Elevated Expression of Interleukins in Lung Adenocarcinomas Induced by N-Nitrosobis(2-hydroxypropyl)amine in Rats

The expression of interleukins (ILs) in lung adenocarcinomas induced by N -nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks old, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNAs were extracted from 14 individual adenocarcinomas and 2 specimens of normal lung tissue of untreated rats. In adenocarcinomas, elevated expression of IL-1α (6/14), IL-1β (14/14), IL-3 (7/14), IL-4 (11/14), IL-5 (9/14), IL-6 (11/14) and IL-10 (8/14) was observed, compared with normal lung tissues. In contrast, no expression of IL-2 was detected in any case. The results suggest that preferential expression of these ILs and their complex networks may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.     

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.     

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.     

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.     

Carcinogenic Potency of N-Nitrosomethyl(2-hydroxypropyl)amine and Other Metabolic Relatives of N-Nitrosobis(2-hydroxypropyl)amine by Single Intraperitoneal Injection on the Lung of Rats

The carcinogenic effects of a single intraperitoneal injection of N-nitrosobis(2-hydroxypropyl)-amine (BHP) or its metabolic relatives, N-nitrosomethyl(2-hydroxypropyl)amine (MHP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitroso-2,6-dimethylmorpholine (NDMM), were studied in male Wistar rats. The main target organ of these nitrosamines proved to be the lung, followed by the thyroid. Lung lesions were induced in a dose-dependent manner with total lung tumor incidences reaching 55% to 100%. BHP, MHP, HPOP and NDMM all caused lung carcinomas to develop (22% to 44% incidence), whereas BOP was only associated with adenomas. On the basis of dose administered and incidence of carcinomas, MHP appeared to be the most potent lung carcinogen of the five nitrosamines investigated. Smaller numbers of neoplasms were also induced in the kidney, urinary bladder, esophagus and intestine at differing rates by these nitrosamines.     

Potentiation of Invasive Capacity of Rat Ascites Hepatoma Cells by Transforming Growth Factor-β

The in vitro invasive capacity of poorly invasive cells (W₁), which were cloned from rat ascites hepatoma cells (AH 130), was potentiated dose- and time-dependently by pretreating the cells with transforming growth factor-β (TGF-β). This potentiation of invasive capacity was completely abolished by anti-TGF-β antibody. When the treated cells were ip inoculated into rats, the cells extensively invaded the peritoneum, and formed penetrating tumor nodules. The effect of TGF-β was reversed by subculturing the treated cells without TGF-β. The potentiation of invasive capacity by TGF-β might participate in platelet-associated enhancement of tumor cell metastasis.     

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.     

Improvement of Macrophage Dysfunction by Administration of Anti-transforming Growth Factor-β Antibody in EL4-bearing Hosts

An experimental therapy for improvement of macrophage dysfunction caused by transforming growth factor-β (TGF-β) was tried in EL4 tumor-bearing mice. TGF-β was detected in cell-free ascitic fluid from EL4-bearers, but not in tbat from normal mice, by western blot analysis. The ascites also showed growth-suppressive activity against MvlLn cells, and the suppressive activity was potentiated by transient acidification. To investigate whether the functions of peritoneal macrophagcs were suppressed in EL4-bearers, the abilities to produce nitric oxide and tumor necrosis factor-α (TNF-α) upon lipopolysaccharide (LPS) stimulation were measured. Both abilities of macrophages in EL4-bearing mice were suppressed remarkably on day 9, and decreased further by day 14, compared with non-tumor-bearing controls. TGF-β activity was abrogated by administration of anti-TGF-α antibody to EL4-bearing mice. While a large amount of TGF-β was detected in ascitic fluid from control EL4-bearers, little TGF-β was detectable in ascites from EL4-bearers given anti-TGF-β antibody. Furthermore, while control macrophages exhibited little or no production of nitric oxide and TNF-α on LPS stimulation in vitro , macrophages from EL4-bearers administered with anti-TGF-β antibody showed the same ability as normal macrophages. These results clearly indicate that TGF-β contributes to macrophage dysfunction and that the administration of specific antibody for TGF-β reverses macrophage dysfunction in EL4-bearing hosts.     

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.     

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.     

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.     

Programmed Cell Death Ligand (PD-L1) Expression in Stage II and III Lung Adenocarcinomas and Nodal Metastases

IntroductionProgrammed death ligand 1 (PD-L1) expression determined by immunohistochemistry (IHC) may serve as a predictive biomarker for anti–PD-1/PD-L1 therapies; however, little is known about intertumoral heterogeneity of PD-L1 expression determined by IHC in lung adenocarcinomas (ADCs), and there have been conflicting results on the prognostic role of PD-L1 expression in ADCs.MethodsPD-L1 expression was evaluated in resected stage II and III ADCs by using various cutoffs and correlated with clinicopathologic parameters and survival. PD-L1 expression was also compared between the primary tumor and lymph node metastases.ResultsThere were 109 study cases. PD-L1 expression was seen in 56 (51%), 43 (39%), and 19 (17%) when cutoffs of at least 1%, at least 5%, and at least 50%, respectively, were used. Abundant intratumoral CD8-positive T cells were a significant predictor of the expression in the primary tumor, with cutoffs of 1% and 5% (p < 0.001 for both) by multivariate analysis, whereas they were a nonsignificant predictor of the expression with a 50% cutoff (p = 0.076). PD-L1 expression was concordant between the primary tumor and nodal metastasis in most of the cases, but it was discrepant in up to 38%. The discrepancy was attributed in part to different predominant histologic patterns between the primary and metastatic tumors. In the entire cohort, PD-L1 expression with all cutoffs had no bearing on 5-year recurrence-free survival.ConclusionsPD-L1 expression is associated with abundant intratumoral CD8-positive T cells in resected ADCs, suggesting a predictive role of PD-L1 expression in anti–PD-1/PD-L1 therapies; however, the possible intertumoral heterogeneity of PD-L1 expression raises a concern about selecting the most appropriate sample for PD-L1 IHC.     

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.     

Transforming Growth Factor-α Expression of Renal Proximal Tubules in Wistar Rats Treated with Ferric and Aluminum Nitrilotriacetate

A high incidence of renal adenocarcinoma has been observed in rats treated with ferric nitrilotriacetate (Fe-NTA) but not in rats treated with aluminum nitrilotriacetate (Al-NTA). Transforming growth factor (TGF)-α is one of the several cytokines that is known to be expressed in human and rat renal adenocarcinomas. However, its role in neoplastic transformation is still questionable. Therefore, we investigated the effect of repeated Fe-NTA and Al-NTA administration on renal TGF-α expression. Male Wistar rats were given Fe-NTA (n = 16, 5–10 mg FeAg) and Al-NTA (n = 19, 1–2 mg Al/kg) i.p., three times a week for 3 or 12 weeks. Another group of rats (n=4) was given Fe-NTA (5–10 mg FeAg) three times a week for 12 weeks and then left untreated for one year. Immunoreactivity for TGF-α was positive in the collecting ducts and on the apical surface of proximal tubules in the outer stripe of the outer medulla in all the animals including NTA-injected control animals. However, TGF-α immunoreactivity in the regenerative proximal tubular epithelium was observed only in the animals treated with Fe-NTA for 12 weeks. Northern blot analysis also showed expression of TGF-α mRNA only in animals treated with Fe-NTA for 12 weeks. The expression of TGF-α mRNA in the kidney was stronger than that in the liver or brain, TGF-α was also positive in renal cell carcinoma found in animals treated with Fe-NTA for 12 weeks and left untreated for one year. These results suggest that TGF-α expression may play an important role in renal carcinogenesis and that it may be a sensitive marker during the induction stage of renal cell carcinoma.     

Inhibitory Effects of Antioxidants on N-Bis(2-hydroxypropyl)nitrosamine-induced Lung Carcinogenesis in Rats

Potential second-stage modifying effects of 8 antioxidants on lung tumorigenesis initiated by N-bis(2-hydroxypropyl)nitrosamine (DHPN) were examined in male F344 rats. After an initial 2-week treatment with DHPN (0.1% in drinking water), rats were administered one of the antioxidants supplemented in the diet for 30 weeks. Although the incidences of lung adenomas were not affected, those of carcinomas were lowered by 2% butylated hydroxyanisole (BHA, 2 rats/20 rats), 1% butylated hydroxytoluene (BHT, 1/20), 0.8% ethoxyquin (EQ, 3/20) and 1%α-tocopherol (α-TP, 2/20) treatments as compared to the control level (9/20), while 5% sodium l -ascorbate (SA), 0.8% catechol (CC), 0.8% resorcinol (RN), and 0.8% hydroquinone (HQ) did not exert any significant effect on incidence. Quantitative analysis of adenomas and carcinomas (numbers and areas of lesions per unit area of lung section) revealed obvious inhibitory effects of SA, CC, and RN as well as BHA, BHT, EQ, and α-TP. Among the antioxidants, BHT exerted the strongest inhibitory activity. In contrast, DHPN-induced thyroid tumorigenesis was significantly enhanced by BHT (14/20) and EQ (20/20) treatments (control=5/20). Thus the antioxidants showed opposite effects on lung and thyroid carcinogenesis in the rat.