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.     

Binding, internalization, and degradation of epidermal growth factor by balb 3T3 and BP3T3 cells: relationship to cell density and the stimulation of cell proliferation.

Epidermal growth factor (EGF) stimulates the growth of both benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) and untransformed Balb 3T3 cells. We describe here the binding, internalization, and degradation of [125I]-EGF by BP3T3 cells and 3T3 cells. Binding of [125I]-EGF reaches a maximum after 45 to 90 minutes incubation at 37 degrees C. In both BP3T3 and 3T3 cells the extent of EGF binding required to stimulate DNA synthesis is density dependent; sparse cultures require a 15-30% occupancy to elicit a maximal response whereas dense cultures require a 70-85% occupancy. At physiological concentrations the total binding of [125I]-EGF to 3T3 cells is higher than to BP3T3 cells, and this difference increases at higher cell densities. The rate of degradation of [125I]-EGF is directly proportional to the total [125I]-EGF binding in each cell type. This supports the hypothesis that one cause of the diminished serum requirement of BP3T3 cells is a reduced rate of utilization of serum growth factors.     

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.     

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells

Summary We have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50–75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.     

The expression of ceruloplasmin, an angiogenic glycoprotein, by mouse embryonic fibroblasts

Balb/c 3T3, Swiss 3T3 and Rous sarcoma virus transformed Balb/c 3T3 mouse embryonic fibroblasts produced ceruloplasmin in vitro, whereas primary cultures prepared from the Balb/c mouse embryos did not produce ceruloplasmin. The amount of ceruloplasmin synthesis by the Balb/c 3T3 cell line is enhanced by Rous sarcoma virus-transformation (1.5-3 fold) and by treatment with dexamethasone (about 2.4 fold). The protein was identified as ceruloplasmin by immunoprecipitation with ceruloplasmin-specific polyclonal antibody, and by similarity of peptide maps, and subunit molecular weight (135,000 dalton) to that of authentic ceruloplasmin from primary cultures of mouse hepatocytes.     

Distinct proteolytic mechanisms in serum-sufficient and serum-restricted fibroblasts. Transformed 3T3 cells fail to regulate proteolysis in relation to culture density only during serum-sufficiency.

Thymidine incorporation (reflecting cell division), degradation of long-half-life proteins and protein synthesis were compared in normal Swiss mouse 3T3 fibroblasts and their counterparts transformed by simian virus 40 at both high and low culture densities (no. of cells/cm2). Normal cells maintained faster proteolysis at high culture density than at low. Degradation was in all conditions enhanced by serum deprivation (1% serum). In serum-sufficient (10%) conditions, there was an inverse correlation between degradation and cell division, but in serum-restricted conditions proteolysis increased substantially as culture density was increased, without change in cell division. Protein synthesis generally changed in a converse sense to protein degradation. In serum-sufficient conditions, transformed 3T3 cells failed to regulate proteolysis in response to culture density. However, in serum-restricted conditions they can regulate proteolysis as do normal cells. Transformed 3T3 cells regulate protein synthesis and thymidine incorporation very poorly in response to culture density in both conditions studied. The failure of regulation of both protein synthesis and degradation may contribute to the exaggerated growth of transformed cells in serum-sufficient conditions. The retention by such cells of regulation of proteolysis during serum restriction may also aid their survival. Studies with several lysosomotropic agents indicated that lysosomes contribute to proteolysis in all conditions studied, but also that its regulation in serum restriction is distinct from that in serum sufficiency, and may involve primarily a non-lysosomal mechanism.     

Intracellular degradation of hemoglobin transferred into fibroblasts by fusion with red blood cells

Hamster fibroblast protein and rabbit hemoglobin were labelled by incubation of fibroblasts (BHK21) or reticulocytes with [³H]leucine. Alternatively, human or rabbit hemoglobin was labelled by carbamoylation of erythrocytes with K¹⁴CNO. The labelled hemoglobins were introduced into fibroblasts by virus-mediated fusion between the blood cells and fibroblasts. The hemoglobins became uniformly distributed throughout the cytoplasm. Degradation was assessed from release of acid-soluble radioactivity into the medium. Radioactivity from [¹⁴C]-carbamoylhemoglobin was released as carbamoylvaline and homocitrulline, and these compounds were not metabolized or reincorporated by the cells. Intermediate degradation products could not be detected. The degradation of hemoglobin followed first-order kinetics. The half-life of both carbamoylated and native rabbit hemoglobin in hamster fibroblasts was 28 h, and the half-life of carbamoylated human hemoglobin was about 150 h in fibroblasts from hamster (BHK21), mouse (Balb/3T3), and man (MRC 5), corresponding to that of the more stable endogenous proteins. Phenylhydrazine increased the intracellular degradation of carbamoylated human hemoglobin about 13 times, whereas the degradation of endogenous proteins was little affected. Hemoglobin was degraded in homogenates at 31% h^?1 at pH 5 and 0.3% h^?1 at pH 7.4. Phenylhydrazine increased these rates to 45% h^?1 and 9.7% h^?1, respectively. Growing hamster fibroblasts, which are brought into quiescence by serum deprivation or by high culture density, increase the degradation of endogenous protein and of hemoglobin in parallel.     

Intracellular protein degradation in serum-deprived human fibroblasts.

IMR90 human fibroblasts were labelled by incubation of cells for 48 h in medium containing 10% serum and [3H]leucine. The labelled protein was degraded at a rate of 1%/h during a subsequent incubation in medium with 10% serum. Incubation in medium without serum caused a transient enhancement of the degradation of endogenous protein, which was also found in cells labelled in medium without serum. The degradation of micro-injected haemoglobin was enhanced by serum deprivation in a non-transient manner. These results suggest that enhanced degradation in serum-free medium occurs only for a subpopulation of cell proteins and that it appears transient because the major part of the pool of susceptible endogenous proteins is being degraded during the first 20-30 h in serum-free unlabelled medium. Protein turnover in various cell compartments was measured by a double-labelling technique. Most of the enhanced degradation in serum-deprived cultures (73-83%) was due to breakdown of cytosolic proteins. The enhanced degradation of cytosolic proteins seemed to affect several proteins irrespective of their molecular mass or metabolic stability.     

Density Dependent Change of Myristoylated Proteins in C3H10T1/2 Fibroblasts and Their Transformants

We have examined the pattern of protein myristoylation in C3H10T1/2 fibroblasts during cell growth. During the growing phase of 10T1/2 cells, several proteins were radiolabelled with [³H]myristate, and among them proteins with molecular masses of 22, 35, a doublet of 42–45 and 67 kDa were labelled predominantly. The extent of myristoylation in each of these proteins changed with cell density. The amount of radioactivity incorporated into the 22 kDa protein in 10T1/2 cells decreased with increasing cell density and remained at a low level during the stationary phase. In contrast, the incorporation into the 67 kDa protein increased parallel to cell density. The density-dependent change of myristoylation was not observed in any of the transformants of 10T1/2 cells thus far examined. The 67 kDa protein was identified as MARCKS (myristoylated alanine-rich C kinase substrate) by immunoprecipitation with an anti-MARCKS antibody. By Western blot analysis, we found that the amount of MARCKS in 10T1/2 cells increased significantly analogous with cell density. Therefore, it is possible that MARCKS and the 22 kDa protein play a role in contact-mediated cell signalling in 10T1/2 cells, but the mechanism is lost in transformed cells. © 1997 John Wiley & Sons, Ltd.     

Platelet-derived growth factor regulation of fos stability correlates with growth induction.

A brief exposure of quiescent, density-arrested Balb/c 3T3 fibroblasts to platelet-derived growth factor (PDGF) results in expression of the proto-oncogene c-fos; furthermore, the translation product of c-fos, p55fos, was shown to have increased stability in cells upon continued exposure to PDGF. The induction of competence or growth initiation requires a longer exposure to PDGF than that necessary for the induction of the immediate-early, growth-related genes. The need for the continued presence of PDGF for growth initiation beyond the time required for the induction of immediate-early gene expression may be due, in part, to PDGF-dependent post-translational stabilizations of gene products. We speculate that a PDGF-mediated event increases p55fos stability, resulting in a continued elevated level of Fos protein, which in turn allows a continued Fos-mediated activity required by Balb/c 3T3 cells to become competent to enter the cell cycle.     

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.     

Protein turnover in senescent cultured chick embryo fibroblasts

The over-all rates of protein synthesis, degradation and net accumulation were estimated in rapidly growing young and slowly doubling old cultures of chick fibroblasts. We find that not only the rate of protein synthesis is reduced in senescent cultures, but the average rate of protein degradation is also slowed down considerably. This decrease in the rate of protein breakdown in aging cells stands in contrast with the previously observed acceleration of this process by other conditions (such as serum deprivation or overcrowding) that lead to the cessation of cellular growth. Though the retarded protein degradation may contribute to the acculation of abnormal proteins in senescent cells we find that the breakdown of grossly abnormal puromycin peptides proceeds equally rapidly in young and old cultures. The protein content of senescent cells increases by 1.8-fold as compared to young cells, while the average cell volume is increased even more (almost 5-fold). By contrast, consideration of the over-all balance of protein metabolism in these cells indicates that the average concentration of metabolically turning-over proteins is somewhat higher in senescent than in young fibroblasts.     

Protein degradation in skin fibroblasts from patients with Duchenne muscular dystrophy.

The rates of degradation of [3H]leucine-labelled proteins have been measured in cultures of skin fibroblasts obtained from normal controls (five subjects) and patients with Duchenne muscular dystrophy (six subjects). Cultures were incubated with [3H]leucine (10 microCi/ml) for 60 min to label "short-lived" proteins, and with [3H]leucine (5 microCi/ml) for 60 h to label "long-lived" proteins. Optimal wash procedures were devised for removal of [3H]leucine from the extracellular space and from cell pools before beginning degradation measurements. Re-utilization of [3H]leucine released from degraded labelled proteins was prevented by supplementing the medium with 4mM-leucine. Rates of degradation did not depend on the growth state of the cells or on cell age over the range used (passages eight-20). Degradation of long-lived proteins was approximately linear over a 24h period, at a rate of 1.0% per h. 30% of short-lived protein was degraded within 6h. No differences were observed between protein degradation in normal fibroblasts and in those from patients with Duchenne muscular dystrophy.     

Cell-cell contact and growth regulation of pinocytosis in 3T3 cells

In sub-confluent cultures of Balb/c-3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet-derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth-inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density-inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40-viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density-dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth-inhibition rather than to any direct physical effects of cell–cell contact.     

Overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene induces cell proliferation and phenotypic transformation in mouse fibroblasts

Na(+)/K(+)/Cl(-) cotransporter activity is stimulated in early G(1) phase of the cell cycle and this stimulation was shown to be an essential event in fibroblast cell proliferation. In order to elucidate further the role of the Na(+)/K(+)/Cl(-) cotransporter in cell proliferation, we overexpressed the gene encoding the Na(+)/K(+)/Cl(-) cotransporter in mouse fibroblasts, and analyzed cellular phenotypic changes. Mouse Balb/c 3T3 cells were stably transfected with the cDNA of the shark rectal gland Na(+)/K(+)/Cl(-) cotransporter gene (NKCC1), and expressed in a mammalian vector under the cytomegalovirus promoter (Balb/c-NKCC1 cells). The transfected cells exhibited up to 10-fold greater bumetanide-sensitive Rb(+) influx compared to the control cells. The Balb/c-NKCC1 cells have acquired a typical transformation phenotype indicated by: (1) Loss of contact inhibition exhibited by growth to a higher cell density in confluent cultures, and formation of cell foci; (2) proliferation in low serum concentrations; and (3) formation of cell colonies in soft agar. The control cells transfected with the NKCC1 gene inserted in the opposite orientation in the vector retained their normal phenotype. Furthermore, the two specific inhibitors of the Na(+)/K(+)/Cl(-) cotransporter activity; bumetanide and furosemide inhibited the clonogenic efficiency in the NKCC1 transfected cells. These control experiments indicate that the apparent transformation phenotype acquired by the Balb/c-NKCC1 cells was not merely associated with the process of transfection and selecting for the neomycin-resistant clones, but rather with the overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene. In order to ascertain that the regulated and normal expression of the Na(+)/K(+)/Cl(-) cotransporter control cell proliferation, the effect of bumetanide a specific inhibitor of the cotransporter, was tested on Balb/c 3T3 cell proliferation, induced by fibroblasts growth factor (FGF) and fetal calf serum (FCS). Bumetanide inhibited synchronized Balb/c 3T3 cell exit from the G(0)/G(1) arrest and entering S-phase. The inhibition was reversible, as removal of bumetanide completely released cell proliferation. Taken together, these results propose that the NKCC1 gene is involved in the control of normal cell proliferation, while its overexpression results in apparent cell transformation, in a manner similar to some protooncogenes.     

Cell density downregulates DNA synthesis and proliferation during osteogenesis in vitro

Abstract. The classical models of in vitro cell culture comprise fibroblasts and epithelial cells. Osteogenic cells represent another interesting cell model; however, it is not known whether during osteogenesis cell density regulates cell growth as seen in cultures of fibroblasts and epithelial cells. We selected MC3T3-E1 cells for study because they are an osteogenic cell line that, when subcultured, grow to confluence and form multilayers of cells in conventional cultures by continued proliferation, as do fibroblasts. Once maximum cell density is obtained, proliferation is down regulated resulting in a mixed population of quiescent and dividing cells. We used this model to determine whether downregulation of proliferation as expressed by cell number and DNA synthesis is cell density-dependent. MC3T3-E1 cells were cultured over a period of 34 days to determine their kinetics, viability, ability to synthesize DNA, distribution within phases of the cell cycle and cell number-response relationships. Our results show that (1) viability ranged between 92% and 96% and the cell number 2.5 x 10⁵ per cm² once cultures reached steady state, (2) most cells entered the G₀/G₁ phase of the cell cycle on day 7, (3) there was no correlation between the proportion of cells in S phase and downregulation of DNA synthesis, (4) a direct relationship exists between cell density and downregulation of DNA synthesis on day 8, (5) the minimum time for cells to be cultured before downregulation of DNA synthesis begins is independent of cell number, and (6) downregulation of DNA synthesis is reversible. These results suggest that density-dependent downregulation of DNA synthesis may be a mechanism of growth control for osteogenic cells in vitro that operates more like density-dependent growth control in cultures of fibroblasts rather than epithelial cells.     

Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

Partial purification of a high molecular weight hepatocyte growth stimulating factor from normal calf serum.

A heparin-binding protein, acting as a potent hepatocyte growth stimulating factor (HGSF) was extracted and partially purified from normal calf serum. HGSF stimulated DNA synthesis and proliferation in primary cultures of adult Balb/c mouse hepatocytes and in two liver-derived epithelial cell lines (C6 and C2.8) plated at low cell density in serum-free medium in the absence of epidermal growth factor. HGSF was non-dialyzable in M(r) 50,000 cutoff membranes, and was purified after chromatofocusing on PBE94 resin, (NH4)2SO4 precipitation (80% salt concentration) of the active fractions eluted at pH 5.7, flow chromatography and elution through Sephacryl S300 HR and HA-Ultrogel columns. The hepatotrophic activity was eluted with a protein fraction that was concentrated approximately 40,000 fold over the starting material. The effect was half maximal at approximately 50 ng/ml on adult hepatocytes in primary culture, HGSF had a molecular weight of 90,000-110,000 by gel filtration, was unstable on heat-treatment and was completely inhibited after trypsin digestion and after reduction with dithiothreitol. HGSF did not stimulate growth in Balb/c 3T3 fibroblasts. When injected into partially (40%) hepatectomized Balb/c mice, HGSF increased hepatic DNA synthesis 2 to 4-fold over the background stimulation, at 20 hours after the hepatectomy.