Difference in transport of leucine in attached and suspended 3T3 cells.

3T3 cells in subconfluent culture take up leucine through a transport system which has a relatively high affinity for leucine (M system). When the culture becomes confluent, the M system is turned off and leucine is transported by another system which has a low affinity for leucine (S system). The M system is reactivated by transferring the cells into subconfluent cultures. In suspension cultures 3T3 cells, initiated from confluent cultures, the M system is not activated and leucine is transported by the S system. In cells suspended from subconfluent culture, the M system continues to operate at a high level for four hours and then is gradually turned off. Tumor virus transformed 3T3 cells (SV3T3 and Py3T3) grow quite well in suspension culture and transport leucine both in monolayer and suspension through a high affinity system, with a high Vmax value. A derivative of 3T3, 3T3/41, which grows in suspension much more slowly than tumor virus transformed 3T3 cells, also takes up leucine through a high affinity transport system both in monolayer and suspension but its Vmax value is lower than that of the transformed cells.     

Enhancement of transformed cell growth in agar by serine protease inhibitors

We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum-free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV-REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3-Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3-Aga cells while having little effect on PyMLV-REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum-free monolayer growth. Under conditions of excess aprotinin, PyMLV-REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor-beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor.     

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.     

A preparative suspension culture system permitting quantitation of anchorage-independent growth by direct radiolabeling of cellular DNA

We have developed a hybrid methylcellulose/agar suspension culture system which permits long-term colony formation of transformed mesenchymal cells. In contrast to traditional agar suspensions, our system allows for recovery of cells and direct biochemical analysis of anchorage-independent growth. The ability to readily radiolabel cellular macromolecules in these preparative cultures permits a quantitative and objective analysis of colony formation by incorporation of [3H]thymidine into newly synthesized DNA.     

Serum protease inhibitors promote anchorage-independent growth of transformed cells in serum-free medium.

The effect of three serum serine protease inhibitors on the serum-free agar growth of an SV40-transformed 3T3 cell line was investigated. Antithrombin III, alpha-2-macroglobulin and alpha-1-antitrypsin were found to potently stimulate colony growth in a semisolid medium because of their anti-proteolytic properties. These results indicate that protease inhibitors can facilitate tumor cell growth in serum-free agar cultures and suggest that the stimulatory effect of serum on the growth of certain transformed cells in agar may at least partially be due to the high levels of protease inhibitors found in serum.     

Control of rat C6 glioma cell proliferation: uncoupling of the inhibitory effects of hydrocortisone hormone in suspension and monolayer cultures

We undertook a comparative study of the effects of the hormone hydrocortisone (Hy) on C6 glioma cells grown in monolayer and in suspension in cultures. We found Hy reversibly renders C6 cells anchorage- and serum-dependent for their growth. In monolayer cultures, Hy was found to inhibit cell cycle traversing exclusively at G1 phase. In agarose suspension, Hy was found to block colony development. Hy-resistant variants were selected and isolated in agarose suspension. Examination of these variants showed that cells selected for Hy-resistance in suspension can be Hy sensitive when anchored to a solid substrate. We conclude that resistance to Hy in suspension and resistance to it in monolayer culture are two independent phenotypes.     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.     

Similarities between fibroblast-derived growth factor and platelet-derived growth factor

Platelet-derived growth factor (PDGF), one of the most potent mitogens in serum for non-transformed cells, shares many biological and physical properties with fibroblast-derived growth factor (FDGF), a polypeptide produced by BHK cells transformed by SV40. Thus FDGF and PDGF have biological activity which is recoverable from sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, at positions indicating similar molecular weights. Further, the biological activity of both factors is heat-stable but sensitive to mercaptoethanol. FDGF and PDGF have similar abilities to induce DNA synthesis synergistically in the presence of either insulin, epidermal growth factor (EGF), vasopressin or colchicine. In contrast to other growth factors, (i) either FDGF or PDGF can induce DNA synthesis in the absence of other mitogens in 3T3 cells maintained in serum-free medium and (ii) a transient exposure of cultures to FDGF or PDGF causes a persistent stimulation of DNA synthesis. Either FDGF or PDGF enhances colony formation of non-transformed cells cultured in suspension in the presence of EGF and serum. FDGF is not PDGF adsorbed by SV40-BHK cells from serum, since SV40-BHK cells plated and grown in the absence of serum still produce FDGF. In view of the similarities between PDGF and FDGF, we suggest that they may belong to the same family of growth factors.     

Morphological transformation, autonomous proliferation and colony formation by chicken heart mesenchymal cells infected with avian sarcoma, erythroblastosis and myelocytomatosis viruses

Normal chicken heart mesenchymal cells at low density in monolayer culture in plasma-containing medium have a polygonal shape and are proliferatively quiescent. The combination of epidermal growth factor and insulin at hyperphysiological concentration, an insulin-like growth factor surrogate, causes these cells to assume a fusiform shape and to increase 40-fold in number during four days of incubation. These mitogenic hormones do not, however, induce normal chicken heart mesenchymal cells to form colonies in agarose suspension culture. Chicken heart mesenchymal cells infected with the Schmidt-Ruppin or Prague-A strains of Rous sarcoma virus or with the Fujinami or Y73 avian sarcoma viruses assume spindle and round shapes, increase 50-100 fold in number during four days of monolayer culture in the absence of mitogenic hormones and form macroscopic colonies during 3-4 days of agarose suspension culture. The autonomous (mitogenic hormone-independent) proliferation, in monolayer culture, of cells infected with temperature-sensitive transformation mutants of Rous sarcoma virus (tsNY68, tsNY72, tsLA24, tsLA29) is temperature-sensitive. Chicken heart mesenchymal cells infected with avian erythroblastosis virus assume spindle shapes and proliferate in monolayer culture at a rate comparable to that of sarcoma virus-infected cells but do not, however, form colonies in agarose suspension culture. Cells infected with the myelocytomatosis virus MC29 assume stellate shapes and increase 18-fold in number during four days of monolayer culture. Cells infected with the myelocytomatosis virus MH2 assume fusiform shapes and increase fourfold in number during four days of monolayer culture. Neither MC29 nor MH2 renders chicken heart mesenchymal cells capable of colony formation in agarose suspension culture. Infection with avian leukosis viruses (RAV-1, RAV-2, RPL-42) or with transformation-defective mutants of Rous sarcoma virus (tdNY105, 107, 109) does not affect the morphology or proliferative behavior of chicken heart mesenchymal cells. Monolayer culture of chicken heart mesenchymal cells in plasma-containing medium appears, therefore, to define the ability of onc genes of acute transforming avian retroviruses to induce autonomous (mitogenic hormone-independent) cell proliferation, the essential characteristic of neoplasia. The differences in transformed morphology and rates of autonomous proliferation between cells infected with different acute transforming retroviruses probably reflects differences in the modes of action of the transforming proteins encoded by the onc genes of the respective viruses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.     

Isolation of clones of hamster embryo cells transformed by the bovine papilloma virus

Primary hamster embryo cells infected with bovine papilloma virus (BPV) or treated with BPV DNA-calcium phosphate precipitates showed striking morphological alterations characteristic of transformed cells. Long, spindle-shaped cells grew into dense foci, eventually overgrowing monolayers of normally shaped cells. Samples of these cells were tested for anchorage-independent growth in dilute agarose medium. Cells were able to grow in agarose to form colonies which, when removed from agarose and transferred to liquid medium, established clones. Mockinfected cultures inoculated with plain medium displayed normal cell morphology and growth properties. This is the first report of BPV-transformed cells demonstrating anchorage-independent growth in agarose and the establishment of BPV transformed clones.     

Characterization of a serum-free culture system comparing growth factor requirements of transformed and untransformed cells

We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [3H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF a or b, or TGF-beta 1) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-beta 1 (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-beta 1 and FGF a or b were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state.     

Studies of long-range density effects on the proliferation of 3T3 and RLCW cells in recirculated medium.

Swiss mouse 3T3 cells and rat liver-derived RLCW cells were grown in monolayers and perfused with culture medium. A flow-rate dependent increase in the growth rate was observed both by 3H-thymidine uptake and by a rise in cell numbers. The characteristics of the response were dependent on the recirculating volume and on whether serum was present in the culture medium. In RLC cultures perfused with serum-supplemented medium the growth promoting effect decreased with increasing density of the cells. In the absence of serum, recirculation of NCTC medium had no effect on RLCs but increased growth was observed in recirculated MEM. In 3T3 cultures, a linear response was observed over a limited density range in the presence of 10% serum-supplemented medium indicating that substances present in the serum substantially modify the behaviour of the monolayer to perfusion. In serum-free medium the effect of perfusion on 3T3 cultures was confined to a small density range and was consistent with the more rapid removal of a diffusible inhibitor from the pericellular environment by recirculating the medium. Treatment of the perfusing medium with immobilised proteinases (trypsin, chymotrypsin, protease) did not alter the response except in the presence of putrescine.     

Overexpression of insulin receptors in fibroblast and ovary cells induces a ligand-mediated transformed phenotype.

To investigate whether overexpression of the insulin receptor results in altered cell growth we used NIH 3T3 cells transfected with a bovine papilloma virus/insulin receptor cDNA construct (3T3/HIR). These cells expressed high numbers of insulin receptors (mean +/- sd, 631.0 +/- 16.7 ng receptors/10(6) cells). Insulin significantly stimulated the growth of 3T3/HIR cells maintained in serum-free medium. Moreover, in these cells, insulin induced marked phenotypic changes, including alterations in cell shape, loss of contact inhibition, and focal growth. In contrast to 3T3/HIR cells, insulin was without effect in either wild-type 3T3 cells (3T3/wt), 3T3 cells transfected with the neomycin resistance gene (3T3/NEO), or the bovine papilloma virus (3T3/BPV). To assess the presence of anchorage-independent growth, cells were seeded in soft agar and inspected for colony formation. 3T3/HIR cells showed absent or minimal colony growth in the absence of insulin. However, there was a dose-dependent insulin-stimulated increase in both colony size and number. Insulin-stimulated colony formation was specifically inhibited by an insulin antagonist, monoclonal antibody MA-10. In the presence of 100 nM insulin, about 3% of cells formed large colonies. Insulin neither stimulated growth nor induced colony formation in 3T3/wt cells or 3T3/NEO cells. Insulin also stimulated colony formation in CHO cells transfected with an insulin receptor cDNA construct. In conclusion, overexpression of normal insulin receptors induces a ligand-dependent transformed phenotype. This phenomenon may have clinical relevance by conferring a selective growth advantage to tumor cells with high numbers of insulin receptors.     

Arrest of 3T3 cells in G1 phase in suspension culture

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

Arrest of 3T3 cells in G1 phase in suspension culture.

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

The effect of recombinant erythropoietin on murine megakaryocyte colony formation

We investigated the effect of a recombinant human erythropoietin preparation (recombinant Epo) on murine megakaryocyte (MK) colony formation in serum-free and serum-containing culture systems, in order to study the relationship between Epo and megakaryopoiesis. Pokeweed mitogen spleen-conditioned medium (PWM-SCM), a standard source of MK colony stimulator, dose-dependently stimulated MK colony formation in the two culture systems. The plating efficiency of serum-free cultures was almost equal to that of cultures containing serum. Recombinant Epo also dose dependently stimulated MK colony formation in serum-containing cultures. However, in serum-free cultures recombinant Epo alone did not stimulate the growth of MK colonies; with the addition of fetal calf serum (FCS) to the serum-free cultures, recombinant Epo induced the growth of MK colonies. Furthermore, recombinant Epo enhanced MK colony formation through the stimulation of PWM-SCM or murine interleukin 3 (IL-3) in serum-free cultures. Our data show that Epo can act as a stimulator of megakaryopoiesis in collaboration with a factor in serum, or with an MK colony stimulator such as IL-3.     

Studies of Long-Range Density Effects on the Proliferation of 3T3 and RLCW Cells in Recirculated Medium

Swiss mouse 3T3 cells and rat liver-derived RLCW cells were grown in monolayers and perfused with culture medium. A flow-rate dependent increase in the growth rate was observed both by ³H-thymidine uptake and by a rise in cell numbers. The characteristics of the response were dependent on the recirculating volume and on whether serum was present in the culture medium. In RLC cultures perfused with serum-supplemented medium the growth promoting effect decreased with increasing density of the cells. In the absence of serum, recirculation of NCTC medium had no effect on RLCs but increased growth was observed in recirculated MEM. In 3T3 cultures, a linear response was observed over a limited density range in the presence of 10% serum-supplemented medium indicating that substances present in the serum substantially modify the behaviour of the monolayer to perfusion. In serum-free medium the effect of perfusion on 3T3 cultures was confined to a small density range and was consistent with the more rapid removal of a diffusible inhibitor from the pericellular environment by recirculating the medium. Treatment of the perfusing medium with immobilised proteinases (trypsin, chymotrypsin, protease) did not alter the response except in the presence of putrescine.     

Behavior of transforming growth factors in serum-free media: An improved assay for transforming growth factors

Summary Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-β alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-β are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity. Editor's Statement This communication describes a modification of the standard assay for transforming growth factors. The techniques employed make use of advantages provided by recent advances in serum-free cell culture to provide a well-defined detection system that is more sensitive than conventional procedures. Experimental approaches described in this article also should be helpful in unraveling differences in cellular behavior encountered under anchorage-dependent vs. anchorage-independent conditions. D. W. Barnes