Expression of epidermal growth factor, transforming growth factor-alpha and their receptor genes in human gastric carcinomas; implication for autocrine growth

The expressions of mRNA for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) 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-alpha genes. TMK-1 and MKN-28 cells also expressed EGF mRNA. Production of EGF, TGF-alpha and EGFR protein by gastric carcinoma cell lines was also confirmed by EGF and TGF-alpha specific monoclonal antibody binding. As for surgical specimens, EGFR and TGF-alpha 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-alpha monoclonal antibodies inhibited the spontaneous 3H-TdR uptake by gastric carcinoma cells. These results suggest that EGF and/or TGF-alpha produced by tumor cells act as autocrine growth factors for gastric carcinomas.     

Induction of Growth Factor-receptor and Metalloproteinase Genes by Epidermal Growth Factor and/or Transforming Growth Factor-α in Human Gastric Carcinoma Cell Line MKN-28

We examined the effects of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) on EGF receptor (EGFR) phosphorylation and the expression of mRNAs for oncogenes, growth factors, their receptors and metalloproteinase genes by MKN-28 gastric carcinoma cells which express EGF, TGF-α and EGFR genes. Both EGF and TGF-α stimulated EGFR phosphorylation. EGF and TGF-α induced FOS, MYC and ERBB-2 oncogene expression. Interestingly, EGF increased the expression of mRNAs for TGF-α and EGFR. On the other hand, TGF-α increased TGF-α mRNA but decreased the expression of mRNAs for EGFR and TGF-β. Furthermore, mRNAs for interstitial collagenase, stromelysin and procollagen type I genes were also enhanced after treatment with EGF and TGF-α. These results indicate that EGF and TGF-α successively evoke cascade phenomena which favor tumor progression, invasion and extracellular matrix formation, acting as autocrine growth regulators for gastric carcinomas.     

EGF and TGF-alpha, the ligands of hyperproduced EGFR in human esophageal carcinoma cells, act as autocrine growth factors

In order to ascertain autocrine growth factors in esophageal carcinomas, we analysed expression of mRNAs and proteins for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in 6 esophageal carcinoma cell lines. Gene alterations were also examined. All of the esophageal carcinoma cell lines expressed mRNA for EGFR and TGF-alpha genes. Interestingly, EGF mRNA of about 5.0 kb was also detected in TE-1, TE-2, and TE-8 cells. Production of protein was also confirmed by binding assay and ELISA on 3 of the 6 cell lines. The cells had a relatively high number of EGFRs and produced TGF-alpha and EGF protein at the same time. Furthermore, anti-EGF (KEM-10) and anti-TGF-alpha (WA-3) monoclonal antibodies (MAbs) inhibited spontaneous uptake of tritiated thymidine (3H-TdR) by TE-1 cells which expressed EGF, TGF-alpha and EGFR mRNA and protein. These results strongly suggest that EGF and/or TGF-alpha produced by carcinoma cells function as autocrine growth factors for human esophageal carcinomas.     

Expression of cripto, a Novel Gene of the Epidermal Growth Factor Family, in Human Gastrointestinal Carcinomas

The expression of mRNA for cripto gene, a novel transforming gene of the epidermal growth factor family, was examined in 20 alimentary tract carcinoma cell lines, 60 surgically resected tumor tissues and their adjacent normal mucosas. Although the cripto mRNA was not detected in esophageal carcinomas or in normal mucosas, it was detected in gastric and colorectal carcinomas. In gastric carcinomas, 2.2 kb cripto mRNA was detected in one cell line, all the gastric carcinoma tissues and their adjacent normal mucosas. Of 23 gastric tumor tissues 8 (34.8%) exhibited a higher mRNA level than normal gastric mucosas. cripto mRNA was detected in 2 out of 6 colorectal carcinoma cell lines. Interestingly, 18 (81.8%) out of 22 colorectal carcinoma specimens expressed a higher level of cripto mRNA than that in normal mucosas. The level of the expression was higher than that in gastric carcinoma tissues. The expression was also correlated to tumor stage of colorectal carcinomas.     

Coexpression of Platelet-derived Growth Factor (PDGF) A-Chain and PDGF Receptor Genes in Human Gastric Carcinomas

In this study we examined the expression of platelet-derived growth factor (PDGF) A-chain and PDGF receptor genes in seven human gastric carcinoma cell lines and 15 gastric carcinoma tissues. Expression of mRNA for PDGF A-chain was found in all gastric cell lines and all gastric carcinoma tissues. Two of the seven gastric carcinoma cell lines expressed PDGF receptor mRNA. Out of the 15 gastric carcinoma tissues, eight showed enhanced expression of PDGF receptor mRNA and all of them demonstrated prominent fibrous stroma. Moreover, the incidence of enhanced expression of PDGF receptor mRNA was higher in scirrhous carcinoma than in well differentiated adenocarcinoma. These results strongly suggest that PDGF produced by tumor cells acts as a paracrine growth factor for production of fibrous stroma in gastric carcinomas     

Quantitative Assay of Epidermal Growth Factor Receptor in Human Squamous Cell Carcinomas of the Oral Region by an Avidin-Biotin Method

A quantitative assay method for epidermal growth factor receptors (EGFRs) of human tumor tissues was established, based on enzyme-labeled avidin-biotin (LAB) interaction with anti-human EGFR monoclonal antibody 52SIgG. A standard calibration curve for EGFR estimation in human tumor tissues was obtained with A431#8 cells cloned from A431 human epidermoid carcinoma cell line. The coefficient of variance for the standard curve was below 35% in the application to tumor tissues from nude mice implanted with human tumor cell lines. The minimum tissue amount required for the quantitative assay was around 0.1 g (wet weight). Using the LAB method, the correlation between the level of EGFR number and tumor malignancy was examined for 14 human squamous cell carcinomas (SCCs) from the oral region. Seven of the SCCs showed a more than two-fold higher EGFR number compared to normal gingival tissues. Three highly aggressive carcinomas with poor prognosis possessed five to ten times higher levels of EGFR number than normal tissues. The elevated EGFR level in the SCCs seems to correlate to increasing tumor size and the stage of SCCs as clinically classified according to the 1987 UICC TNM system.     

Epidermal Growth Factor Receptors in Cancer Tissues of Esophagus, Lung, Pancreas, Colorectum, Breast and Stomach

The levels of epidermal growth factor (EGF) receptors were investigated in surgically resected tumors of various origins including esophagus (n = 33), lung (n = 14), pancreas (n = 9), colorectum (n = 10), breast (n = 23) and stomach (n = 8). The ¹²⁵I-EGF binding capacities of squamous cell carcinomas of esophagus and lung were exceptionally higher than those of the other cancer tissues. Immunohistochemical staining with an anti-EGF receptor monoclonal antibody detected EGF receptors in the basal cells and parabasal cells of normal esophageal epithelium and in all the cancer cells of squamous cell carcinoma tissues of esophagus and lung. DNA replicating cells were examined by the bromodeoxyuridine staining method and it was found that the basal cells and parabasal cells of normal epithelium and peripheral cells of cancer pearls are proliferating. Contrary to this, a tumor antigen TA-4, known as a specific marker for squamous carcinoma, was detected in the differentiated cancer cells and in middle-layer squamous cells. These results strongly suggest that the increase in EGF receptor levels may be associated with the development of human squamous cell cancers of esophagus and lung. Thus, measurement of EGF receptor expression in tumor tissues has diagnostic value and should prove useful for the development of new therapies.     

Coexpression of platelet-derived growth factor (PDGF) A-chain and PDGF receptor genes in human gastric carcinomas

In this study we examined the expression of platelet-derived growth factor (PDGF) A-chain and PDGF receptor genes in seven human gastric carcinoma cell lines and 15 gastric carcinoma tissues. Expression of mRNA for PDGF A-chain was found in all gastric cell lines and all gastric carcinoma tissues. Two of the seven gastric carcinoma cell lines expressed PDGF receptor mRNA. Out of the 15 gastric carcinoma tissues, eight showed enhanced expression of PDGF receptor mRNA and all of them demonstrated prominent fibrous stroma. Moreover, the incidence of enhanced expression of PDGF receptor mRNA was higher in scirrhous carcinoma than in well differentiated adenocarcinoma. These results strongly suggest that PDGF produced by tumor cells acts as a paracrine growth factor for production of fibrous stroma in gastric carcinomas.     

EXPRESSION OF EPIDERMAL GROWTH FACTOR, TRANSFORMINGGROWTH FACTOR-α AND THEIR RECEPTOR IN HUMAN PITUITARY TUMORS

The development of pituitary tumor was regulated by multiple-step signal transduction and their complicated process. A lot of growth factors participated in the cell's proliferation and their function activity, so the abnormal in growth factor and/or its receptor may involve in the pituitary tumorigenesis.[1] To investigate the role of epidermal growth factor receptor (EGFR) and its protein tyrosine kinases signaling conduction in the pathogenesis of pituitary tumors, we examined the expressi…     

慢性胆囊炎和胆囊癌组织中EGF、EGFR的表达及其意义

背景与目的:表皮生长因子(epidermalgrowthfactor,EGF)及其受体(epidermalgrowthfactorreceptor,EGFR)在肿瘤形成过程中起着重要作用,胆结石胆囊炎与胆囊癌关系密切,本研究通过观察EGF、EGFR在慢性胆囊炎、胆囊癌中的表达,探讨两者与胆囊癌发生的关系。方法:采用免疫组化SABC法检测手术切除的41例胆囊癌、26例单纯增生、14例不典型增生和10例正常胆囊组织中EGF、EGFR及增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)的表达。结果:EGF、EGFR阳性表达率在胆囊癌(63.4%、70.7%)、不典型增生(71.4%、85.7%)中分别高于单纯增生(15.4%、27%)和正常胆囊组织(0%、0%)(P<0.01);PCNA评分有随胆囊粘膜病变程度的加重而递增的趋势,在正常胆囊组织、单纯增生、不典型增生和胆囊癌中分别为1.0、1.0、1.9288±0.9972和3.0488±0.669,(P<0.01);EGF、EGFR表达与PCNA的表达有统计学意义(P<0.05),EGF、EGFR、PCNA的表达与胆囊癌TNM分期无统计学意义(P>0.05)。结论:EGF、EGFR的表达参与胆囊癌的发生,与细胞增殖活性相关。     

Co-expression of heparin-binding EGF-like growth factor and related peptides in human gastric carcinoma

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of polypeptide growth factors, which includes EGF, transforming growth factor α (TGF-α), amphiregulin (AR) and betacellulin (BTC). To assess the potential role of HB-EGF in human gastric carcinomas, the expression of HB-EGF and EGF receptor (EGF-R) was examined in normal and cancerous gastric tissues and cultured gastric cancer cell lines. By Northern blot analysis, there was a 4.7-fold increase in HB-EGF mRNA levels in human gastric cancers compared with normal gastric tissues. There was a concomitant 3.9-fold increase in EGF-R mRNA levels in these cancers. Immunostaining revealed co-localization in 72% of the cancer cells of HB-EGF and EGF-R. AR and BTC moieties were not evident by Northern blot analysis. However, using PCR, both AR and BTC mRNA species were demonstrated in normal and cancerous gastric tissues. By Northern blot analysis, HB-EGF, TGF-α, AR, BTC and EGF-R mRNA moieties were co-expressed in KATO III and NCI-N87 gastric cancer cell lines. Furthermore, HB-EGF, EGF and TGF-α enhanced the growth of both cell lines in a dose-dependent manner. Our findings suggest that HB-EGF is relatively abundant in human gastric cancers and that co-expression of the EGF ligand family may lead to excessive activation of EGF-R in this disorder. © 1996 Wiley-Liss, Inc.     

Expression of Amphiregulin, a Novel Gene of the Epidermal Growth Factor Family, in Human Gastric Carcinomas

The expression of mRNA for amphiregulin (AR), a novel gene of the epidermal growth factor family, was examined in 8 human gastric carcinoma cell lines and 32 gastric carcinoma tissues as well as corresponding normal mucosa. Of the 8 gastric carcinoma cell lines, 7 expressed 1.4 kb AR mRNA at various levels. The expression of AR mRNA by TMK-1 and MKN-28 cells was increased by treatment with epidermal growth factor or transforming growth factor α. In surgical cases, all the gastric carcinoma tissues and their adjacent normal mucosa expressed AR mRNA. Interestingly, 20 (62.5%) out of 32 tumors expressed AR mRNA at higher levels than their corresponding normal mucosas (tumor/normal ≥1.2). No obvious correlation was observed between the AR mRNA levels and the histological types or tumor staging of gastric carcinoma. Immunohistochemically, AR protein was localized to the cytoplasm and/or nucleus in tumor cells. These results suggest that AR produced by tumor cells may be involved in the pathogenesis and/or progression of human gastric carcinoma.     

Establishment and characterization of a human gastric scirrhous carcinoma cell line in serum-free chemically defined medium

We have established a human gastric scirrhous carcinoma cell line (designated as HSC-43) in a serum-free chemically defined medium (CDM) without any polypeptide growth factor, from a primary tumor of a 56-year-old male patient. HSC-43 cells grew in vitro in adherence with a population doubling time of 55 hr, and had the cytological properties of mucinous epithelial tumor cells. Cytogenetic analysis of the cells revealed pseudo-tetraploidy, with structural abnormalities of deletion at chromosome Iq25 and with 3 marker chromosomes. Some cells had retained features of signet-ring cells and caused fibroblastic proliferation when transplanted into athymic nude mice. The possible involvement of transforming growth factor-α(TGF-α), and its receptor, the epidermal-growth-factor receptor (EGFR), on the growth of HSC-43 cells was studied. Synthesis and secretion of TGF-α by HSC-43 cells were confirmed by biological assay and enzyme-linked immunosorbent assay. Radioreceptor analysis showed the presence of receptors for EGF in HSC-43 cells. Proliferation of HSC-43 cells was inhibited by antibodies against TGF-α and/or the EGFR. However, neither TGF-α nor epidermal growth factor (EGF) was effective in stimulating the cell growth of HSC-43 cells, irrespective of the cell density when supplemented exogenously. Our data suggest that TGF-α and EGFR play a role in the autocrine growth of HSC-43 cells. This may be another example of growth regulation of gastric carcinoma.     

Relationship between VEGF, EGF and invasion, metastasis of gastric cancer cells

Objective: To investigate the relationship between expression of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and the biological properties of gastric cancer cells such as invasion and metastasis.Methods: RT-PCR was performed to semi-quantitatively detect the mRNA expressions of EGF, EGFR, VEGF and VEGFR in four kinds of gastric cancer cell lines BGC823, MGC803, HGC27 and SGC7901, which were classified by their differentiation degree in our experiment. We obtained cell line growth curves from MTT assays. The migration of gastric cancer cells was observed under inverted phase contrast microscope. The changes of invasion and adhesion were detected by a Transwell assay.Results: The growth rates slowed down sequentially in MGC803, HGC27, BGC823 and SGC7901(P＜0.05). The ability of migration, invasion and adhesion were reduced sequentially, and the difference was significant. The expressions of EGF, EGFR, VEGF and VEGFR were significantly stronger in MGC803 and HGC27 than in BGC823 and SGC7901 cells, and the difference was statistically significant(P＜0.05).Conclusion: The expressions of VEGF and EGF had close relationship with the properties of migration, adhesion and invasion of gastric cancer cells in vitro. Thus, targeting VEGF and EGF may be a potential therapeutic strategy for inhibiting peritoneal metastasis of gastric cancer.     

Epidermal Growth Factor-dependent Dissociation of CrkII Proto-oncogene Product from the Epidermal Growth Factor Receptor in Human Glioma Cells

Human glioma cells frequently overexpress epidermal growth factor receptor (EGFR). We found that the CrkII proto-oncogene product was associated with the EGFR in human glioma cells in the absence of epidermal growth factor (EGF). EGF stimulation of glioma cells induced the phosphorylation of tyrosine 221 of the CrkII protein, which correlates with its dissociation from the EGFR. By contrast, Shc and Grb2 were inducibly associated with the EGFR in response to EGF stimulation of glioma cells. In A431 cells, epidermoid carcinoma cells which overexpress EGFR, CrkII was tyrosine-phosphorylated and associated with the EGFR in an EGF-dependent manner. Therefore, the dissociation of CrkII from the EGFR upon stimulation with EGF appears to be specific to glioma cells. The Cbl oncogene product was also tyrosine-phosphorylated in U87MG glioma cells upon EGF stimulation. However, unlike in other cell lines, CrkII was not inducibly bound to Cbl in U87MG glioma cells. Thus, EGF-dependent binding of CrkII to phosphotyrosine-containing proteins appears to be suppressed in glioma cells. To evaluate the physiological role of dissociation of CrkII from EGFR, we expressed the CrkII-23 mutant in glioma cells. CrkII-23 mutant, which was isolated as a suppressor gene of the EGF-dependent transformation of NRK cells, binds constitutively to EGFR. We found that expression of CrkII-23 inhibited the anchorage-independent growth of the glioma cells in the presence of EGF. Taken together, these data implicate EGF-dependent dissociation of CrkII from EGFR in the oncogenicity of human glioma cells.     

Expression of cripto, a novel gene of the epidermal growth factor family, in human gastrointestinal carcinomas

The expression of mRNA for cripto gene, a novel transforming gene of the epidermal growth factor family, was examined in 20 alimentary tract carcinoma cell lines, 60 surgically resected tumor tissues and their adjacent normal mucosas. Although the cripto mRNA was not detected in esophageal carcinomas or in normal mucosas, it was detected in gastric and colorectal carcinomas. In gastric carcinomas, 2.2 kb cripto mRNA was detected in one cell line, all the gastric carcinoma tissues and their adjacent normal mucosas. Of 23 gastric tumor tissues 8 (34.8%) exhibited a higher mRNA level than normal gastric mucosas. cripto mRNA was detected in 2 out of 6 colorectal carcinoma cell lines. Interestingly, 18 (81.8%) out of 22 colorectal carcinoma specimens expressed a higher level of cripto mRNA than that in normal mucosas. The level of the expression was higher than that in gastric carcinoma tissues. The expression was also correlated to tumor stage of colorectal carcinomas.     

An effect of K-ras gene mutation on epidermal growth factor receptor signal transduction in PANC-1 pancreatic carcinoma cells

The Ras protein is involved in tyrosine kinase signal transduction pathway steps such as EGFR signalling. Most human pancreatic carcinomas harbor a point mutation of K-ras oncogene and overexpress transforming TGF-α. We studied how K-ras gene mutation could influence the EGFR signal transduction mechanism and the autonomous proliferation of pancreatic carcinoma cells, using PANC-1 human pancreatic carcinoma line and WI-38 normal human fibroblast cell line as a control. PANC-1 cells responded to neither EGF nor exogenous TGF-α, although anti-TGF-α MAb suppressed their growth. Expression of TGF-α mRNA was detected only in PANC-1 cells, which confirmed EGFR being within an autocrine loop. Ras protein and MAP kinase were constitutively activated in PANC-1 cells so that the cells did not respond to treatment with staurosporine or herbimycin A, and exhibited slight response to EGF stimulation. PANC-1 cells harbored K-ras gene mutation in codon 12. In contrast, EGF stimulation induced an elevation of GTP-bound ratio to Ras protein and an activation of MAP kinase with accelerated growth in WI-38 cells. From these findings, we concluded that K-ras gene mutation possibly plays an important role in the autonomous proliferation of PANC-1 pancreatic carcinoma cells, and that an autocrine loop represented by TGF-α and EGFR may further accelerate the growth of PANC-1 cells. © 1996 Wiley-Liss, Inc.     

Expression of TGF-beta and procollagen type I and type III in human gastric carcinomas

To elucidate the difference between scirrhous and non-scirrhous gastric carcinomas, we examined the expressions of TGF-beta, procollagen type I and type III in 7 gastric carcinoma cell lines and 37 gastric carcinoma tissues, and also examined the effect of TGF-beta on the expression of procollagen mRNA by TMK-I cells. TGF-beta mRNA was detected in all the tumors examined in vivo and in vitro. Interestingly, 9 (90%) of 10 scirrhous gastric carcinomas revealed higher levels of TGF-beta mRNA than normal tissues, while 8 (38%) of 21 well-differentiated adenocarcinomas had higher TGF-beta mRNA levels than normal tissues. As for procollagen mRNA, most of the human gastric carcinoma cell lines expressed type-I procollagen mRNA and MKN-I expressed type-III procollagen mRNA. Furthermore, procollagen type-I mRNA accumulation in TMK-I cells was increased by exogenous TGF-beta. Most of the tumor tissues from surgical specimens expressed higher procollagen mRNA than normal tissues. These results indicate that TGF-beta produced by carcinoma cells might stimulate collagen synthesis not only by fibroblasts but also by carcinoma cells themselves, leading to diffuse fibrosis of scirrhous gastric carcinomas.