Activation of Nonexpressed Endogenous Ecotropic Murine Leukemia Virus by Transfection of Genomic DNA into Embryo Cells

We studied the infectivity of endogenous ecotropic murine leukemia virus genomes contained in high-molecular-weight DNA prepared from virus-free cells of the AKR-2B line, and from RF, BALB/c, B6, and (BALB/c x B6)F(1) mouse embryo cells. When DNA prepared from virus-free AKR-2B cells was transfected into NIH-3T3 cells, no virus-positive cultures were observed, a result consistent with previous reports. However, when DNAs from virus-free AKR-2B cells or virus-free cells containing the RF/J or BALB/c ecotropic proviruses were transfected into chicken embryo cells that were then cocultivated with SC-1 (mouse) cells, virus-positive cultures were recovered. The specific infectivities of the AKR provirus(es) contained in virus-free cells and the molecularly cloned Akv-1 provirus were similar when chicken embryo cells were used as primary recipients. Virus-positive cultures were also observed when secondary mouse embryo cells were used as recipients for DNA from virus-free AKR-2B and RF/J cells. The transfected chicken embryo-SC-1 cultures produced XC-positive murine leukemia virus that is N-tropic. Virus-positive recipient cultures were observed 10- to 100-fold more frequently when AKR-2B DNA was used than when BALB/c DNA was used as the donor DNA. Our studies indicate that some nonexpressed ecotropic murine leukemia virus proviruses are activated upon transfection into chicken embryo cells. Such studies suggest that there are different factors governing the expression of murine leukemia virus after transfection into established cell lines (NIH-3T3) and into nonestablished secondary cultures (chicken and mouse).     

Bifunctional activity of transforming growth factor type beta on the growth of NRK-49F cells, normal and transformed by Kirsten murine sarcoma virus

Transformation of rat NRK-49F cells (49F) by Kirsten murine sarcoma virus (Ki-MSV) renders these cells (Ki-49F cells) capable of autonomous anchorage independent (AI) growth. As compared to nontransformed 49F cells, the transformation by Ki-MSV does not modify the cell response to transforming growth factor-beta (TGF-beta) in monolayer conditions, but alters it in A I growth conditions. The growth of nontransformed or Ki-MSV-transformed adherent 49F cells is slowed down by porcine TGF-beta, and this effect is reversed by epidermal growth factor (EGF). This decrease in the cell growth rate, induced by TGF-beta, does not affect the cloning efficiency of untransformed and transformed adherent 49F cells. Contrarily, porcine TGF-beta decreases the A I cloning efficiency of Ki-49F cells in agar-gelled medium; this effect is only partly reversed by EGF, which does not synergise with TGF-beta to enhance the A I growth as in the case of untransformed 49F cells. Media conditioned by 49F cells, Ki-49F cells, and chicken embryo fibroblasts contain a latent TGF-beta whose capacity to promote the A I growth of 49F cells and to inhibit that of Ki-49F cells is unmasked by acidification. The same situation exists concerning TGF-beta from human platelets. Neutral extracts are inefficient in both tests of promotion and inhibition of A I growth and contain an acid-activable component with an apparent molecular weight of 600 kd. In acid extracts, a 5-9 kd apparent molecular weight component is responsible for the A I growth enhancement of 49F cells and the A I growth inhibition of Ki-49F cells. Further purification by reverse phase chromatography shows that both activities strictly coelute at the same point (32%) of an acetonitrile gradient. These results indicate that TGF-beta is present in physiological conditions as a latent form which requires activation for inhibiting the A I growth of transformed cells as well as for enhancing that of 49F cells.     

Cells transformed by Rous sarcoma virus release transforming growth factors

Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage-independent colony formation in gelified medium, at the restrictive temperature (41 degrees 5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.     

Tissue-type plasminogen activator acts as a cytokine that triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression

Tissue-type plasminogen activator (tPA), a serine protease well known for generating plasmin, has been demonstrated to induce matrix metalloproteinase-9 (MMP-9) gene expression and protein secretion in renal interstitial fibroblasts. However, exactly how tPA transduces its signal into the nucleus to control gene expression is unknown. This study investigated the mechanism by which tPA induces MMP-9 gene expression. Both wild-type and non-enzymatic mutant tPA were found to induce MMP-9 expression in rat kidney interstitial fibroblasts (NRK-49F), indicating that the actions of tPA are independent of its proteolytic activity. tPA bound to the low density lipoprotein receptor-related protein-1 (LRP-1) in NRK-49F cells, and this binding was competitively abrogated by the LRP-1 antagonist, the receptor-associated protein. In mouse embryonic fibroblasts (PEA-13) lacking LRP-1, tPA failed to induce MMP-9 expression. Furthermore, tPA induced rapid tyrosine phosphorylation on the beta subunit of LRP-1, which was followed by the activation of Mek1 and its downstream Erk-1 and -2. Blockade of Erk-1/2 activation by the Mek1 inhibitor abolished MMP-9 induction by tPA in NRK-49F cells. Conversely, overexpression of constitutively activated Mek1 induced Erk-1/2 phosphorylation and MMP-9 expression. In mouse obstructed kidney, tPA, LRP-1, and MMP-9 were concomitantly induced in the renal interstitium. Collectively, these results suggest that besides its classical proteolytic activity, tPA acts as a cytokine that binds to the cell membrane receptor LRP-1, induces its tyrosine phosphorylation, and triggers intracellular signal transduction, thereby inducing specific gene expression in renal interstitial fibroblasts.     

Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor

Medium conditioned by BRL-3A cells, a known source of insulin-like growth factor II (IGF-II), induced phenotypic transformation (anchorage-independent proliferation) of mouse BALB/c 3T3 fibroblasts but not rat NRK-49F fibroblasts, in the presence of 10% calf serum. A specific radioreceptor assay and a bioassay indicated that BRL-3A conditioned medium contained 0.5-1 ng/ml of type beta transforming growth factor (beta TGF). Purified IGF-II and beta TGF acting together reconstituted the transforming activity of BRL-3A conditioned medium on BALB/c 3T3 cells. Insulin was 5-10% as potent as IGF-II in supporting the transforming action of beta TGF on BALB/c 3T3 cells. NRK-49F cells were phenotypically transformed by beta TGF in the presence of EGF and 10% calf serum as the sole source of IGFs. However, transformation of NRK-49F cells under these conditions was inhibited by addition of purified IGF-binding protein. Addition of an excess of IGF-II prevented the inhibitory action of IGF-binding protein. The different sensitivity of the two cell lines to IGFs was correlated with lower levels of type I IGF receptor and higher levels of type II IGF receptor in NRK-49F cells as compared with BALB/c 3T3 cells. The results suggest that cellular stimulation by IGFs is a prerequisite for transformation of rodent fibroblasts by beta TGF. We propose that transformation of fibroblasts by beta TGF requires concomitant stimulation by the set of growth factors that support normal cell proliferation.     

Conversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions

A latent beta-TGF activity is spontaneously released into serum-free culture medium by chicken embryo fibroblasts. Anchorage-independent growth activity measured on NRK-49F indicator cells, of this latent beta-TGF can be revealed by four different treatments: acidification, alkalinisation, exposure to urea, and heating to 100 degrees C for 3 minutes. This lact activating treatment indicates that latent beta-TGF activation in vitro is non-enzymatic. Active beta-TGF exists in a low molecular weight form 16 Kd (apparent) in 1M acetic acid, which elutes on reverse phase (FPLC) between 33-35% acetonitrile. Under neutral conditions only a high molecular weight form excluded on Biogel P60 is observed. This form is poorly active on NRK-49F for anchorage independent growth but can be fully activated by prior acidification. Rechromatography of the latent beta-TGF-containing fractions under acidic conditions converts the high molecular weight form to an apparent 16 Kd active form. We suggest that the high molecular weight form may correspond to a complex of a beta-TGF associated with a carrier or binding protein.     

Fibroblast growth factor induces the soft agar growth of two non-transformed celllines

Summary Recent studies have determined that fibroblast growth factor (FGF) potentiates the soft agar growth responses of NRK-49F cells to several combinations of transforming growth factors (TGFs). In the current study, two other non-transformed cell lines, NR-6 and AKR-2B, which do not spontaneously form colonies in soft agar, were examined for their soft agar growth responses to FGF. Both the acidic form and basic form of FGF were found to induce the soft agar growth of these cells. In the case of NR-6 cells, the effects of TGF-β were also examined. TFG-β potentiated the soft agar growth response of NR-6 cells to FGF, but on its own did not induce soft agar growth. Attempts to identify other factors capable of modulating the response of these cells to FGF, led to the finding that both 12-0-tetra-decanoylphorbol-13-acetate and retinoic acid suppress FGF-induced soft agar growth of NR-6 cells and AKRR-2B cells. The finding that FGF induces the soft agar growth of both non-transformed cell lines, together with the findings of others that both forms of FGF are angiogenic, lends further support to the suggestion that FGF plays a significant role in the in vivo growth of some, and possibly many, tumors. This work was supported by grants from the Nebraska Department of Health (86-11R, 87-38), the National Institute of Child Health and Human Development (HD 19837, HD 21568) and the National Cancer Institute (Laboratory Cancer Research Center Support Grant CA 36727). Editor's Statement The last several years have seen extraordinary advances in the understanding of the biochemistry and physiology of heparin-binding growth factors. Among the activities of these peptides that may be of significance for neoplasia and wound healingin vivo is ability to promote anchorage independent growth of some cell types. In this study the interactions among several stimulatory and inhibitory factors are examined in a soft agar growth assay. An appreciation of these interactions is critical in attempts to relatein vitro effects to those in the intact organism.     

Production and significance of TGF-beta in AT-3 metastatic cell line established from the Dunning rat prostatic adenocarcinoma

A colony formation assay using NRK-49F cells revealed that a metastatic cell line, AT-3, established from the Dunning prostatic carcinoma could produce TGF-beta in a latent form. TGF-beta at a concentration as low as 0.05 ng/ml either stimulated the attachment or detachment of AT-3 cells depending on the kind of culture media. Acid extracts from conditioned medium (5 micrograms/ml) showed the activity comparable to that of TGF-beta (5 ng/ml). The detached cells were able to grow in suspension. TGF-beta (0.1 ng/ml) could also stimulate the growth of MC3T3-El osteoblasts established from mouse calvaria. These results suggest that TGF-beta is a key growth factor for osteoblastic bony metastasis of prostate cancer.     

Autocrine growth regulation of W12 and GCA cells in culture

Two rat kidney cell lines transformed by two strains of ASV virus were investigated. It was demonstrated that these two lines (1) showed density-independent growth, (2) had a decreased requirement for serum in the culture medium, (3) had the ability to grow in a chemically defined medium (without serum), and the rate of this growth had increased with the increase in starting density of cells, and (4) had the ability of anchrage-independent growth, even without serum. These results confirmed autostimulation of growth of W12 and GCA cells. It was also shown that the crude conditioned media contained autocrine growth factors, which could be extracted with 1M acetic acid. The extracts (AEs) stimulated the growth of the parental cells and NRK-49F cells almost as well as 5% calf serum and the extraction resulted in several-fold purification of mitogenic substances. These substances were not only specific to parental lines, but also stimulated growth of other transformed lines and normal NRK-49F cells. Extracts from the conditioned media of W12 and GCA cells intensified the rate of anchorage-independent growth in the concentration-dependent manner. In AE-W12, two peaks of mitogenic activity were detected (F1, F2) and similarly in AE-GCA (F3, F4). Fractions F2 (approximately 8 kDa), F3 (approximately 25 kDa) and F4 (approximately 12 kDa) were thermostable but F1 (approximately 45 kDa) was thermolabile. All four fractions were sensitive to trypsin and DTT treatment, and were acid-stable. Using ELISA kit it was shown that W12 and GCA cells released TGFbeta1 and GCA cells released very small quantities of bFGF. These results confirmed the autocrine regulation of growth in both cell lines.     

Supplementary factors required for serum-free culture of rat kidney cells of line NRK-49F

Factors required as supplements to basal tissue culture medium for the multiplication of cells of the cloned rat fibroblast line called normal rat kidney 49F (NRK-49F) were identified as epidermal growth factor, fibronectin, insulin, and retinoic acid. The requirement for fibronectin was manifested on a clean glass surface but not on the polystyrene plastic surface tested. This set of required factors differs substantially from the factor sets required by the Madin-Darby canine kidney (MDCK) and LLC-PK1 pig kidney lines of epithelial cells and the baby hamster kidney 21 (BHK-21) line of fibroblasts. The serum-free medium supplemented with the four factors supported rapid growth of NRK-49F cells when the initial cell population density was about 8,000 cells/cm2 or greater. At lower initial densities, cell multiplication was markedly increased by adding serum-free medium that had been conditioned by NRK-49F cells. Cell growth rate in the defined serum-free medium stayed high through two serial passages but declined in the third serial passage unless the cell-conditioned medium was added.     

Development of a retroviral vector for inducible expression of transforming growth factor beta 1.

A retroviral vector system for the expression of exogenous genes under the control of an inducible promoter was developed. By utilizing this system, the cDNA for human transforming growth factor beta 1 (TGF-beta 1) was inserted into a retroviral vector under the control of an internal mouse metallothionein promoter and introduced via infection into normal rat kidney fibroblasts (NRK-49F) and epithelial cells (NRK-52E), Chinese hamster ovary cells (CHO), and the human monocytic cell line U937. Control of TGF-beta 1 expression, achieved by Cd2+ induction of vector-encoded TGF-beta 1 mRNA, was cell line specific and resulted in a concomitant increase in neutralizable TGF-beta 1 production by the cells. Autocrine stimulation of vector-containing cells by vector-encoded TGF-beta 1 was detected by an increase in soft-agar colony formation of NRK-49F infectants compared with that of the control cells. In addition, the use of a second internal promoter in a retroviral vector of similar design allowed isolation of stable infectants from a cell line (CHO) in which the viral long terminal repeat does not function efficiently.     

Purification and characterisation of a growth factor from porcine bone

A growth factor was extracted from porcine bone matrix by demineralisation and purified by heat and acid treatment, hydroxyapatite chromatography and gel filtration under dissociative conditions and reverse-phase HPLC. Using the mitogenic response of osteoblast-progenitor cells from embryonic chicken, a mitogenic activity was purified 3000-fold. The mitogenic protein thus purified shows an apparent molecular mass of 13.5 kDa in both the nonreduced and reduced form on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The mitogenic activity is sensitive to proteinase K, dithiothreitol, and resistant to DNAse, RNase, heat (70 degrees C) and pH (3-10). The factor stimulates the proliferation of osteoblast-progenitor cells from embryonic chick at a concentration of 1 ng/ml. It is active on cells from skin, periosteum and sternum and has no or little activity on cells of the calvaria, intestine or kidney of embryonic chick or on mouse AKR-2B/Balb c/3T3 cell line.     

Adenomatous polyposis coli localization is both cell type and cell context dependent

The adenomatous polyposis coli (APC) tumor suppressor protein is mutated in most colorectal carcinomas. In addition to its role in WNT signaling it is proposed to be involved in both cell migration and mitosis. Although a variety of studies have shown an APC localization along lateral membranes of adjacent epithelial cells the existence of a cortical APC localization in mammalian cells remains controversial. To address this we have used matched rat epithelial (NRK-52E) and fibroblast (NRK-49F) cell lines to investigate the localization of APC. Subconfluent cultures of NRK-52E and -49F cells displayed microtubule-associated APC populations by immunostaining. However, confluent NRK-52E, but not -49F monolayers, exhibited a cortical APC distribution. Cortical APC localized in close proximity to a number of cell junction proteins in a microtubule-independent manner while calcium switch experiments suggested that APC was recruited to the cortex only when junction assembly was complete. Confluent NRK-49F and -52E cells also showed contrasting APC localizations in response to monolayer wounding. Our data suggests APC cortical localization is a feature of confluent epithelioid cells and that the subcellular distribution of APC is therefore dependent upon both cell type and context.     

Supplementary factors required for serum-free culture of rat kidney cells of line NRK-49F

Summary Factors requred as supplements to basal tissue culture medium for the multiplication of cells of the cloned rat fibroblast line called normal rat kidney 49F (NRK-49F) were identified as epidermal growth factor, fibronectin, insulin, and retinoic acid. The requirement for fibronectin was manifested on a clean glass surface but not on the polystyrene plastic surface tested. This set of required factors differs substantially from the factor sets required by the Madin-Darby, canine kidney (MDCK) and LLC-PK₁ pig kidney lines of epithelial cells and the baby hamster kidney 21 (BHK-21) line of fibroblasts. The serum-free medium supplemented with the four factors supported rapid growth of NRK-49F cells when the initial cell population density was about 8,000 cells/cm² or greater. At lower initial densities, cell multiplication was markedly increased by adding serum-free medium that had been conditioned by NRK-49F cells. Cell growth rate in the defined serum-free medium stayed high through two serial passages but declined in the third serial passage unless the cell-conditioned medium was added.     

Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo

Unilateral ischemia reperfusion injury (UIRI) with longer ischemia time is associated with an increased risk of acute renal injury and chronic kidney disease. Exosomes can transport lipid, protein, mRNA, and miRNA to corresponding target cells and mediate intercellular information exchange. In this study, we aimed to investigate whether exosome-derived miRNA mediates epithelial-mesenchymal cell communication relevant to renal fibrosis after UIRI. The secretion of exosomes increased remarkably in the kidney after UIRI and in rat renal tubular epithelium cells (NRK-52E) after hypoxia treatment. The inhibition of exosome secretion by Rab27a knockout or GW4869 treatment ameliorates renal fibrosis following UIRI in vivo. Purified exosomes from NRK-52E cells after hypoxia treatment could activate rat kidney fibroblasts (NRK-49F). The inhibition of exosome secretion in hypoxic NRK-52E cells through Rab27a knockdown or GW4869 treatment abolished NRK-49F cell activation. Interestingly, exosomal miRNA array analysis revealed that miR-150-5p expression was increased after hypoxia compared with the control group. The inhibition of exosomal miR-150-5p abolished the ability of hypoxic NRK-52E cells to promote NRK-49F cell activation in vitro, injections of miR-150-5p enriched exosomes from hypoxic NRK-52E cells aggravated renal fibrosis following UIRI, and renal fibrosis after UIRI was alleviated by miR-150-5p-deficient exosome in vivo. Furthermore, tubular cell-derived exosomal miR-150-5p could negatively regulate the expression of suppressor of cytokine signaling 1 to activate fibroblast. Thus, our results suggest that the blockade of exosomal miR-150-5p mediated tubular epithelial cell-fibroblast communication may provide a novel therapeutic target to prevents UIRI progression to renal fibrosis.     

Differential regulation of the expression of transforming growth factor-beta s 1 and 2 by retinoic acid, epidermal growth factor, and dexamethasone in NRK-49F and A549 cells

Although most biological activities of transforming growth factor-beta s 1 and 2 (TGF-beta 1 and TGF-beta 2) examined in vitro are similar or identical, recent studies suggest that each of these factors may be independently regulated in vivo. In this study we have used highly sensitive and specific sandwich enzyme-linked immunosorbent assays for TGF-beta 1 and TGF-beta 2 to examine the effects of a variety of treatments on expression of these two TGF-beta isoforms. We show that epidermal growth factor (EGF) induces secretion of TGF-beta 1 and not TGF-beta 2, whereas retinoic acid (RA) induces secretion of TGF-beta 2 and not TGF-beta 1 in NRK-49F normal rat kidney fibroblasts and A549 human lung carcinoma cells. Moreover, treatment with EGF diminishes the levels of TGF-beta 2, while RA decreases the levels of TGF-beta 1 in both cell lines. Dexamethasone (Dex), on the other hand, inhibits the secretion of both TGF-beta 1 and TGF-beta 2 in A549 cells, while selectively inhibiting TGF-beta 1 secretion in NRK-49F cells. The interactive effects of EGF, RA, and Dex on the production of TGF-beta 1 and TGF-beta 2, which were studied on NRK-49F cells, demonstrate that EGF blocks the induction of TGF-beta 2 mRNA and peptide by RA, while Dex inhibits the induction of TGF-beta 1 mRNA and peptide by EGF. These results demonstrate that RA, EGF and Dex are each unique, differential, and interactive regulators of the expression of TGF-beta s 1 and 2.     

Cadmium induces hypertrophy accompanied by increased myc mRNA accumulation in NRK-49F cells

Previous studies showed that Cd⁺⁺ inhibits EGF-induced DNA synthesis that not EGF-induced myc mRNA accumulation or amino acid incorporation into protein in serum-starved NRK-49F cells. In this study, flow cytometry was used to analyze the DNA and protein content of individual cells stimulated with Cd⁺⁺ and/or epidermal growth factor (EGF). myc oncogene expression in these cells was also measured. It was found that, in both parental NRK-49F cells and in a clonal subpopulation, N1, Cd⁺⁺ induces an hypertrophic response. In parental NRK-49F cells, however, lower doses of Cd⁺⁺ (0.5 M) induced more pronounced hypertrophic responses than did higher doses (4 M); whereas in N1 cells, the Cd⁺⁺-induced hypertrophic response shows a pattern of increasing response with doses of Cd⁺⁺ from 0.5 to 4 M. myc mRNA accumulation measured 2 hours after stimulation correlated with the hypertrophic responses in both NRK-49F cells and in N1 cells. The results show that Cd⁺⁺-induced hypertrophy in NRK-49F cells is associated with increased myc oncogene mRNA accumulation, indicating that cell proliferation and cell hypertrophy may in part share common activation pathways.Abbreviations EGF Epidermal growth factor - FCM Flow cytometric analysis - FITC fluorescein isothiocyanate - PI propidium iodide     

Specific induction of secreted proteins by transforming growth factor-beta and 12-O-tetradecanoylphorbol-13-acetate. Relationship with an inhibitor of plasminogen activator

To examine the mechanisms by which transforming growth factors (TGFs) regulate the proliferation of eukaryotic cells, five cell lines, from different species and tissues, were treated with three agents that inhibit DNA synthesis and proliferation: BSC-1 cell-derived growth inhibitor (GI/TGF-beta), platelet-derived transforming growth factor-beta (TGF-beta), and 12-O-tetradecanoylphorbol-13-acetate. The cell lines tested were mink lung CCL 64 epithelial cells, Maloney sarcoma virus-transformed CCL 64.1, monkey kidney BSC-1 epithelial cells, human epidermoid A431 cells, and mouse embryo AKR-2B (clone 84A) cells. All cell lines responded to one or more of these agents by synthesizing and secreting a 48 to 51-kDa protein (IIP48). The TGF-beta s and 12-O-tetradecanoylphorbol-13-acetate had little or no effect on the incorporation of [35S] methionine into other secreted proteins or on the pattern of [35S]methionine-labeled intracellular proteins analyzed by one-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maximum increase in induction of IIP48 varied from 2-fold to greater than 800-fold compared with the controls and occurred within 6 h of adding GI/TGF-beta to CCL 64 cells. Actinomycin D, alpha-amanitin, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole selectively decreased both the control and induced levels of IIP48 even after as little as 6 h of incubation. Thus, it appears that IIP48 mRNA turns over rapidly. Induction of IIP48 was dissociated from the inhibition of DNA synthesis by GI/TGF-beta. However, we found that epidermal growth factor and GI/TGF-beta act synergistically to increase the secreted level of IIP48. Others have shown that epidermal growth factor and TGF-beta act synergistically to stimulate growth of cells in agar. IIP48 from CCL 64, BSC-1, and AKR-2B cells is specifically immunoprecipitated by antibody to bovine plasminogen activator inhibitor. We found previously that TGF-beta also inhibits the production of major excreted protein, a thiol protease. It is proposed that TGF-beta is able to promote anchorage-independent growth of untransformed cells because of its ability to inhibit the production of secreted proteases and to increase the production of protease inhibitors.