Human teratocarcinoma stem cells: glycolipid antigen expression and modulation during differentiation

Teratocarcinomas are germ cell tumors in which pluripotent stem cells, embryonal carcinoma (EC) cells, undergo differentiation along the pathways resembling those occurring during early embryogenesis. Human EC cell lines established in vitro provide a model for studying embryonic cellular differentiation in a way that is pertinent to early human development. The predominant glycolipid antigens expressed by EC cells of both humans and mice have globoseries core structures; in humans they are terminally modified to yield the monoclonal antibody-defined stage-specific embryonic antigens SSEA-3 and SSEA-4, and also globo-ABH antigens; in the mouse terminal modification yields the Forssman antigen rather than SSEA-3 and -4. These observations focus attention on the possible role of the P-blood group system, which regulates synthesis of globoseries oligosaccharides, in the behavior of cells in the early embryo and in teratocarcinomas. Marked changes in the core structures of the cell surface glycolipids occur as the EC cells differentiate; thus globoseries structures rapidly diminish and are replaced by lactoseries and then by ganglioseries glycolipids. During differentiation of the NTERA-2 line of pluripotent human EC cells into neurons and other cell types, the various subsets of differentiated cells that arise are distinguished by their differential expression of new glycolipid antigens, particularly ganglioside GT3 (recognized by antibody A2B5), and ganglioside 9-0-acetyl GD3 (recognized by antibody ME311). Neurons are found among the A2B5+/ME311- cells.     

A novel human T cell antigen preferentially expressed on mature T cells and shared by both well and poorly differentiated B cell leukemias and lymphomas

A new lymphocyte differentiation antigen shared by all normal T cells and some malignant B cells was defined by a monoclonal antibody designated 12.1. This antibody reacted with all peripheral blood T cells but not with normal B cells and B cell lines. Analysis with a fluorescence activated cell sorter showed that the expression of 12.1 antigen changes during T cell maturation. Most thymocytes, blasts of acute T cell leukemia, and cells from established leukemic T cell lines bear a small amount of 12.1 antigen. In contrast the majority of peripheral blood T cells, activated T cells, and leukemic T cells of the Sezary syndrome bear a large amount of 12.1 antigen. Unexpectedly, antibody 12.1 reacted with leukemic cells from most patients with B-type chronic lymphocytic leukemia (CLL) and some patients with lymphosarcoma cell leukemia (LSCL). Among these leukemias, expression of the 12.1 antigen was not correlated with the stage of B cell maturation, with the amount of surface immunoglobulin on the cells, or with the presence or absence of monoclonal gammapathy. In a comparative serologic analysis the antigen defined by antibody 12.1 was distinct from the p67 T cell antigen (defined by monoclonal antibody 10.2) that is also known to be expressed by B-type CLL cells.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Novel neutral glycolipid associated with adult T-cell leukemia and its pathological biochemistry

During attempts to make antibodies cross-reactive with human lymphocytes, we established a monoclonal antibody (VJ-41) from the alloimmunization of mice, that is, B10. A (3R) anti-B10. A (5R). VJ-41 reacted with all cases of freshly isolated adult T-cell leukemia cells (36 cases) but not with cells from other hematological disorders (more than 50 cases). Human T-cell leukemia virus type I healthy carriers also seemed to possess these VJ-41 antigen positive cells. However, in vitro established adult T-cell leukemia cell lines did not show the reactivity with VJ-41. Normal lymphocytes from humans or mice apparently did not carry this antigen, but mitogen activated lymphocytes or some in vitro maintained malignant cell lines of both human and mouse origins showed positive reaction. Having established solid phase radioimmunoassay to detect the VJ-41 antigen in plasma, it was found that healthy human T-cell leukemia virus type I carriers, but not the majority of adult T-cell leukemia patients, predominantly possessed this antigen. Even though immunochemical characterizations of cellular materials were unsuccessful, a certain neutral glycolipid was detected from healthy human T-cell leukemia virus type I carrier plasma by using thin-layer chromatography and immunostaining with the VJ-41 antibody.     

Interaction of human embryonal carcinoma cells and differentiated derivatives in vitro with simian virus 40, human adenovirus type 7, or PARA

Polyclonal antibodies were used to assay human embryonal carcinoma (EC), differentiating EC, yolk sac carcinoma, and teratoma cells for expression of viral early antigen (T-Ag) after infection with simian virus 40 (SV40). Cells of four EC lines were induced to differentiate by cultivation at low density or by exposure to retinoic acid or dimethyl sulfoxide. After infection with SV40, T-Ag was expressed by 1%, or less, of the cells (presumed to be differentiated derivatives) in only some EC cultures whereas the antigen was synthesized by a significant percentage of the yolk sac carcinoma, teratoma, and differentiating EC cells. Also, viral late proteins were produced by EC cells infected with human adenovirus type 7 (Ad7), and SV40 T-Ag was expressed by EC cells after infection with PARA, which is an Ad7-SV40 hybrid virus containing the SV40 T-Ag sequence controlled by Ad7 late regulatory sequences. Thus, T-Ag is not synthesized by the parental EC cells infected with SV40, but it is expressed in cultures of infected differentiated derivatives. The EC cells produce T-Ag, however, when expression of the viral protein is controlled by the Ad7 regulatory sequences in PARA particles. These results demonstrate that expression of T-Ag after infection with SV40 is an indicator of EC cell differentiation and also raise the possibility that, as in mouse EC cells infected with the virus, the SV40 regulatory sequences controlling T-Ag synthesis are not active in human EC cells.     

Identification of a glycoprotein, gp21, of adult T cell leukemia virus by monoclonal antibody

A mouse hybridoma cell line producing monoclonal antibody, F10, was established from mice hyperimmunized with cells bearing adult T cell leukemia (ATL) virus (ATLV). F10 antibody reacted with an ATLV structural polypeptide ( gp21 ) with a m.w. of 21,000 that was glycosylated on cell surfaces. The gp21 was expressed on cell surfaces of all ATL-associated antigen (ATLA)-positive human cell lines but not on ATLA-negative cell lines nor peripheral blood leukocytes stimulated with mitogens. The gp21 was also detected with anti-ATLA-positive human serum, and the binding of F10 antibody to ATLA-positive cell surfaces was significantly blocked by pretreatment with anti-ATLA-positive human sera. Double immunofluorescence staining with F10 antibody and anti-ATLA-positive human serum caused co-capping on cell surfaces, which suggests that gp21 co-exists with other ATLV antigens expressed on cell surfaces. Immunoprecipitation studies also suggested that the gp21 is a minor component of the ATLV envelope.     

Characterization of a xenogeneic antiserum raised against the fetal germ cells of the mouse: cross-reactivity with embryonal carcinoma cells.

A xenogeneic antiserum (PG-1) raised against the germ cells of the 13.5d p.c. mouse conceptus reacts with the fetal germ cells of both sexes and adult male germ cells, but not with any nongerminal tissue that has been tested. PG-1 can also react with the EC cells of four different teratocarcinoma cell lines. There are, however, marked differences in the absorptive capacity of the different EC cell lines. The antigen(s) recognized by the antiserum are confined to only a proportion of cells in some EC lines. This antiserum has a number of potential applications in studies of the origin and development of the mouse germ cell lineage.     

Development and distribution of a human B cell subpopulation identified by the HB-4 monoclonal antibody

Monoclonal antibodies have been successfully used to identify B cell differentiation antigens, few of which mark discrete B cell subpopulations. We have produced a monoclonal antibody, HB-4, against a cell surface antigen on the human B cell line, BJAB, which has an unusual distribution on normal lymphoid cells. HB-4, an IgM antibody, was found to react with an antigen that is expressed by a subpopulation of B cells, approximately 50% of natural killer cells, and not by other types of cells in bone marrow, blood, and lymphoid tissues. In two-color immunofluorescence assays, the HB-4-reactive antigen was found on less than 5% of immature IgM+ B cells in fetal liver and bone marrow and on 25% of B cells in fetal spleen. The HB-4 antibody reacted with 40% of IgM+ cells in newborn blood and 60% of B cells in adult blood. In contrast, only 2 to 26% of IgM+ B cells in the peripheral lymphoid tissues of adults were HB-4+. HB-4+ B cells could be induced to proliferate by cross-linkage of their surface immunoglobulins but not by T cell-derived growth factors. The subpopulation of activated B cells that is responsive to T cell-derived differentiation factors was HB-4-, as were plasma cells. The HB-4 antibody was reactive with some but not all B cell malignancies and cell lines, and not with malignancies or cell lines of other lineages. The HB-4 antigen may therefore serve as a useful nonimmunoglobulin marker for the identification of a subpopulation of mature resting B cells that are present in the highest frequency in the circulation.     

Expression of markers shared between human natural killer cells and neuroblastoma lines

Summary Neuroblastoma is a tumor of neuroectodermal origin arising most commonly from the adrenal medulla. We have examined the ability of several monoclonal antibodies which recognize markers predominantly expressed on human natural killer (NK) cells to react with neuroblastoma cell lines in vivo derived sections of tumor. HNK-1 (Leu 7) is a monoclonal IgM antibody which recognizes a carbohydrate epitope on NK cells and a wide range of tumor cell types. We have shown that HNK-1 recognizes the human neuroblastoma lines SMS-KCNR, SMS-KAN, NMB/N7, and IMR/5. Expression of this antigen on cell lines can be slightly increased by retinoic acid-induced differentiation of the cells. N901 (NKH1), a monoclonal antibody raised against interleukin 2-dependent human NK cell lines also recognizes all human neuroblastoma cell lines examined. This expression is independent of differentiation induction and levels remain unaltered following retinoic acid treatment of the cell lines. Lastly, with monoclonal antibody 49H.8, it has been found that reactivity of the lines is weak until induction of differentiation, after which highly significant increases of reactivity are seen. 49H.8 recognizes several cryptic carbohydrate antigens with varying affinities, shown to identify mouse and rat NK cells. In contrast to other NK markers, human neuroblastoma cell lines did not express significant reactivity with B73.1, Leu 11b, or Leu 18. Immunohistochemical staining of sections of human neuroblastoma tumors correlated with the in vitro findings; however, staining with N901 and 49H.8 was only seen on frozen sections, not paraffin-embedded. The significance of shared NK cell-neuroblastoma/neuron antigens is currently under investigation.     

Differentiation of human germ cell tumor cells

Human germ cell tumors are an excellent model for investigating the mechanism of human early embryogenesis as well as cellular differentiation. Three human EC cell lines, NCR-G 2, 3 and 4 were newly established from testicular mixed embryonal carcinomas in vitro, G3 and G4 cells were capable of somatic cell differentiation. The G3 cells demonstrated the most noticeable antigenetical changes with the administration of retinoic acid. SSEA-1 appeared on some cells whereas expression of HLA-A, B, C as well as 2H2, 2D7 and 5D4 antigens tended to be reduced in G3 cell line. 2H2, 2D7 and 5D4 antigens which we recently produced were immature human EC specific cell surface antigens, defined by mouse monoclonal antibodies, obtained immunization with G2 cells. The production of hCG, high molecular weight cytokeratin and intercellular matrices such as type IV collagen and laminin were inducible in G3 cells. Thus, G3 cells are thought to be one of the most pluripotent human EC cells. These findings clearly indicate that the EC cell lines we established provide an opportunity to study differentiation mechanism of human germ cell tumors and also human somatic cells.     

Relationship between the rearrangement of immunoglobulin genes, the appearance of a B lymphocyte antigen, and immunoglobulin synthesis in murine pre-B cell lines

Eighteen Abelson virus-transformed immature B cell lines were established and immunoglobulin biosynthesis, expression of a B lymphocyte antigen detected by a monoclonal antibody, and rearrangement of immunoglobulin genes in these cell lines were studied. Only one cell line (A1) synthesized micro-chains but no light chains, and the other cell lines synthesized no detectable immunoglobulins. None of the cell lines established had detectable membrane-associated IgM. Fifteen cell lines expressed a B lymphocyte antigen on their cell surfaces. In three cell lines, however, the majority (greater than 99%) of cells did not express this antigen. Heavy chain genes were rearranged on both chromosomes in all the cell lines, although one heavy chain gene was deleted in three cell lines. In 12 of 18 cell lines, one or both kappa-chain genes were rearranged. In six cell lines, however, both kappa-chain genes remained in embryonic form; lambda-chain genes were in embryonic form in all the cell lines. These results suggested the hierarchy of Ig gene rearrangements, beginning with mu and proceeding to kappa and then to lambda. JH rearrangement was also shown to precede the appearance of a B lymphocyte antigen. In three cell lines (A1-A3), which were considered subclones derived from a single common precursor, it was suggested that one rearranged JH gene was functional, and the other was nonfunctional, indicating that allelic exclusion already operated in pre-B cells.     

A New Monoclonal Antibody that Recognizes 180 kDa Polypeptide Expressed on Early Mouse Embryos and Mouse Embryonal Carcinoma Cells

A hybridoma clone 1F7 producing a monoclonal antibody against OTF9-63 mouse embryonal carcinoma cell line was isolated with the aid of the intrasplenic primary immunization technique. This antibody was capable of recognizing embryonal carcinoma cell lines originated from certain mouse strains, including 129/Sv, but not those which were established from other strains as well as human and other murine cell lines except FM3A, a mouse mammary carcinoma cell line. Indirect immunofluorescence study revealed that 1F7 antigen was expressed on early mouse embryos in a stage specific manner. In order to characterize the 1F7 antigenic molecules, we analyzed both glycoshingolipids and glycoproteins recovered from OTF9-63 by means of immunostaining after thin layer chromatography and SDS-polyacrylamide gel electrophoresis followed by Western blotting, respectively. It was concluded that 1F7 antigenic determinants were carried by 180 kDa polypeptides. One of the most interesting characteristics of 1F7 antigen is complete failure to express on the embryonic ectoderm of 5.5d and 6.5d embryos, one of cell types most closely related to embryonal carcinoma cells. 1F7 antibody may help analyse the process of teratocarcinogenesis in 129/Sv mice.     

A monoclonal antibody to a differentiation antigen present on mature human macrophages and absent from monocytes

A monoclonal antibody is described that has been generated in the mouse against cultured human blood monocytes/macrophages. The antibody, designated 25F9, belongs to the IgG1 subclass, detects antigens of m.w. 86,000, and does not react with freshly isolated blood monocytes but reacts with monocytes after 3 days of culture. The expression of the 25F9 antigen on macrophages increases with culture time. Furthermore, the antibody is negative on platelets, granulocytes, lymphocytes, and a large number of human cell lines except the two melanoma lines MeWo and Mel 57. In cryostat sections of normal human tissue (skin, lung, liver, thymus) and of inflammatory or neoplastic tissue (cutaneous lymphoma, eczema, BCG-granuloma, and melanoma), the antibody reacts with scattered macrophages in the dermis but not with epidermal Langerhans cells, with alveolar macrophages, with liver Kupffer cells, and with scattered macrophages in the cortex and medulla of thymus. In eczema, BCG-granuloma, and cutaneous lymphoma, only a few infiltrating macrophages were stained. On the other hand, a large number of macrophages and melanophages reacted positively in melanoma. In some cases melanoma cells also stained weakly positive. Thus, the antibody detects a differentiation antigen preferentially expressed on mature, tissue-fixed macrophages and absent from blood monocytes.     

A novel cytotoxic T-cell clone that exhibits differential modes of recognition in the proliferative and cytolytic phase

A T-cell clone (1G8-H7) cytotoxic to P815Y mastocytoma (H-2d) has been established from spleen cells of a C3H/He mouse (H-2k) primed with P815Y cells by means of in vitro stimulation with irradiated C3H.H-2o(H-2KdDk) spleen cells. The clone 1G8-H7 was an interleukin 2 (IL-2)-dependent and H-2Kd antigen-dependent CTL clone and it killed P815Y cells but not Concanavalin A-induced spleen blast cells bearing H-2Kd antigen. The involvement of H-2Kd antigen in the cytolytic recognition mechanism was shown by the inhibition of lysis by anti-H-2Kd monoclonal antibody and also by the cold inhibition experiment that employed H-2Kd-bearing spleen cells. Comparison of cytotoxic activities between 1G8-H7 and Kd-specific CTL clones showed that the killing of P815Y cells by clone 1G8-H7 was not explained by the susceptibility to cell-mediated cytolysis of P815Y cells. These results suggest that H-2Kd antigen on the stimulating cell is sufficient to deliver a proliferation signal in the proliferative phase of this clone, but in the cytolytic phase an additional interaction with surface structure on the target cell other than that with H-2Kd antigen is required for the induction of cytolysis. Possible elucidations for the differential modes of recognition are discussed.     

Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.     

High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives

Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-cAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines committed to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.     

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical markers of the progress of differentiation in cloned teratocarcinoma cell lines.

The progeny of single teratocarcinoma cells will give rise to several different cell types in vitro, and the latter were shown to be functionally differentiated by biochemical criteria. In all these studies, cloned lines of mouse teratocarcinoma cells were assayed during the course of differentiation for some biochemical products characteristic of the tissues formed. The carcinoembryonic protein, alpha-foetoprotein, was not synthesized by undifferentiated embryonal carcinoma (EC) cells, but was synthesized in increasing amounts during their differentiation to endoderm-type cells in suspension culture. alpha-Foetoprotein was shown to be a product of endoderm cells, but not all endoderm cells synthesized this protein. During the course of further differentiation when EC cells or aggregates were grown in tissue-culture dishes, other biochemical products appeared. In cultures containing predominantly nerve-type cells, there was a 30-fold increase in the specific activity of acetylcholinesterase, with concomitant appearance of the aldolase isoenzyme characteristic of mouse brain. In some cultures, a small amount of muscle-type cell formation was marked by the appearance of the MB isoenzyme of creatine phosphokinase. Generally, biochemical differentiation was immature.     

Comparison of the effects of transforming growth factor beta, N,N-dimethylformamide, and retinoic acid on transformed and nontransformed fibroblasts

In order to compare the effects of transforming growth factor (TGF beta) with those of the differentiation promoters N,N-dimethylformamide (DMF) and retinoic acid (RA), the antiproliferative and fibronectin-inducing activities of the three agents were examined. AKR-2B mouse embryo fibroblasts and their chemically transformed counterpart AKR-MCA cells were used as the model system. Growth in monolayer culture of both cell lines was inhibited by TGF beta (EC50 approximately 1 ng/ml), DMF (EC50 approximately 0.5%), and RA (EC50 approximately 1 microM) in a concentration-dependent manner. Time-dependent elevation in fibronectin expression was also observed with all three agents. The EC50 for growth inhibition of both cell lines by TGF beta agreed well with that obtained for stimulation of fibronectin synthesis. A 3-h exposure to TGF beta is sufficient to obtain the maximal fibronectin level observed at 48 h in AKR-2 B cells but not in AKR-MCA cells. Our results indicate that in this system the effects of TGF beta are similar to those of the chemical differentiation inducers DMF and RA. Furthermore, our data also suggest that the TGF beta signal may be processed differently by nontransformed and transformed fibroblasts.