While replication-defective retroviral vectors provide excellent vehicles for the long-term expression of therapeutic genes, they also harbor the potential to induce undesired genetic changes by random insertions into the host genome. The rate of insertional mutagenesis for retroviral vectors has been determined in several different assay systems; however, the rate at which such events induce cellular transformation has not been directly determined. Such measurements are critical to determining the actual risk of carcinogenesis resulting from retroviral gene therapy. In this study, the ability of a replication-defective retroviral vector, G1nBgSvNa, to induce cellular transformation in the BALB/c-3T3 in vitro transformation assay was assessed. The transformation frequency observed in vector-transduced BALB/c-3T3 cells, which contained one to six copies of integrated provirus, was not significantly different from that of untreated control cells. The finding that G1nBgSvNa was nontransforming in this assay indicates that the rate of transformation induced by retroviral insertions is less than the spontaneous rate of cellular transformation by BALB/c-3T3 cells, or less than 1.1 x 10(-5). These results are the first to define an upper limit for the rate of transformation induced by retroviral vectors.

Mouse embryo-derived AKR-2B fibroblasts and murine fibrosarcoma cells (the 1591 cell line) were transfected with a murine transforming growth factor-beta 1 (TGF beta 1) cDNA under the transcriptional control of either the simian virus-40 early promoter or the cytomegalovirus promoter/enhancer. Selected clones secreted 2- to 4-fold more TGF beta-competing activity into their media than the parental cell line or neomycin-transfected controls. The TGF beta 1 released into the cell-conditioned medium was latent. Despite the latency of the overexpressed TGF beta 1, TGF beta 1-transfected cells exhibited phenotypic features of TGF beta 1-treated cells. When confluent, the TGF beta 1-transfected cells had the morphological characteristics of the parental cells that have been treated with active TGF beta 1. AKR-2B cells that expressed higher levels of TGF beta 1 also expressed high levels of c-sis and c-myc mRNAs and decreased TGF beta 2 and TGF beta 3 mRNAs in the same manner as parental AKR-2B cells that had been treated with active TGF beta 1. The transfected 1591 cells that overexpressed TGF beta 1 bound less [125I]TGF beta 1 than did parental 1591 cells, but after a mild acid wash demonstrated an increase in [125I]TGF beta 1 binding. Our results suggest that these TGF beta 1-transfected fibroblast and fibrosarcoma cells have the capacity to activate TGF beta; however, as very little activated TGF beta is detected in the medium, it is hypothesized that these cells activate latent TGF beta 1 and bind the activated TGF beta 1, thus acquiring a phenotype consistent with TGF beta 1-treated cells.

A serotonin-secreting human pancreatic carcinoid cell line (BON) is demonstrated to express transcripts for all three mammalian types of transforming growth factor beta (TGF beta 1, 2, and 3). Similarly, freshly excised carcinoid tumors from six patients were also found to express mRNA for all three of the type-beta TGFs. Medium conditioned by BON cells had detectable TGF beta activity, although most of the activity was latent as determined by radioreceptor assay with and without prior acid treatment. However, nonactivated BON-conditioned medium stimulated DNA synthesis, soft agar growth, and an increase in TGF beta 1 and fibronectin mRNA expression in AKR-2B fibroblasts. In addition, BON-conditioned medium had a potent endothelial cell growth-stimulatory activity. Since the TGF beta s inhibit growth of endothelial cells, the presence of other growth factors was suspected. TGF alpha, c-sis, and basic fibroblast growth factor transcripts were also found to be expressed by the BON carcinoid cells. These data indicate that multiple peptide growth factors may have a paracrine role in the desmoplastic reaction accompanying carcinoid tumors.

A murine fibroblast cell line (AKR-2B clone 84A) and an epithelial cell line (BALB/MK) were compared for their ability to bind different transforming growth factor-beta (TGF beta) species. The results of competitive binding assays indicated that the epithelial cells had a higher affinity for TGF beta than the fibroblasts. This difference may be the basis for the sensitivity of epithelial cells to much lower concentrations of TGF beta than fibroblasts. Affinity cross-linking studies showed that both cell types express the three cell surface TGF beta-binding molecules that have been previously described for a variety of cell types. The complexity of these cell surface binding proteins was further evaluated using all possible combinations of radiolabeled ligands in competition with each of the three unlabeled TGF beta species. Differences in the ability of specific TGF beta types to compete with radiolabeled TGF beta 2 for binding to the type I and II receptors were observed, with TGF beta 1 being more potent for epithelial cells, and TGF beta 2 being more potent for fibroblasts. In addition, a difference in the ability of different TGF beta species to compete the [125I]TGF beta 3 from epithelial cell surface receptors was apparent. TGF beta 2 was not able to compete with [125I]TGF beta 3 for binding to the type II receptor at any concentration tested, while TGF beta 1 and TGF beta 3 were about equally potent in competition for this receptor type. These differences in cell surface receptor binding of structurally and biologically similar molecules may reflect different functions for these molecules.

BALB/MK (MK) is a continuous murine keratinocyte line whose cells are strictly dependent on exogenous epidermal growth factor (EGF) for growth in culture. A derivative cell, KC, resulted from Kirsten murine sar coma virus transformation, and these cells no longer require EGF for their growth. Despite differences in MK and KC growth conditions, both cell lines are growth inhibited by picomolar concentrations of transforming growth factor‐β (TGF‐β). When MK and KC cells were maintained in the presence of TGF‐β, resistant variants eventually proliferated only from the KC population. In an attempt to determine the mechanism of development of TGF‐β resistance, the TGF‐β‐resistant cells (KCR cells) were compared with TGF‐β‐sensitive KC cells with regard to growth properties, TGF‐β1 binding characteristics, and gene expression. KCR cells continued to synthesize DNA and proliferated in the presence of TGF‐β1 concentrations up to 2 nM, which was 500‐fold greater than the ED50 for the sensitive cells. Although the KCR cells possess similar receptor numbers and affinity for TGF‐β1, we observed differences in affinity cross‐linking studies. The KCR cells expressed more of the type III, high molecular weight cell surface binding protein and less of the type II than the KC cells. The type I moiety was clearly altered to a smaller size in some, but not all, KCR cells. In gene regulation studies, there was no apparent difference in c‐Ki‐ras and v‐Ki‐ras mRNA levels in the KC and KCR cells. Additionally, expression of TGF‐α and TGF‐β, mRNA was similar in MK, KC, and KCR cells. The expression of proliferation‐associated genes, such as c‐myc and MGSA/c‐gro/kc, which were markedly decreased by TGF‐β1, in the MK and KC cells, was not altered by TGF‐β1 in the KCR cells. The data suggest that the loss of TGF‐β1 responsiveness in the KCR cells was due to an alteration in the TGF‐β receptor that did not permit signal transduction, although the existence of postreceptor alterations cannot be excluded.

Exogenous TGF beta inhibits the proliferation of human breast cancer cells in vitro. These cells synthesize and secrete TGF beta into their medium predominantly in a latent form. With neutralizing antibodies against native, biologically active TGF beta (278ab and 282ab), we have examined whether HS578T and MDA-231 breast cancer cells utilize their endogenous TGF beta for growth regulation. Low levels of TGF beta activity were detectable in conditioned medium from confluent monolayers of both cell lines in the absence of acid or protease treatment as measured by radioreceptor assay. When added to subconfluent monolayers of the respective cell line, this untreated conditioned medium inhibited DNA synthesis and cell proliferation. This inhibition was blocked by anti-TGF beta antibodies, whereas nonimmune rabbit IgG had no effect. Similar to exogenous TGF beta 1, this conditioned medium induced a dose-dependent increase in steady-state TGF beta 1 mRNA levels when added to subconfluent HS578T cells; this increase was blocked by the 278ab. Consistent with the above, preincubation of either cell line with anti-TGF beta antibodies increased subsequent specific binding of 125I-TGF beta to cell surface receptors without changing binding affinity. Addition of 278ab to quiescent HS578T or MDA-231 cells induced a dose-dependent increase in [3H]thymidine incorporation. Both antibodies stimulated cell proliferation in serum-free medium and anchorage-independent growth of both cell lines. Finally, incubation of HS578T cells with 278ab under serum-free conditions decreased the basal level of TGF beta 1 message expression. These data indicate that cultured human breast cancer cells utilize endogenously produced TGF beta as an autocrine negative growth regulator.

TGF-β1 is demonstrated to inhibit skin keratinocyte proliferation when added during the G1 phase of the cell cycle. Human foreskin keratinocytes transformed with either HPV-16 or -18 or SV40, however, were resistant to the growth inhibitory effects of TGF-β1. Since TGF-β1 appears to inhibit keratinocyte growth through down-regulation of c-myc, it was hypothesized that these DNA tumor viruses might be modulating the response to TGF-β1 via this pathway. Transient expression of proteins HPV-16 E7, adenovirus type 5 E1A, and SV40 large T antigen is demonstrated to block TGF-β1 suppression of c-myc transcription. This effect was not observed with DNA tumor virus transforming proteins mutated in their pRB binding domain. These observations indicate that pRB or another protein that interacts with this binding domain mediates TGF-β1 regulation of c-myc gene expression and growth inhibition.

The TGF beta family of polypeptide growth factors regulates a remarkable diversity of cellular functions, many of which are not directly associated with cell growth. The present review has summarized many of the recent studies that have just begun to conceptually integrate this expanding array of TGF beta functions into the context of a three-dimensional, multicellular organ or tissue, be it normal or diseased. This fascinating research strongly implicates TGF beta as a key modulator of a wide variety of important physiologic and pathophysiologic processes.

The number of different growth regulatory molecules which have been isolated and characterized is continuing to increase. As more information is obtained, it has become apparent that the cooperative actions of many factors with distinct activities is necessary for appropriate proliferative responses. An interplay of both growth stimulatory and growth inhibitory factors is essential for normal growth. Of crucial importance, therefore, is the appropriate regulation of growth factors. Unregulated expression, synthesis, posttranslational processing or activation of either positive or negative growth signals may contribute to neoplastic transformation (Fig. 3). Altered responses to normally positive or negative signals by transformed cells have been demonstrated by several investigators [64, 79, 84]. While altered growth factor responses in transformed cells are well documented, the mechanisms responsible for the loss of growth control are poorly understood and are likely to be both complex and numerous. Continued efforts to dissect and comprehend fully growth factor action on normal cells will be necessary before an understanding of neoplastic transformation can be achieved.

Two human pancreatic adenocarcinoma cell lines (PANC 1 and MIA PACA 2) were examined for expression of growth factors that could potentially play a role either in growth regulation of the tumor cells, or in cells that comprise the stromal elements of tumors. Both cell lines expressed transforming growth factor-α (TGFα), basic fibroblast growth factor (bFGF), c-sis (PDGF B chain), TGFβ1, and TGFβ3 mRNA by Northern blot analysis. Only the PANC 1 cells, however, expressed the TGFβ2 transcript. TGFP-like competing activity was found in medium conditioned by either cell line, but TGFα-like [epidermal growth factor (EGF)-competing] activity was not detected in the medium from either cell line by radioreceptor assay. TGFα and EGF caused concentration-dependent stimulation of soft agar colony growth of the MIA PACA 2 cells, while only TGFα caused a significant but less dramatic stimulation of soft agar growth of the PANC 1 cells. Insulin stimulated the anchorage-independent growth of MIA PACA 2 but not PANC 1 cells. Likewise, bFGF also caused a concentration-dependent stimulation of MIA PACA 2 but not PANC 1 growth in soft agar, and PDGF had no effect on the growth of either cell line. TGFβ had no inhibitory or stimulatory effect on soft agar colony growth of either the PANC 1 or the MIA PACA 2 cells, although both cell lines exhibited high affhity, saturable TGFβ binding sites, and TGFβl was capable of autoinduction of TGFβ1 mRNA expression in PANC 1 cells. The ability to continue to respond to positive growth regulatory factors coupled with the loss of responsiveness to negative growth factors may be important in the pathogenicity of these aggressive tumors.

Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.

Chronic pulmonary hypertension is associated with extensive structural remodeling of the pulmonary arterial bed. The structural changes in the arterial walls include increased production of extracellular matrix components and smooth muscle cell hypertrophy, changes that have been similarly induced by transforming growth factor-beta (TGF-beta) in culture. In the present study, experiments were performed to determine whether TGF-beta is present in sheep lung lymph, and whether TGF-beta levels were altered in an animal model of chronic pulmonary hypertension induced by continuous air embolization. Several standard biological assays for TGF-beta activity were used for these determinations including soft agar assays, inhibition of epithelial cell proliferation, and a TGF-beta-specific radioreceptor assay. In each case, control lung lymph contained high concentrations of TGF-beta (100 ng/ml) which required transient acidification for detection. Samples of lung lymph from hypertensive sheep showed a transient and early two- to threefold increase in concentrations of latent TGF-beta. This activity could be partially blocked by TGF-beta antibodies. These studies indicate that sheep lung lymph contains TGF-beta and that the level of TGF-beta increases early during the development of pulmonary hypertension. Thus, TGF-beta may contribute to the development of the structural changes in the pulmonary arteries that occur during the onset of chronic pulmonary hypertension.

Transforming growth factors (TGFs) were originally defined by their biological effects on fibroblastic cells (for review see Goustin et al. 1986). These effects included induction of morphological transformation in monolayer culture and Stimulation of colony formation in soft agar. While the early studies with TGFα were somewhat misleading with respect to the function of these factors, they did lead to the purification and cloning of two important growth-regulatory molecules, TGFα and TGFβ. Interestingly, one of these factors (TGFα) is a potent mitogen for a wide variety of cell types, while the other (TGFβ) is the most potent growth-inhibitory Polypeptide known for most cell types (Goustin et al. 1986).

Transfection of C3H/10T1/2 cells with either a c-myc or an activated c-Ha-ras gene decreased the cellular dependence for serum-derived factors to proliferate in monolayer. The c-myc-transfected cells did, however, require a high plasma concentration for significant growth, while the ras transfectants grew extremely well in either low or high concentrations of either plasma or serum. Stimulation of quiescent cultures with purified growth factors demonstrated that c-myc transfection did not alter qualitatively or quantitatively the requirement for both epidermal growth factor (EGF) and insulin to progress to DNA synthesis. Cells transfected with either a ras gene alone or a combination of ras plus c-myc lost their dependence on EGF for DNA synthesis; cultures became committed to S phase in serum-free medium supplemented with insulin alone. The ras transfectants arrested in mid-G1, 6 h prior to S phase. The EGF independence of the ras transfectants is consistent with the mid-G1 arrest of these cells at a point(s) distal to the primary action of EGF in early G0-G1.

Recent cDNA characterization has predicted the existence of a new member of the transforming growth factor family, transforming growth factor-beta 3 (TGF beta 3). However, nothing is known about the biological activities of the TGF beta 3 protein, since it has not been purified from any natural sources. We report here the recombinant expression in mammalian cells and the purification to apparent homogeneity of human TGF beta 3. The TGF beta 3 was evaluated in comparison with purified TGF beta 1 and TGF beta 2 in several assays for its effects on stimulation or inhibition of proliferation of mammalian cells. These analyses revealed that TGF beta 3 exerts activities similar to the two other TGF beta species, but that there are distinct differences in potencies between the different TGF beta forms depending on the cell type and assay used.

Transforming growth factor beta is a polypeptide growth factor with a multiplicity of diverse biologic effects. Increasingly, data support a role for TGF beta in the autocrine regulation of normal epithelial cell growth (Figure 1). Definition of the normal pathways for growth stimulation and inhibition of epithelial cell growth by autocrine peptides like TGF beta and TGF alpha undoubtedly will increase understanding of normal growth and development, embryogenesis, wound repair, and tumorigenesis.

Transforming growth factor-beta (TGF beta) is produced by most cultured cells in an inactive form. Potential activation mechanisms of latent TGF beta were studied using fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model. Active TGF beta was monitored by radioreceptor and soft agar assays as well as by antibody inhibition and immunoprecipitation. Little or no TGF beta was detected in untreated conditioned medium. Treatment of the medium with extremes of pH (1.5 or 12) resulted in significant activation of TGF beta as shown by radioreceptor assays, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved by pH 1.5. In an effort to define more physiological means of TGF beta activation, the effects of some proteases were tested. Plasmin and cathepsin D were found to generate 25-kD bands corresponding to the active form of TGF beta as shown by immunoprecipitation analysis of radiolabeled cell-conditioned medium. Plasmin treatment of the medium resulted in activity that was quantitatively similar to that of mild acid treatment as measured by radioreceptor and soft agar assays. In addition, the plasmin-generated activity was inhibited by anti-TGF beta antibodies. Sequential treatments of AKR-MCA cell-conditioned medium with mild acid followed by plasmin or plasmin followed by mild acid gave activation comparable to either treatment alone. The data suggest that conditioned medium may contain at least two different pools of latent TGF beta. One pool is resistant to mild acid and/or plasmin and requires strong acid or alkali treatment for activation. A second pool is activated by mild pH change and/or plasmin. Activation of this form of latent TGF beta may take place by dissociation or proteolytic digestion from a precursor molecule or hypothetical TGF beta-binding protein complex.