Serum Factor Requirements of Normal and Simian Virus 40-Transformed 3T3 Mouse Fibroblasts

Evidence is presented to show the presence in normal rat serum of four different serum factors essential for growth of 3T3 or SV40-transformed 3T3 mouse fibroblasts: a factor that specifically promotes growth of normal 3T3 cells; two factors that specifically promote growth of transformed 3T3 cells; and a factor that sustains viability of both normal and transformed 3T3 cells in serum-free medium, probably without inducing growth of the cells. These factors are separated and partially purified.     

Control of growth of benzo(a)pyrene-transformed 3T3 cells.

The growth controls observed in benzo[a]pyrene-transformed 3T3 cells (BP3T3) are compared with those of virus-transformed and normal 3T3 cells. Superficially, the chemically transformed BP3T3 cells have the same behavior as virus-transformed SV3T3 cells. Both grow to high cell density in culture medium with 10% serum, both form colonies in Methocel, and both are tumorigenic. Closer examination, however, has disclosed that BP3T3 cells exhibit "normal" growth controls at low serum concentrations. In contrast to the behavior of SV3T3 cells, the initiation of DNA synthesis in BP3T3 cells is still dependent on a serum factor. If BP3T3 cells are grown in medium with 0.2% serum, the cells become quiescent, with growth arrested in the Gu or G0 phase of the cell cycle. The addition of serum or the fibroblast growth factor (FGF) to such quiescent cells leads to the initiation of DNA synthesis and the resumption of growth. As with normal 3T3 cells, if the growth rate of BP3T3 cells is limited by a suboptimal concentration of serum, the growth rate of the cells is increased by the addition of FGF. Also, BP3T3 cells show density-dependent regulation of growth, if the medium contains a low concentration of serum. BP3T3 cells, therefore, have the behavior of "transformed" cells when cultured in medium with 10% serum, but behave as "normal" cells in medium with low serum. In comparison with normal 3T3 cells, the difference in growth behavior of BP3T3 cells appears to be due to a substantial decrease in the cells' requirement for a serum growth factor of the FGF type. Exploration of possible causes of this substantial decrease indicates that the primary cause is a lower rate of depletion of the serum growth factor from the culture medium by BP3T3 cells. The decrease in rate of depletion is sufficient to account for the uncontrolled growth of BP3T3 cells in medium with 10% serum. It is suggested that a decreased rate of depletion of a growth factor may contribute to tumorigenicity of cells in vivo.     

Effect of a Fibroblast Growth Factor, Insulin, Dexamethasone, and Serum on the Morphology of BALB/c 3T3 Cells

The effects of serum, fibroblast growth factor, dexamethasone, and insulin on the morphology of two lines of BALB/c 3T3 cells are described and illustrated. Fibroblast growth factor, a polypeptide purified from bovine brain and pituitary glands, stimulates DNA synthesis and cell division in both sparse and confluent cultures of quiescent 3T3 cells. When cells are grown in the presence of the factor, they go through one or two cycles of division and their morphology differs from that of cells either growing in 10% serum or maintained in a quiescent state in low serum; they appear rounded with long processes and show no sign of contact inhibition. When dexamethasone is present with the growth factor, the cells grow to a high density and are not contact-inhibited; they have the appearance of transformed cells. Addition of insulin or dibutyryl cyclic AMP to the cells results in a morphology similar to that of cells growing in the presence of serum. Addition of growth factor or growth factor plus dexamethasone to cells that have reached confluency in 10% serum and that are clearly contact-inhibited results in the resumption of growth. The final cell density reached is similar to that of cells growing in the presence of 30% serum with daily fluid changes. No contact inhibition is observed in either of these situations.     

The human transforming growth factor type alpha coding sequence is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.

A peptide secreted by some tumor cells in vitro imparts anchorage-independent growth to normal rat kidney (NRK) cells and has been termed transforming growth factor type alpha (TGF-alpha). To directly investigate the transforming properties of this factor, the human sequence coding for TGF-alpha was placed under the control of either a metallothionein promoter or a retroviral long terminal repeat. These constructs failed to induce morphological transformation upon transfection of NIH 3T3 cells, whereas viral oncogenes encoding a truncated form of its cognate receptor, the EGF receptor, or another growth factor, sis/platelet-derived growth factor 2, efficiently induced transformed foci. When NIH 3T3 clonal sublines were selected by transfection of TGF-alpha expression vectors in the presence of a dominant selectable marker, they were shown to secrete large amounts of TGF-alpha into the medium, to have downregulated EGF receptors, and to be inhibited in growth by TGF-alpha monoclonal antibody. These results indicated that secreted TGF-alpha interacts with its receptor at a cell surface location. Single cell-derived TGF-alpha-expressing sublines grew to high saturation density in culture. However, when plated as single cells on contact-inhibited monolayers of NIH 3T3 cells, they failed to form colonies, whereas v-sis- and v-erbB-transfected cells formed transformed colonies under the same conditions. Moreover, TGF-alpha-expressing sublines were not tumorigenic in nude mice. These and other results imply that TGF-alpha exerts a growth-promoting effect on the entire NIH 3T3 cell population after secretion into the medium but little, if any, effect on the individual cell synthesizing this factor. It is concluded that the normal coding sequence for TGF-alpha is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.     

Transforming growth factor(s) production enables cells to grow in the absence of serum: an autocrine system.

Kirsten murine sarcoma virus (KiMSV)-transformed rat-1, normal rat kidney (NRK), and BALB/c 3T3 cells are capable of continual growth in a serum-free medium supplemented with transferrin and insulin but with no exogenous mitogenic growth factors. Cells transformed by a mutant of KiMSV that is temperature sensitive for the maintenance of transformation grow in this medium at the permissive temperature only. At the nonpermissive temperature, growth is dependent upon the presence of serum-free conditioned medium from the transformed cells. Normal rat-1 cells are also dependent upon factors from the transformed cells for growth in this serum-free/mitogen-free medium. The serum-derived growth factors, epidermal growth factor, and fibroblast growth factor have no effect on the transformed cells, although epidermal growth factor can replace transforming growth factors produced by KiMSV-transformed cells for the growth of rat-1 cells. Growth of the transformed cells in serum-free medium at clonal densities is dependent upon the presence of conditioned medium collected from the same cells grown to high densities. These results show that (i) growth in serum-free/mitogen-free medium is a general property of KiMSV-transformed cells and (ii) growth of the transformed cells in this medium is dependent upon the presence of growth factors known to be produced by the cells, and they provide support for the hypothesis that serum-free growth of KiMSV-transformed cells is dependent upon ectopically produced growth factors.     

Mouse NIH 3T3 cells expressing human colony-stimulating factor 1 (CSF-1) receptors overgrow in serum-free medium containing human CSF-1 as their only growth factor.

Mouse NIH 3T3 cells expressing the human c-fms protooncogene encoding the receptor for colony-stimulating factor 1 (CSF-1) are able to proliferate in serum-free medium containing platelet-derived growth factor (PDGF), insulin, transferrin, and albumin as the only exogenous proteins. When PDGF and insulin were replaced by purified human recombinant CSF-1, the cells became spindle shaped and refractile, were no longer contact inhibited, and proliferated to high densities. Thus, transduction of the human c-fms gene into mouse fibroblasts can not only reprogram their growth factor requirements but can also induce ligand-dependent features of cell transformation. NIH 3T3 cells stably transformed by the feline v-fms oncogene or by a mutated, oncogenic human c-fms gene were able to proliferate in the absence of exogenous growth factors. A monoclonal antibody that prevents signal transduction by the human CSF-1 receptor inhibited the growth of cells transformed by the activated c-fms oncogene, confirming that CSF-1 receptor function was required to abrogate growth factor requirements and to maintain the transformed state.     

Loss of epidermal growth factor requirement and malignant transformation

Serum provides growth factors that regulate and limit the growth of normal cells in tissue culture. Animal cells that are malignantly transformed usually exhibit diminished serum requirements for growth in culture. We have used a defined, serum-free medium to determine which of these growth factors becomes dispensable for the growth of transformed Syrian and Chinese hamster fibroblast cells. The medium's four growth factors-epidermal growth factor (EGF), insulin, fibroblast growth factor, and transferrin-were added or omitted as desired. A decreased requirement for EGF was most closely related to tumorigenicity of chemically (ethyl methanesulfonate) transformed cells in nude mice. All lines examined retained their requirement for transferrin, which is needed throughout the growth cycle, in contrast to the other factors, which are needed primarily in G(1) phase. Lines that had lost their EGF requirement but had retained their insulin requirement were arrested in G(1) by insulin deficiency, indicating that their growth control system remained. Mutagenesis with ethyl methanesulfonate can also create requirements of the transformed cells for unknown factors in serum. We conclude that an initial step that reduces the serum requirement in culture, and in tumorigenesis, is relaxation of the growth-regulatory function of EGF.     

Insulin and growth factor effects on c-fos expression in normal and protein kinase C-deficient 3T3-L1 fibroblasts and adipocytes.

We investigated the expression of the protooncogene c-fos in 3T3-L1 fibroblasts and adipocytes in response to a variety of growth-promoting agents in normal cells and in cells preincubated with phorbol esters to deplete them of protein kinase C. There was a rapid accumulation of c-fos mRNA in fibroblasts and adipocytes treated with phorbol 12-myristate 13-acetate, platelet-derived growth factor, fibroblast growth factor, fetal calf serum, bombesin, and insulin, especially in the adipocytes. Phorbol 12-myristate 13-acetate pretreatment abolished the increase in c-fos mRNA due to additional phorbol 12-myristate 13-acetate treatment and decreased but did not eliminate the ability of platelet-derived growth factor, fibroblast growth factor, fetal calf serum, bombesin, and insulin to stimulate c-fos mRNA. These data suggested that c-fos mRNA could be induced in serum-deprived 3T3-L1 fibroblasts and adipocytes by at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C. In the very insulin-sensitive 3T3-L1 adipocytes, insulin rapidly and transiently increased c-fos expression (c-fos mRNA appeared by 15 min and disappeared after 60 min) via interaction with its own cellular receptor, rather than by interacting with receptors for one of the insulin-like growth factors. Cycloheximide treatment in combination with insulin or phorbol 12-myristate 13-acetate resulted in superinduction of c-fos mRNA. We conclude that insulin can rapidly stimulate c-fos mRNA accumulation in 3T3-L1 adipocytes and that part of the growth factor-stimulated increase in this mRNA that occurs in protein kinase C-deficient cells may be due to activation of a pathway similar or identical to that activated by insulin.     

Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells.

The ras gene product (p21) is a GTP-binding protein and is thought to play an important role in signal transduction of growth and differentiation in many types of mammalian cells. The p21.GTP complex is an active conformation, as described previously for polypeptide chain elongation factors (EF-Tu and EF-G) and heterotrimeric GTP-binding proteins (G proteins). In the study reported here, we measured the amounts of p21-bound guanine nucleotides under various conditions in the G54 cell line, a derivative of Swiss 3T3 cells that overexpresses normal c-Ha-ras. More p21.GTP complexes were present in growing cells than in quiescent cells. When quiescent cells were stimulated with fetal bovine serum to promote DNA synthesis, p21.GTP increased approximately 2-fold. Among a number of purified growth factors, platelet-derived growth factor enhanced the formation of p21.GTP, whereas the combination of bombesin and insulin, which also induces DNA synthesis, did not. These results strongly suggest that p21 is a transducer of the growth signal from the platelet-derived growth factor receptor in Swiss 3T3 cells and that the signal is transmitted through a p21.GTP complex.     

Steroid hormones mediate reversible phenotypic transition between transformed and untransformed states in mouse fibroblasts.

Hydrocortisone at physiological concentrations reversibly inhibits DNA synthesis in ST1 cells (a line of mouse fibroblasts possessing 40 chromosomes and derived from Swiss 3T3 cells). This inhibitory activity is a property of glucocorticoids, but the beta-OH of C-11 of glucocorticoids is not essential for the inhibition. The steroid hormone restores to ST1 cells dependency on serum, density, and anchorage for growth. When injected into nude mice, ST1 cells generated malignant invasive fibrosarcoma. Injections of dexamethasone into tumor-bearing animals blocked tumor growth. The steroid hormone seems to induce a reversible transition between a transformed and a "normal" phenotype. ST1 cells treated or untreated with hydrocotisone are not responsive to fibroblast growth factor, epidermal growth factor, or prostaglandin F2alpha whereas they are responsive to a factor that is a contaminant in bovine serum albumin.     

Detection and Quantitation of Simian Virus 40 Genetic Material in Abortively Transformed BALB/3T3 Clones

Infection with simian virus 40 is known to induce many cells to synthesize DNA and to divide in a medium lacking serum protein growth factor(s) that is essential for growth of uninfected cells (factor-free medium). Cells infected under these conditions then go through several rounds of division, since colonies containing more than 100 cells are formed. Many of these colonies are abortively transformed since, upon subsequent passage of the cells in standard medium, they can no longer grow in factor-free medium and show no other properties of viral transformation. We have examined these abortively transformed cells for the presence of simian virus 40 DNA sequences. Of the three clones tested, two were found to contain viral genetic material despite the fact that they were phenotypically normal.The number of simian virus 40 genome equivalents present was determined by measurement of DNA reassociation kinetics on hydroxyapatite. Two of the abortively transformed lines contained approximately five viral genome equivalents per diploid cell, while the DNA from a third abortive transformant was indistinguishable from that of uninfected BALB/3T3 cells. A standard simian virus 40 transformant, isolated under similar conditions, contained two copies of the viral genome per cell. The abortive transformants also appear to contain the entire viral genome rather than multiple partial copies. Subclones of one abortively transformed line containing five copies per cell had 2.7-10 copies of viral genetic material per diploid cell.     

Potent stimulation of vascular endothelial cell growth by differentiated 3T3 adipocytes.

3T3 cells that have undergone adipose differentiation in vitro secrete into the culture medium a potent growth stimulatory activity for bovine aortic endothelial cells. When medium containing 2% fetal calf serum, which does not support significant endothelial cell growth, is conditioned by 3T3-F442A adipocytes, the endothelial cells grow rapidly (doubling time, 24 hr) at a rate equal to the growth rate in 20% fetal calf serum. The potency of the conditioned medium is further shown by the fact that it can be diluted 1:5 with little apparent loss of activity and shows a half-maximal stimulation at 10 microliter/ml. Serum is not required for either the secretion of this mitogen by the adipocytes or its action on the endothelial cells, as shown by the fact that the latter are stimulated to divide in serum-free medium conditioned by the adipocytes. The growth stimulatory activity appears to be specific for vascular endothelial cells in that no other cell type examined, including vascular smooth muscle cells and pericytes, are significantly stimulated by medium conditioned by 3T3-F442A cells. Similarly, medium conditioned by no other cell type examined has more than 10% of the activity of medium conditioned by the adipocytes. The specificity and potency of the adipocyte-derived factor suggest that it may play a role in the vascularization of this tissue during development. Preliminary biochemical analysis indicates that the adipocyte factor is nondialyzable and is not inactivated by heat or proteases. The protease insensitivity distinguishes the adipocyte growth stimulatory activity from the low levels of activity secreted by fibroblasts and preadipocytes, suggesting that the adipocyte mitogen is a product specifically related to the differentiation process.     

Migration of Mouse 3T3 Fibroblasts in Response to a Serum Factor

A serum factor that promotes migration of (mouse) 3T3 fibroblast cells is shown to be distinct from the growth-promoting and cell-survival factors of serum. The factor promotes migration of cells from the edge of a wound in confluent 3T3 cells, but cell migration under these conditions does not lead to the initiation of DNA synthesis. Subsequent addition of serum initiates DNA synthesis in the migrating cells. The results establish that breaking contacts between quiescent 3T3 cells is not sufficient to initiate DNA synthesis. The DNA synthesis observed in migrating 3T3 cells in the typical "woundhealing" experiment presumably results because the migrating cells have an increased ability to use serum factors. Serum-factor requirements for "wound healing" in cultured 3T3 cells are discussed.     

Selective Inhibition of Growth of Transformed Cells by Protease Inhibitors

Five protease inhibitors with different modes of action were found to reduce the growth of transformed mouse (Py3T3, SV3T3, and 3T12) and hamster (PyBHK) cells. Some of these inhibitors caused the transformed cells to cease growth at saturation densities characteristic for nontransformed cells.The protease inhibitors were strikingly selective with regard to the transformed cells; they had essentially no effect on the growth of the nontransformed cells. From this result, it is concluded that the inhibitors block a protease-like activity that is required for the unrestrained growth of transformed cells.The inhibitors exerted their effect directly on the cells; they did not affect growth by interacting with serum components of the medium.     

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3′-kinase in NIH 3T3 cells

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21^ras) and phosphatidylinositol kinase. p21^ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21^ras-bound GTP to p21^ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21^ras. Quiescent cells had dramatically reduced levels of activated p21^ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3′-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.     

Growth hormone and adipose differentiation: growth hormone-induced antimitogenic state in 3T3-F442A preadipose cells.

An additional activity for pituitary growth hormone is described--i.e., the in vitro induction of an antimitogenic state in murine 3T3-F442A preadipocyte fibroblasts. We previously developed a serum-free, hormonally defined medium permissive for the adipose differentiation of 3T3-F442A cells. When 3T3-F442A fibroblasts were maintained in serum-free medium without insulin but with growth hormone (2 nM), typical adipose differentiation did not occur. However, we found that growth hormone induced a state of cellular refractoriness to the mitogenic stimulus of fetal bovine serum as assayed by de novo DNA synthesis. The mitogen refractory condition (i.e., the antimitogenic state) was time-dependent (half maximal at approximately 2.5 days) and growth hormone concentration-dependent (half maximal and maximal at approximately 0.05 and 2.0 nM, respectively). The antimitogenic state was specifically induced by growth hormone and was not mediated by insulin-like growth factor I or prolactin. The growth hormone-induced antimitogenic state was completely reversible. The antimitogenic state was not induced by growth hormone in 3T3-C2 cells, a sister clone of 3T3 cells that exhibits essentially no adipose conversion. The kinetics for growth hormone-dependent commitment to adipose differentiation and induction of the antimitogenic state were similar. We suggest a relationship of growth hormone-induced antimitogenic state and the growth hormone-induced adipose differentiation of 3T3-F442A cells.     

Coordinate control of 3T3 cell proliferation by platelet-derived growth factor and plasma components.

DNA synthesis and cell division were measured in Swiss mouse 3T3 cells cultured in different concentrations of cell-free plasma-derived serum and increasing amounts of a platelet-derived growth factor. In plasma-derived serum alone, the cells were quiescent and they were arrested in the Go/G1 phase of the cell cycle. Addition of a platelet-derived growth factor to quiescent cells maintained in plasma-derived serum stimulated both DNA synthesis and cell division. When plasma components were present at high concentration (5%, vol/vol), the amount of platelet factor added to the cultures determined the number of cell doublings. Plasma-derived molecules were required for the platelet factor to stimulate DNA synthesis and cell division in the maximal number of cells. In addition, plasma components had to be present for recently divided cells to respond to the platelet factor. When 3T3 cells were cultured in excess platelet factor and limiting amounts of plasma-derived serum (0.5%, vol/vol), the cells underwent one doubling and then ceased to proliferate. Addition of fresh plasma-derived serum to these cells induced a second round of cell division. Plasma components and the platelet-derived growth factor acted in a coordinate fashion to regulate the proliferation of Swiss 3T3 cells.     

Radioimmunoassay of a human serum growth factor for Balb/c-3T3 cells: derivation from platelets.

A radioimmunoassay has been developed for the detection and quantification of a human serum polypeptide that has growth-promoting activity for confluent Balb/c-3T3 cells. Antiserum to this growth factor does not recognize antigens in mouse, guinea pig, or bovine serum but does detect some crossreacting antigen in the serum of the New World monkey Cebus albifrons and more in the serum of the Old World rhesus monkeys Macaca mulatta and M. fascicularis, demonstrating that the antigenic determinants of the growth factor have a degree of species specificity. Serum derived from whole human blood contains approximately 770 pg of the growth factor per mg of protein; serum derived from platelet-poor blood contains about 112 pg of the growth factor per mg of protein. As much as 1 microng of the growth factor per mg of protein has been recovered from human platelets by heating them at 100 degrees for 2 min. Approximately 1-2 ng of the growth factor, in either whole serum or platelets, stimulates 5 to 10 X 10(3) confluent Balb/c-3T3 cells to replicate. The heat treatment of platelets allows the purification and quantitative recovery of the growth factor from blood.