Potentiation of Metastatic Capacity by Transforming Growth Factor-β1 Gene Transfection

This study was designed to assess whether the excessive secretion of transforming growth factor-β1 (TGF-β1) by Chinese hamster ovary (CHO) cells transfected with TGF-β1 gene may be linked to the development of a metastatic phenotype. We observed large numbers of metastatic colonies in the lungs of nude mice inoculated with the transfected CHO cells. The tumors derived from these transfected cells demonstrated marked angiogenesis. We postulate that the overproduction of TGF-β1 by these tumors may participate in the metastatic progression following establishment of angiogenesis at the primary tumor site.     

Transforming Growth Factor-β and Hepatocyte Growth Factor Produced by Gastric Fibroblasts Stimulate the Invasiveness of Scirrhous Gastric Cancer Cells

Scirrhous gastric carcinoma is characterized by cancer cells that infiltrate rapidly in the stroma with extensive growth of fibroblasts. In the present study, we examined the effect of gastric fibroblasts on the invasiveness of a Scirrhous gastric cancer cell line, OCUM-2D, using an invasion assay. Gastric fibroblast-derived conditioned medium (CM) significantly stimulated the invasiveness of OCUM-2D cells, as did transforming growth factor- β (TGF- β ) and hepatocyte growth factor (HGF). The stimulating activity of gastric fibroblast-derived CM was inhibited significantly by anti-TGF- β neutralizing antibody or anti-HGF neutralizing antibody. TGF- β and HGF were detected in the gastric fibroblast-derived CM, and TGF- β receptor and C-met (HGF receptor) were expressed on OCUM-2D cells. Thus, TGF- β and HGF produced by gastric fibroblasts appear to affect the invasiveness of scirrhous gastric cancer cells. TGF- β was also detected in the conditioned medium derived from OCUM-2D cells, though HGF was not. TGF- β appears to affect the invasiveness of OCUM-2D cells in both paracrine and autocrine fashions.     

Inhibitory Action of Transforming Growth Factor-β on Induction of Differentiation of Myeloid Leukemia Cells

The effect of transforming growth factor-β (TGF-β) on induction of differentiation of mouse myeloid leukemia M1 cells was investigated. TGF-β1 induced adherence of M1 cells to plastic dishes and inhibited their proliferation. However, it did not induce differentiation-associated properties, such as phagocytic activity, lysozyme activity or morphological maturation. TGF-β1 also caused dosedependent inhibition of dexamethasone-induced differentiation of M1 cells. The inhibitory activity of TGF-β1 was 20 times that of TGF-β2 on M1 cells. These results suggest that TGF-β1 inhibits proliferation and dexamethasone-induced differentiation of M1 cells by interacting with receptors that can distinguish between TGF-β1 and TGF-β2. TGF-β1 had a much lower inhibitory effect on the growth of a variant M1 cell clone, which was resistant to differentiation inducers, and it did not induce adherence of the resistant M1 cells.     

Inhibition of Tumor Necrosis Factor-α and β Secretion by Lymphokine Activated Killer Cells by Transforming Growth Factor-β

Transforming growth factor- β (TGF- β ) has a variety of immunosuppressive properties. We investigated the effect of TGF- β secreted by glioblastoma (T98G) cells on the secretion of tumor necrosis factor- α and - β (TNFs) by lymphokine activated killer (LAK) cells stimulated with tumor cells. The supernatant from T98G cells was preincubated with anti-TGF- β l and - β 2 neutralizing antibodies or untreated, and added to a coculture of LAK and Daudi cells. The neutralizing antibodies were added to LAK/Daudi and LAK culture, and natural human TGF- β and recombinant human TGF- β were also added to the LAK/Daudi culture. LAK cells were also cultured with T98G cells, of which the supernatant contained both active and latent forms of TGF-/ β 1 and TGF- β 2, and the neutralizing antibodies were added to the coculture. TNFs activity in the supernatants from LAK/ Daudi cultures was examined by a specific bioassay. Addition of the supernatant from T98G cells to LAK/Daudi culture resulted in the inhibition of TNFs secretion by LAK cells. The inhibition was abrogated by the pretreatment of the supernatants with the anti-TGF- β antibodies. Addition of TGF- β and TGF- β to LAK/Daudi culture inhibited TNFs secretion by LAK cells in a dose-dependent manner. Addition of anti-TGF β - antibodies to LAK culture resulted in an increase of TNFs secretion. These results suggest that, if tumor cells have the capacity to convert TGF- β from a latent to an active form, the active TGF- β suppresses TNFs secretion by LAK cells stimulated with the tumor cells, and that TGF- β secreted and activated by glioblastoma cells suppresses the propagation of immune reaction by inhibiting TNFs secretion by activated lymphocytes adjacent to tumor cells.     

Elevated Levels of Plasma Transforming Growth Factor-β in Patients with Hepatocellular Carcinoma

We measured the plasma transforming growth factor-β (TGF-β) concentration in 14 patients with human hepatocellular carcinoma (HCC) and 9 age-matched normal subjects using growth inhibition assay of mink lung epithelial cells. The calculated plasma TGF-β concentration in the patients with HCC was 28.6 ± 27.9 ng/ml (mean± SE), showing significant elevation compared with that in 9 normal subjects (5.3 ± 3.3 ng/ml, P<0.01). In three cases, we could measure plasma TGF-β levels before and after their treatment for HCC. The plasma TGF-β levels decreased from 59.0 to 18.2 ng/ml after hepatic resection in one case, and from 24.0 to 10.7 ng/ml and from 12.4 to 3.4 ng/ml after transhepatic arterial embolization in the other two cases. These data indicate that plasma TGF-β level is elevated in patients with HCC, probably due to release from HCC tissues.     

Enhancement of Tumorigenicity and Invasion Capacity of Rat Mammary Adenocarcinoma Cells by Epidermal Growth Factor and Transforming Growth Factor-β

We have studied the effects of growth factors and cytokines on the tumorigenicity and invasion capacity of tumor cells by using regressor and progressor tumor cell lines (ER-1 and ERpP, respectively) derived from an SHR rat mammary adenocarcinoma. ER-1 cells regress spontaneously whereas ERpP cells show invasive growth and high metastasis to lung and other organs in syngeneic SHR rats. When ER-1 cells were pretreated with either epidermal growth factor (EGF) or transforming growth factor-β (TGF-β) for 24 h in vitro , and intraperitoneally transplanted into SHR rats, they grew and killed the host, whereas ER-1 cells pretreated with tumor necrosis factor-α did not. Tumorigenicity and invasion capacity of ERpP cells were also enhanced by treatment with EGF and TGF-β. The ER-1 cells pretreated with EGF, once grown in vivo , had acquired irreversible tumorigenicity and invasion capacity without requiring further EGF treatment, and the enhanced malignancy was irreversible. These findings suggest that growth factors play an important role in acquisition of malignancy of tumor cells.     

In vitro Invasion of Endothelial Cell Monolayer by Rat Ascites Hepatoma Cells

To study the tissue preference of invasion, we developed an assay system for the invasion of endothelial cells as a modification of the previously established assay of tumor cell invasion of meso-thelial cells. Rat ascites hepatoma cells (AH 130) that had been seeded on a monolayer of cultured endothelial cells penetrated and formed tumor cell colonies under the monolayer. The penetration was time-dependent and the number of penetrated tumor cells and colonies was proportional to the number of tumor cells seeded. Comparison of the in vitro tumor cell invasion of endothelial cell monolayer with that of cultured mesothelial cell layer showed that a clone from the tumor cells (CI-30) which was highly penetrative into the mesothelial cell layer had only a limited ability to penetrate the endothelial cell layer.     

Modulation of c-myc Expression by Transforming Growth Factor β1 in Human Hepatoma Cell Lines

The effects of transforming growth factor β1 (TGF-β1) on cell proliferation of human hepatoma cell lines, PLC/PRF/5 and Mahlavu, were investigated under serum-free conditions. DNA synthesis was strongly inhibited in the PLC/PRF/5 cells by addition of TGF-β1 (0.5 to 4.0 ng/ml), but remained unchanged in the Mahlavu cells. Also the expression of c- myc mRNA was suppressed by the addition of TGF-β1 in the PLC/PRF/5 cells but not in the Mahlavu cells. These results indicate that TGF-β1 might regulate cell growth, in part, by modulating c- myc expression, although there is no direct proof that c- myc expression is really relevant to DNA synthesis mediated by TGF-β1.     

Transforming Growth Factor-β CD4+ T cells Tumor-bearing state Immunosuppression Enhanced Production of TGF-β and a Progressive Increase in TGF-β Susceptibility of Anti-tumor CD4+ T Cell Function

The present study deals with the effect of transforming growth factor-β (TGF-β) on anti-tumor immune responsiveness at various stages of the tumor-bearing state. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1–3 wk after inoculation with CSA1M cells produced interleukin-2 (IL-2) and macrophage-activating factor (MAF)/interferon-γ (IFN-γ) upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4⁺ T cells and antigen-presenting cells that had been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. The IL-2-producing capacity of CD4⁺ T cells reached the maximal level as early as one week after tumor implantation but decreased with the progress of tumor-bearing stages. In contrast, the capacity of CD4⁺ T cells to produce MAF/IFN-γ was not affected but was maintained at high levels even late in the tumor-bearing state. The addition of recombinant TGF-β (rTGF-β) to cultures of spleen cells from various tumor-bearing stages resulted in the suppression of lymphokine production. However, the magnitude of the TGF-β-induced suppression varied depending on which tumor-bearing stages of splenic cells were tested as a responding cell population; it was slight in cells from early (1–3 wk) tumor-bearing stages but increased in cells from donor mice at later tumor-bearing stages. Thus, spleen cells from late tumor-bearing stages with weak but significant IL-2-producing and considerable MAF/IFN-γ producing capacities failed to produce these lymphokines when rTGF-β was present in cultures. A progressive increase in the TGF-β susceptibility was also observed for IL-4-producing Th2 as well as IL-2/MAF-producing Th1 cells. In addition, increased levels of TGF-β were detected in plasma from tumor-bearing mice at late stages. Taken together, these results indicate that tumor-bearing mice exhibit enhanced production of TGF-β as well as a progressive increase in the susceptibility of anti-tumor CD4⁺ T cells to TGF-β-induced suppressive mechanisms.     

Gene Expressions of Protein Tyrosine Phosphatases in Regenerating Rat Liver and Rat Ascites Hepatoma Cells

mRNA levels for ten protein tyrosine phosphatases (PTPs), PTP-S, PTPH1, PTP-1, GLEPP1, LRP, PTP1D, PTPG1, PTPγ, PTPδ, and LAR, were determined during regeneration of rat liver, and mRNA levels for 5 PTPs, PTP-S, PTP-1, PTPγ, PTPδ, and LRP, were determined in three lines of rat ascites hepatoma cells. In regenerating rat liver, the expression patterns of PTP genes after partial hepatectomy could be classified into four groups. In group 1 (PTP-S and PTPH1), the mRNA levels increased rapidly, reached a maximum 7 h after partial hepatectomy, remained at a plateau for 1–2 days and then decreased gradually. In group 2 (PTP-1, GLEPP1, and LRP), the mRNA levels showed two peaks on days 1 and 5, and then decreased gradually. In group 3 (PTP1D and PTPG1), the mRNA levels increased rapidly, reached a maximum at 7 h, remained high for several days, and then did not decrease but rather increased after day 7. In group 4 (PTPγ, PTPδ, and LAR), the mRNA levels remained constant for the first 5 days and increased over the control levels after day 7. In rat ascites hepatomas, gene expression of non-receptor-like PTPs (PTP-S and PTP-1) showed various neoplastic alterations, whereas mRNAs of receptor-like PTPs (PTPγ, PTPdL, and LRP) were lost or drastically decreased.     

Expression of Epidermal Growth Factor, Transforming Growth Factor-α and Their Receptor Genes in Human Gastric Carcinomas; Implication for Autocrine Growth

The expressions of mRNA for epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and EGF receptor (EGFR) genes were examined in 7 human gastric carcinoma cell lines and 15 gastric carcinoma tissues and the corresponding normal mucosas. All of the gastric carcinoma cell lines expressed mRNA for EGFR and TGF-α genes. TMK-1 and MKN-28 cells also expressed EGF mRNA. Production of EGF, TGF-α and EGFR protein by gastric carcinoma cell lines was also confirmed by EGF and TGF-α specific monoclonal antibody binding. As for surgical specimens, EGFR and TGF-α mRNA were detected at high levels in all the tumor tissues. Interestingly, EGF mRNA was detected in 5 (33.3%) of the 15 gastric carcinomas but it was not detected in normal tissues. Moreover, anti-EGF and anti-TGF-α monoclonal antibodies inhibited the spontaneous ³H-TdR uptake by gastric carcinoma cells. These results suggest that EGF and/or TGF-α produced by tumor cells act as autocrine growth factors for gastric carcinomas.     

Distribution of Transforming Growth Factor-β and Its Receptors in Gastric Carcinoma Tissue

The distribution of the three mammalian isoforms of transforming growth factor (TGF)-β (TGF-β1,-β2, and -β3) as well as their signaling receptors, TGF-β type I and type II receptors (TβR-I and TβR-II, respectively), in gastric carcinoma tissue was examined by immunohistochemistry using specific antibodies. Tissue specimens were obtained from 25 cases of gastric carcinoma, which were classified into two groups according to Lauren's classification, i.e. 15 cases of diffuse carcinoma and 10 cases of intestinal carcinoma. In normal gastric mucosa apart from carcinoma nests, all of TGF-β1, -β2, -β3, TβR-I and TβR-II were clearly demonstrated in fundic glands. In sharp contrast, none of them was detectable in surface mucous cells. In carcinoma cells, strong staining for TGF-β1, -β2 and β3 was obtained only in diffuse-type carcinoma. In particular, carcinoma cells scattered as single cells or small nests had a tendency to show strong staining for TGF-βs. The receptors tended to be distributed concomitantly with the ligands, and diffuse-type carcinoma showed stronger receptor staining than intestinal-type carcinoma. In cancer stroma, TGF-βs and receptors were detected in both diffuse and intestinal types, but the area with positive staining was wider and more dispersed in diffuse-type carcinoma than in intestinal carcinoma. These results suggest that TGF-β may contribute in part to the variety of histogenesis and mode of progression of gastric carcinoma.     

Serum Requirement for in vitro Invasion by Tumor Cells

The effect of fetal calf serum (FCS) on in vitro invasion by rat ascites hepatoma cells (AH130) was studied by using the in vitro invasion assay. Although the coculture of the highly invasive clone (MM1) of AH130 cells and the mesothelial cell layer or endothelial cell layer in modified minimum essential medium supplemented with 10% PCS resulted in extensive penetration of the layer by the tumor cells, the omission of PCS resulted in an almost complete elimination of the in vitro invasion. The in vitro invasiveness by human small cell lung cancer cells (OCIO) was also remarkably reduced by the omission of PCS from the assay medium, suggesting a requirement of serum for the in vitro tumor cell invasion. When 10% PCS was added to the medium 2 h after the tumor cell seeding in FCS-free invasion assay system, penetration by MM1 cells was observed within an hour. This rate of penetration was almost the same as that when 10% PCS was added at the time of tumor cell seeding. PCS was also required for the penetration of a mesothelial cell monolayer by MM1 cells in a defined growth medium (SFM-101), in which MM1 cells were well maintained. The invasion-inducing activity appears to be independent of the growth-stimulating activity in serum.     

Transforming Growth Factor-α Induces the Differentiation of Sarcomatoid Cholangiocarcinoma Cells

A sarcomatoid cholangiocarcinoma cell line, ETK-1, was established from a patient. Phenotypically, the cells corresponded to immature biliary epithelial cells. Because a small number of ETK-1 cells appeared to differentiate spontaneously along a biliary epithelial lineage in continuous culture, we examined the factors that initiate and/or promote the differentiation of the cells. Transforming growth factor-α (TGFα) induced significant changes in ETK-1 cells. After stimulation with the factor, ETK-1 cells displayed morphologic transformation at a much higher frequency, with the appearance of many large cells with intracytoplasmic vacuoles, and the production of mucinous substances. These morphologically transformed cells were phenotypically similar to welldifferentiated adenocarcinoma cells. The expression pattern of integrins after TGFα treatment also supported the maturation of the ETK-1 cells. The antibody against the receptor of TGFα inhibited these changes by TGFα. Moreover, the proliferation rate of ETK-1 cells was suppressed by TGFα. Our data suggest that TGFα can act as a differentiation factor along a biliary epithelial lineage.     

Secretion of Transforming Growth Factor-ß by Human Myelogenous Leukemic Cells and Its Possible Role in Proliferation of the Leukemic Cells

Transforming growth factor(TGF)-ß activity was found in the neutral extracts of human myelogenous leukemic cells or K562 cells and the conditioned medium from K562 cell culture. BALB/c 3T3 cells grown in soft agar in the presence of TGF-ß₁ produced an activity that stimulated the growth of K562 cells. This activity was non-dialyzable. acid-stable, heat-sensitive and partially Inactivated by pronase treatment. These results suggest a mutual growth reliance between the leukemic cells and fibroblasts mediated by paracrine growth factors produced by these cells.     

Detection of Active Form of Transforming Growth Factor-β in Cerebrospinal Fluid of Patients with Glioma

We examined transforming growth factor-β (TGF-β) activity in cerebrospinal fluid of 39 patients with various brain tumors, and found it in 10 glioma cases that had lesions related to subarachnoid space or ventricle. In one glioma case, TGF-β detected on admission disappeared after radiation and chemotherapy. We confirmed that five glioma cell lines produced TGF-β, and that four of them produced active form of TGF-β directly. The active form of TGF-β was also identified from cerebrospinal fluid before the acidification treatment in two cases. The calculated contents were 110 ng/ml and 18 ng/ml. These results indicate that active form of TGF-β is directly produced by tumor cells in patients with glioma, and may contribute to immunodeficiency of the host.     

Mutations and Reduced Expression of the Transforming Growth Factor-β Receptor II Gene in Rat Lung Adenocarcinomas Induced by N-Nitrosobis-(2-hydroxypropyl)amine

Mutations and expression of the transforming growth factor-β receptor type II (TGF-βRII) gene were investigated in lung adenocarcinomas induced by N -nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-βRII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-βRII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-βRII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.     

Close Correlation between the Dephosphorylation of p53 and Growth Suppression by Transforming Growth Factor-β1 in Nasopharyngeal Carcinoma Cells Transduced with Adenovirus Early Region Genes

The mechanism of growth inhibition by transforming growth factor (TGF)-β1 was investigated. We examined the growth inhibitory effects of TGF-β1 on human nasopharyngeal carcinoma (KB) cells which constitutively expressed p53. TGF-β1 suppressed the DNA synthesis of KB cells in a dose-dependent manner. It had minimal effect on adenovirus-2-transduced KB cells expressing either adenovirus early region 1B (E1B) or 1A (E1A) product, which respectively binds to p53 or Rb product and inhibits its function, and no growth inhibition at all was observed with KB cells expressing both E1B and E1A products. Dephosphorylation of the p53 was promoted by TGF-β1 stimulation in KB cells, but not in E1B-producing KB cells, which sequestrate the function of p53. The growth inhibition of KB cells by TGF-β1 was significantly reduced by treatment with okadaic acid. These results suggest that p53 transduces the antiproliferative signal of TGF-β1 possibly through its dephosphorylation.