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.     

Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor beta

A mutant clone (MO-5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF): binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-beta efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-beta while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-beta showed low colony formation capacity in soft agar in the absence of TGF-beta. Colonies of MO-5 formed by TGF-beta in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-beta. Pretreatment of MO-5 with TGF-beta induced secretion of TGF-beta-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-beta-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the "transformed" phenotype in MO-5 is discussed.     

Epidermal growth factor (EGF) receptor density controls mitogenic activation of normal rat kidney (NRK) cells by EGF

Normal rat kidney (NRK) fibroblasts are immortalized cells that are strictly dependent on externally added growth factors for proliferation. When cultured in the presence of epidermal growth factor (EGF) as the only growth stimulating hormone, these cells have a normal phenotype and undergo density-dependent growth inhibition. It has been postulated that this density-arrest results from a decrease of EGF receptor levels below a threshold level which makes these cells unresponsive to stimulation by EGF. In the present study, we show that NRK cells, made quiescent by serum-deprivation at submaximum density, are mitogenically still responsive to EGF, but show enhanced mitogenic stimulation after 8 hr pre-treatment with either transforming growth factor β (TGFβ) or retinoic acid (RA), while prostaglandin F_2α (PGF_2α) and bradykinin (BK) enhance the mitogenic stimulation by EGF only slightly under these conditions. Addition of TGFβ or RA results in an increase of both ¹²⁵I-EGF-binding capacity and EGF receptor mRNA levels. Using flow cytometric analysis, we show that pre-treatment with TGFβ or RA increases the percentage of cells entering the cell cycle as a function of time. Furthermore, pre-treatment of the cells with TGFβ or RA increases the rate of mitogen-activated protein kinase (MAPK) phosphorylation by EGF. PGF_2α and BK also increase EGF receptor levels, but only with delayed kinetics. These results show that already in serum-deprived quiescent NRK cells, EGF receptor levels limit EGF-induced mitogenic stimulation. This observation provides further evidence for the regulating role of the EGF receptor in density-dependent growth control of NRK cells. J. Cell. Physiol. 174:9–17, 1998. © 1998 Wiley-Liss, Inc.     

A transforming growth factor-beta like growth factor in mouse embryonal carcinoma cells

Our previous study indicated that polypeptides isolated from acid/ethanol extracts of solid tumors of a cloned F9-3 embryonal carcinoma cells by Bio-Gel P60 column chromatography were found to be able to stimulate anchorage independent growth of either NIH 3T3 cells or NRK 49 F cells in soft agar. The major peak of active elute had a molecular weight of about 15 kDa as determined by SDS-polyacrylamide gel electrophoresis. In the present report we further isolated and purified the active compound corresponding to molecular weight of 15 kDa by gel filteration on Bio-Gel P10 column (Fig. 1) and then by high pressure liquid chromatography (Fig. 2). It was found that the purified 15 kDa molecules showed some properties similar to transforming growth factor-beta (TGF-beta): 1. Colony-stimulating activity in soft agar can be induced in NRK 49 F cells only in the presence of mouse epidermal growth factor (EGF) (Plate I); 2. Increase in relative uptake of 3H-thymidine in NRK 49 F cells occurred in the presence of EGF, but with the same amount of EGF, not much change in 3H-thymidine incorporation could be found with further increasing amounts of purified 15 kDa molecules (Fig. 3); 3. Like human blood platelets derived TGF-beta, inhibition effect on the growth of mink lung epithelial cells (CCL/64) can also be exhibited by purified 15 kDa molecules (Fig. 4). In addition, using ELISA procedure, we have also demonstrated that the 15 kDa molecules had immunological reactivity with the antibody raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta 1 from human blood platelets (Fig.5). Thus, the 15 kDa molecules isolated from mouse F9-3 embryonal carcinoma cells appeared to share some common antigenic determinants with human TGF-beta 1 molecule. These results taken together provide strong support for the existence of TGF-beta like growth factor in mouse embryonal carcinoma cells.     

Transforming growth factor-beta modulates the high-affinity receptors for epidermal growth factor and transforming growth factor-alpha

The epidermal growth factor (EGF) receptor mediates the induction of a transformed phenotype in normal rat kidney (NRK) cells by transforming growth factors (TGFs). The ability of EGF and its analogue TGF-alpha to induce the transformed phenotype in NRK cells is greatly potentiated by TGF-beta, a polypeptide that does not interact directly with binding sites for EGF or TGF-alpha. Our evidence indicates that TGF-beta purified from retrovirally transformed rat embryo cells and human platelets induces a rapid (t 1/2 = 0.3 h) decrease in the binding of EGF and TGF-alpha to high-affinity cell surface receptors in NRK cells. No change due to TGF-beta was observed in the binding of EGF or TGF-alpha to lower affinity sites also present in NRK cells. The effect of TGF-beta on EGF/TGF-alpha receptors was observed at concentrations (0.5-20 pM) similar to those at which TGF-beta is active in promoting proliferation of NRK cells in monolayer culture and semisolid medium. Affinity labeling of NRK cells and membranes by cross-linking with receptor-bound 125I-TGF-alpha and 125I-EGF indicated that both factors interact with a common 170-kD receptor structure. Treatment of cells with TGF-beta decreased the intensity of affinity-labeling of this receptor structure. These data suggest that the 170 kD high-affinity receptors for EGF and TGF-alpha in NRK cells are a target for rapid modulation by TGF-beta.     

Effects of transforming growth factors and regulation of their mRNA levels in two human endometrial adenocarcinoma cell lines.

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.     

Transforming growth factor-beta 1 stimulates glucose uptake and the expression of glucose transporter mRNA in quiescent Swiss mouse 3T3 cells

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional polypeptide that regulates the proliferation and differentiation of various types of animal cells. TGF-beta 1 stimulated glucose uptake and the expression of a brain-type glucose transporter (GLUT1) mRNA in quiescent mouse 3T3 cells. TGF-beta 1 also synergistically stimulated these activities when given together with calf serum, phorbol ester, fibroblast growth factor, or epidermal growth factor. The increases in glucose uptake and the GLUT1 mRNA level were induced by picomolar concentrations of TGF-beta 1 within 3 h of stimulation, reached a peak between 6 and 9 h, and then decreased gradually to basal levels before an increase in DNA synthesis. The stimulation of GLUT1 mRNA expression was completely abolished by actinomycin D, but was not affected by cycloheximide, suggesting that new protein synthesis was not required for the expression of GLUT1 mRNA. TGF-beta 1 had little mitogenic activity and did not affect serum-induced DNA synthesis in quiescent 3T3 cells. However, it stimulated DNA synthesis synergistically when given with fibroblast growth factor, epidermal growth factor, phorbol ester, or insulin. These results suggest that TGF-beta 1 mediates the stimulation of glucose uptake, GLUT1 mRNA expression, and DNA synthesis via a pathway(s) and cellular components distinct from those for other growth factors. The possible role of the TGF-beta 1-induced stimulation of glucose transport activity in the control of mouse fibroblast proliferation is also discussed.     

Biphasic effects of type beta transforming growth factor on epidermal growth factor receptors in NRK fibroblasts. Functional consequences for epidermal growth factor-stimulated mitosis

Exposure of confluent NRK cells to transforming growth factor-beta (TGF-beta) results in distinct alterations in subpopulations of plasma membrane epidermal growth factor (EGF) receptors. The low affinity sites increase in number, whereas the high affinity sites undergo a transient decrease in affinity followed by a prolonged increase in number. Cycloheximide inhibits both of these effects. Functional assays measuring EGF-stimulated thymidine incorporation in the presence of TGF-beta show that the resulting long-term stimulation of EGF receptor binding is associated with an increased sensitivity to EGF. Similarly, the initial, transient decrease in EGF binding is associated with a temporary inhibition of EGF-stimulated thymidine incorporation. The results describe a bifunctional effect of TGF-beta at the biochemical level consistent with the action of this peptide on NRK cell growth.     

Cell-mediated co-action of transforming growth factors: incubation of type beta with normal rat kidney cells produces a soluble activity that prolongs the ruffling response to type alpha

Intense, continuous ruffling is a characteristic of many transformed cells, but untransformed cells ruffle intensely only briefly after exposure to growth factors. We reported previously that cells of a normal rat kidney (NRK) cell line transformed by Kirsten murine sarcoma virus secrete their own ruffle-inducing agent(s) that cause sustained ruffling in either themselves or untransformed NRK cells. In the present study, we examined the roles of the transforming growth factors TGF-alpha and TGF-beta in the induction and maintenance of ruffling in untransformed NRK cells and observed the following: TGF-alpha caused a transient epidermal growth factor (EGF)-like response, which could be blocked by prior exposure of cells to EGF or by antiserum directed against the COOH-terminus of TGF-alpha. TGF-beta caused no ruffling and did not itself prolong TGF-alpha ruffling. A new, buffer-soluble (transferable) mediator activity produced by incubation of TGF-beta with NRK cells for 6-h extended the duration of maximal TGF-alpha-induced ruffling by several-fold. This study demonstrates that TGF-alpha alone causes an EGF-like, transient ruffling response, but neither TGF-alpha or TGF-beta alone, nor the two together, cause transformation-associated sustained ruffling. Rather, TGF-alpha acts in concert with a new, TGF-beta-dependent activity. This new activity appears to inhibit normal cellular off-regulation of TGF-alpha-induced ruffling. Inhibition of the cellular off-regulation of a growth factor response could play a key role in the unregulated growth associated with malignancy.     

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.     

Inhibition of normal rat kidney cell growth by transforming growth factor-beta is mediated by collagen

We have investigated the mechanism of inhibition of the serum-free monolayer growth of normal rat kidney (NRK) cells by transforming growth factor-beta (TGF-beta). NRK cells grown on fibronectin-coated dishes exhibited a biphasic response to TGF-beta. Monolayer growth was slightly stimulated by subpicomolar concentrations, while picomolar concentrations of TGF-beta inhibited NRK cell growth in the presence or absence of epidermal growth factor. NRK cells exhibited a similar biphasic growth response to exogenous type I collagen. TGF-beta induced a 3-5-fold increase in the deposition of type I collagen-like proteins into the extracellular matrix of NRK cells during serum-free growth. Type I collagen-like proteins were identified by their sensitivity to degradation by purified bacterial collagenase and by Western blot analysis. The TGF-beta dose-response curves for induction of extracellular matrix-localized collagen and inhibition of NRK cell growth were similar. Finally, the inclusion of a purified bacterial collagenase, which did not degrade TGF-beta or TGF-beta receptors, or alter control NRK growth, prevented exogenous collagen or TGF-beta from inhibiting the serum-free growth of NRK cells. Our results demonstrate that an increase in collagen secretion plays an important role in the inhibition of the growth of NRK cells by TGF-beta.     

Platelet-derived growth factor or basic fibroblast growth factor induce anchorage-independent growth of human fibroblasts

Anchorage-independent growth, i.e., growth in semi-solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage-independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage-independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage-independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet-derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage-independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF-beta) decreased the growth induced by PDGF. EGF combined with TGF-beta induced colony formation in semi-solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage-independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF-beta can stimulate anchorage-independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.     

Transforming growth factor beta (TGF-beta) inhibits hepatocyte DNA synthesis independently of EGF binding and EGF receptor autophosphorylation

Subpicomolar concentrations of human platelet-derived transforming growth factor beta (TGF-beta) inhibited growth factor-stimulated DNA synthesis in primary cultures of adult rat hepatocytes. This inhibition was not the result of changes in the size of intracellular pools of 3H-thymidine and was not dependent on the state of confluence of the cells. A 24-hr exposure to TGF-beta either before or after insulin/EGF stimulation was as inhibitory on DNA synthesis between 48 and 72 hr of culture as was TGF-beta present throughout 72 hr of culture. From 12 hr in culture to 24 hr, hepatocyte EGF binding sites dropped from about 230,000 to 85,000 per cell with no significant change in Kd, but with a loss in capacity for EGF-induced receptor down-regulation. Maximally inhibitory concentrations of TGF-beta did not compete with EGF for the EGF receptor, and a 4- to 24-hr exposure to TGF-beta did not alter subsequent EGF binding. Coincubation of hepatocytes with TGF-beta and EGF did not influence the 60% reduction in EGF binding sites produced by EGF alone. In addition, TGF-beta did not prevent EGF-induced autophosphorylation of the 170,000 dalton EGF receptor in membranes from whole liver. Our studies suggest that TGF-beta regulates hepatocyte growth independently of changes in EGF receptor number, ligand affinity, or postbinding autophosphorylation.     

Characterization of transforming growth factors produced by the insulin-independent teratoma-derived cell line 1246-3A

The 1246-3A cell line is an insulin-independent variant derived from the adipogenic cell line 1246. Data presented in this paper indicate that the 1246-3A cell line releases in its culture medium two types of transforming growth factors, TGF-alpha- and TGF-beta-like polypeptides, and a growth inhibitor. TGF-alpha like polypeptide eluted from Biogel P60 column into two fractions with an apparent molecular weight of 50 kDa and 13 kDa. These high-molecular-weight TGF-alpha-like factors competed with 125I-EGF for binding to epidermal growth factor (EGF) receptors and were specifically immunoprecipitated by incubation with antirat TGF-alpha antibody, not by incubation with anti-EGF antibody. Both fractions promoted anchorage-independent growth of normal rat kidney NRK cells in the absence of EGF and stimulated DNA synthesis in quiescent Balb/c-3T3 cells in a fashion similar to EGF and synthetic TGF-alpha. In addition to secreting TGF-alpha-like polypeptides, 1246-3A cells produce TGF-beta. This polypeptide, eluted from Biogel P60 chromatography with an apparent molecular weight of 25 kDa, promoted anchorage-independent growth of NRK cells in the presence of EGF and was growth inhibitory for Chinese hamster lung fibroblasts CCL 39 cells. Interestingly, another growth inhibitory activity was detected in Biogel P60 fractions and eluted with an apparent molecular weight of between 9.5-11 kDa. This fraction was different from TGF-beta and TGF-alpha as determined by specific radioreceptor competition assays. TGF-alpha and TGF-beta-like polypeptides could represent autocrine growth stimulators for the insulin-independent 1246-3A cells and act in synergy with insulin-related factor (IRF) for an optimal stimulation of 1246-3A cell proliferation in serum-free medium.     

Epidermal growth factor (EGF) stimulates EGF receptor synthesis

Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis.     

Transforming growth factor-beta controls receptor levels for epidermal growth factor in NRK fibroblasts

NRK fibroblasts exposed to transforming growth factor-beta (TGF-beta) show increased binding of radiolabeled epidermal growth factor (EGF) relative to untreated cells. The binding of another growth factor, rat insulin-like growth factor-II, is unaffected. The increase in EGF binding induced by TGF-beta is not due to inhibition of EGF processing nor to an alteration in the affinity of plasma membrane EGF receptors. However, treatment of the cells with TGF-beta does cause a rapid increase in the number of plasma membrane receptors for EGF. TGF-beta has little effect on the rate of overall protein synthesis, but the increase it induces in EGF binding can be completely inhibited by cycloheximide and tunicamycin. Thus a selective synthetic mechanism underlies TGF-beta action. Cells incubated with TGF-beta also show altered down regulation of their EGF receptors in response to the ligand; concentrations of EGF that can induce strong biological responses no longer decrease the plasma membrane receptor level below the basal state. These results agree well with the known specificity and synergism of the interaction between TGF-beta and EGF. Moreover, they describe a mechanism of growth control in which bioactive peptides act coordinately through a regulatory effect on the number of cell-surface receptors.