells. Thus, enhanced glucose transport appears not to be a specific consequence of transformation, but a reflection of the active growth state of the cell.

Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth.

We have quantitated by autoradiography the binding of [125I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G1 phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high serum) also reduce growth inhibitory activity. [125I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.

Fibroblast growth regulatory factor inhibits DNA synthesis in BALB/c 3T3 cells by arresting in G1

A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated fibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late G1 or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early G1 showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the G1. Further, the regulatory factor delayed the progression of G0 G1 -arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early G1, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.

Polyadenylate-protein complexes in resting and growing 3T3 cells

The properties of the ribonuclease resistant cytoplasmic ribonucleoprotein particles were studied in contact-inhibited and serum induced proliferating 3T3 cells. The RNP particles were fractionated by oligo (dT)-cellulose chromatography and banded in CsSO 4 gradients. The main RNP fraction, eluted with 25% formamide, contained the major ribonuclease resistant RNA sequences in both resting and growing cells. The protein component of this fraction had a molecular weight of about 72,000 in contact-inhibited cells and 81,000 in serum induced cells.

Increase in plasma membrane phosphodiesterase activity in contact-inhibited 3T3 cells and in phenotypically reverted SV-3T3 cells

Contact-inhibited 3T3 mouse fibroblast cells, in contrast to logarithmically growing 3T3 cells and SV-3T3 transformed cells, have increased levels of plasma membrane-bound phosphodiesterase (oligonucleotidase, E.C.3.1.4.19; nucleotide pyrophosphatase, E.C. 3.6.1.9) activity. The increase in enzyme, recorded as increased specific activity, is reversible, as evidenced by the return to normal values following dilution of confluent 3T2 cells and re-initiation of growth. Increased enzyme activity is induced again when the cells regain the confluent state. Transformed SV-3T3 cells can be induced to mimic the contact inhibited state, including increased plasma membrane phosphodiesterase activity, by exposure to a combination of: (i) agents that are known to induce increased intracellular cAMP levels and (ii) additions of purified 3T3 or SV-3T3 plasma membranes. Additions of either alone fails to induce the increase in membrane phosphodiesterase activity, although each alone can significantly suppress cell growth, as measured by incorporation of 3H amino acids. We suggest that the elevation of plasma membrane phosphodiesterase activity may serve as a measure of conversion to the contact-inhibited state in both normal cells and phenotypically reverted transformed cells.

Control of DNA synthesis in growing BALB/c 3T3 mouse cells by a fibroblast growth regulatory factor

A cell surface component from quiescent BALB/c 3T3 mouse cells that inhibits DNA synthesis and cell division when added to a culture of growing 3T3 cells has been detected. The inhibition of DNA synthesis by this factor was dependent on concentration and time of incubation; a transient exposure of cells to the factor followed by incubation in its absence for 20 hr was sufficient to elicit its inhibitory effect. The active component appears to be protein in nature, as judged by heat inactivation and trypsin sensitivity. Extracts obtained in an identical manner from quiescent 3T3 cells that had been preincubated in situ with uridine diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc) did not inhibit DNA synthesis. The effect was specific for UDP-GlcNAc: incubation with three other nucleotide sugars yielded active component. Incubation of the inactive component from UDP-GlcNAc-treated cells with purified N-acetyl-beta-D-glucosaminidase in vitro restored its inhibitory property. Extracts from growing cells failed to inhibit DNA synthesis. These results suggest that reversible glycosylation with N-acetyl-D-glucosamine residues may serve as a regulatory signal for the conversion of the active factor to its inactive form. We propose that the onset of quiescence of 3T3 cells is due to a casual relationship between depletion of growth factors in the culture medium and the presence of the active regulatory factor on the cell surface that inhibits DNA synthesis; conversion of the regulatory factor to its inactive form under favorable nutritional status may be viewed as a switch that allows DNA synthesis to resume.

Murine cytomegalovirus gene expression during nonproductive infection in go-phase 3T3 cells

Murine 3T3 cells, maintained in the Go-phase by deprivation of serum, were infected with murine cytomegalovirus (MCMV) and examined for the synthesis of viral DNA, RNA, and proteins. No viral DNA replication could be detected, although the viral genome remained viable since infectious centers persisted. Viral RNA was analyzed by reassociation kinetics with 125I-MCMV DNA. At early and late times after infection, the percentage of viral DNA transcribed amounted to 18% and 21%, respectively (comprising one abundance class), in contrast to 26% and 38%, respectively (two abundance classes), at corresponding times during productive infection in growing 3T3 cells. Five viral polypeptides were detected in the Go-phase cells, but these did not include structural polypeptides. When infected Go-phase cells were exposed to serum and fresh medium, viral DNA synthesis occurred, and progeny virions appeared subsequently. Thus this system may prove useful for analyzing a latent herpes infection in vitro.

Epidermal growth factor. Relationship between receptor regulation and mitogenesis in 3T3 cells.

Exposure of confluent nondividing 3T3 cells to 10 nm mouse epidermal growth factor (EGF) at 37/sup 0/, followed by incubation for 4.5 h at 37/sup 0/, leads to a 70 to 85% reduction in the binding capacity for /sup 125/I-EGF. Scatchard analysis of the binding data indicate that the reduction of /sup 125/I-EGF binding is due to a decrease in the number of available EGF-receptors per cell, without any change in the affinity of the receptors for EGF. This modulation of the EGF-receptor by the growth factor, termed ''down regulation,'' is dependent on temperature, EGF concentration, time, and the physiological state of the cell. Receptor loss occurs at physiological EGF concentrations (0.1 to 10 nm) which span the concentration range which is mitogenic for 3T3 cells. Maximal stimulation of either (/sup 3/H)thymidine uptake or cell division occurs at 1 nm EGF, a concentration at which only 20% of the EGF-receptor sites are occupied and down regulation is only 55% complete. Low EGF concentrations (< or = to 1 nm) result in down regulation of unoccupied EGF-receptors. Down regulation of the EGF-receptor also occurs in SV 40-transformed 3T3 cells. Growing 3T3 cells exposed to EGF also loose available EGF-receptors. In contrastmore » to confluent cells, dividing 3T3 cells rapidly replace EGF-receptors on the surface of the cell, in the presence of EGF.« less

Calcium stimulation of plasminogen activator secretion/production by swiss 3T3 cells.

Actively growing Swiss 3T3 cells secret high levels of plasminogen activator which decreases after the cells become confluent. In contrast, simian virus 40-transformed 3T3 cells secrete large amounts of plasminogen activator independent of cell density (Chou, I.-N., O'Donnel, S.P., Black, P.H., and Roblin, R.O. (1977) J. Cell. Physiol. 91, 31-38). These results suggest a correlation between active cell multiplication and plasminogen activator secretion in both 3T3 and simian virus-transformed 3T3 cells. The data reported herein indicate that treatment of both subconfluent and confluent Swiss 3T3 cells with high concentrations of Ca2+ (final 3.0 to 4.9 mM) increases the amounts of both secreted and cell-associated plasminogen activator in a dose-dependent manner. In addition, the ionophore A23187 (19 to 95 nM) in the presence of a normal level of Ca2+ (1.8 mM) stimulates both production and secretion of plasminogen activator from growing 3T3 cells. The Ca2+ stimulation of plasminogen activator production/secretion may be related to the mitogenic effect of Ca2+.

Effect of exogenous ATP on the permeability properties of transformed cultures of mouse cell lines.

HEPPEL,~ AND ILAN FRIEDBERG§ From the Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, England External ATP produces an extensive change in the mem- brane permeability of transformed cultured mouse cells, leading to a depletion of intracellular pools labeled with 13Hluridine, L3H1adenosine, ““Rb, or Z-deoxy[“HIglucose. For example, when 3T6 (a cell line derived from 3T3 by spontaneous transformation), SV3T3 (3T3 cells transformed by simian vacuolating virus 40), and PY3T3 (3T3 cells trans- formed by polyoma virus) cells are exposed to as little as 0.2 mM ATP in buffered saline medium for several minutes, there results a 20- to 30-fold increase in rate of efflux of the acid-soluble uridine nucleotide pool. A much smaller stimu- lation (from 0- to 3-fold) is seen with resting or growing 3T3 cells or with secondary cultures of mouse embryo fibro- blasts. The increase in permeability produced by ATP is highly specific; it was not obtained with GTP, CTP, UTP, ADP, inorganic phosphate, and various chelating agents. The effect of ATP in increasing permeability is pa-depend- ent and occurs much faster at 37.5” than at 20”. Stimulation of efflux persists after removal of ATP and the enhanced rate of efflux is the same at 20” as at 37.5”. The effect of ATP is seen in various buffered saline media. When the solutions are removed and replaced with Dul- becco’s modified Eagle’s serum-free medium (Smith, J. D., Freeman, G., Vogt, D., and Dulbecco, R. (1960) Virology 12, 185-196), the increased rate of efflux is quickly suppressed. The presence of bicarbonate, divalent cations, and a more neutral pH all contribute to restore a normal permeability barrier. Upon addition of serum, the acid-soluble pools are restored in 1 h and the process can be repeated. This treat- ment with ATP does not affect the subsequent appearance of the cultured cells or their rate of growth.

Inhibition of DNA synthesis in cultures of 3T3 cells by isolated surface membranes.

When added to a sparse culture of 3T3 cells, a surface membrane-enriched fraction from 3T3 cells inhibited the rate of DNA synthesis in a time- and concentration-dependent manner. The membrane preparation had no effect on the rate of DNA synthesis of simian virus 40-transformed 3T3 cells. A similar membrane preparation from transformed cells had a lesser inhibitory effect on 3T3 cells and no effect on transformed cells. The inhibition by membranes was reversible. The data suggest that, when added to growing 3T3 cells, 3T3 surface membranes can mimic the effect of increasing cell density on DNA synthesis.