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.     

Anti-immunoglobulin treatment of murine B-cell lymphomas induces active transforming growth factor beta but pRB hypophosphorylation is transforming growth factor beta independent.

Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, leading to apoptosis and cell death in the immature lymphomas WEHI-231 and CH31, but not in the CH12 B-cell line. In this system, which has been used as a model for B-cell tolerance, we have established that these lymphomas produce active transforming growth factor beta (TGF-beta) when treated with anti-Ig and that their hierarchy of sensitivity to TGF-beta generally correlates with their growth inhibition by anti-Ig. TGF-beta, in turn, has been shown to interfere with the phosphorylation of the retinoblastoma gene product, pRB. Herein, we also demonstrate that in WEHI-231 B-lymphoma cells treated with anti-Ig for 24 h, the pRB protein is found to be predominantly in the underphosphorylated form, as previously reported for cells arrested by the exogenous addition of TGF-beta. However, neutralizing antibodies to TGF-beta failed to prevent growth inhibition by anti-Ig in WEHI-231 and CH31. When WEHI-231 lymphoma cells were selected for growth in TGF-beta, the majority of the TGF-beta-resistant clones remained sensitive to anti-Ig-mediated growth inhibition. In these clones, the retinoblastoma gene product was found to be in the underphosphorylated form after 24-h treatment with anti-Ig, but not with TGF-beta. These data show that anti-Ig treatment of murine B-cell lymphomas stimulates the production of active TGF-beta but that a TGF-beta-independent pathway may be responsible for the pRB underphosphorylation and cell cycle blockade.     

Altered metabolic and adhesive properties and increased tumorigenesis associated with increased expression of transforming growth factor beta 1

Transforming growth factor-beta (TGF-beta) is a potent mediator of cell proliferation and extracellular matrix formation, depending on the cell type and the physiological conditions. TGF-beta is usually secreted in a "latent" complex that needs activation before it can exert its effects. Several observations correlate increased expression of TGF-beta 1 with tumorigenesis. To evaluate the physiological relevance of increased TGF-beta 1 synthesis in tumor cells we established cell clones overexpressing TGF-beta 1 and observed the resulting physiological changes in TGF-beta overproducing cells in vitro and in vivo. As a model system we used the human E1A-transformed 293 tumor cells, which are insensitive to the direct growth modulatory effects of TGF-beta. The selection of this cell line allows an assessment of physiological alterations independent of TGF-beta induced proliferative changes. The use of two TGF-beta 1 expression vectors containing either the natural or a modified TGF-beta 1 precursor cDNA permitted the establishment of separate 293 cell lines overexpressing latent or active TGF-beta. Comparison of the resulting changes in glycolytic rate, adhesiveness and integrin and plasminogen activator expression established that, in vitro, both types of clones behaved similarly, indicating that expression of latent TGF-beta induces autocrine changes in the tumor cells and thus suggesting that some level of cell-associated activation occurs. TGF-beta overexpression resulted in an increased metabolic rate due to enhanced glycolysis, a property long associated with tumor cells. This increased glycolysis was not associated with altered proliferation. Cells overexpressing TGF-beta also displayed enhanced fibronectin mRNA and plasminogen activator synthesis and increased adhesiveness in vitro. They showed enhanced survival when plated sparsely on plastic in the absence of serum, and attached more readily to laminin. In addition, synthesis of several beta 1 integrins, in particular the alpha 1/beta 1, alpha 2/beta 1, and alpha 3/beta 1, all of which recognize laminin, were enhanced. Finally, cells overexpressing active TGF-beta, but not latent TGF-beta, also showed increased tumorigenicity in nude mice. Thus, an increase in endogenous TGF-beta synthesis confers several proliferation-independent phenotypic changes which may be of significance for the survival of the tumor cell inoculum or its subsequent growth, and for tumor formation and development. In the case of cells expressing active TGF-beta, the release of active TGF-beta into the vicinity of the tumor cells may also result in a more hospitable environment for tumor growth.     

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.     

Predominant Th2/Tc2 polarity of tumor-infiltrating lymphocytes in human cervical cancer

Cytotoxic T lymphocytes (Tc) play a central role in cellular immunity against cancers. The cytotoxic potential of freshly isolated tumor-infiltrating lymphocytes (TILs) is usually not expressed. This suggests the possible existence of as yet unspecified and perhaps complex immunosuppressive factors or cytokines that affect the anti-tumor capacity of these TILs in the tumor milieu. In the present study, we demonstrated for the first time that TILs derived from human cervical cancer tissue consist mainly of Th2/Tc2 phenotypes. In vitro kinetic assays further revealed that cancer cells could direct the tumor-encountered T cells toward the Th2/Tc2 polarity. Cancer cells promote the production of IL-4 and down-regulate the production of IFN-gamma in cancer-encountered T cells. The regulatory effects of cervical cancer cells are mediated mainly by IL-10, and TGF-beta plays only a synergistic role. The cancer-derived effects can be reversed by neutralizing anti-IL-10 and anti-TGF-beta Abs. IL-10 and TGF-beta are present in cancer tissue and weakly expressed in precancerous tissue, but not in normal cervical epithelial cells. Our study strongly suggests important regulatory roles of IL-10 and TGF-beta in cancer-mediated immunosuppression.     

Transforming growth factor-beta and cellular immune responses in synovial fluids

Mononuclear cells in synovial fluids (SF) from patients with rheumatoid arthritis and other arthropathies are characterized by functional and phenotypic changes, including impaired mitogen responsiveness and inverted ratios of CD4+/CD8+ T lymphocytes. This is related to previously described activities in synovial fluids that inhibit proliferation of lymphocytes induced by mitogens and cytokines. The present study examines the relationship of these activities and transforming growth factor beta (TGF-beta), which is now known as the most potent endogenous inhibitor of lymphocyte function. It is shown that most of the activity in SF that inhibits IL-1-induced thymocyte or T cell proliferation is neutralized by a specific antibody to TGF-beta. Analysis of the SF in the CCL64 assay, a standard test for TGF-beta, showed a close correlation between the levels of immunosuppressive activity and TGF-beta. SF contain spontaneously active inhibitors of T cell function and this is caused by the presence of active TGF-beta. Higher titers are found after transient acidification, which is known to activate the latent form of TGF-beta. Characterization of the TGF-beta isoforms showed that most of the material in SF is TGF-beta 2. Analysis of TGF-beta effects on T cell subsets demonstrated that it completely inhibits proliferation of CD4+ cells whereas at the same concentrations of purified or rTGF-beta CD8+ cells are only inhibited by maximally 31.1%. SF also preferentially inhibit CD4+ Th cell proliferation and this effect is neutralized by antibody to TGF-beta. Collectively these results indicate that the presence of TGF-beta accounts for most of the immunosuppressive activities in SF and that this factor may be responsible for functional and phenotypic changes of SF lymphocytes.     

Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200

Background and Objective Immune escape by tumors can occur by multiple mechanisms, each a significant barrier to immunotherapy. We previously demonstrated that upregulation of the immunosuppressive molecule CD200 on chronic lymphocytic leukemia cells inhibits Th1 cytokine production required for an effective cytotoxic T cell response. CD200 expression on human tumor cells in animal models prevents human lymphocytes from rejecting the tumor; treatment with an antagonistic anti-CD200 antibody restored lymphocyte-mediated tumor growth inhibition. The current study evaluated CD200 expression on solid cancers, and its effect on immune response in vitro. Methods and Results CD200 protein was expressed on the surface of 5/8 ovarian cancer, 2/4 melanoma, 2/2 neuroblastoma and 2/3 renal carcinoma cell lines tested, but CD200 was absent on prostate, lung, breast, astrocytoma, or glioblastoma cell lines. Evaluation of patient samples by immunohistochemistry showed strong, membrane-associated CD200 staining on malignant cells of melanoma (4/4), ovarian cancer (3/3) and clear cell renal cell carcinoma (ccRCC) (2/3), but also on normal ovary and kidney. CD200 expression on melanoma metastases was determined by RT-QPCR, and was found to be significantly higher in jejunum metastases (2/2) and lung metastases (2/6) than in normal samples. Addition of CD200-expressing, but not CD200-negative solid tumor cell lines to mixed lymphocyte reactions downregulated the production of Th1 cytokines. Inclusion of antagonistic anti-CD200 antibody restored Th1 cytokine responses. Conclusion These data suggest that melanoma, ccRCC and ovarian tumor cells can express CD200, thereby potentially suppressing anti-tumor immune responses. CD200 blockade with an antagonistic antibody may permit an effective anti-tumor immune response in these solid tumor types.     

Autocrine transforming growth factor-beta signaling mediates Smad-independent motility in human cancer cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor that plays a critical role in modulating cell growth, differentiation, and plasticity. There is increasing evidence that after cells lose their sensitivity to TGF-beta-mediated growth inhibition, autocrine TGF-beta signaling may potentially promote tumor cell motility and invasiveness. To understand the molecular mechanisms by which autocrine TGF-beta may selectively contribute to tumor cell motility, we have generated MDA-MB-231 breast cancer cells stably expressing a kinase-inactive type II TGF-beta receptor (T beta RII-K277R). Our data indicate that T beta RII-K277R is expressed, can associate with the type I TGF-beta receptor, and block both Smad-dependent and -independent signaling pathways activated by TGF-beta. In addition, wound closure and transwell migration assays indicated that the basal migratory potential of T beta RII-K277R expressing cells was impaired. The impaired motility of T beta RII-K277R cells could be restored by reconstituting TGF-beta signaling with a constitutively active TGF-beta type I receptor (ALK5(TD)) but not by reconstituting Smad signaling with Smad2/4 or Smad3/4 expression. In addition, the levels of ALK5(TD) expression sufficient to restore motility in the cells expressing T beta RII-K277R were associated with an increase in phosphorylation of Akt and extracellular signal-regulated kinase 1/2 but not Smad2. These data indicate that different signaling pathways require different thresholds of TGF-beta activation and suggest that TGF-beta promotes motility through mechanisms independent of Smad signaling, possibly involving activation of the phosphatidylinositol 3-kinase/Akt and/or mitogen-activated protein kinase pathways.     

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.     

Evidence that transforming growth factor-beta is a hormonally regulated negative growth factor in human breast cancer cells

The hormone-dependent human breast cancer cell line MCF-7 secretes transforming growth factor-beta (TGF-beta), which can be detected in the culture medium in a biologically active form. These polypeptides compete with human platelet-derived TGF-beta for binding to its receptor, are biologically active in TGF-beta-specific growth assays, and are recognized and inactivated by TGF-beta-specific antibodies. Secretion of active TGF-beta is induced 8 to 27-fold under treatment of MCF-7 cells with growth inhibitory concentrations of antiestrogens. Antiestrogen-induced TGF-beta from MCF-7 cells inhibits the growth of an estrogen receptor-negative human breast cancer cell line in coculture experiments; growth inhibition is reversed with anti-TGF-beta antibodies. We conclude that in MCF-7 cells, TGF-beta is a hormonally regulated growth inhibitor with possible autocrine and paracrine functions in breast cancer cells.     

Transforming growth factor-β and p-21: multiple molecular targets of decorin-mediated suppression of neoplastic growth

Decorin is a member of the small leucine-rich proteoglycan (SLRP) gene family that has recently become a focus in various areas of cancer research. The decorin protein consists of a core protein and a covalently linked glycosaminoglycan chain. Decorin binds to collagens type I, II and IV in vivo and promotes the formation of fibers with increased stability and changes in solubility. Further, the decorin core protein binds to growth factors, including transforming growth factor-beta (TGF-beta), to other intercellular matrix molecules such as fibronectin and thrombospondin, and to the decorin endocytosis receptor. Decorin may directly interfere with the cell cycle via the induction of p21WAF1/CIP1 (p21), a potent inhibitor of cyclin-dependent kinases (CDKs). Here, we discuss interactions of decorin with TGF-beta and with p21, both of which are relevant to carcinogenesis and tumor progression. TGF-beta is released by tumors of various histogenetic origins and promotes immunosuppression in the host and tumor immune escape by induction of growth arrest and apoptosis in immune cells, by downregulation of MHC II antigen expression and by changes in the cytokine release profiles of immune and tumor cells. Moreover, TGF-beta may modulate tumor growth in an autocrine and paracrine fashion, may mediate drug resistance, and may facilitate tumor angiogenesis. Decorin binds to TGF-beta, thus inhibiting its bioactivity, and is a direct or indirect negative modulator of TGF-beta synthesis. Ectopic expression of decorin results in the regression of rat C6 gliomas, an antineoplastic effect attributed to the reversal of TGF-beta-induced immunosuppression. On the other hand, de novo expression of decorin in colon cancer cells and some other tumor cells, even though not in glioma cells, results in an upregulation of p21 expression and a cell cycle arrest, presumably in a TGF-beta-independent manner. Decorin expression is downregulated in many tumors but upregulated in the peritumoral stroma. By virtue of its growth regulatory and immunomodulatory properties, decorin promises to become a novel target for the experimental therapy of human cancers.     

Multiple suppressive effects of transforming growth factor beta 1 on the immune response in chickens

The immunosuppressive effect of human recombinant TGF-beta 1 on chicken immune responses in vitro was evaluated. TGF-beta 1 at 1-10 ng/ml reduced T cell proliferation in response to concanavalin A by 50-80% and B cell proliferation in response to LPS by greater than 90%. In contrast, when added to immune spleen cells, it reduced the secondary PFC response to sheep erythrocytes by less than 50%, particularly when added at the same time as antigen on Day 2 of incubation. When TGF-beta 1 was added during a 2-day incubation to nylon wool-nonadherent immune or normal spleen cells, it caused the maintenance and/or appearance of suppressor cells. These suppressor cells, in coculture with immune spleen cells, inhibited the secondary PFC response in vitro without any further exposure to TGF-beta 1. The phenotype of the cells giving rise to suppressor cells under the influence of TGF-beta 1 was CT8+, TCR2+(alpha,beta), CT4-, TCR1-(gamma,delta) cells. The results suggest that, in addition to direct suppressive effects on the proliferation of B cells and of some T cells, TGF-beta 1 may suppress immune responses by maintaining or by promoting the development of suppressor T cells.     

Transforming growth factor-beta: an important mediator of immunoregulation

Transforming growth factor-beta (TGF-beta) is synthesized and secreted by a wide variety of cells, including cells of the immune system. Lymphocytes and monocytes possess high affinity TGF-beta receptors and the addition of TGF-beta to in vitro cell cultures results in significant modulation of immune function. TGF-beta inhibits the proliferation of thymocytes, T cells, B cells, and natural killer cells. Additionally, it inhibits certain differentiative functions of lymphocytes including a marked inhibition of immunoglobulin production by human B lymphocytes. TGF-beta has dichotomous actions on monocytes. It is a potent chemoattractant for monocytes and induces interleukin 1 mRNA expression while inhibiting generation of reactive oxygen intermediates and monocyte killing. Evidence is accumulating that TGF-beta regulates immune function in vivo and that overproduction of TGF-beta may be associated with immunosuppression.     

Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness

Type beta transforming growth factor (TGF-beta) is a unique polypeptide that has been isolated from a number of different tissues and can induce the phenotypic transformation of non-neoplastic fibroblasts as measured by the stimulation of their growth in soft agar. Recently, TGF-beta has been demonstrated to exert profound inhibitory effects on T and B lymphocyte proliferation. In this study, the effects of TGF-beta on natural killer (NK) cell function were investigated. After 20 hr of culture in the presence of TGF-beta, the NK activity of peripheral blood lymphocytes (PBL) was significantly reduced compared with PBL cultured in medium alone. Similarly, TGF-beta produced a significant depression in the cytolytic activity of highly enriched large granular lymphocytes (LGL). This effect of TGF-beta appeared to be mediated directly on the effector cells, because cultivation of the K562 target cells in TGF-beta did not affect target cell susceptibility to lysis. Binding studies with 125I-TGF-beta indicated that LGL possess approximately 1400 high-affinity (Kd = 1PM) receptors/cell, which represents a considerably higher affinity receptor for TGF-beta than that found on fibroblasts. Culturing of PBL and LGL in TGF-beta resulted in a marked blunting of the boosting of NK cytolysis by interferon-alpha but not by interleukin 2, which suggested that TGF-beta may down-regulate interferon-alpha receptors on NK cells. These results, indicate that in addition to inhibitory effects on T and B cells, TGF-beta also inhibits NK cell function. Although the in vivo role of TGF-beta is presently undefined, it may be an important immunoregulatory protein that has a negative influence on lymphocyte activation.