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.     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

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.     

Interleukin-1β Stimulates Mitogen-Activated Protein Kinase in U373 Astrocytoma Cells without the Production of Lipid Second Messengers

IL-1 is one of the cytokines known to affect astroglial cells in normal brain development, brain injury and neurodegenerative diseases. IL-1 causes astrocytes to become more reactive, alter the expression and release of molecules and in some cases to proliferate. We have investigated the mitogenic effect and signal transduction pathway induced by IL-1 in U373 cells, a human astrocytoma cell-line. Recombinant human IL-1 induced mitogenesis of U373 cells in a dose-dependent fashion as assessed by tritiated thymidine incorporation. The following signal transduction mechanisms, reported to be induced in other systems by IL-1, were investigated in U373 cells: (1) activation of phosphatidylcholine-specific phospholipase C as assayed by incorporation of tritiated choline into cellular phospholipids, (2) production of diacylglycerol, a lipid second messenger, (3) activation of sphingomyelinase, and (4) activation of mitogen-activated protein kinase (MAPK). Of these, IL-1 activated only MAPK. In cultured rat astrocytes, IL-1 caused activation of MAPK without inducing proliferation.     

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.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

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.     

Stretch-induced paracrine hypertrophic stimuli increase TGF-β1 expression in cardiomyocytes

Cardiac hypertrophy refers to the abnormal growth of cardiomyocytes, and is often caused by valvular heart disease and hypertension. It involves the activation of growth, including increased protein synthesis and changes in gene expression. Transforming growth factor-₁ (TGF-₁) may play a central role in protecting the heart during the hypertrophic response by helping to restore normal functions of the affected myocardium. We tested the hypothesis that cardiomyocytes respond to stretch-induced paracrine hypertrophic stimuli with increased expression of TGF-₁. To that purpose, we investigated whether angiotensin II (AII), endothelin-1 (ET-1) and TGF-, secreted by stretched cardiac and vascular cells, are involved in the paracrine mechanisms of stretch-induced changes of TGF-₁ mRNA expression in stationary (i.e. non-stretched) cardiomyocytes.Our results indicated that TGF-₁ mRNA expression in stationary cardiomyocytes was increased by AII release from cardiomyocytes that had been stretched for 30–60 min. Furthermore, it is likely that ET-1 and TGF- were released by stretched cardiac fibroblasts and endothelial cells to induce TGF-₁ mRNA expression in stationary cardiomyocytes. Stretched vascular smooth muscle cells did not influence TGF-₁ mRNA expression in stationary cardiomyocytes. These results indicate that AII, ET-1 and TGF-, released by cardiac cell types, act as paracrine mediators of TGF-₁ mRNA expression in cardiomyocytes. Therefore, we conclude that in stretched myocardium the cardiomyocytes, cardiac fibroblasts and endothelial cells take part in intercellular interactions contributing to cardiomyocyte hypertrophy.     

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.     

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.     

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.     

The Primary Structure of Water Buffalo αs1- and β-Casein: Identification of Phosphorylation Sites and Characterization of a Novel β-Casein Variant

The primary structure of water buffalo _s1-casein and of -casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a -elimination/thiol derivatization. Water buffalo _s1-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine _s1-casein C variant, the water buffalo _s1-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine A₂-casein variant, the two water buffalo -casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo -casein variants seem to be homologous to bovine A₂-casein.     

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.     

Transforming growth factor-β-mediated down-regulation of antitumor cytotoxicity of spleen cells from MOPC-315 tumor-bearing mice engaged in tumor eradication following low-dose melphalan therapy

We have previously shown that treatment of mice bearing a large MOPC-315 plasmacytoma with a low dose of the anticancer drug melphalan (l-phenylalanine mustard;l-PAM) results in the acquisition of a potent CD8⁺ T-cell-mediated anti-MOPC-315 cytotoxic T lymphocyte (CTL) activity by the hitherto immunosuppressed tumor bearers, and this immunity contributes to complete tumor eradication. In the studies presented here, we sought to determine how the acquisition of this antitumor immunity following low-dose chemotherapy is possible, in light of the report that MOPC-315 tumor cells produce transforming growth factor- (TGF-), an immunosuppressive cytokine that can down-regulate the generation of CTL responses. We found that the acquisition of CTL activity following low-dosel-PAM therapy is not due to a chemotherapy-induced decrease in the sensitivity of MOPC-315 tumor bearer spleen cells to TGF--mediated inhibition of CTL-generation. Moreover, even spleen cells from MOPC-315 tumor-bearing mice, which had receivedl-PAM therapy 7 days earlier and had acquired CTL activity in vivo, were sensitive to the inhibitory activity of TGF- upon culture for as little as 1 day, with or without stimulator tumor cells. However, the production of TGF- by MOPC-315 tumors decreased drastically as a consequence of the low-dose chemotherapy. Thus, the curative effectiveness of low-dosel-PAM therapy for MOPC-315 tumor-bearing mice may be due, at least in part, to a reduction in TGF- production that enables the development of tumor-eradicating immunity.Work was supported by research grant IM-435 from the American Cancer Society and research grant CA54413 from the National Cancer Institute.In partial fulfillment of the requirements for the Doctor of Philosophy DegreeSupported in part by the Dorothea Fleming Cancer Research Fellowship Award     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Anti-(transforming growth factor β) antibodies with predefined specificity inhibit metastasis of highly tumorigenic human xenotransplants in nu/nu mice

Monoclonal antibodies (mAb) were prepared against conjugated transforming growth factor 1 (TGF1) peptides: amino acid positions 48–60 and positions 86–101. Two antibodies, mAb 16-3G1 [anti-(48–60)] and mAb 5-2G6 [anti-(86–101)] cross-reacted with native TGF1,-2 and-3 (16-3G1) or only with native TGF1 (5-2G6). Both mAb were used to characterize TGF-mediated effects on the metastatic potential in nude mice of human carcinoma cell line SLU-1 and its metastatic subline SLU-M1. Autocrine TGF1-mediated up-regulation of cell proliferation and its suppression by anti-TGF antibodies in vitro was recorded for SLU-M1 cells whereas SLU-1 cell proliferation in vitro appeared to be refractory to anti-TGF antibodies and exogenous TGF-1. However, the potential of s.c. tumours to develop distant metastases in nude mice was about the same for both cell lines. Development of primary tumours and distant metastases could be suppressed by treatment of mice with anti-TGF antibodies. Thus we assume that the metastatic potential of tumour cells is independent of TGF-mediated growth-regulation effects in vitro. The anti-TGF-induced suppression of tumour progression and metastasis in nude mice might rather result from stimulation of the immune surveillance. TGF-mediated autocrine down-regulation of MHC-unrestricted cytotoxicity of activated human monocytes and CD56⁺ LAK cells and its reversion by anti-TGF antibodies could be readily demonstrated. In all our experimental series, the neutralizing potential of both anti-TGF antibodies, though directed against opposite sites of the TGF1 molecule, was very similar.     

The TF1-ATPase and ATPase activities of assembled α3β3γ, α3β3γδ, and α3β3γε complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the α3β3, and α3β3δ complexes

The ATPase activity of the F₁-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 µM where 70% stimulation is observed at 36°C. Half maximal stimulation is observed at about 3 µM dye. At rhodamine 6G concentrations greater than 10 µM, ATPase activity declines with 50% inhibition observed at about 75 µM dye. The ATPase activities of the ₃₃ and ₃₃ complexes assembled from isolated subunits of TF₁ expressed inE. coli deleted of theunc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF₁. In contrast, the ATPase activities of the ₃₃ and ₃₃ complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 µM dye at 36°C. The ATPase activity of TF₁ is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF₁ is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 µM dye at 30°C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF₁ stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.     

The α/β Interfaces of α1β1, α3β3, and F1: Domain Motions and Elastic Energy Stored during γ Rotation

ATP synthase (F_oF₁) consists of F₁ (ATP-driven motor) and F_o (H⁺-driven motor). F₁ is a complex of ₃₃ subunits, and is the rotating cam in ₃₃. Thermophilic F₁ (TF₁) is exceptional in that it can be crystallized as a monomer and an ₃₃ oligomer, and it is sufficiently stable to allow refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified or . The nucleotide-dependent open–close conversion of conformation is an inherent property of an isolated and energy and signals are transferred through / interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F₁ (MF₁) and TF₁ were analyzed by an atom search within the limits of 0.40 nm across the interfaces. Seven (plus thermophilic loop in TF₁) contact areas are located at both the catalytic and noncatalytic interfaces on the open form. The number of contact areas on closed increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in . The notion of elastic energy in F_oF₁ has been revised. X-ray crystallography of F₁ is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of F_oF₁. The domain motion and elastic energy in F_oF₁ will be elucidated by time-resolved crystallography.