Demonstration of transforming growth factor activity in mammary epithelial tissues

Transforming growth factor (TGF) activity has been demonstrated in acid-ethanol extracts of bovine mammary gland, of Ehrlich Ascites Mammary Carcinoma Cells, and of the ascites fluid. The extracts differ in their activity in the soft agar test when using either human or rat fibroblasts. The most active extract obtained from mammary gland tissue was chromatographed and the TGF activity shown to be coeluting with EGF receptor-competing activity. The present data and our previous reports show that TGFs and a growth inhibitor for mammary epithelial cells coexist in bovine mammary gland as separate growth factors.     

Hepatocyte growth factor induces tubulogenesis of primary renal proximal tubular epithelial cells

Hepatocyte growth factor (HGF)-induced tubulogenesis has been demonstrated with renal epithelial cell lines grown in collagen gels but not with primary cultured renal proximal tubular epithelial cells (RPTEs). We show that HGF selectively induces proliferation and branching morphogenesis of primary cultured rat RPTEs. Additional growth factors including fibroblast growth factor (FGF)-1, epidermal growth factor (EGF), FGF-7, or insulin-like growth factor-1 (IGF-1) did not selectively induce tubulogenesis. However, when administered in combination, these factors initiated branching morphogenesis comparable to HGF alone and greatly augmented HGF-induced proliferation and branching. Microscopic analysis revealed that branching RPTEs were undergoing tubulogenesis and formed a polarized epithelium. TGF-β1 blocked HGF- or growth factor cocktail (GFC; HGF, FGF-1, EGF, IGF-1)-induced proliferation and branching morphogenesis. Adding TGF-β1 after GFC-induced tubulogenesis had occurred caused a progressive regression of the tubular structures, a response associated with an increase in apoptosis of the RPTEs. Primary cultured RPTEs are capable of undergoing HGF-induced tubulogenesis. Unlike cell lines, combinations of growth factors differentially augment the response. J. Cell. Physiol. 180:81–90, 1999. © 1999 Wiley-Liss, Inc.     

Regulation of vimentin expression in cultured human mammary epithelial cells

Using five different monoclonal antibodies to vimentin, we have examined the expression of vimentin in cryostat sections and serum-free cultures of normal human breast tissue. In cryostat sections, myoepithelial cells as well as stromal cells showed immunoreactivity to vimentin, irrespective of the antibody used. In contrast, luminal epithelial cells were negative for vimentin, but positive for keratin K18. In culture, myoepithelial cells showed immunoreactivity to vimentin from their first appearance in monolayer. Moreover, a fraction of luminal epithelial cells expressed vimentin in addition to keratin K18. We found a clear, reversible correlation between proliferation, determined by incorporation of [3H]-TdR, and induction of vimentin in the luminal epithelial cells. Thus, in growth-stimulated cultures on a medium containing cholera toxin (CT), epidermal growth factor (EGF), transferrin (Tf), hydrocortisone (H) and insulin (I), the fraction of vimentin-positive luminal epithelial cells increased, while it decreased within 14 days from approximately 36% to 3% on a medium containing CT and EGF, only. We therefore conclude: (1) vimentin is constantly expressed in myoepithelial cells in situ and in vitro, and (2) expression of vimentin in luminal epithelial cells in vitro is not a result of monolayer cultivation as such, but rather associated with the increased growth rate seen in culture.     

Raf and RhoA cooperate to transform intestinal epithelial cells and induce growth resistance to transforming growth factor beta

Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) beta. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E2 levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGFalpha expression, or TGFbeta sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFbeta resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFbeta sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.     

Pre-clinical studies of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 (TG-C)

Background aimsThe aim was to evaluate cartilage regeneration in animal models involving induced knee joint damage. Through cell-mediated gene therapy methods, a cell mixture comprising a 3:1 ratio of genetically unmodified human chondrocytes and transforming growth factor beta-1 (TGF-β1)-secreting human chondrocytes (TG-C), generated via retroviral transduction, resulted in successful cartilage proliferation in damaged regions.MethodsNon-clinical toxicology assessments for efficacy, biodistribution and local/systemic toxicity of single intra-articular administration of the cell mixture in mice, rabbits and goats was conducted.ResultsAdministration of the mixture was tolerated well in all of the species. There was evidence of cartilage proliferation in rabbits and goats. As an additional precautionary step, the efficacy of TGF-β1 secretion in irradiated human chondrocytes was also demonstrated.ConclusionsFour studies in rabbits and goats demonstrated the safety and efficacy of TG-C following direct intra-articular administration in animal models involving induced knee joint damage. Based on these pre-clinical studies authorization has been received from the USA Food and Drug Administration (FDA) to proceed with an initial phase I clinical study of TG-C for degenerative arthritis.     

Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland

Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates cell proliferation, apoptosis and immune system responses. In the breast, the mammary epithelium is the primary source of TGFB1 and increased expression is associated with increased breast cancer risk. This study was conducted to investigate the roles of epithelial cell-derived TGFB1 in regulation of epithelial cell activity and macrophage phenotype in the mammary gland. Tgfb1 null mutant and wildtype mammary epithelium was transplanted into contra-lateral sides of the cleared mammary gland of TGFB1 replete scid mice. Transplanted tissue was analysed for markers of proliferation and apoptosis to determine the effect of Tgfb1 null mutation on epithelial cell turnover, and was analysed by immunohistochemistry to investigate the location, abundance and phenotype of macrophages. The number of proliferating and dying ductal epithelial cells, determined by BrdU and TUNEL, was increased by 35% and 3.3-fold respectively in mammary gland transplanted with Tgfb1 null epithelium compared to wildtype epithelium (p < 0.05). Abundance of F4/80+ macrophages in between Tgfb1 null epithelial cells compared to wildtype epithelial cells was increased by 50%. The number of iNOS+ and CCR7+ cells in the stroma surrounding Tgfb1 null alveolar epithelium was increased by 78% and 2-fold respectively, and dendriform MHC class II+ cells within ductal epithelium were decreased by 30%. We conclude that epithelial cell-derived TGFB1 in the mammary gland has two functions: (1) regulation of cellular turnover of epithelial cells, and (2) regulation of local macrophage phenotype. These findings shed new light on the diversity of roles of TGFB1 in the mammary gland which are likely to impact on breast cancer risk.     

Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells

CD44, a receptor for hyaluronan (HA), has been implicated in tumor growth and metastasis. Most CD44-positive cells fail to exhibit constitutive HA receptor function but CD44-mediated HA binding on hematopoetic cells can be induced by antibody cross-linking of the receptor and by physiologic stimuli, including cytokines. We now demonstrate that oncostatin M (OSM) and transforming growth factor-beta1, cytokines known to regulate the growth of tumor cells, stimulate HA binding in lung epithelial-derived tumor cells. In lung epithelial-derived tumor cells, cytokine-induced binding resulted from post-translational modification of the receptor. OSM-induced HA binding was associated with a reduction in N-linked carbohydrate content of CD44. In addition, OSM induced HA binding via a novel mechanism requiring sulfation of chondroitin sulfate chains linked to CD44. The mechanism underlying transforming growth factor-beta1 induced HA binding was distinct from the effects of OSM. The data presented indicate that modulation of the glycosylation and sulfation of CD44 by cytokines provides mechanisms for regulating cell adhesion during tumor growth and metastasis.     

Altered structure of the hybrid cell surface proteoglycan of mammary epithelial cells in response to transforming growth factor-beta

Transforming growth factor beta (TGF-beta) is a polypeptide growth factor that affects the accumulation of extracellular matrix by many cell types. We have examined the ability of mouse mammary epithelial (NMuMG) cells to respond to TGF-beta and assessed the effect of the growth factor on the expression of their cell surface heparan sulfate/chondroitin sulfate hybrid proteoglycan. NMuMG cells respond maximally to 3 ng/ml TGF-beta and the response is consistent with occupancy of the type III receptor. However, cells that are polarized, as shown by sequestration of the cell surface PG at their basolateral surfaces, must have the growth factor supplied to that site for maximal response. Immunological quantification of proteoglycan core protein on treated cells suggests that the cells have an unchanging number of this proteoglycan at their cell surface. Nonetheless, metabolic labeling with radiosulfate shows a approximately 2.5-fold increase in 35SO4-glycosaminoglycans in this proteoglycan fraction, defined either by its lipophilic, antigenic, or cell surface properties. Kinetic studies indicate that the enhanced radiolabeling is due to augmented synthesis, rather than slower degradation. Analysis of the glycosaminoglycan composition of the proteoglycan shows an increased amount of chondroitin sulfate, suggesting that the increased labeling per cell may be attributed to an augmented synthesis of chondroitin sulfate glycosaminoglycan on the core protein that also bears heparan sulfate, thus altering the proportions of these two glycosaminoglycans on this hybrid proteoglycan. We conclude that TGF-beta may affect NMuMG cell behavior by altering the structure and thus the activity of this proteoglycan.     

Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells

The growth of human epithelial cells is stimulated by cholera toxin and analogues of cyclic AMP, while the growth of breast derived fibroblasts is inhibited. These compounds have little effect on DNA synthesis in the absence of other mitogens but show a synergistic effect with serum and/or EGF. The results suggest that high intracellular levels of cyclic AMP in human mammary epithelial cells increase the growth response of the cell to mitogens.     

Thrombospondin-1-dependent mechanism of transforming growth factor beta-1 activation in cultured human endothelial cells

Using two immortal human umbilical vein endothelial-derived cell lines we investigated the thrombospondin-1 (TSP-1)-dependent mechanism of the transforming growth factor beta-1 (TGFbeta1) activation. Exogenous human platelet TSP-1 was demonstrated to increase (1.7-2.0-folds) the concentration of active TGFbeta1 produced by both cell lines, but did not influence the concentration of total TGFbeta1. The process of TGFbeta1 activation is known to be regulated by G418GWSHW423 fragment of TSP-1 molecule. The simultaneous addition of TSP-1 and 100-fold molar excess (about TSP-1) of the synthetic GGWSHW peptide abolished TSP-1-induced TGFbeta1 activation. GGWSHW peptide inhibited the basal activation of TGFbeta1 in endothelial cells, whereas the secretion of total TGFbeta1 and TSP-1 was not altered. We conclude that TSP-1-dependent mechanism of endogenous TGFbeta1 activation is operation in human endothelial cells.     

Initial phase I safety of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 in degenerative arthritis patients

Background aimsTissueGene-C (TG-C) represents a cell-mediated gene therapy for localized delivery of allogeneic chondrocytes expressing transforming growth factor (TGF)-β1 directly to the damaged knee joint. Untransduced human chondrocytes (hChonJ cells) have also been incorporated into the TG-C product at a 3:1 ratio with TGF-β1-expressing chondrocytes (hChonJb#7) in order to help fill in the defect and as target cells for the actions of the expressed TGF-β1.MethodsA phase I dose-escalating clinical trial was performed to evaluate the safety and biologic activity of TG-C in patients with advanced osteoarthritis of the knee joint (full thickness cartilage defect) that was refractory to existing non-operative therapies. Following a single intra-articular injection into the joint space of the damaged knee, patients were monitored for safety, and an evaluation was performed to assess the pharmacokinetics and biologic activity of TG-C.ResultsThere were no treatment-related serious adverse events. Swelling, effusion and minor localized reactions such as warming sensation or itching were observed in a dose-dependent manner at the injection site. Knee evaluation scores seemed to indicate a dose-dependent trend toward efficacy; however, patient numbers were not sufficient to determine statistical significance.ConclusionsOverall, there were no significant safety issues related to the administration of TG-C, with only some minor injection site reactions observed. Additionally, knee scoring analyzes indicated a possibility that TG-C may contribute to improvement of arthritic symptoms. More study is warranted to evaluate further the safety and determine the potential efficacy of TG-C.     

Induction of transforming growth factor alpha expression in mouse mammary epithelial cells after transformation with a point-mutated c-Ha-ras protooncogene

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.     

Response of cultured normal human mammary epithelial cells to X rays

The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique.     

Inhibition of proliferation of cultured rat liver epithelial cells at specific cell cycle stages by transforming growth factor-beta

Proliferation of early-passage propagable cultured rat liver epithelial cells derived from normal adult rats is markedly inhibited by transforming growth factor-beta (TGF-beta). Inhibition, which is completely reversible, is effected at two distinct points of the cell cycle, the G1/S border and the G0 or early G1 phase. With increasing passages in culture, hepatic epithelial cells progressively become less sensitive to the inhibitory effect of TGF-beta.     

Methionine protects against hyperthermia-induced cell injury in cultured bovine mammary epithelial cells

The aim of this study was to investigate the effects of methionine on cell proliferation, antioxidant activity, apoptosis, the expression levels of related genes (HSF-1, HSP70, Bax and Bcl-2) and the expression levels of protein (HSP70) in mammary epithelial cells, after heat treatment. Methionine (60 mg/L) increased the viability and attenuated morphological damage in hyperthermia-treated bovine mammary epithelial cells (BMECs). Additionally, methionine significantly reduced lactate dehydrogenase leakage, malondialdehyde formation, nitric oxide, and nitric oxide synthase activity. Superoxide dismutase, catalase, and glutathione peroxidase enzymatic activity was increased significantly in the presence of methionine. Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 h, and the expression level of the HSP70 gene and protein further increased with incubation at 42 °C for 30 min. Compared to the control, the expression of HSF-1 mRNA increased, and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress. Methionine also increased survival and decreased early apoptosis of hyperthermia-treated BMECs. Thus, methionine has cytoprotective effects on hyperthermia-induced damage in BMECs.