Anchorage-independent growth of RSV-transformed rat (GCA, W 12 and XC) cells

Three RSV-transformed rat cell lines: GCA, W12 and XC were characterized as to their ability to anchorage-independent growth in comparison to normal rat kidney (NRK-49F) cells. Differences in the threshold density (TD) and colony forming efficiency (CFE) of the investigated cells are described. The ability of virally transformed cells to stimulation of soft agar colony formation of NRK cells in coculture assay was presented. The production of TGFs-like factors by GCA, W12 and XC cells was suggested.     

3H-thymidine incorporation method versus colony forming assay in the determination of anchorage-independent cell growth of rat sarcoma (XC) and Morris hepatoma 7777 (MH) cells.

The modification of 3H-thymidine incorporation method of Tanigawa was used to the estimation of anchorage-independent growth of virally and chemically transformed rat cells. The relationship between colony forming assay and 3H-TdR incorporation test was determined, depends on the composition of culture medium and the period of incubation of rat sarcoma (XC) cells with thymidine. The influence of exogenous mitogens (RFG, TGF beta 1 and insulin) and autocrine factor (at different step of purification) on the growth of Morris hepatoma 7777 (MH) cells was estimated by both methods. Regression analysis comparing the results of colony counting and thymidine incorporation revealed good correlation between the two methods. The modification can be used the determination of growth stimulating or growth inhibiting activity and in multistep purification procedure of autocrine growth factors.     

Anchorage-dependent growth factor(s) produced by rat sarcoma (XC) cells.

The autocrine growth factor(s) was isolated from serumfree conditioned medium of rat sarcoma (XC) cells. Autocrine activity was enriched by ultrafiltration using Amicon YM 10 membrane, extraction with 1 M acetic acid and partially purified (650-fold) by chromatography on Bio-Gel P-100 and P-60. The final recovery of the autocrine factor(s) was 4 micrograms from 1800 ml of the conditioned medium (a yield of 6%). The factor(s) with molecular weight 6-10 kDa was heat and acid stable but inactivated by trypsin and dithiothreitol. It stimulated anchorage-dependent (but not anchorage-independent) growth of XC cells as well as untransformed, established lines of rat (NRK) and mouse (3T3) cells. The results obtained may suggest that autocrine factor(s) produced by XC cells can be one of EGF-like or/and insulin-like growth factors.     

Cellular responsiveness to growth factors correlates with a cell's ability to express the transformed phenotype

Five random subclones of the rat fibroblast line F2408 vary in their frequency of transformation by the unrelated Kirsten murine sarcoma virus and Abelson murine leukemia virus. The same pattern of sensitivity is displayed when the cells are induced to anchorage-independent growth (transformed) by epidermal, platelet-derived, and sarcoma growth factors, or by whole serum. Our results demonstrate that a growth factor's ability to render cells anchorage independent is not unique to transforming growth factors, but common to many growth factors; anchorage-independent growth is a function of the total growth factor concentration in the medium; cells vary in their inherent responsiveness to growth-factor-induced anchorage-independent growth; and cells resistant to growth-factor-induced anchorage-independent growth are also resistant to transformation by a variety of tumor viruses. We conclude that the way a cell responds to growth factors plays a central role in the expression of the transformed phenotype.     

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     

Continuous maintenance of transformed fibroblasts under reduced serum conditions: utility as a model system for investigating growth factor-specific effects in nonquiescent cells

We report the continuous growth maintenance of untransformed and chemically transformed fibroblasts (AKR-2B, AKR-MCA cells) in low concentrations of serum (0.1% FBS). The cell lines established (AKR-0.1F, MCA-0.1F) proliferated at rates comparable to cells maintained under high serum conditions (10% FBS). Complete removal of serum from the cells did not induce quiescence. The MCA-0.1F cells were more similar to the untransformed AKR-2B fibroblasts in their morphology, saturation density, inability to form colonies under anchorage-independent conditions, steady-state level of c-myc expression, and kinetics of induction of c-myc in response to specific growth factors. This report demonstrates the utility of this cell line as a nonquiescent model system for investigating growth factor-specific effects in serum-free, cycling cells. Addition of transforming growth factor-beta (TGF-beta) (5 ng/ml) to proliferating MCA-0.1F cells, in the absence of any serum, induced a multilayered growth pattern at confluency, similar to that of AKR-MCA cells maintained in 10% FBS. Other growth factors tested did not elicit this effect. The induction of this growth pattern by TGF-beta was associated with a sustained induction of the c-myc proto-oncogene at confluency, but not with a restoration of anchorage-independent growth. The data suggest that TGF-beta may play a role in the up-regulation of c-myc at confluency previously described for AKR-MCA cells maintained in 10% serum.     

Integrin-linked kinase is involved in lactoferrin-induced anchorage-independent cell growth and survival in PC12 cells

AimsBovine lactoferrin (bLf) causes anchorage-independent cell growth in PC12 cells. The present study investigated the mechanisms involved in bLf-induced anchorage-independent cell growth and survival in PC12 cells.Main methodsThe number of adherent cells and suspended cells was estimated separately by using a methyl thiazol tetrazolium (MTT) assay, and the sum of both optical density (O.D.) (570 nm) values was used as a measure of the total number of cells.Key findingsIntegrin-linked kinase (ILK) plays an important role in integrin and growth factor signaling pathways. Stable transfection of PC12 cells with a dominant negative kinase-deficient mutant of ILK (DN-ILK) inhibited bLf-induced anchorage-independent cell growth. The ILK activity in the parental cells was transiently activated after addition of bLf, whereas bLf-induced activation of ILK was blocked in DN-ILK-transfected cells. bLf also activated p38 mitogen-activated protein kinase (MAPK); however, the p38 MAPK activation was inhibited by stable DN-ILK transfection. Moreover, cell viability in the suspended cells by bLf strongly decreased after treatment with SB203580, an inhibitor of p38 MAPK.SignificanceThese results suggest that ILK is involved in bLf-induced anchorage-independent cell growth and viability via activation of p38 MAPK.     

Neoplastic transformation of mouse mammary epithelial cells by deregulated myc expression.

A spontaneously immortalized, nontumorigenic mouse mammary epithelial cell line (MMEC) was transfected with an activated myc construct by electroporation. Constitutive expression of myc in MMEC resulted in anchorage independence in soft agar and tumorigenicity in nude mice. The myc-expressing MMEC showed higher saturation density, faster growth rate, and partial abrogation of serum-derived growth factor(s) requirement compared with parent MMEC. Epidermal growth factor or transforming growth factor alpha stimulated the anchorage-independent growth, but not the anchorage-dependent growth, of MMEC-myc cells. Type 1 transforming growth factor beta, on the other hand, inhibited both the anchorage-independent and anchorage-dependent growth of MMEC-myc cells. These results demonstrate that deregulated expression of myc results in neoplastic transformation iin mammary epithelial cells. Accompanying the transformation is altered sensitivity to polypeptide growth factors.     

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.     

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.     

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.     

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.     

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.     

Orthovanadate both mimics and antagonizes the transforming growth factor β action on normal rat kidney cells

Normal rat kidney [NRK] cells grown in the presence of epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) have a normal phenotype and undergo density-dependent growth inhibition, whereas in the presence of multiple growth factors, density arrest is lost and the cells become phenotypically transformed. We studied the influence of the protein tyrosine phosphatease (PTPase) inhibitor sodium orthovanadate on the mitogenic stimulation of NRK cells by growth factors and on transformation-linked properties as loss of density-dependent growth inhibition and anchorage-independent growth. The fraction of cells in serum-deprived monolayer cultures that is induced to proliferate upon mitogenic stimulation by EGF or PDGF is only slightly enhanced upon addition of low concentrations (25–50 μM) of vanadate. Addition of vanadate per se induces proliferation of only a very limited amount of cells, but results in a shift of the dose-response curves for other growth factors to lower concentrations. Vanadate added in combination with EGF or PDGF is able to mimic the effect of transforming growth factor β (TGFβ) in inducing phenotypic transformation. In monolayer cultures density-dependent growth inhibition is lost and anchorage-independent proliferation is observed on dishes coated with poly(2-hydroxy-ethyl methacrylate) (polyHEMA). The extent of these changes is similar to that induced by TGFβ. However, the morphology of the obtained colonies in polyHEMA-coated dishes is quite different. Cells transformed by TGFβ in the presence of EGF form rather amorphous colonies, whereas in the presence of orthovanadate colonies are formed that tend to fall apart in loose cells. The effect of vanadate on cell transformation is dependent on the growth factor conditions in a bimodal way. When a suboptimal dose of growth factor(s) is used, 25 μM vanadate is very effective in preventing density-induced growth inhibition and stimulating anchorage-independent proliferation. However, the same concentration of vandate is inhibitory when cells are maximally stimulated and antagonizes the transforming effect of TGFβ added in combination with other growth factors. It is hypothesized that vanadate acts on a set of different protein tyrosine phosphatases. Some of these are positive and others negative regulators of growth. © 1993 Wiley-Liss, Inc.     

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.     

Conditioned medium of human nasopharyngeal carcinoma epithelioid cell line CNE 1 contained the activities of transformed growth-inhibiting factor(s)

The hormone defined serum free conditioned medium (SFCM) of human nasopharyngeal carcinoma epithelioid cell line (CNE 1) was assaied by both the [3H] thymidine incorporation test and the soft agar test. It was found that the SFCM can stimulate the growth of long-term serum free cultured CNE 1 cells in accordence with the fact that the growth rate of long-term serum free cultured CNE 1 cells was directly proportional to the plating density. Alternatively 5% SFCM can inhibit the growth of short-term serum free cultured CNE 1 cells by 51% in which the indicator cell may remained the responsive state of growing in the serum supplemented medium to the effector of interest. Furthermore, SFCM resulted in inhibition of anchorage-independent growth of CNE 1 cells and A431 cells. Also in soft agar test, SFCM can reduce the colony formation of NRK-49F cells in the presence of EGF or EGF plus TGF-beta. These finding suggested that CNE 1 can autocrine growth stimulating factor(s) and growth inhibiting factor(s) in the serum free medium, the latter can strongly reverse malignant phenotypies of CNE 1 and A431 cells in serum supplemented surrounding.     

Anchorage-independent colony formation of SV40 transformed BALB/c-3T3 cells in serum-free medium: role of cell- and serum-derived factors

The colony-forming response of SV40 transformed BALB/c-3T3 cells in agarose suspension culture was studied in a serum-free medium (with insulin, transferrin and serum albumin as the only macromolecular supplements) that was optimized for colony formation of fibronectin-attached monolayer cultures. In this serum-free medium, the SV3T3 cells fail to form colonies in agarose suspension. However, they can be induced to anchorage-independent colony formation by the growth factors that are additionally required by their untransformed counterparts for proliferation in monolayer culture. The SV3T3 cells are also rendered anchorage-independent for colony formation in serum-free medium by conditioned medium from dense monolayer serum-free SV3T3 cultures. These experiments suggest that it is the cell-substrate interaction that is responsible for the growth factor autonomy of fibronectin-attached transformed cells.     

Matrix-independent activation of phosphatidylinositol 3-kinase,Stat3, and cyclin A-associated Cdk2 Is essential for anchorage-independent growth of v-Ros-transformed chicken embryo fibroblasts

The question remains open whether the signaling pathways shown to be important for growth and transformation in adherent cultures proceed similarly and play similar roles for cells grown under anchorage-independent conditions. Chicken embryo fibroblasts (CEF) infected with the avian sarcoma virus UR2, encoding the oncogenic receptor protein-tyrosine kinase (RPTK) v-Ros, or with two of its transformation-impaired mutants were grown in nonadherent conditions in methylcellulose (MC)-containing medium, and the signaling functions essential for Ros-induced anchorage-independent growth were analyzed. We found that the overall tyrosine phosphorylation of cellular proteins in CEF transformed by v-Ros or by two oncogenic nonreceptor protein-tyrosine kinases (PTKs), v-Src and v-Yes, was dramatically reduced in nonadherent conditions compared with that in adherent conditions, indicating that cell adhesion to the extracellular matrix plays an important role in efficient substrate phosphorylation by these constitutively activated PTKs. The UR2 transformation-defective mutants were differentially impaired compared with UR2 in the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and Stat3 in nonadherent conditions. Consistently, the constitutively activated mutants of PI 3-kinase and Stat3 rescued the ability of the UR2 mutants to promote anchorage-independent growth. Conversely, dominant negative mutants of PI 3-kinase and Stat3 inhibited UR2-induced anchorage-independent growth. UR2-infected CEF grown in nonadherent conditions displayed faster cell cycle progression than the control or the UR2 mutant-infected cells, and this appeared to correlate with a PI 3-kinase-dependent increase in cyclin A-associated Cdk2 activity. Treatment of UR2-infected cells with Cdk2 inhibitors led to the loss of the anchorage-independent growth-promoting activity of UR2. In conclusion, we have adopted an experimental system enabling us to study the signaling pathways in cells grown under anchorage-independent conditions and have identified matrix-independent activation of PI 3-kinase and Stat3 signaling functions, as well as the PI 3-kinase-dependent increase of cyclin A-associated Cdk2 kinase activity, to be critical for the Ros-PTK-induced anchorage-independent growth.