Neuroblastoma cells express c-sis and produce a transforming growth factor antigenically related to the platelet-derived growth factor.

Mouse neuroblastoma Neuro-2A cells produce transforming growth factors during exponential growth in a defined hormone-free medium, which, on Bio-Gel columns in 1 M HAc, elute at a molecular size of 15 to 20 kilodaltons (kDa). These neuroblastoma-derived transforming growth factors have strong mitogenic activity, but they do not compete with epidermal growth factor for receptor binding (E. J. J. van Zoelen, D. R. Twardzik, T. M. J. van Oostwaard, P. T. van der Saag, S. W. de Laat, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 81:4085-4089, 1984). In this study approximately 80% of the mitogenic activity was immunoprecipitated by antibodies raised against platelet-derived growth factor (PDGF). Immunoblotting indicated a true molecular size of 32 kDa for this PDGF-like growth factor. Analysis of poly(A)+ RNA from Neuro-2A cells demonstrated the expression of the c-sis oncogene in this cell line, whereas in vitro translation of the RNA yielded a 20-kDa protein recognized by anti-PDGF antibodies. Separation by reverse-phase high-pressure liquid chromatography demonstrated the presence of two distinct mitogenic activities in neuroblastoma-derived transforming growth factor preparations, one of which is antigenically related to PDGF. Both activities had the ability to induce anchorage-independent growth in normal rat kidney cells, both in the presence and in the absence of epidermal growth factor. It is concluded that Neuro-2A cells express c-sis with concomitant production and secretion of a PDGF-like growth factor, which plays a role in the induction of phenotypic transformation on normal rat kidney cells.     

Phenotypic transformation of normal rat kidney cells by transforming growth factor beta is not paralleled by enhanced production of a platelet-derived growth factor.

Phenotypic transformation of normal rat kidney (NRK) cells requires the concerted action of multiple polypeptide growth factors. Serum-deprived NRK cells cultured in the presence of epidermal growth factor (EGF) become density-inhibited at confluence, but they can be restimulated by a number of defined polypeptide growth factors, resulting in phenotypic cellular transformation. Kinetic data show that restimulation by transforming growth factor beta (TGF-beta) and retinoic acid is delayed when compared to induction by platelet-derived growth factor (PDGF), indicating that both TGF beta and retinoic acid may exert their growth-stimulating action by an indirect mechanism. Northern blot analysis shows that NRK cells express the genes for various polypeptide growth factors, including TGF beta 1, PDGF A-chain and basic fibroblast growth factor, but that the levels of expression are not affected by TGF beta or retinoic acid treatment. NRK cells also secrete low amounts of a PDGF-like growth factor into their extracellular medium, but the levels of secretion are insufficient to induce mitogenic stimulation and are unaffected by agents inducing phenotypic transformation. In combination with studies on the effects of anti-PDGF antibodies, it is concluded that phenotypic transformation of NRK cells by TGF beta and retinoic acid is not the result of enhanced production of a PDGF-like growth factor.     

Phenotypic transformation of normal rat kidney cells in a growth-factor-defined medium: induction by a neuroblastoma-derived transforming growth factor independently of the EGF receptor

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth-factor-inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth-factor-defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth-factor-defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage-independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.     

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.     

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.     

Transforming growth factor activity of bovine brain-derived growth factor

Bovine brain-derived growth factor (BDGF), whose biochemical properties resemble those of endothelial cell growth factor (ECGF) and brain-derived acidic fibroblast growth factor (acidic FGF), is able to promote colony formation of normal rat kidney fibroblasts (NRK cells) in soft agar. As in the case of transforming growth factor beta (TGF beta), EGF potentiates the anchorage-independent growth promoting activity of BDGF. In the presence of EGF (5 ng/ml), the optimal concentration of BDGF for stimulation of anchorage-independent of NRK cells is approximately 0.5 ng/ml. At higher concentrations, BDGF becomes inhibitory. The anchorage-independent cell growth promoting activity of BDGF differs from that of TGF beta in acid and reducing agent stability.     

Altered response to growth factors or retinoic acid in phenotypic transformation of normal rat kidney cells expressing human c-fos gene.

Anchorage-independent growth of normal rat kidney (NRK) fibroblast in soft agar depends on both transforming growth factor beta (TGF beta) and epidermal growth factor (EGF). To examine whether c-fos protein is involved in phenotypic transformation of NRK cells, we have transfected and isolated several NRK cell lines that carry the human c-fos gene fused to the metallothionein IIA promoter. A transfectant, Nf-1, had constitutive levels of the human c-fos expression. Anchorage-independent growth of Nf-1 was already stimulated by EGF alone, and the colony sizes of Nf-1 were comparable to those of the parental NRK in the presence of both EGF and TGF beta. Anchorage-independent growth of NRK could be observed in the presence of TGF beta or retinoic acid or platelet derived growth factor (PDGF) and EGF. No growth of NRK in soft agar appeared when basic fibroblast growth factor (bFGF) and EGF were present. By contrast, anchorage-independent growth of Nf-1 was surprisingly enhanced by EGF and TGF beta or retinoic acid or PDGF or bFGF. Expression of the human c-fos gene may compensate the signal to phenotypic transformation induced by TGF beta as well as retinoic acid or PDGF or bFGF.     

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

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-beta 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-beta 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.     

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     

Fibronectin-associated transforming growth factor

We have studied the ability of fibronectins to induce anchorage-independent growth of NRK-49F cells in serum-free medium. Cells were seeded in soft agar in the presence of various concentrations of plasma fibronectins, and colonies were counted after 10 days. It was found that, with some exceptions, human plasma fibronectins induced anchorage-independent growth at concentrations in 20-100 micrograms/ml range. The ability of exogenously supplied fibronectins to promote anchorage-independent growth of NRK cells is attributed to a transforming growth factor (TGF) activity associated with gelatin-agarose affinity purified plasma fibronectins. This TGF activity required epidermal growth factor (EGF) in our serum-free assay system. The TGF-like activity appears to either co-purify or to be associated with fibronectin at neutral pH during molecular sieve chromatography and during ultracentrifugation through sucrose density gradients. The TGF activity "dissociates" from fibronectin at extremes of pH, however, and can be separated from fibronectin by molecular sieve chromatography in 1 M acetic acid. Under these conditions, the TGF-like activity chromatographed as a single peak with an apparent molecular weight of approximately 30 kDa. The physical-chemical properties, chromatographic behavior, and biological activity of this TGF suggest that it is type-beta transforming growth factor/growth inhibitor (beta-TGF/GI). The TGF activity has been observed in fibronectin isolated from fresh human plasma as well as in fibronectins from several other species obtained from commercial suppliers. Our results would suggest that caution be applied in the interpretation of experiments in which gelatin affinity purified fibronectins are used at micrograms/ml concentrations.     

The role of polypeptide growth factors in phenotypic transformation of normal rat kidney cells

A serum-free assay has been established for studying the role of polypeptide growth factors in inducing loss of density-dependent inhibition of growth of normal rat kidney (NRK) cells. The process has been characterized by measuring the time course of [3H]thymidine incorporation into confluent, quiescent NRK cultures stimulated by defined polypeptide growth factors, in combination with cell counting studies, increases in DNA content, and cell cycle analysis by means of a fluorescence-activated cell sorter. It is shown that none of the growth factors tested (epidermal growth factor, platelet-derived growth factor, transforming growth factor-beta, and retinoic acid) is able to induce loss of density-dependent inhibition of growth by itself, but strong synergism was observed when combinations of growth factors were tested. None of the above factors was found to be essential, however, since any combination of three of the above four growth factors strongly induced the process. Strong parallels were observed between the growth factor requirements for inducing loss of density-dependent inhibition of growth under serum-free conditions and the requirements for induction of anchorage-independent proliferation under growth factor-defined assay conditions. This indicates that most likely the same cellular processes underlie these two aspects of phenotypic transformation, although data indicate that anchorage-independent proliferation may be a more restricted property of phenotypic transformation than loss of density dependence of proliferation. It is concluded that phenotypic transformation of NRK cells does not require specific polypeptide growth factors, but reflects the ability of these cells to respond to multiple growth factors.     

Transforming growth factor-beta enhances the extent of intercellular communication between normal rat kidney cells

Normal rat kidney (NRK) cells cultured in the presence of epidermal growth factor are contact-inhibited at confluent densities. In the additional presence of transforming growth factor (TGF)-beta, however, cells undergo phenotypic transformation which is accompanied by a loss of contact inhibition. In this study, we show by means of the fluorescence photobleaching recovery technique and a scrape-loading dye transfer technique that quiescent confluent cultures of NRK cells do not show extensive gap junction-mediated intercellular communication. Cells contact-inhibited in the presence of epidermal growth factor also show only limited intercellular communication, although with an enhanced permeability coefficient. Cells phenotypically transformed upon addition of TGF beta, however, show extensive intercellular communication, with a similarly enhanced permeability coefficient. This enhanced intercellular communication induced by TGF beta is paralleled by an increase in intracellular pH. It is concluded that in contrast to what has been observed during tumorigenic transformation, phenotypic transformation of NRK cells induced by TGF beta results in an enhancement of the extent of gap junction-mediated intercellular communication.     

Transforming growth factor-beta overrides the adhesion requirement for surface expression of alpha(5)beta(1) integrin in normal rat kidney fibroblasts. A necessary effect for induction of anchorage-independent growth.

We have previously shown that the expression of alpha(5)beta(1) integrin on the cell surface is dependent upon cell adhesion to the extracellular matrix, and we report here that transforming growth factor-beta (TGF-beta) overcomes this requirement in normal rat kidney (NRK) fibroblasts. Thus, suspended NRK cells treated with TGF-beta show levels of surface alpha(5)beta(1) integrin that are equivalent to those seen in adherent cells. Moreover, several experiments showed that this effect is necessary for the induction of anchorage-independent growth by TGF-beta. First, a kinetic analysis showed that surface expression of alpha(5)beta(1) integrin was restored in TGF-beta-treated NRK cells prior to the induction of anchorage-independent growth. Second, NRK cell mutants that have lost their TGF-beta requirement for surface expression of alpha(5)beta(1) integrin were anchorage-independent in the absence of TGF-beta. Third, an antisense oligonucleotide to the beta(1) integrin subunit or, fourth, stable expression of an alpha(5)-antisense cDNA blocked the ability of TGF-beta to stimulate anchorage-independent growth. Thus, TGF-beta overrides the adhesion requirement for surface expression of alpha(5)beta(1) integrin in NRK cells, and this effect is necessary for the induction of anchorage-independent growth.     

Identification and initial characterization of transforming growth factor-like mitogen(s) in human anterior pituitary

Transforming growth factor (TGFs) are a family of peptide(s) defined by their ability to induce anchorage-independent growth of non-neoplastic indicator cells in soft agar. We found that acid-ethanol extracts of human anterior pituitary tissues were able to stimulate colony growth of normal rat kidney fibroblasts in soft agar. When subjected to gel-filtration on a column of Bio-Gel P-60 in 1 M acetic acid, the majority of TGF activity eluted in fractions corresponding to an apparent mol wt 15,000. The activity was heat- and acid-stable, but was inactivated by treatment with trypsin and dithiothreitol. Pituitary TGF-like materials did not compete with epidermal growth factor (EGF) for receptor binding and did not require EGF for colony-forming activity. Thus, human pituitary TGF was not like type alpha or type beta TGF.     

Reciprocal effects of epidermal growth factor and transforming growth factor beta on the anchorage-dependent and -independent growth of A431 epidermoid carcinoma cells

The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.     

Transforming growth factor-beta induces platelet-derived growth factor (PDGF) messenger RNA and PDGF secretion while inhibiting growth in normal human mammary epithelial cells

Platelet-derived growth factor (PDGF) is a potent mitogen in human serum which specifically stimulates the proliferation of mesenchymal cells. We have now examined normal human mammary epithelial cells (HMEC) derived from reduction mammaplasties and grown in a serum-free defined medium. Medium conditioned by HMEC contained a PDGF-like activity that competed with [125I]PDGF for binding to PDGF receptors in normal human fibroblasts. When conditioned media were incubated with antiserum specific for either PDGF-A or PDGF-B, only PDGF-A antiserum was capable of inhibiting binding of conditioned media to PDGF receptors. Using an RNase protection assay, mRNA from normal HMEC was probed for both the PDGF-A and PDGF-B chains. Little or no PDGF-B was found in HMEC strains, while a strong signal was seen with the PDGF-A probe. When HMEC were grown in the presence of transforming growth factor-beta (TGF beta) for 48 h, inhibition of growth was observed in association with a 20- to 40-fold stimulation of PDGF-B mRNA and a 2-fold stimulation of PDGF-A mRNA. This mRNA induction was extremely rapid (within 1 h), and secreted PDGF activity was induced 2- to 3-fold. Two other HMEC growth inhibitors and differentiating agents, sodium butyrate and phorbol ester 12-O-tetradecanoylphorbol-13-acetate, had no effect on PDGF mRNA regulation. The current study suggests that PDGF gene induction is an extremely rapid and specific indicator of TGF beta function regardless of whether TGF beta is acting in a growth stimulatory or inhibitory manner. Any role of PDGF-B in TGF beta modulation of differentiation of normal or malignant mammary gland remains to be determined.     

Transforming growth factor-beta induction of type-1 plasminogen activator inhibitor. Pericellular deposition and sensitivity to exogenous urokinase

The human tumor cell line HT-1080 was used as a model system to study the effects of transforming growth factor-beta (TGF beta) on polypeptide synthesis and proteolytic activity of malignant cells. Confluent cultures were exposed to TGF beta under serum-free conditions, and alterations in the production of proteins were examined by metabolic labeling and polypeptide analysis. TGF beta induced the synthesis and secretion of the Mr 47,000 endothelial type plasminogen activator inhibitor (PAI-1) as shown by reverse zymography, immunblotting, and immunoprecipitation analyses. TGF beta-induced PAI-1 was rapidly deposited in the growth substratum of the cells as shown by metabolic labeling and extraction of the cultures with sodium deoxycholate. Using pulse-chase experiments, we found a relatively fast turnover of substratum-associated PAI-1. Exogenously added urokinase released PAI-1 from the substratum even in the presence of the plasmin inhibitor aprotinin, suggesting a direct effect of urokinase. Immunoreactive complexes of higher molecular weight were subsequently detected in the medium. Epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor, and insulin did not elicit similar effects on the amount of PAI-1. TGF beta also inhibited the anchorage-independent growth of HT-1080 cells at the same concentrations at which it induced PAI-1. These results indicate that TGF beta can modulate the extracellular proteolytic activity of cultured cells by enhancing the secretion and deposition of PAI-1 into their microenvironment. It remains to be established whether TGF beta inhibition of anchorage-independent growth of these cells is associated with the induction of PAI-1.     

Partial purification of an immunosuppressive protein from a human tumor cell line and analysis of its relationship to transforming growth factor beta

The A673 human rhabdomyosarcoma cell line constitutively produces an acid-soluble, potent immunosuppressive factor (ISF), which inhibits T-cell proliferation. We have partially purified this factor from the culture supernatant of A673 cells by a sequence of acid extraction, gel filtration, cation exchange chromatography, and reverse-phase HPLC. Characterization studies indicate that ISF is similar or identical to transforming growth factor beta (TGF beta). ISF exhibits a molecular weight of 25 kDa in sodium dodecyl sulfate-polyacrylamide gels. ISF, like TGF beta, is a very basic protein (pI = 9.5) that is sensitive to reduction. Anti-TGF beta 1 antibodies completely block ISF activity in the thymocyte assay. Furthermore, ISF, like TGF beta, stimulated the anchorage-independent growth of normal rat kidney fibroblasts in soft agar.     

Induction of anchorage-independent growth by transforming growth factor-beta linked to anchorage-independent expression of cyclin D1

Transforming growth factor-beta (TGF-beta) was originally identified, characterized, and named on the basis of its ability to induce anchorage-independent growth (phenotypic transformation). This effect has received little attention in recent years, probably because the induction of anchorage-independent growth by TGF-beta has been observed only in a few cell lines, of which NRK fibroblasts are among the best studied. We have previously reported that normal rat kidney cells have lost their normal adhesion requirement for expression of cyclin D1, and we now show that this loss is causal for the induction of anchorage-independent growth by TGF-beta. First, we show that TGF-beta fails to induce anchorage-independent growth in NIH-3T3 cells and human fibroblasts that have retained their adhesion requirement for expression of cyclin D1. Second, we show that TGF-beta complements rather than affects cyclin D-cdk4/6 kinase activity in NRK cells. Third, we show that forced expression of cyclin D1 in suspended 3T3 cells renders them susceptible to transformation by TGF-beta. These results may explain why the induction of anchorage-independent growth by TGF-beta is a rare event and yet also describe a molecular scenario in which the mesenchymal response to TGF-beta could indeed involve the acquisition of an anchorage-independent phenotype.     

Growth of cells on a perfluorocarbon-medium interphase: A quantitative assay for anchorage-independent cell growth

Summary A high density, purified, nontoxic solvent, heptacosafluorotributylamine (FC43), was successfully used as a culture surface for growing several normal and oncogene-transformed cell lines under anchorage-independent conditions. Normal rat kidney (NRK) fibroblasts and the normal mammary epithelial cell lines NMuMG and A1, clone N4, of murine and human origin, respectively, failed to grow at a FC43 growth medium interphase or in soft agar in the absence of transforming growth factor alpha (TGFα) and transforming growth factor beta (TGFβ). However, NRK fibroblasts transformed with the Kirstenras viral oncogene (K-NRK) or NMuMG cells transformed with a point-mutated c-Harvey-ras proto-oncogene or polyoma middle T-transforming gene (NMuMG-ras ^H and NMuMG-pyt, respectively) exhibited rapid growth and formed large colonies when cultured on an FC43-medium interphase. In addition, NRK cells treated with TGFα and TGFβ and K-NRK cells grown on FC43 exhibited a sensitivity to the growth inhibitory effects of 4-cis-L-hydroxyproline comparable to that observed for the same cells grown in soft agar. These results demonstrated that the two-phase assay system (FC43-growth medium interphase) may be superior to soft agar for monitoring the anchorage-independent growth of cells because of the ease of cell plating, the ability to recover cells and secreted products from the upper aqueous phase, and the shorter growth period required to complete the assay (3–4 days). Editor's Statement This report describes the application of a novel technique that provides an alternative approach to assay of anchorage independence with some unique advantages over conventional methods.