Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta).

The effects of transforming growth factor beta (TGF-beta) on interferon gamma-mediated killing of the intracellular protozoan parasite Trypanosoma cruzi and on the course of T. cruzi infection in mice were investigated. Spleen cells from mice with acute T. cruzi infections were found to produce elevated levels of biologically active TGF-beta in vitro, and the possibility that TGF-beta may mediate certain aspects of T. cruzi infection was then addressed. When mouse peritoneal macrophages were treated with TGF-beta in vitro, the ability of IFN-gamma to activate intracellular inhibition of the parasite was blocked. This occurred whether cells were treated with TGF-beta either before or after IFN-gamma treatment. TGF-beta treatment also blocked the T. cruzi-inhibiting effects of IGN-gamma on human macrophages. Additionally, treatment of human macrophages with TGF-beta alone led to increased parasite replication in these cells. The effects of TGF-beta on T. cruzi infection in vivo were then investigated. Susceptible C57BL/6 mice developed higher parasitemias and died earlier when treated with TGF-beta during the course of infection. Resistant C57BL/6 x DBA/2 F1 mice treated with TGF-beta also had increased parasitemias, and 50% mortality, compared with no mortality in infected, saline-treated controls. A single dose of TGF-beta, given at the time of infection, was sufficient to significantly decrease resistance to infection in F1 mice and to exacerbate infection in susceptible C57BL/6 mice. Furthermore, a single injection of TGF-beta was sufficient to counter the in vivo protective effects of IFN-gamma. We conclude that TGF-beta, produced during acute T. cruzi infection in mice, is a potent inhibitor of the effects of macrophage activating cytokines in vivo and in vitro and may play a role in regulating infection.

Cell-mediated co-action of transforming growth factors: incubation of type beta with normal rat kidney cells produces a soluble activity that prolongs the ruffling response to type alpha.

Intense, continuous ruffling is a characteristic of many transformed cells, but untransformed cells ruffle intensely only briefly after exposure to growth factors. We reported previously that cells of a normal rat kidney (NRK) cell line transformed by Kirsten murine sarcoma virus secrete their own ruffle-inducing agent(s) that cause sustained ruffling in either themselves or untransformed NRK cells. In the present study, we examined the roles of the transforming growth factors TGF-alpha and TGF-beta in the induction and maintenance of ruffling in untransformed NRK cells and observed the following: TGF-alpha caused a transient epidermal growth factor (EGF)-like response, which could be blocked by prior exposure of cells to EGF or by antiserum directed against the COOH-terminus of TGF-alpha. TGF-beta caused no ruffling and did not itself prolong TGF-alpha ruffling. A new, buffer-soluble (transferable) mediator activity produced by incubation of TGF-beta with NRK cells for 6-h extended the duration of maximal TGF-alpha-induced ruffling by several-fold. This study demonstrates that TGF-alpha alone causes an EGF-like, transient ruffling response, but neither TGF-alpha or TGF-beta alone, nor the two together, cause transformation-associated sustained ruffling. Rather, TGF-alpha acts in concert with a new, TGF-beta-dependent activity. This new activity appears to inhibit normal cellular off-regulation of TGF-alpha-induced ruffling. Inhibition of the cellular off-regulation of a growth factor response could play a key role in the unregulated growth associated with malignancy.

A 60-kD protein mediates the binding of transforming growth factor-beta to cell surface and extracellular matrix proteoglycans.

The biological activity of many cytokines is regulated by binding proteins present at the cell surface, in extracellular matrices or in soluble phase. We describe here a TGF-beta binding protein that is both an extracellular matrix and a cell surface protein. When intact extracellular matrices of HEP-G2 cells were affinity cross-linked with 125I-TGF-beta 1, two major binding components were seen: a 250-kD, proteoglycan-like molecule, presumed to be betaglycan, and a 60-kD protein. The 60-kD TGF-beta-binding protein was also present at the cell surface. It could be released from the cell surface by treating cells with high salt, heparin, chondroitin sulfate, heparitinase, or chondroitinase, indicating that it is bound to heparan sulfate and chondroitin sulfate proteoglycans. The 60-kD protein bound TGF-beta 1 with an apparent dissociation constant of 1.6 nM, and there were 30,000 binding sites per cell at the cell surface. In addition to the HEP-G2 cells and another hepatoma cell line, the 60-kD protein was also found in a human colon carcinoma (HT-29) cell line but not in rat kidney (NRK-49F) or human fibroblast (HUT-12) cell lines. The 60-kD protein could be extracted from cells containing it and transferred to the surface of previously negative cells. The 60-kD protein may serve to regulate the binding of TGF-beta to its signal transducing receptors by targeting TGF-beta to appropriate locations in the microenvironment of cells.

Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets.

Damage to the vessel wall is a signal for endothelial migration and replication and for platelet release at the site of injury. Addition of transforming growth factor-beta (TGF-beta) purified from platelets to growing aortic endothelial cells inhibited [3H]thymidine incorporation in a concentration-dependent manner. A transient inhibition of DNA synthesis was also observed in response to wounding; cell migration and replication are inhibited during the first 24 hours after wounding. By 48 hours after wounding both TGF-beta-treated and -untreated cultures showed similar responses. Flow microfluorimetric analysis of cell cycle distribution indicated that after 24 hours of exposure to TGF-beta the cells were blocked from entering S phase, and the fraction of cells in G1 was increased. The inhibition of the initiation of regeneration by TGF-beta could allow time for recruitment of smooth muscle cells into the site of injury by other platelet components.

Transformation induced by Abelson murine leukemia virus involves production of a polypeptide growth factor.

Rat embryo fibroblasts transformed by Abelson murine leukemia virus (MuLV) produce and release a transforming growth factor (TGF). Production of this factor is correlated with a tyrosine-specific protein kinase that is functionally active and is associated with the major Abelson MuLV gene product, P120. Transformation-defective mutants of Abelson MuLV do not transform cells, do not have their virus coded transforming gene product phosphorylated in tyrosine, and do not induce TGF production. Abelson MuLV-induced TGF morphologically transforms cells in culture, competes with 125I-labeled epidermal growth factor (EGF) for binding to cell receptors, and induces phosphorylation of tyrosine acceptor sites in the 160,000-dalton EGF membrane receptor. After purification to homogeneity, Abelson virus-induced TGF migrates as a single polypeptide with an apparent size of 7400 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Transforming growth factor-beta in leishmanial infection: a parasite escape mechanism.

The course of infection with the protozoan parasite Leishmania is determined in part by their early replication in macrophages, the exclusive host cells for these organisms. Although factors contributing to the survival of Leishmania are not well understood, cytokines influence the course of infection. Transforming growth factor-beta (TGF-beta) is a multipotential cytokine with diverse effects on cells of the immune system, including down-regulation of certain macrophage functions. Leishmanial infection induced the production of active TGF-beta, both in vitro and in vivo. TGF-beta was important for determining in vivo susceptibility to experimental leishmanial infection.

Stimulation of bone resorption in vitro by synthetic transforming growth factor-alpha.

Experiments were conducted to test the hypothesis that tumor-derived transforming growth factor-alpha (TGF-alpha) is responsible for the increased bone resorption and hypercalcemia seen in some malignant diseases. Homogeneous synthetic TGF-alpha prepared by the solid-phase synthesis method stimulated bone resorption directly in vitro in a concentration-dependent manner. Incubation times of 72 hours or more were required to stimulate resorption, which is similar to the time course of bone resorption by epidermal growth factor.

Epithelial wound healing enhanced by transforming growth factor-alpha and vaccinia growth factor.

Epidermal regeneration following middermal injuries to skin requires both proliferation and migration of keratinocytes. Epidermal growth factor (EGF) stimulates the proliferation of keratinocytes in culture, and topical administration of EGF accelerates epidermal regeneration of partial thickness burns or split-thickness incisions in vivo. Transforming growth factor-alpha (TGF-alpha) and vaccinia growth factor (VGF) have substantial sequence homology with EGF, and all appear to bind to the same receptor protein. Whether TGF-alpha or VGF can affect epidermal wound healing in vivo is not known. The present studies show that topical administration of TGF-alpha or VGF in antibiotic cream to partial thickness burns (second degree) accelerated epidermal regeneration in comparison with untreated or vehicle-treated burns. Low levels of both TGF-alpha and VGF (0.1 microgram per milliliter) appeared to be more effective than EGF in stimulating epidermal regeneration. Regenerated epithelium from burns treated with TGF-alpha or VGF appeared normal histologically. This finding suggests that topical application of selected growth factors may be useful in accelerating healing of partial thickness injuries.

Production of transforming growth factor beta 1 during repair of arterial injury.

Repair of arterial injury produced by balloon angioplasty leads to the formation of a neointima and a narrowing of the vascular lumen. In this study, we examined the possibility that smooth muscle cells (SMC) in injured rat carotid arteries are stimulated to produce type-1 transforming growth factor-beta (TGF-beta 1) during neointima formation in vivo. Levels of TGF-beta 1 transcripts (2.4 kb) were significantly increased within 6 h after carotid injury and reached a maximum (five to sevenfold) by 24 h. Regenerating left carotids had sustained increases in TGF-beta 1 mRNA levels (about fivefold) over the next 2 wk, during which time a substantial neointimal thickening was formed. No changes in basal TGF-beta 1 mRNA levels were found in contralateral uninjured carotids at any of the times examined. Immunohistochemical studies showed that a large majority of neointimal SMC were stained for TGF-beta 1 protein in an intracellular pattern, consistent with active TGF-beta 1 synthesis in this tissue. Neointima formation and TGF-beta 1 immunoreactivity were correlated with increases in fibronectin, collagen alpha 2(I), and collagen alpha 1(III) gene expression. Infusion of purified, recombinant TGF-beta 1 into rats with a preexisting neointima produced a significant stimulation of carotid neointimal SMC DNA synthesis. These results suggest that TGF-beta 1 plays an important role as an endogenous growth regulatory factor produced by neointimal SMC themselves during progressive neointimal thickening after balloon angioplasty.

Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors.

Infection of A431 cells with vaccinia virus, or exposure to a mitogenic polypeptide secreted by vaccinia virus-infected cells, induces tyrosine phosphorylation of epidermal growth factor receptors.

Expression and characterization of transforming growth factor alpha precursor protein in transfected mammalian cells.

Analysis of a cDNA clone derived from retrovirus-transformed rat fibroblasts has recently suggested that the mature 50-amino-acid form of transforming growth factor alpha (TGF alpha) is derived from a 159-amino-acid transmembrane precursor by proteolytic cleavage. To understand the processing of the TGF alpha precursor molecule in more detail, we have expressed this protein in baby hamster kidney (BHK) fibroblasts under control of the metal-ion-inducible metallothionein promoter and characterized the expressed precursor with site-specific antipeptide antibodies. One of the BHK transfectants, termed 5:2, expressed the TGF alpha mRNA in a cadmium- and zinc-inducible manner. The TGF alpha precursor protein was detected by immunoprecipitation analysis of radiolabeled cell cultures. In the induced 5:2 cells, a polypeptide of Mr 13,000 to 17,000 was readily identified by peptide antisera made to three different regions of the TGF alpha precursor protein. No such protein species were observed in BHK cells treated with cadmium and zinc or in uninduced 5:2 cells. However, two cell lines known to produce TGF alpha naturally, Leydig testicular tumor cells and Snyder-Theilan feline sarcoma virus-transformed Fisher rat embryo fibroblasts, possessed detectable levels of immunologically related Mr 13,000 to 17,000 proteins. Cell fractionation studies indicate that the Mr 13,000 to 17,000 species expressed in induced 5:2 cells is membrane associated, consistent with predictions based on the cDNA sequence of the TGF alpha precursor. Media conditioned by induced 5:2 cells contained epidermal growth factor receptor-competing activity, which, upon size fractionation, was similar in size to the mature processed form of TGF alpha. These data show that these nontransformed BHK cells possess the ability to process the TGF alpha precursor molecule into its native form.

Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells.

Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-beta and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-beta nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants. The major proteins secreted by these cells consisted of latent TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.

Transforming growth factors in the urine of normal, pregnant, and tumor-bearing humans.

Transforming growth factor (TGF) activities could be detected in the urine of normal, pregnant, and tumor-bearing humans. These acid- and heat-stable polypeptides competed for binding to epidermal growth factor (EGF) membrane receptors and promoted the anchorage-independent growth of nontransformed rodent cells. They differed from human EGF in their apparent molecular weights and soft-agar growth-stimulating activity. The urine from pregnant females contained TGF activities with apparent molecular weight(s) (relative) (Mr) of 10,000 ad 17,000--20,000. In the case of a lung cancer patient, an additional major activity of approximately 30,000--35,000 Mr was found. All urine specimens examined also contained a "common" 8,000-Mr soft-agar growth-stimulating activity, which competed for binding to EGF membrane receptors and which was chromatographically separable from EGF (urogastrone). Thus urine may provide a convenient and readily available source for the biochemical characterization of these TGF-like activities, some of which may be clinically useful biologic markers for certain types of cancer.

Protection against 7, 12-dimethylbenz[a]anthracene-induced rat mammary carcinoma by infection with mouse xenotropic type C virus.

A single ip inoculation of female, outbred Sprague-Dawley rats with a viable mouse xenotropic type C virus significantly reduced the incidence and/or retarded the development of mammary carcinoma induced by 7, 12-dimethylbenz[a]anthracene administered orally 7 days after virus. Although infectious virus could not be isolated from organs of infected rats, high titers of circulating and tumor-associated antibodies were detected against the viral internal core protein p30, and a low-grade antibody response to intact virus or envelope glycoprotein was found. Moreover, a cell-mediated immune response, measured by lymphocyte transformation, was detected with the use of intact virus but not with p30 antigen. No immunity developed after a single inoculation of UV-inactivated virus. These data indicated that inoculation of adult individuals of heterologous species with viable xenotropic mouse type C virus resulted in the rapid disappearance of infectious virus from the recipient, followed by the development of both humoral and cellular immunity to virion constituents. These events led, by unknown mechanisms, to the effective retardation of chemical carcinogenesis when infection preceded carcinogen administration.

Inhibition and promotion of differentiated-like phenotype of a human lung carcinoma in athymic mice by natural and recombinant forms of transforming growth factor-beta.

Both structurally related forms of transforming growth factor-beta (TGF-beta types I and II) are potent inhibitors of tumor cell growth in vitro and can also modulate the differentiation of some cells in culture. In this study, we describe the effects of natural and recombinant TGF-betas on the growth and differentiation of a xenograft of human lung adenocarcinoma A549 in male athymic BALB/c mice. Subcutaneous, peritumoral injection of both forms of TGF-beta inhibited, in a dose-dependent manner, the growth of established human lung tumors. Histologically, tumors inhibited by TGF-beta appeared more differentiated, as judged by reduced mitotic activity and a predominance of highly specialized mucus-secreting goblet-like cell types. These findings suggest that TGF-betas can be useful in the development of novel, perhaps less cytotoxic, cancer therapeutic strategies.

Differential expression of transforming growth factor-alpha during prenatal development of the mouse.

Transforming growth factors (TGFs) are differentially expressed in the mouse during neonatal development. Highest levels are seen early at Day 7, and lower levels, at Day 13. Both small- and large-molecular-weight forms of TGF are found; they share many biochemical properties with rat TGF-alpha, including a similar high-pressure liquid chromatography elution profile. Although the embryo-derived activities compete with epidermal growth factor for binding to epidermal growth factor membrane receptors, they are immunologically distinct from epidermal growth factor. These embryonic polypeptides, however, do cross-react in a competitive radioimmunoassay developed using a synthetic peptide corresponding to the carboxy-17 amino acids of rat TGF-alpha as the immunogen. The highly conserved TGF-alpha family of peptides produced by some tumor cells may therefore represent derepressed forms of these embryonic growth factors. A functional role in neonatal development is proposed.

Mouse embryonic transforming growth factors related to those isolated from tumor cells.

Growth factors with the two characteristic properties of sarcoma growth factor, the ability to stimulate anchorage-independent growth of normal mouse or rat fibroblasts and the ability to compete with 125I-labeled epidermal growth factor for receptor binding, can be isolated from 12- to 13-day-old normal mouse embryos of various strains. Two size classes of the transforming factor from embryo cells can be isolated with apparent molecular weights of 20,000 and 10,000. The lower-molecular-weight factor has been purified several hundred-fold and has the same properties as the peptides with a molecular weight of 10,000 produced by mouse sarcoma virus-transformed 3T3 cells. Whole-mouse embryos contain approximately 10% as much sarcoma growth factor per g of tissue as do the murine sarcoma virus-transformed cells; how it is distributed among the embryonic tissues remains to be determined.

Type C retrovirus activation and possible functions in the normal and tumor-bearing host.

The pathological consequences of tumor virus infection, transformation, and tumor development in certain experimental animals is a well-established and accepted fact. More recently, it has been suggested that these viruses may also have a physiological function participating in such processes as cellular differentiation, immune recognition, and embryogenesis. This paper delineates the current information giving some credence to physiological function for type C viruses.

Comparison of growth factors functionally related to epidermal growth factor in the urine of normal and human tumor-bearing athymic mice.

Urine from nude mice contains epidermal growth factor (EGF) and a minor acid-stable component with an apparent molecular weight of 20,000, which competes with EGF for binding to EGF membrane receptors and which promotes colony formation by normal rat kidney cells in soft agar. The levels of this Mr 20,000 urine-derived growth factor are increased approximately 4- to 10-fold in nude mice bearing tumors following s.c. injection of cultured human tumor cells. Following removal of the primary tumor, the concentration of this factor is reduced to basal levels, and thus, elevated levels of this growth factor appear to be dependent on tumor burden. The Mr 20,000 urinary component is separable into four EGF competing activities by high-performance liquid chromatography; the major species is immunologically related to mouse submaxillary gland EGF and therefore appears to be of host origin. However, in addition to elevated levels of host growth factor, urine from tumor-bearing mice also contains transforming growth factor activity in amounts comparable to that released by the tumor cells in culture. The tumor-derived urinary transforming growth factor activity is immunologically unrelated to EGF but is immunoreactive with an antiserum to transforming growth factor-alpha. We propose that the nude mouse may be a useful model to examine the role of both host- and tumor-derived growth factors in tumorigenesis and the usefulness of these factors as biological markers of response to therapy and tumor progression.

High-molecular-weight transforming growth factor activity in the urine of patients with disseminated cancer.

Urine from 22 patients with a variety of disseminated cancers and from an equivalent number of nonmalignant controls of similar age and sex was tested for the presence of transforming growth factor (TGF) activity as measured by the ability to promote the growth in soft agar of nontransformed indicator cells. Cancer patients included those with carcinomas of the lung, breast, colon, and ovary, as well as melanomas and sarcomas. The nonmalignant controls included both normals and individuals with a variety of inflammatory and infectious disorders. Aliquots of unfrozen urine were acid extracted, chromatographed on a Bio-Gel P-30 column, and then tested for TGF activity using normal rat kidney fibroblasts and epidermal growth factor (EGF)-competing activity with human carcinoma A431 cells. These assays revealed that a high-molecular-weight TGF activity (Mr 30,000 to 35,000) which coelutes with EGF-competing activity was present in 18 of 22 cancer patients but present in only five of 22 nonmalignant controls (p less than 0.01). In contrast, a low-molecular-weight TGF activity (Mr 6000 to 8000) which does not coelute with EGF-competing activity was found in all urines tested. These results indicate that an EGF-related, high-molecular-weight TGF activity is found in the urine of cancer patients and may be a useful tumor marker. Unlike other tumor markers described previously, high-molecular-weight TGF activity has a biological activity which is related to the expression of the transformed phenotype.