Unusual antiproliferative effects of transforming growth factors-β 1 andβ 2 against primary cells from human tumors

Transforming growth factors 1 and2 (TGF-1 and2), tested in a clonogenic assay against primary cells from human tumors, suppress proliferation to different extents. In nineteen of twenty-six cell cultures, proliferation was < 50% of control with factor at 0.04 or 0.4 nM. Of these, TGF- 2 was more active than TGF-1 in fourteen; and TGF-1 was more active than TGF-2 in five. In seven of the nineteen, proliferation was 0% with one or the other factor. In contrast, cisplatin was much less effective in inhibiting proliferation of some of the same cells even at 1,000 or more times the molar concentration of the factors. Surprisingly, when TGF- 1 and TGF-2 were combined at equal concentrations, the antiproliferative effect of one was cancelled or markedly inhibited by the other.     

Localization of Transforming Growth Factor β in Association with Neuromuscular Junctions in Adult Human Muscle

Transforming growth factors- 1, 2, and 3 are known for their regulatory function in embryogenesis, fibrogenesis, and tissue repair of different cell types. A trophic function of TGF- subclasses for motoneurons has been shown in vitro. TGF- 1 is a potent survival factor for cultured embryonic rat motoneurons. In addition, TGF- 1 stimulates proliferation of rat Schwann cells. Recently, TGF- 2 has been reported to be associated with the subsynaptic nuclei of mature rat neuromuscular junctions. In this study, we investigated the expression of TGF- 1, 2, and 3 at neuromuscular junctions in skeletal muscle of 11 adults without neuromuscular disease. On muscle biopsies, neuromuscular junctions were depicted by acetylcholine esterase reaction and acetylcholine receptor antibodies. TGF- 1, 2, and 3 were stained immunohistochemically with monoclonal antibodies. Some muscle fibers showed low levels of inhomogeneous immunoreactivity for both TGF- 1 and TGF- 3. Intense immunoreactivity of TGF- 1 and 3 was shown at the postsynaptic area of neuromuscular junctions. TGF- 2 was expressed in the same subcellular distribution, but less strongly. In conclusion, the colocalization of TGF- with neuromuscular junctions may suggest a significant function in neuromuscular communication.     

Comparative Functional Analysis of Rat <Emphasis Type="Italic">TGF-β1</Emphasis> and <Emphasis Type="Italic">Xenopus laevis</Emphasis><Emphasis Type="Italic">TGF-β5</Emphasis> Promoters Suggest Differential Regulations

We have carried out a comparative functional analysis of the rat TGF-1 and Xenopus laevis TGF-5 promoters across several mammalian and amphibian cell lines. Progressive deletion constructs of both the promoters have been made using a PCR based approach and the basal promoter activities studied in Xenopus tadpole cell line (XTC), Xenopus adult kidney fibroblast cell line (A6), human hepatoma cell line (HepG2), normal rat kidney cell line (NRK), and Chinese hamster ovary cell line (CHO). Data suggests that the basal promoter activity of TGF-1 is low as compared to TGF-5 promoter in XTC cells but comparable in A6 cells, while TGF-5 promoter shows nearly negligible activity as compared to TGF-5 promoter in all the tested mammalian cell lines. Moreover, TGF-5 promoter is found to be repressed in XTC cells on treatment with TGF-5 protein. Thus, the regulation of TGF-1 and TGF-5 promoters is distinct in amphibian and mammalian species. We therefore suggest that contrary to the suggested functional equivalence of TGF-1 and TGF-5 proteins, TGF-1 and TGF-5 genes have distinct functions in their respective species. Present address (Kartiki V. Desai): Laboratory of Cell Regulation and Carcinogenesis, NCI, NIH Bldg 41, Room C619, Bethesda, MD 20892, USA     

Effect of transforming growth factor beta (TGF-β) on ATP citrate lyase in isolated hepatocytes

Summary Transforming growth factor beta (TGF-) activates ATP citrate lyase in freshly isolated rat liver hepatocytes in a time dependent manner. Maximal stimulation of the enzyme occurred with less than thirty minutes of incubation of the cells with TGF-. The half maximal effect on the enzyme determined in hepatocytes incubated with TGF- for 10 min at 37°C was elicited by TGF- concentrations in the 10^–11 – 10^–12 M range. The potential role of TGF- stimulation of ATP citrate lyase activity in new membrane synthesis is discussed.     

Cellular origin and distribution of transforming growth factor-β1 in the normal rat myocardium

Summary Transforming growth factor- (TGF-) is a biologically active polypeptide present in normal tissues as well as transformed cells. Two structurally related forms of this peptide are TGF- ₁ and TGF- ₂. Using freshly isolated cardiomyocytes and non-myocyte heart cells, and a [³²P]-labelled cDNA probe to human TGF- ₁, we demonstrated that mRNA for TGF- ₁ could be detected only in the nonmyocyte fraction of heart cells. In the present study, the distribution of TGF- ₁ in the heart was determined by immunofluorescence staining by use of a polyclonal antibody to porcine TGF- ₁ in cryostat sections of rat heart. Immunofluorescence staining was intense around the blood vessels and radially diffuse in the surrounding myocardium.     

Bcl-2 blocks apoptotic signal of transforming growth factor-β in human hepatoma cells

Transforming growth factor- (TGF-) has been shown to induce apoptosis on normal hepatocytes and hepatoma cells both in vitro and in vivo. However, how the TGF- induces apoptosis is still not clear. We examined the expression of anti-apoptosis proteins and sensitivity to TGF- in three well differentiated human hepatoma cell lines. Two TGF- sensitive cell lines Hep3B and HuH7 totally lacked Bcl-2. In contrast, the TGF- resistant HepG2 cells expressed a substantial amount of Bcl-2. All three cell lines expressed equal amounts of Bcl-X_L, Bcl-X_S and Bax. Overexpression of Bcl-2 in Hep3B and HuH7 cells protected them from TGF--induced apoptosis. TGF- treatment increased intracellular peroxide production and suppressed the expression of glutathione-S-transferase in the Hep3B cells, and these effects were partially suppressed by the overexpression of Bcl-2. These results suggest that Bcl-2 may protect cell from TGF--F-induced apoptosis by interfering TGF- generated signals leading to induce reactive oxygen species production.     

Transforming growth factor-β1 immobilises dendritic cells within skin tumours and facilitates tumour escape from the immune system

Human skin tumours often regress spontaneously due to immune rejection. Murine skin tumours model this behaviour; some regress and others progress in syngeneic immunocompetent hosts. Previous studies have shown that progressor but not regressor skin tumours inhibit dendritic cell (DC) migration from the tumour to draining lymph nodes, and transforming growth factor-₁ (TGF-₁) has been identified as a responsible factor. To determine whether increased production of TGF-₁ in the absence of other differences inhibits DC migration from the tumour and enables it to evade immune destruction, a murine regressor squamous cell carcinoma clone was transfected with the gene for TGF-₁. This enhanced growth in vitro and in vivo, causing it to become a progressor. TGF-₁ transfection reduced the number of infiltrating DCs by about 25%. Quantitation of CD11c⁺ E-cadherin⁺ (epidermally derived) DCs in lymph nodes determined that TGF-₁ reduced the number of DCs that migrated from the tumour to undetectable levels. This was supported by showing that TGF-₁ reduced DC migration from cultured tumour explants by greater than tenfold. TGF-₁ transfection also reduced the number of infiltrating CD4 and CD8 T cells. Thus, TGF-₁ production by skin tumours is sufficient to immobilise DCs within the tumour, preventing their migration to lymph nodes. This reduces the number of T cells that infiltrate the tumour, preventing regression. Thus, TGF-₁ is a key regulator of whether skin tumours regress or progress.     

Exogenous glycosaminoglycans (GAG) differentially modulate GAG synthesis by anchorage-independent cultures of the outer cells from neonatal rat calvaria in the absence and presence of TGF-β

In anchorage-dependent (AD) cultures of the outer cell population (OCP) from neonatal rat calvaria, transforming growth factor-₁ (TGF-) specifically upregulated the synthesis of chondroitin sulfate (CS) proteoglycan (PG) and uncoupled the inhibitory effect of increasing cell density on CS PG synthesis (reference #30). Utilizing the same cell population, we have further examined the possibility that glycosaminoglycans (GAG) known to be synthesized and secreted by bone cells might exert feedback effects on GAG synthesis and/or its stimulation by TGF-. Although addition of TGF- alone stimulated net synthesis of HA and CS in both AD and anchorage-independent (AI) cultures, significant alterations of basal and TGF--stimulated GAG synthesis by exogenous GAGs were observed only in AI cultures. In AI cultures exogenously added hyaluronic acid (HA) markedly enhanced the basal synthesis of HA and CS while heparin (H) suppressed the basal synthesis of HA, CS as well as dermatan sulfate (DS). Also, the addition of HA markedly potentiated the stimulation by TGF- of HA and CS synthesis as did heparan sulfate (HS) for CS and DS synthesis. H suppressed the stimulation of the synthesis of HA, CS and DS by TGF-. Overall, our results indicate specific effects of individual GAGs on basal and TGF--stimulated GAG synthesis in OCP cultures. We suggest that some of the GAGs in the OCP microenvironment (which with the exception of HA are covalently linked to protein cores of secreted PGs), acting in concert with TGF-, may serve as an amplification system for upregulating GAG synthesis in the rapidly growing neonatal calvarium.     

Glycosylphosphatidylinositol (GPI) hydrolysis by Transforming Growth Factor-β1 (TGF-β1) as a potential early step in the inhibition of epithelial cell proliferation

Glycosylphosphatidylinositol (GPI) was previously identified in rabbit articular chondrocytes as being a precursor of inositolphosphate glycan (IPG), released upon (Transforming Growth Factor-) (TGF-) exposure, and capable of mimicking the proliferative effects of the growth factor. Here, using mink lung epithelial cells (CCL 64), which are known to be growth-inhibited by TGF-, we studied the potential role of GPI-derived molecules in the antiproliferative effect of TGF-1. We first identified an endogenous pool of GPI material and three different anionic forms of IPG in epithelial cells pre-labeled with [3H] glucosamine. Shortly (8 min) after TGF-1 addition, the cells responded by a rapid and transient hydrolysis of GPI, accompanied by the release of the most anionic form of IPG. This TGF--released IPG, after partial purification, was shown to decrease the proliferation of CCL 64 cells. Moreover, anti-IPG antibodies reduced the effects of TGF- and blocked the effects of partially purified IPG. These data strongly suggest that GPI hydrolysis may be an early step of the TGF- signalling pathway involved in growth inhibition of epithelial cells.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Dual Function of Transforming Growth Factor-β in the Control of Proliferation and Apoptosis of Cells of the Nervous System

Transforming growth factor- (TGF-) is an agent that gave the name to an extensive superfamily of congeneric cytokines playing important roles in numerous physiological and pathological processes. TGF- is involved in a few signal pathways controlling growth, differentiation, and death (apoptosis) of the nerve cells. Yet, it was found that the role of TGF- in each of these processes is dual: it can act either as their stimulator or as an inhibitor. This review describes examples and principal mechanisms of the dual functions of TGF- in its regulatory influences realized in the mammalian nervous system.     

Antifibrotic effects of <Emphasis Type="Italic">Salvia miltiorrhiza </Emphasis>on dimethylnitrosamine-intoxicated rats

Excessive oxidative stress is implicated in hepatic fibrogenesis. Extracts of Salvia miltiorrhiza (Sm) have been shown to protect cells against oxidative stress. In this study we investigated the in vitro and in vivo effects of Sm on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-1 (TGF-1). The inhibitory effects of Sm (50~400 g/ml) on TGF-1-induced -smooth muscle actin (-SMA) secretion and the mRNA expressions of fibrosis-related genes, including -SMA, connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats. DMN-treated rats were randomly assigned to 1 of 4 groups: saline, Sm (20 mg/kg), Sm (100 mg/kg), or silymarin (100 mg/kg), each given by gavage twice daily for 5 weeks starting from the onset of DMN administration. Sm (200 and 400 g/ml) significantly inhibited TGF-1-stimulated -SMA secretion and the mRNA expressions of -SMA, CTGF, and TIMP-1 in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with either a low (1.8 ± 0.2) or high (1.8 ± 0.1) dose of Sm, or silymarin (1.4 ± 0.2) were significantly reduced in comparison with DMN-treated rats receiving saline (3.1 ± 0.1). Hepatic collagen contents were also significantly reduced by either Sm or silymarin treatment. The mRNA expression levels of -SMA, TGF-1, and procollagen I were all attenuated in Sm- and silymarin-treated rats. Moreover, levels of plasma aspartate transaminase activities were reduced by Sm and silymarin treatment. In conclusion, our results show that Sm exerted antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.     

Characterization of Baboon (Papio hamadryas) Milk Proteins

The major proteins of baboon milk were identified as -lactoglobulin (LG), -lactalbumin (LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human LA, lysozyme, and albumin and bovine LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon LG are identical to those of macaque (Macaca fasicularis) LG except for a (D/N) polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of LG were elucidated using RT-PCR amplification of poly(A)⁺ mRNA purified from lactating mammary gland. Baboon LG consists of 168 amino acid residues (M_r 20,750) and is the longest LG identified to date. LG and LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4–6, of individual baboon milk samples at varying stages of lactation.     

Transforming Growth Factor-β and Ischemic Brain Injury

1. Necrosis and apoptosis are the two fundamental hallmarks of neuronal death in stroke. Nevertheless, thrombolysis, by using the recombinant serine protease t-PA, remains until now the only approved treatment of stroke in man.2. Over the last years, the cytokine termed Transforming Growth Factor-1 (TGF-1) has been found to be strongly up-regulated in the central nervous system following ischemia-induced brain damage.3. Recent studies have shown a neuroprotective activity of TGF-1 against ischemia-induced neuronal death. In vitro, TGF-1 protects neurons against excitotoxicity by inhibiting the t-PA-potentiated NMDA-induced neuronal death through a mechanism involving the up-regulation of the type-1 plasminogen activator inhibitor (PAI-1) in astrocytes.4. In addition, TGF-1 has been recently characterized as an antiapoptotic factor in a model of staurosporine-induced neuronal death through a mechanism involving activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and a concomitant increase phosphorylation of the antiapoptotic protein Bad.5. Altogether, these observations suggest that either TGF- signaling or TGF-1-modulated genes could be good targets for the development of new therapeutic strategies for stroke in man.     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Syndecan-4 as a molecule involved in defense mechanisms

Syndecan-4 is a transmembrane heparan sulfate proteoglycan belonging to the syndecan family. Syndecan-4-deficient [(Synd4(–/–)] mice were produced to clarify the in vivo role of syndecan-4. Synd4(–/–) mice were more susceptible to -carrageenan-induced nephropathy, and the placental labyrinth from the deficient embryos exhibited more thrombi than wild-type ones. Importantly, Synd4(–/–) mice were more susceptible to endotoxin shock. Further analysis revealed that the mechanism to suppress excessive production of interleukin-1 (1L-1) by transforming growth factor- (TGF-) was impaired in the deficient mice. TGF-, one of the cytokines involved in the suppression mechanism, bound to heparan sulfate chain of syndecan-4, which was induced in macrophages and the microvasculature after administration of lipopolysaccharide. Therefore, augmentation of TGF- function by induced syndecan-4 was suggested as a mechanism of the suppressive action of syndecan-4 against endotoxin shock. Published in 2003.     

Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.     

Role of tumor-derived transforming growth factor-β1 (TGF-β1) in site-dependent tumorigenicity of murine ascitic lymphosarcoma

An ascitic lymphosarcoma (LS-A) of Swiss mice that regressed spontaneously on subcutaneous (s.c.) transplantation was investigated for the mechanism of its progressive growth and host mortality on intraperitoneal (i.p.) transplantation. In vitro studies indicated significant inhibition of LS-A proliferation seeded at higher cell density (>10⁴/ml). Culture supernatants of LS-A caused bi-modal growth effects, the early supernatants (24 h) caused stimulation and the late (72 h) supernatants inhibited LS-A proliferation. The 72-h supernatants also suppressed T and B cell response to mitogens in a dose-dependent manner. Pan anti-transforming growth factor- antibody abrogated the inhibitory effects of supernatants. The supernatants contained both latent as well as bio-active form of transforming growth factor-1 (TGF-1) as determined by ELISA. Mice bearing i.p. ascites tumor had elevated serum TGF-1, hemoglobulinemia, splenic lymphopenia, impaired response of the T cells to mitogen and reduced expression of transferrin receptor (CD71) on the bone marrow cells. However, mice which rejected s.c. transplants, did not show significant changes in these parameters. Our studies indicated profound influence of site of tumor growth on tumor progression and host immune system mediated by tumor-derived TGF-1. It is possible that human tumors which secrete TGF-1 may exhibit similar patho-physiological effects in the host depending on the anatomical site of the tumor.     

Filamentous Marine Fungi as Producers of <Emphasis Type="Italic">O</Emphasis>-Glycosylhydrolases: β-1,3-Glucanase from <Emphasis Type="Italic">Chaetomium indicum</Emphasis>

Ninety fungal strains (42 species) isolated from marine habitats were studied for their ability to produce extracellular enzymes. Cultural filtrates of these strains were shown to contain a series of glycosidases (-glucosidases, N-acetyl--glucosaminidases, -galactosidases -mannosidases) and glucanases (1,3--glucanases, amylases) which varied with habitat. The level of activity depended on the species of fungi. Several promising strains capable of producing both individual enzymes and a set of enzymes for splitting carbohydrate-containing compound have been isolated. Optimal conditions for growth of Chaetomium indicum and for biosynthesis of -1,3-glucanase were determined. -1,3-Glucanase was isolated using ion-exchange chromatography, ultrafiltration, and gel filtration. The presence of 2 enzyme forms was shown; both forms were exo--1,3-glucanases.     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.