Growth regulation of primary rat tracheal epithelial cell cultures by endogenous transforming growth factor-βs

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to secrete transforming growth factor-β (TGFβ) and to be growth inhibited by exogenous TGFβ. The purpose of the present studies was to determine whether the endogenous TGFβ(s) were regulating the growth of RTE cell cultures and, if so, which isoforms were involved. Neutralizing antibodies specific to TGFβ1 and TGFβ2 were added to cultures, and their effects on several growth parameters were measured. Addition of antibodies to early cultures (day 1), resulted in 1.8-and 3-fold increases in colony formation and cell number, respectively, above control IgG-treated cultures. Antibody dose-response experiments revealed that TGFβ2 was the predominant isoform inhibiting early RTE cell growth. The antibody treatments resulted in similar stimulation of early growth at low and high seeding densities, suggesting that the endogenous TGFβs were acting locally. Anti-TGFβ1 treatment of cultures at various stages of growth resulted in 1.2–1.7-fold increases in DNA synthesis above controls, whereas anti-TGFβ2 treatment resulted in increased DNA synthesis only in early and late cultures (1.7- and 2.5-fold, respectively), but not during midlogarithmic growth. Continuous treatment with a combination of both antibodies resulted in increased growth and decreased exfoliation in early cultures, but had no effect on the slow down of growth in late cultures. Thus endogenous TGFβs inhibited primarily early growth and contributed to, but did not appear to be responsible for, plateau of growth in late stage cultures. Antibody treatment of secondary cultures resulted in 4–70-fold increases in colony formation, depending on the age of the primary cultures when replated, indicating that endogenous production of both TGFβ1 and TGFβ2 greatly inhibits the subculturability of primary RTE cells. Other experiments suggested that cholera toxin enhances RTE cell growth in part by counteracting the inhibitory effects of endogenous TGFβs. © 1993 Wiley-Liss, Inc.     

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.     

Role of TGF alpha and its receptor in proliferation of immortalized rat tracheal epithelial cells: studies with tyrphostin and TGF alpha antisera

We have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGF alpha and do so regardless of the mechanism by which they were transformed. In order to determine whether TGF alpha is an autocrine growth regulator of immortalized RTE cells, we have examined the function of TGF alpha/EGF receptors and the growth requirements for TGF alpha in these cells. The level of immunoprecipitated TGF alpha/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGF alpha/EGF receptors. Scatchard analysis of TGF alpha/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high-affinity (Kd = 0.4 nM) and low-affinity (Kd = 9.8 nM) binding sites. A tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor decreased in a dose-dependent manner the proliferation as well as EGF-induced autophosphorylation of the TGF alpha/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF-dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGF alpha antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGF alpha and its receptor.     

Role of receptor complexes in resistance or sensitivity to growth inhibition by TGFβ in intestinal epithelial cell clones

Untransformed rat intestinal epithelial cells (IEC-18) were chemically mutagenized, selected in the presence of TGFβ₁, and cloned by limiting dilution. Two clones (4–5, 4–6) were resistant to growth inhibition by both TGFβ₁ and TGFβ₂. Another clone (4–1) was more sensitive to both TGFβ isoforms (relative to parental IEC-18 cells). IC₅₀ values for TGFβ_{1 and 2} in the 4–1 cells were at least 1/9 those of the parental cells; growth rates were reduced by 49% for TGFβ₁ and by 26% for TGFβ₂ in this clone. This increased sensitivity to TGFβ was explained by the 5- to 10-fold increase, relative to parental cells, in binding of TGFβ₁ and TGFβ₂ to both the type I and II receptors. In contrast, the resistance to growth inhibition by TGFβ in the 4–5 and 4–6 cells could not be explained by a decrease in either TGFβ binding affinities or in total number of receptors expressed, by the presence of serum binding components, or by occupation of receptor binding sites with autocrine TGF-β₁. However, in comparison to TGFβ-sensitive cells (IEC-18, 4–1), the resistant cells displayed a higher ratio of type II relative to type I receptor binding by TGF-β₁. Thus, a critical ratio of binding to receptor subtypes correlated with growth inhibition by TGF-β₁. Resistance to TGF-β₂ in the same clones did not appear to be receptor related. Thus different mechanisms for resistance to TGF-β₁ and TGF-β₂ were observed within a given clone. © 1993 Wiley-Liss, Inc.     

The effect of epidermal growth factor and transforming growth factor β1 on proliferation and differentiation of urothelial cells in urinary bladder explant culture

The effects of two growth factors, EGF and TGFβ₁, on growth and differentiation of different populations of urothelial cells in explant cultures of mouse urinary bladder have been studied by electron microscopy and lectin analysis. In an explant culture 10 days after the implantation three different populations of urothelial cells can be distinguished. Original urothelial cells, which are integral part of the explant, new urothelial cells, which cover the baso-lateral sides of the explant and are organized in a multilayer epithelium, and new urothelial cells, which are not any more in direct contact with the explant and grow over the membrane in epithelium-like structure. Exogenously added EGF or TGFβ₁ did not affect either the formation or the thickness of multilayered urothelium, so cells were proliferating on the free surfaces of stroma as well as on the epithelium expanding over the membrane. In the absence of growth factors in medium, the newly formed baso-lateral multilayered epithelium bordering the stroma shows ultrastructural signs of terminal differentiation suggesting that for cell proliferation and differentiation the action of stroma is of crucial importance. On the other hand, the differentiation of the epithelium spreading over the membrane, but not its thickness, is affected by exogenously added TGFβ₁. Solely in TGFβ₁-treated cultures a differentiation sirnilar to that in vivo takes place. The apoptosis of the urothelial cells was not increased by TGFβ₁. The lectin analysis by WGA and ConA conjugated with ferritin revealed that ConA-ferritin combines only with the surface cells which grow over the membrane in the absence of TGFβ₁.     

Transforming growth factor β1 increases the phosphoenolpyruvate carboxykinase mRNA level in cultured rat hepatocytes

Transforming growth factor β1 (TGFβ1) elevated the phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in primary cultures of rat hepatocytes. Although this increase was not as large as the rise in PEPCK gene expression induced by the cAMP-elevating agents glucagon or isoproterenol, the effect of TGFβ1 was several-fold and concentration-dependent, with ED₅₀ at about 2.5 pM, which is in the same concentration range as the previously found growth-inhibitory effect of TGFβ. The data show that the level of mRNA for PEPCK, an enzyme typically expressed in the liver, can be regulated in the same direction by TGFβ1 and cAMP.     

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.     

Induction of Smooth Muscle Cell Phenotype in Cultured Human Prostatic Stromal Cells

Stromal cells are key regulators of growth and differentiation in the adult human prostate. Alterations in the stroma are believed to initiate the development of benign prostatic hyperplasia, and stromal–epithelial interactions may have a role in malignant progression. The prostatic stroma is composed of two major cell types, smooth muscle cells and fibroblasts. Cell cultures from the prostatic stroma have been established by several investigators, but the phenotype of these cells has not been extensively characterized and it is not clear whether they are fibroblastic or smooth muscle-like. In this study, the response of stromal cells cultured from normal prostatic tissues to transforming growth factor-β (TGFβ) was investigated. We confirmed a previous report that TGFβ inhibited the growth of prostatic stromal cells in serum-containing medium, and showed that inhibition also occurred in serum-free medium. Growth inhibition by TGFβ was irreversible after 24 to 72 h of exposure. In the absence of TGFβ, cells were fibroblastic and expressed vimentin and fibronectin but little α-smooth muscle actin. After 3 days of exposure to 1 ng/ml of TGFβ, the majority of cells expressed α-smooth muscle actin and desmin, as demonstrated by immunocytochemistry and immunoblot analysis. This effect was specific and α-smooth muscle actin was not induced by two other growth-inhibitory factors, retinoic acid or 1,25-dihydroxyvitamin D₃. These results suggest that TGFβ is an important regulator of growth and differentiation of prostatic stromal cells and that a smooth muscle cell phenotype is promoted in the presence of TGFβ.     

Secretion of rat tracheal epithelial cells induces mesenchymal stem cells to differentiate into epithelial cells

Bone marrow MSCs (mesenchymal stem cells) can differentiate into various tissue cells, including epithelial cells. This presents interesting possibilities for cellular therapy, but the differentiation efficiency of MSCs is very low. We have explored specific inducing factors to improve the epithelial differentiation efficiency of MSCs. Under inducing conditions, MSCs differentiated into epithelial cells and expressed several airway epithelial markers using RTE (rat tracheal epithelial) cell secretions. Rat cytokine antibody array was used to detect cytokines of the RTE secretion components, in which 32 kinds of protein were found. Seven proteins [TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), VEGF (vascular endothelial growth factor), BDNF (brain-derived neurotrophic factor), TGFβ1 (transforming growth factor β1), MMP-2 (metalloproteinases-2), OPN (osteopontin) and activin A in RTE secretions] were assayed using ELISA kits. The four growth factors (VEGF, BDNF, TGFβ1 and activin A) were involved in regulating stem cell growth and differentiation. We speculated that these four play a vital role in the differentiation of MSCs into epithelial cells by triggering appropriate signalling pathways. To induce epithelial differentiation, MSCs were cultured using VEGF, BDNF, TGFβ1 and activin A. Differentiated MSCs were characterized both morphologically and functionally by their capacity to express specific markers for epithelial cells. The data demonstrated that MSCs can differentiate into epithelial cells induced by these growth factors.     

Endothelium-dependent epithelial–mesenchymal transition of tumor cells: Exclusive roles of transforming growth factor β1 and β2

Background

Induction of epithelial–mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not.

Methods

Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor β (TGFβ) antibodies were used to neutralize TGFβ₁, TGFβ₂ and TGFβ₃. Expression and release of TGFβ proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively.

Results

Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFβ₁ and TGFβ₂ proteins and much lower level of TGFβ₃ protein. Conditioned medium from these endothelial cells contained TGFβ₁ and TGFβ₂, but TGFβ₃ could not be detected. Neutralizing antibody against each of TGFβ₁ or TGFβ₂ did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFβ₁ and TGFβ₂ completely abolished it.

Conclusions

Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFβ₁ and TGFβ₂.

General significance

The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium.     

High glucose induced endothelial cell growth inhibition is associated with an increase in TGFbeta1 secretion and inhibition of Ras prenylation via suppression of the mevalonate pathway

ObjectiveRas proteins are known to affect cellular growth and function. The influence of the prenylation status of Ras on the observed changes in endothelial cell growth under high glucose conditions has not previously been examined.MethodsHuman umbilical vein endothelial cells were exposed to normal or high glucose conditions for 72 h. They were then examined for proliferative and hypertrophic effects, transforming growth factor β₁ (TGFβ₁) release, and phosphorylated p38 expression. The importance of prenylation was explored by the addition of mevalonate, isoprenoids or farnesyltransferase inhibitors to control the high glucose media and by measuring changes induced by high glucose and exogenous TGFβ₁ in Ras prenylation and farnesyltransferase activity. Kidneys from diabetic rats treated with atorvastatin were also compared to specimens from untreated animals and the expression of the Ras effector p-Akt examined.ResultsHigh glucose conditions caused a reduction in cell number. This was reversed in the presence of mevalonate or farnesylpyrophosphate (FPP), suggesting that the cell growth abnormalities observed are due to high glucose induced inhibition of the mevalonate pathway and subsequent prenylation of proteins. Endothelial cells exposed to high glucose increased their secretion of TGFβ₁ and the phosphorylation of p38 both of which were reversed by concurrent exposure to FPP. A reduction in farnesyltransferase activity was observed after exposure to both high glucose and TGFβ₁. Exposure to a farnesyltransferase inhibitor in control conditions mimicked the growth response observed with high glucose exposure and prenylated Ras was reduced by exposure to both high glucose and TGFβ₁. Finally, interruption of the mevalonate pathway with a statin reduced the expression of p-Akt in diabetic rat kidneys.ConclusionThis study demonstrates that high glucose induced significant alterations in endothelial cell growth by inhibition of the mevalonate pathway, which subsequently mediates the increase in TGFβ₁ and inhibition of Ras prenylation.     

The anti-proliferative action of transforming growth factor-β1 on a rat intestinal epithelial cell line (rie-1) is dependent on cell population density and culture passage number

• 1.1. The proliferation of RIE-1 rat intestinal epithelial cells was potently but reversibly inhibited by transforming growth factor-β₁ (TGF-β₁). In early-passage cultures, complete growth arrest was observed when sparse cultures were treated with TGF-β₁ (1 ng/ml).
• 2.2. However, increasing the initial cell culture density resulted in decreased TGF-β₁-mediated inhibition of cell proliferation.
• 3.3. Independent of this population density effect, RIE-1 cells also exhibit a marked phenotypic transition around passage-8 to -10, such that later-passage cells were less responsive to growth inhibition by TGF-β₁].

     

Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGF beta

In this paper we examined the effects of transforming growth factor beta (TGF beta) on the proliferation and differentiation of rabbit tracheal epithelial cells in primary culture. Treatment of these cells with TGF beta inhibits cell proliferation in a time- and dose-dependent manner; concentrations as low as 1 pM are able to inhibit cell growth. Concomitantly, TGF beta causes cells to accumulate in the G0/G1 phase of the cell cycle and a sharp reduction in the ability of the cells to form colonies after subculture at clonal density. These results indicate that TGF beta induces terminal cell division in these cells. The inhibition of cell growth is accompanied by changes in cell morphology and a stimulation of the formation of cross-linked envelopes. TGF beta enhances the levels of transglutaminase activity and cholesterol sulfate, two markers of squamous differentiation. Our results indicate that TGF beta induces terminal squamous cell differentiation in rabbit tracheal epithelial cells. Retinoic acid (RA) does not affect the commitment to terminal cell division induced by TGF beta, but inhibits the expression of the squamous phenotype. Growth of normal human bronchial epithelial cells was affected by TGF beta in a way similar to that of rabbit tracheal epithelial cells. Several carcinoma cell lines tested were quite resistant to TGF beta, whereas growth of one carcinoma cell line was stimulated by TGF beta. These results indicate that a modified response to TGF beta could be one mechanism involved in the aberrant growth control of malignant cells.     

TGFβ induction of extracellular matrix associated proteins in normal and transformed human mammary epithelial cells in culture is independent of growth effects

We have previously characterized a human mammary epithelial cell (HMEC) culture system for the effects of TGFβ1 on cell growth. In the current report, the effects of TGFβ 1 on synthesis and secretion of proteins associated with the extracellular matrix and proteolysis were examined. In particular, we compared the TGFβ responses of normal finite lifespan HMEC, which are growth inhibited by TGFβ, to two immortally transformed cell lines derived from the normal HMEC. One of these lines maintains active growth in the presence of TGFβ and the other shows partial growth inhibition. In contrast to the differing effects of TGFβ on cell growth, we found that all these cell types showed strong induction of most of the mRNA and protein species examined, including fibronectin, collagen IV, laminin, type IV collagenase, urokinase type plasminogen activator (uPA), and plasminogen activator inhibitor 1 (PAI-1). The profile of TGFβ 1 binding proteins was the same in HMEC that were, and were not growth suppressed by TFGβ. Therefore, the effects of TGFβ on cell growth could be dissociated from its effects on specialized responses, indicating that within this one cell type there must be at least two independent pathways for TGFβ activity, one which leads to cessation of proliferation and one which induces a specific set of cellular responses. This cell system may be useful for examining the pathway of TGFβ induced growth inhibition using closely matched cells which vary in their growth-induced response but retain similar specialized responses to TGFβ. © 1993 Wiley-Liss, Inc.     

Heterobicyclic inhibitors of transforming growth factor beta receptor I (TGFβRI)

The TGFβ-TGFβR signaling pathway has been reported to play a protective role in the later stages of tumorigenesis via increasing immunosuppressive Treg cells and facilitating the epithelial to mesenchymal transition (EMT). Therefore, inhibition of TGFβR has the potential to enhance antitumor immunity. Herein we disclose the identification and optimization of novel heterobicyclic inhibitors of TGFβRI that demonstrate potent inhibition of SMAD phosphorylation. Application of structure-based drug design to the novel pyrrolotriazine chemotype resulted in improved binding affinity (Ki apparent?=?0.14?nM), long residence time (T_1/2?>?120?min) and significantly improved potency in the PSMAD cellular assay (IC₅₀?=?24?nM). Several analogs inhibited phosphorylation of SMAD both in vitro and in vivo. Additionally, inhibition of TGFβ-stimulated phospho-SMAD was observed in primary human T cells.     

Potential role of follicle-stimulating hormone (FSH) and transforming growth factor (TGFβ1) in the regulation of ovarian angiogenesis

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)β1 is known to facilitate FSH‐induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFβ1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin‐primed immature rats and treated once with FSH and/or TGFβ1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFβ1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFβ1 > FSH ≈ TGFβ1 > control) that was partly attributed to the increased secretion of pro‐angiogenic factors VEGF and PDGF‐B. This is further supported by the evidence that pre‐treatment with inhibitor of VEGF receptor‐2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2‐day aortic ring culture period suppressed microvessel growth in GCCM‐treated groups, and also inhibited the FSH + TGFβ1‐GCCM‐stimulated release of matrix remodeling‐associated gelatinase activities. Interestingly, pre‐treatment of AG1296 at late stage suppressed GCCM‐induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFβ1 increased the secretion of VEGF and PDGF‐B, and that in turn up‐regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFβ1 play important roles in regulation of ovarian angiogenesis during follicle development. J. Cell. Physiol. 226: 1608–1619, 2011. © 2010 Wiley‐Liss, Inc.     

Transforming growth factor beta 1-specific binding proteins on human vascular endothelial cells.

Transforming growth factor beta (TGF beta) regulates the growth of human umbilical vein endothelial cells (HUVEC) differently depending on the isoform of TGF beta and the culture conditions. The cells are resistant to growth inhibition by TGF beta when the cells are cultured on substratum coated with gelatin. However, the proliferation of HUVEC cultured on substratum without a gelatin coating is inhibited by TGF beta, depending on the isoform and concentration of TGF beta. Binding assays with 125I-TGF beta 1 reveal that HUVEC contain a single class of high-affinity (Kd = 4.4 pM) TGF beta 1 binding sites with 8500 sites per cell. Affinity cross-linking studies demonstrate that HUVEC express 180 and 80 kDa TGF beta 1 binding sites that do not bind TGF beta 2. The reduction and the removal of glycosaminoglycans does not affect the electrophoretic mobility of the 180-kDa binding protein cross-linked with 125I-TGF beta 1. Therefore, the 180-kDa TGF beta 1 binding protein is not related to the type III TGF beta receptor, but might be a novel TGF beta 1-specific receptor/binding protein expressed on vascular endothelial cells. The expression of TGF beta 1 binding sites is not affected by the presence or absence of the gelatin coating on the culture substratum. The data suggest that a gelatin coating does not regulate the susceptibility of HUVEC to TGF beta 1 at the level of the receptor/binding proteins, and that growth inhibition of HUVEC by TGF beta 1 is linked to the regulation of extracellular matrices required for the interaction between the cells and the substratum.     

Divergent action of transforming growth factor beta on DNA synthesis in human foetal liver cells

Purified human transforming growth factor beta (TGF beta) was found to be a potent inhibitor of DNA synthesis in human foetal hepatocytes. Half-maximal inhibition occurred at 0.5-1 pM and was reversible, with an increase in DNA synthesis within 24 h following removal of TGF beta. By contrast, in the same cultures, 'fibroblast-like' non-hepatocytes retained the ability to synthesize DNA in the presence of up to 200 fold higher doses of TGF beta. This differential response to TGF beta suggests that it may act as an important cell growth regulator in the human foetal liver.