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Role of Transforming Growth Factor-ß1 in Invasion and Metastasis in Gastric Carcinoma

From the Cancer Center, Department of Surgery II, and Department of Medical Informatics, Faculty of Medicine, Kyushu University; and National Kyushu Cancer Center, Fukuoka, Japan.

Address reprint requests to Yoshihiko Maehara, MD, Department of Surgery II, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Transforming growth factor-beta1 (TGF-ß1) is a major modulator of cellular proliferation and extracellular matrix formation. We determined the role of TGF-ß1 in invasion and metastasis in gastric cancer.

MATERIALS AND METHODS: We detected TGF-ß1 expression in primary and lymph node metastatic lesions of gastric cancer, using an antibody and in situ hybridization. The plasma TGF-ß1 levels in the peripheral vein and in the tumor drainage vein were assayed.

RESULTS: In the cytoplasm of cancer cells, TGF-ß1 was immunostained in 35.9% (78 of 217) of primary gastric carcinomas, and this expression was confirmed by in situ hybridization. Of 59 gastric carcinomas with a TGF-ß1–negative primary tumor, metastatic lymph nodes were positive for TGF-ß1 staining in 32 cases (54.2%). Positive staining of TGF-ß1 in gastric cancer tissues was closely related to serosal invasion, infiltrative growth, and lymph node metastasis. Multivariate analysis showed that the expression of TGF-ß1 was an independent risk factor for serosal invasion and infiltrative growth of the tumor. The plasma level of TGF-ß1 did not differ between TGF-ß1–negative and –positive groups. There were also no differences in plasma TGF-ß1 levels among each tumor stage, between the peripheral and the tumor drainage veins, and between preoperative and postoperative testings.

CONCLUSION: Transforming growth factor-ß1 is closely related to the invasion and metastasis of gastric cancer, and production of TGF-ß1 in the tumor does not contribute to the total amount of TGF-ß1 in the blood circulation. We interpret our observations to mean that in a tumor microenvironment, TGF-ß1 alters the biologic behavior of the tumor.

	INTRODUCTION

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TRANSFORMING GROWTH FACTOR-beta (TGF-ß) is a family of 25-kd homodimeric polypeptides that were initially identified by their potential to induce growth of mesenchymal cells in soft agar.^1,2 This growth factor regulates cell growth and differentiation in both normal and transformed cells.^3,4 Transforming growth factor-ß1, the most widely studied protein of the three TGF-ß isoforms, derived from a 390-amino acid precursor cleaved to produce a 112-amino acid carboxy-terminal peptide,⁵ is the predominant form in humans.^6,7 Transforming growth factor-ß1 was found to inhibit the growth of normal and neoplastic cells.⁸ In normal cells, TGF-ß1 generally enhances adhesion through increased matrix production and decreased proteolysis.⁹ Resistance to the negative growth-regulating properties of TGF-ß has been observed in epithelial and mesenchymal tumors.¹⁰ Tumor cell lines that lack TGF-ß receptors lose responsiveness to TGF-ß, and the escape of cells from TGF-ß-mediated negative regulation is linked to tumor progression.^11,12

On the other hand, the more aggressive forms of tumors are growth-stimulated by TGF-ß.^13,14 Treatment of the murine mammary carcinomas with TGF-ß1 enhanced their invasion and the rate of pulmonary metastasis.¹⁵ Metastatic fibrosarcomas derived by transfection of fibroblasts with oncogenes were reported to release TGF-ß1 in its active form at significantly higher levels than in corresponding nontransfected cells.^16,17 Tumor cell lines that constitutively secrete active TGF-ß1 are more tumorigenic than those that do not.¹⁸ Tumor cells that produce active TGF-ß1 make use of proteolysis coupled with motility to achieve invasion.^19,20 Huang et al⁶ reported that TGF-ß1 is an autocrine-positive regulator of colon cancer cells because transfection of a TGF-ß1 antisense expression plasmid decreased the tumorigenicity. In cases of breast cancer, expression of TGF-ß1 was positively associated with invasion and metastasis, but expression of TGF-ß2 and TGF-ß3 was not.^21,22 These observations suggest that TGF-ß1 synthesized by the tumor cells may facilitate tumorigenesis and, hence, regulate tumor cell behavior.

Gastric cancer is the leading cause of cancer deaths in Japan, and the survival time of patients with gastric cancer has improved with advances in diagnostics and treatment.²³ This cancer is invasive and metastasization to different organs follows.²⁴ Gastric cancer cells were reported to produce and secrete TGF-ß1 protein, which is responsible for the observed collagen deposition, particularly in cases of scirrhous carcinoma.^25,26 The latent form of TGF-ß1 becomes the active form in the presence of serine protease, extracellularly.^26,27 The stroma developed by TGF-ß1 could provide a hospitable environment for tumor growth, and TGF-ß1 plays a crucial role in tumor extension.^28,29 We examined the expression of TGF-ß1 in primary gastric cancer and tissues in lymph node metastases, as well as plasma TGF-ß1 levels in the peripheral and tumor-drainage veins. Our objective was to determine the clinical significance of TGF-ß1 in advance of a gastric tumor.

	MATERIALS AND METHODS

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Patients
To determine the 5-year survival rate for primary gastric cancer, we studied 217 Japanese adult men and women treated from 1989 to 1992. Immunohistochemistry, in situ hybridization, clinicopathologic, and survival analyses were done. For 48 patients treated in 1992, plasma TGF-ß1 assays were performed. All of these patients underwent gastric resection in the Department of Surgery II, Kyushu University, and the National Kyushu Cancer Center. The following standardized procedures were performed: (1) gastric resection (the resection line was 3 cm from the macroscopic edge for localized tumors and 6 cm for infiltrative tumors); (2) prophylactic lymph node dissection of more than D2 resection; and (3) complete excision of invaded organs, irrespective of the number of sites on the organs, when there was no evidence of incurable factors, such as peritoneal dissemination, liver metastasis, and widespread nodal involvement. Pathologic diagnoses and classification of the resected gastric cancer tissues were made according to the Japanese classification of gastric carcinoma.³⁰ The lymph nodes of groups 1, 2, and 3 are referred to as n1, n2, and n3, respectively, on the basis of the extension of lymph node metastasis. Gastric resection on the basis of lymph node dissection was classified as follows: D1, gastric resection with complete removal of the group 1 lymph node alone; D2, gastric resection with complete removal of group 1 and 2 lymph nodes; and D3, gastric resection with complete removal of group 1, 2, and 3 lymph nodes. "Curability A" indicates that there were no residual tumors and but there was a high probability of cure, under the following conditions: no serosal invasion; n0 treated by D1, D2, D3, or n1 treated by D2, D3; M0, P0, H0; and proximal and distal margins of more than 10 mm. "Curability B" indicates that there were no residual tumors but the patient could not be assessed as Curability A. "Curability C" indicates that there was definite residual tumor. Of the 217 patients, 137 are alive at the time of this writing. Recurrences of the gastric cancer and death occurred in 62 patients, and 18 patients died with another disease. Data from patients who died of causes other than gastric cancer were censored in the statistical analysis.³¹ No patient died within 30 postoperative days. Written informed consent was obtained from each patient.

TGF-ß1 Staining
Tissue sections were immunostained with a polyclonal antibody against human TGF-ß1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).³² Xylene was used to remove paraffin from the sections, and then the sections were progressively hydrated in decreasing concentrations of ethanol. The slides were placed in a thermoresistant beaker filled with 0.1 M phosphate-buffered saline (PBS) (pH 7.4) and autoclaved at 121°C to allow the fixed embedded tissue antigen to react with the monoclonal antibody. The sections were then cooled down to room temperature for about 20 min and rinsed in PBS. These sections were then covered with normal rabbit serum for 15 min to reduce nonspecific staining and incubated with a 1:100 dilution of primary antibody at room temperature for 1 hour. Next, the sections were washed with PBS, incubated with a 1:600 dilution of biotinylated goat anti-rabbit immunoglobulin G (DAKO, Copenhagen, Denmark) at room temperature for 30 min, and then covered with a 1:1,000 dilution of labeled streptavidin peroxidase (DAKO) at room temperature for 30 minutes. The antibody was localized with 3,3'-diaminobenzidine tetrahydrochloride, and 0.065% sodium azide was used to block endogenous peroxidase. Immunostaining was nil when the slides were processed in the absence of a primary antibody or by applying nonimmune sera instead of specific antibodies.

In Situ Hybridization
Transforming growth factor-ß1 in situ hybridization probes were prepared from a pBluescript 2KB+ plasmid containing a 985-base pair fragment of rat TGF-ß1 cDNA insert (American Type Culture Collection, Rockville, MD).³³ The plasmid was linearized with restriction enzyme XbaI for T3 RNA polymerase (antisense probe) or HindIII for T7 RNA polymerase (sense probe) mRNA runoffs incorporating digoxigenin-labeled UTP, according to instructions in the Genius RNA labeling kit (Boehringer Mannheim, Mannheim, Germany).³⁴ All steps were performed at room temperature unless otherwise specified.

Formalin-fixed, paraffin-embedded tissue sections of 5 µm were deparaffinized in xylene, hydrated in a graded series of ethanol to PBS, postfixed with 4% paraformaldehyde for 15 minutes, digested with 10 µg/mL proteinase K (Boehringer Mannheim) for 15 minutes, and postfixed again with 4% paraformaldehyde for 10 minutes. Sections were acidified with 0.2 N HCl for 10 minutes and acetylated with 0.1 M triethanolamine-HCl (pH 8.0)/acetic anhydride for 10 minutes. After the sections were dehydrated with ethanol, they were incubated overnight at 50°C with the 500-ng/mL digoxigenin–labeled RNA probe in hybridization buffer (10 mM Tris-HCl [pH 7.6] containing 50% deionized formamide, 10 µg/mL yeast tRNA, 1x Denhardt's solution, 10% dextran sulfate, 600 mM NaCl, 0.25% sodium dodecyl sulfate, and 1 mM EDTA [pH 8.0]). After hybridization, sections were washed in 2x standard saline citrate (SSC) for 30 minutes at 50°C, TNE buffer (10 mM Tris-HCl, 500 mM NaCl, and 1 mM EDTA [pH 7.6]) for 10 min at 37°C, and TNE buffer containing 10 µg/mL RNase for 30 minutes at 37°C. The sections were then washed in 2x SSC for 20 minutes at 50°C and finally in 0.2x SSC for 2 minutes at 50°C. Slides were washed for 5 minutes in DIG 1 buffer (10 mM Tris-HCl and 150 mM NaCl [pH 7.5]) before incubation with alkaline phosphatase–conjugated antidigoxigenin antibody (Boehringer Mannheim) at 1:500 dilution for 30 minutes. After a wash in DIG 1 buffer, nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate toluidinum solution in DIG 3 buffer (10 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCl₂ [pH 9.5]) was applied, according to the Boehringer Mannheim protocol, in a dark, humid chamber. The bluish-purple color development was allowed to occur over the next 48 hours. The color reaction was stopped with EDTA solution (10 mM Tris-HCl and 1 mM EDTA [pH 7.6]). Controls run in parallel with each experiment included tissue sections that were pretreated with RNase to degrade all mRNA and incubated in hybridization mix without the probe. Probed TGF-ß1 sense and antisense mRNA were run on sequential sections of each specimen.

Preparation of Plasma and TGF-ß1 Assay
One to 2 ml of blood sample, taken from the medial cubital vein before surgery and 3 weeks after surgery and from the tumor drainage vein (left gastric vein) during surgery, was put into silicone-coated test tubes containing sodium-EDTA. After centrifugation at 1,000 x g for 30 min, the plasma was prepared and stored at -80°C until needed for assays. The active and total (active plus latent) TGF-ß1 were assayed using human TGF-ß1 immunoassay kits (R & D Systems, Minneapolis, MN).³⁵ This assay involves use of the quantitative sandwich enzyme immunoassay technique. Transforming growth factor-ß soluble receptor type 2, which binds TGF-ß1, was coated onto the microtiter plate contained in the kit. Standards and samples were pipetted into the wells, and any TGF-ß1 present was bound by the immobilized receptor. After unbound substances were washed away, an enzyme-linked polyclonal antibody specific for TGF-ß1 was added to the wells to sandwich the TGF-ß1 immobilized during the first incubation. The selectivity of the immunoassay is ensured by use of the polyclonal antibody specific for TGF-ß1. After a wash in wash buffer to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and the color developed in proportion to the amount of TGF-ß1 bound in the initial step. When color development ceased, the intensity of the color was measured at 450 nm. To measure total TGF-ß1 (active plus latent TGF-ß1), samples were acidified using 1 N HCl for 10 min. After incubation, acidified samples were neutralized to pH 7.2 to 7.6 with 1.2 N NaOH/0.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

Statistical Analysis
The BMDP Statistical Package program (BMDP, Los Angeles, CA) for the IBM (Armonk, NY) 3090 mainframe computer was used for all analyses.³⁶ The BMDP 4F and 3S programs were used for the ² test and the Kruskal-Wallis test to compare patient data between the groups. The BMDP 1L program was used to analyze survival time, by the Kaplan-Meier method. The Mantel-Cox test was used to test for equality of the survival time. The BMDP 2L program was used to determine independent prognostic factors for simultaneous multivariate adjustment of all covariates, by Cox regression analysis with the forward stepwise model.³⁷ The BMDP LR program was used to determine independent risk factors for simultaneous multivariate adjustment of each covariate, using a backward stepwise logistic regression analysis.^38,39 The level of significance was P less than .05.

	RESULTS

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TGF-ß1 Expression Determined by Immunohistochemical Methods and Based on Clinicopathologic Factors
Gastric cancer tissues excised from 217 patients were examined using a monoclonal antibody directed against TGF-ß1 protein. The TGF-ß1 protein was stained mainly in the cytoplasm of cancer cells, as shown in Figure 1, and was occasionally evident in fibroblasts and smooth muscle cells. The staining pattern in the normal epithelium showed no staining whatsoever or only weak reactivity, whereas cancer cell reactivity was prominent. We then examined different levels of TGF-ß1-positive cells to determine whether the expression of TGF-ß1 might serve as a predictor of behavior of a tumor. Staining was classified as negative if less than 10% of cells were positive and as positive if more than 10% were positive. Seventy-eight (35.9%) of 217 tissues from patients were positive for TGF-ß1 staining.

View larger version (161K): [in this window] [in a new window]   Fig 1. Photomicrograph of TGF-ß1 staining in human primary gastric cancer. TGF-ß1 staining of gastric cancer tissues was evident in the cytoplasm of cancer cells, as shown in this high-power photomicrograph (original magnification, x200).

Positive findings of TGF-ß1 staining in gastric cancer cells did not depend on age, sex, tumor size, location of the tumor, or histology. In cases of TGF-ß1-positive cases, serosal invasion was more prominent and the infiltrative growth was more common. The rate of lymph node metastasis was higher in TGF-ß1-positive cases, but the presence of lymphatic and vascular involvement, peritoneal dissemination, and liver metastasis showed no relation with TGF-ß1 staining (Table 1).

Surgical management was also compared between the groups. There were no differences with regard to the extent of the gastrectomy, the extent of lymph node dissection, and operative curability (Table 2).

TGF-ß1 Expression in Gastric Cancer Tissues Determined by In Situ Hybridization
We next examined the expression of TGF-ß1 mRNA in 20 gastric cancer tissues. Varying degrees of cytoplasmic expression of TGF-ß1 mRNA were observed in gastric cancer cell specimens used for immunohistochemical staining of TGF-ß1 (Fig 2A and 2B). The degree of staining was prominent in cases of TGF-ß1-positive staining, but it was rare for negative staining cases. The pattern of distribution of TGF-ß1 mRNA matched that seen with immunohistochemical staining.

View larger version (81K): [in this window] [in a new window]   Fig 2. Detection of TGF-ß1 mRNA in gastric cancer tissues, using in situ hybridization: (A) hematoxylin and eosin; (B) in situ hybridization study, using a probe to TGF-ß1 mRNA. The cytoplasmic signal is evident in the gastric cancer cells (original magnification, x200).

Relationship of TGF-ß1 Expression Between Primary and Lymph Node Metastasis Lesions
We determined the relationship between the staining of TGF-ß1 in the primary lesion and metastatic lesions in the lymph nodes, using 108 pairs of tissues. The positive rate was 45.4% (49 of 108) for primary lesions and 69.4% (75 of 108) for metastatic lesions in the lymph nodes. Of 59 cases with no staining in the primary lesion, 32 cases (54.2%) showed TGF-ß1 staining in the metastatic lesion (Table 3).

Survival Rates
At the time these data were analyzed, the median follow-up time for the 137 survivors was 93.6 months (range, 6.3 to 109.2 months). The postoperative survival curves for patients with TGF-ß1–negative and TGF-ß1–positive tumors are shown in Fig 3. The survival time was shorter for the TGF-ß1–positive patients, compared with the survival time for TGF-ß1–negative subjects. The 5-year survival rate was 69.2% for TGF-ß1–negative patients and 53.3% for TGF-ß1–positive patients (P < .05).

View larger version (10K): [in this window] [in a new window]   Fig 3. Survival curves for patients with TGF-ß1–stained positive cells (bottom line) was lower compared with findings with no TGF-ß1 staining (top line), with a significant difference (P < .05).

Next, we determined survival rates for the patients with regard to TGF-ß1–negative and TGF-ß1–positive tumors and serosal invasion, histologic growth pattern, and lymph node metastasis (Table 4). Survival curves for patients with TGF-ß1–positive gastric cancers were lower in each subgroup with serosal invasion, a histologic growth pattern, and lymph node metastasis, and there was a statistical difference only between TGF-ß1–negative and –positive groups of serosal invasion–positive cases; the patient number here was smaller in each subgroup and the statistical power was lower.

Multivariate Analysis
Cox regression analysis of all factors listed in Table 1 revealed that lymph node metastasis, serosal invasion, and peritoneal dissemination were independent factors related to the poorer prognosis of the patients (P < .01) but not the expression of TGF-ß1. Logistic regression analysis revealed that the expression of TGF-ß1 is an independent risk factor for serosal invasion and infiltrative growth of the tumor (P < .05).

Plasma TGF-ß1 Levels
The circulating TGF-ß1 levels were assayed for 48 patients (Table 5). The form of TGF-ß1 was almost all latent. The level varied in each case, and there was no difference in plasma TGF-ß1 levels between the TGF-ß1 stain–positive and –negative groups. The TGF-ß1 levels did not change with each stage of tumor serosal invasion and between peripheral vein and drainage vein of the tumor. Surgical removal of the tumor did not alter the plasma level of TGF-ß1, as assayed 3 weeks after surgery. Thus, the TGF-ß1 level in the blood circulation has no apparent relationship with the presence of tumor burden in patients with gastric cancer.

	DISCUSSION

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Gastric cancer cells were positive for TGF-ß1 mRNA and protein expression, and the TGF-ß1 levels were higher in cancer cells than in normal cells.^25-27,40 Mahara et al²⁶ reported that scirrhous cell lines secreted mainly active TGF-ß1, whereas nonscirrhous cells secreted latent TGF-ß1 in vitro.²⁵ Horimoto et al²⁷ identified a serine protease from gastric cancer cell line KATO III, which alters latent TGF-ß1 to the active form extracellularly. The TGF-ß1 produced by carcinoma cells stimulated collagen synthesis in both fibroblasts and cancer cells, which leads to diffuse fibrosis of scirrhous gastric carcinomas.^25,41 Grégoire and Lieubeau²⁹ reported that TGF-ß stimulates the differentiation of fibroblasts into myofibroblasts, accumulates extracellular matrix production, and protects cancer cells from immune cytotoxic effects. In light of these results, it seemed necessary to determine the relationship between TGF-ß1 expression and the biologic behavior of gastric cancer cells.

The expression of TGF-ß1 in gastric cancer cells was closely related to infiltrative growth of the cancer and to the higher rate of lymph node metastasis. The preferential expression of TGF-ß1 in lymph node metastases suggests a clonal selection of tumor cells with TGF-ß1 expression, specific for the higher potential of lymph node metastasis in tumor advance, and TGF-ß1 has a role related to the malignant progression of gastric cancer. Yashiro et al⁴² reported that TGF-ß1 increased the migratory activity of gastric cancer cells in vitro. Highly expressed TGF-ß1 was also reported to be a determining factor for tumor progression and a poorer prognosis in cases of human colorectal and breast cancers.^7,21,43

We compared the plasma levels of TGF-ß1 at the different tumor stages and between the tumor drainage vein and peripheral vein to determine whether the tumor was the main source of circulating TGF-ß1. We also examined the plasma levels of TGF-ß1 before and after surgery. Among the TGF-ß species, TGF-ß1 has been mainly recognized in the blood of humans, compared with TGF-ß2 and TGF-ß3.^44,45 The half-life of TGF-ß1 in plasma was reported to be about 2 hours for the latent form and 2 to 3 minutes for the active form⁴⁶; therefore the level in the third postoperative week should reveal the effects of surgical resection of the tumor. Tsushima et al⁷ reported that plasma TGF-ß1 levels reflected overexpression of the gene in colorectal cancer tissues and were associated with disease progression. Resection of the tumor decreased the plasma level of TGF-ß1 in these colorectal cancers. Ivanovic et al⁴⁷ reported that the plasma TGF-ß1 level in prostatic adenocarcinoma with invasive disease was significantly elevated compared with that of benign prostatic hyperplasia or nonhyperplasia. Plasma TGF-ß1 levels were also seen to be elevated in patients with hepatocellular carcinoma.⁴⁸ In breast cancer patients, TGF-ß1 levels in the peripheral vein were higher and the level was normalized after surgical removal of the tumor.⁴⁴ However, Wakefield et al⁴⁵ reported that of 28 patients with advanced metastatic breast cancer, only two had greatly elevated levels of TGF-ß1. We found that the level of TGF-ß1 varied with the patient, but no differences in plasma levels of TGF-ß1 were noted between the TGF-ß1 stain–positive and –negative groups, between each tumor stage, and between the tumor drainage vein and the peripheral vein, in a larger number of patients. Surgical resection of the tumor did not decrease the plasma level of TGF-ß1. It was reported that secretion of TGF-ß1 by the tumor does not contribute significantly to the total TGF-ß1 in the blood circulation⁴⁵ and that TGF-ß1 is not elevated systematically through platelet activation, which has been proposed to occur in patients with clinically active cancer.⁴⁹ The source of plasma TGF-ß1 has not been identified; therefore, TGF-ß1 produced by cancer cells probably functions in an autocrine and paracrine manner only in the microenvironment in the tumor tissues of gastric cancer. Although the latent form of TGF-ß1 is activated to active form extracellularly, active TGF-ß1 was unlikely to be present in plasma because this form has a short half-life in the blood circulation.⁵⁰

Cells overexpressing active TGF-ß1 by gene transfection showed increased metastatic progression.⁴⁷ Transforming growth factor-ß1 exerts action via binding to specific cell surface receptors. In most cell types, three different types of TGF-ß receptors, ie, types I, 2, and 2I, can be observed.^5,51 We examined the relationship of expressions of TGF-ß1 and TGF-ß receptor I in 50 gastric cancer tissues, and noted a correlation rate of 88% (44 of 50) in the stainings of cancer cells (data not shown). Transformed cell clones that secreted active TGF-ß1 were more tumorigenic than were cells that secreted latent TGF-ß1.¹⁸ The moderately or highly metastatic fibrosarcomas were growth-stimulated by TGF-ß1, whereas nonmetastatic transformed cells of identical cell lineage were growth-inhibited; these findings were similar to those in the nontransformed parental cell lines.^16,17 The in vitro invasive capacity of less invasive cells cloned from rat ascites hepatoma cells was potentiated dose and time dependently by pretreatment of the cells with TGF-ß1.⁵² Induction of DNA synthesis in established cell lines by TGF-ß1 is preceded by induction of platelet-derived growth factor B chain mRNA as well as other mRNAs associated with cell replication, such as fos, jun, and myc.^19,53 Thus, alteration in the responsiveness to TGF-ß1 can be detected immediately after immortalization, an event that may contribute to the increased proliferative potential of these cells. We also detected the expression of platelet-derived growth factor B chain protein in the gastric cancer cells of TGF-ß1–positive cases, using immunohistochemical methods (data not shown). In normal cells, collagenases, cathepsin L, and transin/stromelysin were suppressed by TGF-ß1⁵⁴; however, in tumor cells, the synthesis and activity of these and other proteases were enhanced by TGF-ß1.^15,19,20 Therefore, TGF-ß1 is considered to enhance motility and protease production through autocrine activation, and the invasive and metastatic potential is increased.

Our findings showed that TGF-ß1 is closely related to invasion and metastasis and that in a tumor microenvironment, TGF-ß1 alters the biologic behavior of a malignant gastric lesion.

	ACKNOWLEDGMENTS

 
Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture and Sports in Japan.

We thank M. Ohara for comments on the manuscript.

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Submitted March 9, 1998; accepted October 15, 1998.

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