Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family.

Human glioblastoma cells secrete a peptide, termed glioblastoma-derived T cell suppressor factor (G-TsF), which has suppressive effects on interleukin-2-dependent T cell growth. As shown here, complementary DNA for G-TsF reveals that G-TsF shares 71% amino acid homology with transforming growth factor-beta (TGF-beta). In analogy to TGF-beta it is apparently synthesized as the carboxy-terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino-acid-long mature form of G-TsF. Comparison of the amino-terminal sequence of G-TsF with that of porcine TGF-beta 2 and bovine cartilage-inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G-TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.     

Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.     

Dominant negative mutants of transforming growth factor-beta 1 inhibit the secretion of different transforming growth factor-beta isoforms.

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide factor that is thought to play a major role in the regulation of proliferation of many cell types and various differentiation processes. Several related isoforms have been structurally characterized, three of which, TGF-beta 1, -beta 2, and -beta 3, have been detected in mammalian cells and tissues. Each TGF-beta form is a homodimer of a 112-amino-acid polypeptide which is encoded as a larger polypeptide precursor. We have introduced several mutations in the TGF-beta 1 precursor domain, resulting in an inhibition of TGF-beta 1 secretion. Coexpression of these mutants with wild-type TGF-beta 1, -beta 2, and -beta 3 results in a competitive and specific inhibition of the secretion of different TFG-beta forms, indicating that these mutated versions act as dominant negative mutants for TGF-beta secretion. Overexpression of dominant negative mutants can thus be used to abolish endogenous secretion of TGF-beta and structurally related family members, both in vitro and in vivo, and to probe in this way the physiological functions of the members of the TGF-beta superfamily.     

Sequence analysis of the promoter region of the glioblastoma derived T cell suppressor factor/transforming growth factor (TGF)-beta 2 gene reveals striking differences to the TGF-beta 1 and -beta 3 genes

Human glioblastoma cells secrete a factor termed glioblastoma derived T cell suppressor factor (G-TsF) or transforming growth factor beta 2 (TGF-beta 2) which inhibits the response of T cells to mitogenic or antigenic stimulation. In the present study we isolated the promoter region of the G-TsF/TGF-beta 2 gene. The promoter region shares no homology to the promoter of the TGF-beta 1 or the 5' region of the TGF-beta 3 gene and harbours several familiar DNA motifs, including the cytokine-1 region, an octamer-like sequence, Sp1- and AP-2-like elements and a putative NF-kappa B site. In contrast to the TGF-beta 1 gene, the G-TsF/TGF-beta 2 gene contains three TATA-like sequences but lacks an AP-1 site. To understand the cell type specificity of expression of G-TsF/TGF-beta 2, the individual contribution of the DNA elements detected in the promoter has to be analysed in further studies.     

The murine transforming growth factor-beta precursor

Transforming growth factor-beta (TGF-beta) is a homodimeric polypeptide which can act, often in cooperation with other growth factors, as a mitogenic factor for a variety of cells. TGF-beta can also exert growth inhibitory activity on many other cell lines. We have isolated cDNAs coding for the murine TGF-beta cDNA precursor. The deduced amino acid sequence localizes the 112-amino acid long TGF-beta monomer to the C terminus of the precursor. Two areas of the precursor exhibit a marked degree of homology to the human counterpart. One of these regions comprises the mature TGF-beta monomer, while the other corresponds to the NH2 terminus of the precursor and suggests an important biological function for this area. Northern hybridization results identify a major 2.5-kilobase TGF-beta mRNA and several minor TGF-beta mRNA species.     

Protease inhibitors interfere with the transforming growth factor-beta-dependent but not the transforming growth factor-beta-independent pathway of tumor cell-mediated immunosuppression.

Tumor cells have been reported to exert inhibitory effects on the activation of T lymphocytes in vitro. We show that the IL-2-stimulated proliferation of a Th cell line is suppressed when the T cells are cocultured with human glioblastoma and melanoma cell lines. The use of two Th cell clones that differ in their responsiveness to growth-inhibition by transforming growth factor-beta (TGF-beta) and the analysis of tumor cell-derived supernatants as well as of TGF-beta 1/TGF-beta 2 gene expression allowed to distinguish two pathways of tumor-induced immunosuppression. Glioblastoma cells exert their immunosuppressive effects by producing biologically active TGF-beta 2, whereas the immunosuppressive state induced by melanoma cells is TGF-beta-independent and requires direct contact between tumor cell and T cell. The TGF-beta-dependent immunosuppression is down-regulated by various protease inhibitors and up-regulated by estradiol via modulation of the production of biologically active TGF-beta 2 by glioblastoma cells leaving total activatable TGF-beta 2 unaffected. No such modulation is functional for the TGF-beta-independent pathway of immunosuppression. We conclude that the production of active TGF-beta by tumor cells is regulated at a posttranslational level by the coordinated action of several proteolytic enzymes.     

Expression of transforming growth factor-beta s 1-4 in chicken embryo chondrocytes and myocytes

cDNA probes and antibodies for TGF-beta s 1, 2, 3, and 4 were used to study the expression of these different TGF-beta isoforms in cultured chicken embryo chondrocytes and cardiac myocytes, as well as in developing cartilage and heart tissues. TGF-beta s 2, 3, and 4 mRNAs, but not TGF-beta 1 mRNA, were detected in cultured chondrocytes and myocytes. Expression of TGF-beta s 2 and 4 mRNAs increased with age, while expression of TGF-beta 3 mRNA was independent of age in chondrocytes cultured from 12- to 17-day-old embryos. In contrast, expression of TGF-beta s 2, 3, and 4 mRNAs was constitutive in myocytes cultured from 7- to 9-day-old embryonic hearts; expression of TGF-beta s 3 and 4 mRNAs increased, while expression of TGF-beta 2 mRNA remained unchanged in myocytes from 10-day-old embryos. Immunoprecipitation studies demonstrated expression of TGF-beta in both the conditioned media and the cell lysates of metabolically labeled chondrocyte and myocyte cell cultures. Immunohistochemical staining of cultured chondrocytes and myocytes and of cartilage and heart tissues of developing chicken embryos with antibodies specific for each TGF-beta isoform showed immunoreactive TGF-beta s 1, 2, 3, and 4. Our results demonstrate coordinate expression of these four TGF-beta isoforms in chicken embryo chondrocytes and myocytes, both in vitro and in vivo, with expression of TGF-beta s 2, 3, and 4 mRNA and protein more prominent than that of TGF-beta 1.     

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.     

Differential sensitivity of subclasses of human colon carcinoma cell lines to the growth inhibitory effects of transforming growth factor-beta 1

In this study we have employed a model system comprising three groups of colon carcinoma cell lines to examine the growth-inhibitory effects of two molecular forms of transforming growth factor-beta (TGF-beta), TGF-beta 1 and TGF-beta 2. Aggressive, poorly differentiated colon carcinoma cells of group I did not respond to growth inhibitory effects of TGF-beta 1 or TGF-beta 2, while less aggressive, well-differentiated cells of group III displayed marked sensitivity to both TGF-beta 1 and TGF-beta 2 in monolayer culture as well as in soft agarose. One moderately well-differentiated cell line from group II which has intermediate growth characteristics failed to respond to TGF-beta 1 or TGF-beta 2, but the growth of two other cell lines in this group was inhibited. TGF-beta 1 and TGF-beta 2 were equally potent, 50% growth inhibition for responsive cell lines being observed at a concentration of 1 ng/ml (40 pM). Antiproliferative effects of TGF-beta 1 and TGF-beta 2 in responsive cell lines of groups II and III were associated with morphological alterations and enhanced, concentration-dependent secretion of carcinoembryonic antigen. Radiolabeled TGF-beta 1 bound to all three groups of colon carcinoma cells with high affinity (Kd between 42 and 64 pM). These data indicate for the first time a strong correlation between the degree of differentiation of colon carcinoma cell lines and sensitivity to the antiproliferative and differentiation-promoting effects of TGF-beta 1 and TGF-beta 2.     

Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes

This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.     

Inhibition of growth by transforming growth factor-beta following fusion of two nonresponsive human carcinoma cell lines. Implication of the type II receptor in growth inhibitory responses.

Loss of growth regulation by transforming growth factor-beta (TGF-beta) may be an important step in carcinogenesis. We have used a cell fusion system to show that inhibition of growth by TGF-beta can be restored to carcinoma cell lines that are unresponsive to the inhibitory effects of TGF-beta. In a previous study, the EJ bladder carcinoma line was fused to the SW480 colon adenocarcinoma line and found to produce nontumorigenic hybrid cells along with one hybrid cell clone of low tumorigenicity. Here we show that the capacity of the nontumorigenic hybrid cells to respond to either TGF-beta 1 or TGF-beta 2 has been restored, while the parental or tumorigenic hybrid cells show little or no inhibition of growth following TGF-beta treatment. Cross-linking analyses with labeled TGF-beta 1 demonstrated much higher levels of the type II (85 kDa) receptor in the hybrid cells compared with the parental tumor lines. Both the parental and tumorigenic hybrid cell lines were capable of responding to TGF-beta as evidenced by increased levels of mRNA for fibronectin, type IV collagenase, and plasminogen activator inhibitor after treatment with TGF-beta 1. These results suggest that the type II receptor is necessary for mediating the effects of TGF-beta on inhibition of growth but not on gene activation of the hybrid cells.     

Decreased transforming growth factor-beta response and binding in insulin-independent teratoma-derived cell lines with increased tumorigenic properties.

The mouse C3H teratoma-derived cell line 1246 is an adipogenic cell line which stringently requires insulin to proliferate and differentiate in defined medium. From this cell line an insulin-independent cell line called 1246-3A was isolated. It was found that, in contrast to 1246 cells, 1246-3A cells had lost the ability to differentiate and became tumorigenic when injected at a density of 10(6) cells/mouse into syngeneic host C3H mice. In addition, they produce in their culture medium transforming growth factor alpha- and beta-like polypeptides which stimulate their proliferation. Highly tumorigenic insulin-independent cell lines able to give rise to tumor when injected at a density of 10(4) cells/mouse were isolated by using an in vitro-in vivo shuttle technique. The highly tumorigenic cell lines have lost the response to TGF-beta 1. The binding of TGF-beta 1 to the nontumorigenic parent cell line or to cells displaying increased tumorigenic properties was investigated. The data presented here indicate that the increased tumorigenicity is accompanied by a progressive decrease of specific binding of TGF-beta 1 to the cells. However, the decreased number of cell surface TGF-beta 1 binding sites in the highly tumorigenic cells did not correlate with an increase in the secretion of TGF-beta protein by the tumorigenic cells, as all of TGF-beta produced by the cells was in a latent form. Affinity cross-linking experiments indicated that the 1246 cell line displayed several TGF-beta cross-linked molecular species (MW 140, 92, and 70 kDa). Increase of tumorigenicity was accompanied by a marked decrease in the intensity of the cross-linked bands, particularly of the 92 and 70 kDa species.     

Regulation of transforming growth factor-beta secretion by human peritoneal mesothelial and ovarian carcinoma cells

This study was conducted to compare the secretion of TGF-beta isoforms by human ovarian carcinoma (OVCA) cell lines (n=12) and human peritoneal mesothelial cells (HPMC;n=6) and to examine the regulation of their production by inflammatory cytokines. TGF-beta isoforms were furthermore analysed in OVCA-associated ascitic fluids. HPMC constitutively produced considerable amounts of TGF-beta1 (median 42 pg/10(5)cells; range 7-98) but only minimal amounts of TGF-beta2 (median 0.8 pg/10(5)cells; range 0-1.5). Treatment of HPMC with IL-1beta (10 ng/ml) resulted in a significant elevation of the secretion of both TGF-beta1 (median 187 pg/10(5)cells; range 71-264;P<0.001) and TGF-beta2 (median 1.8 pg/10(5)cells; range 0-13;P<0.01). In OVCA TGF-beta1 and TGF-beta2 were detected in 7/12 and 11/12 of the cell lines, respectively. The levels detected varied widely for TGF-beta1 (median 25 pg/10(5)cells; range 0-410) as well as for TGF-beta2 (median 14 pg/10(5)cells; range 0-419) and there was no correlation between the two isoforms. In contrast to HPMC, TGF-beta secretion by OVCA was not affected by any of the inflammatory cytokines tested. TGF-beta3 could not be detected in supernatants, neither in OVCA nor in HPMC. In ascitic fluids the median level of TGF-beta1 (median 5443 pg/ml; range 737-14687) was 10-fold higher than the level of TGF-beta2 (median 545 pg/ml; range 172-3537). The present data provide a model for the analysis of the molecular mechanisms of aberrant TGF-beta production by OVCA and support the hypothesis that HPMC are an important source of ascitic TGF-beta.