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.     

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.     

Epidermal Growth Factor Induces the Expression of Its Receptor Gene in Human Gastric Carcinoma Cell Line TMK-1

To ascertain the possible autocrine pathway in the growth promotion of gastric carcinomas, a study was made on the effects of exogenous human epidermal growth factor (hEGF) on the expression of mRNA for EGF, transforming growth factor-α (TGF-α), EGF receptor, FOS and MYC genes by TMK-1 cells. Exogenous hEGF increased FOS and MYC mRNA levels 30 min and 1 h after the treatment, respectively. TMK-1 cells accumulated the mRNA for EGF receptor about 7- to 8-fold by 3 h after treatment Expressions of mRNA for EGF and TGF-α genes were detected, but the amounts of the mRNA of these genes in TMK-1 cells were not altered after the treatment.     

Altered Expression of Epidermal Growth Factor Receptor Gene in a Classical Multidrug-resistant Variant of a Human Cancer Cell Line, KB

A variant clone resistant to high doses of colchicine (KB-C1) derived from human cancer KB cell line is resistant to various anticancer agents. The KB-C1 cells were much more resistant to epidermal growth factor and a chimeric toxin, EGF-Pseudomottas exotoxin (PE), than the parental KB cells. KB-C1 cells have decreased numbers of EGF-receptors, though the affinity of the receptors is similar to that in the parental KB cells. A drug-sensitive revertant (C1-R2) partially recovered its EGF-receptor activity. Northern blot analysis showed a decreased level of EGF-receptor mRNA in KB-C1 cells, while the multidrug-resistance gene, mdr-1 , was expressed at very high levels in KB-C1 cells, but not in KB or C1-R2 cells. The drug-resistant cells were less tumorigenic than the parental cells when injected into nude mice. A decreased expression of EGF-receptor in these cells may be one of the pleiotropic properties of multidrug-resistant cells and may perhaps represent the basis for their reduced tumorigenicity.     

Effect of Human Epidermal Growth Factor on Cell Growth and Its Receptor in Human Gastric Carcinoma Cell Lines

The effect of human epidermal growth factor (hEGF) on the growthof various histological types of six human gastric carcinomacell lines was examined. The cell lines had relatively highaffinity EGF receptors (dissociation constant Kd = 10^–9to 10^–10 M). One gastric cancer cell line, MKN-74 (welldifferentiated adenocarcinoma) showed no response to hEGF, incell growth, DNA synthesis or ¹²⁵I-hEGF cell binding. Therewere no apparent correlations between histological type andcell growth, DNA synthesis or number of EGF receptors in thesecells. The number of EGF receptors and the Kd value of the gastriccarcinoma cell lines varied with their internal and externalenvironments. hEGF concentrations corresponding to maximum stimulationin DNA synthesis varied between cell lines. The results suggestsome gastric carcinoma cells to have EGF receptors and theirgrowth seemingly to be stimulated by EGF in vitro. There are,however, no obvious correlations between the effect of hEGFon the growth of human gastric carcinoma cell lines or theirhistological type.     

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     

血管内皮生长因子的表达对人胃癌细胞生物学表型的影响

 目的　探讨血管内皮生长因子对人胃癌细胞生物学表型的影响。方法　将 VEGF16 5正、反义 RNA表达载体导入人胃癌细胞 ,并观察其细胞周期、增殖、裸鼠致瘤生长等生物学指标。结果　与 VEGF正义转染细胞相比 ,在反义转染细胞的细胞周期中 G1期细胞数增加了 1 7.3% ,而S期细胞减少了 43.6 % ;正义转染细胞的克隆形成率明显高于反义转染细胞 ;正义转染细胞组的肿瘤生长速度及瘤体积明显高于其他组。结论　 VEGF可以加强肿瘤组织血管生成 ;VEGF反义RNA可以防治肿瘤生长 ;VEGF可能与肿瘤细胞增殖能力有关.     

Increased Midkine Gene Expression in Human Gastrointestinal Cancers

Midkine (MK) is a product of a retinoic acid-responsive gene, and is a novel growth differentiation factor. We examined the expression of the MK gene in specimens of 47 surgically removed human carcinomas of the gastrointestinal organs, namely, gastric, colorectal, hepatocellular, pancreatic, esophageal, ampullary duodenal and bile duct carcinomas. In most cases, the MK mRNA level was higher in cancer specimens than in the corresponding non-cancerous tissues. Furthermore, MK mRNA was more highly expressed in the colon adenocarcinoma lesion than in the adenoma lesions, in the two familial polyposis cases. While MK mRNA was not detected in the normal liver, it became detectable in cirrhotic tissues in 2 of 4 cases, and its expression was increased in the cancerous tissues. Thus, the increase of MK mRNA level is a phenomenon seen in many human gastrointestinal carcinomas. The increased expression of the MK gene in gastric carcinoma was significantly more prominent in well and moderately differentiated adenocarcinomas than in poorly differentiated adenocarcinomas and signet ring cell carcinomas.     

Evidence that Expression of a Mutated p53 Gene Attenuates Apoptotic Cell Death in Human Gastric Intestinal-type Carcinomas in vivo

To examine in vivo the validity of the results of experiments in vitro , we analyzed the relationship between p53 gene status and apoptotic cell death of human gastric intestinal-type adenocarcinomas. Surgical specimens were classified into two categories: 18 gastric cancers with nuclear p53 protein (A), and 17 gastric cancers without nuclear p53 protein (B). Polyraerase chain reaction-single strand conformation polymorphism disclosed a shifted band that corresponded to a mutation in the p53 gene in 13 cases (72%) in category A and 3 cases (18%) in category B, the frequency being significantly higher in the former ( P <0.05). Apoptotic cells were identified from routinely stained sections and by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The TUNEL index [TI: (the number of TUNEL-positive apoptotic cells/the total number of tumor cells) X100] was 3.8 ±1.4% in category A and 4.9 ±1.2% in category B, the value being significantly lower in the former ( P < 0.05). The proliferating cell nuclear antigen index, defined similarly to the TI, was 56.4±16.3% in category A, and it was significantly higher than that in category B ( P <0.05). The immunohistochemically detected expression of P21^CIP1/WAF1 did not differ between the two categories, while Bax-positive tumor cells were more frequently detected in category A. These results indicate that (1) expression of a mutated p53 gene attenuates apoptotic cell death of gastric cancer, in accordance with the previous in vitro finding that p53 gene mutation provides a possible selective advantage for tumor cell proliferation, and (2) apoptosis is related not only to expression of p53 and the stage of the cell cycle, but also to p53-independent and cell cycle-independent events.     

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.     

Reduced Expression of nm23 Is Associated with Metastasis of Human Gastric Carcinomas

Reduced expression of nm23 gene is implicated in high metastatic potential In a variety of malignancies. To elucidate the role of nm23 in human gastric carcinomas, we examined loss of heterozygosity (LOH) of nm23 gene by Southern blotting, nm23 mRNA expression by Northern blotting and nm23 protein expression by Western blotting as well as immunohistochemistry in both primary and metastatic tumors. LOH of nm23 gene was found in 2 (8%) out of the 23 informative gastric carcinomas. Twenty-two (84%) out of the 26 cases expressed nm23 mRNA at higher levels in primary tumor tissue than in corresponding non-neoplastic mucosa. No obvious correlation was observed between clinico-pathological features and LOH of nm23 gene or nm23 mRNA expression. On the other hand, 52% of the gastric carcinomas showed reduction of nm23 immunoreactivity in the metastatic tumor of regional lymph nodes, as compared to the primary tumor. Interestingly, 71% of the gastric carcinomas showed weaker nm23 immunoreactivity in the liver metastasis than in the primary tumor. These results suggest that nm23 overexpression is linked with development of gastric carcinomas and the decrease in expression of nm23 participates in metastasis.     

血管内皮生长因子在胃癌和癌前病变组织中的表达及其临床意义

目的 :探讨血管内皮生长因子 (全文简称VEGF)在胃癌及癌前病变组织中的表达及临床价值。方法 :采用免疫组化S -P法检测胃癌手术标本 5 2例 ,慢性萎缩性胃炎伴肠上皮化生 14例、不典型增生 18例、胃腺瘤 12例组织中的VEGF蛋白表达和胃癌标本的微血管计数 (MVD)。结果 :慢性萎缩性胃炎伴肠化生患者VEGF表达为 35 71% ,不典型增生为 38 89% ,胃腺瘤为 4 1 6 7% ,三者之间无显著性差异 (P <0 0 5 )。胃癌的表达为 6 3 4 6 % ,与癌前病变组相比有显著性差异(P <0 0 5 )。VEGF表达阳性的肿瘤组织其MVD明显高于阴性者 (P <0 0 5 )。VEGF的高表达与胃癌的浸润深度、淋巴结转移有明显相关性 (P <0 0 5 ) ,与癌组织中不同分化程度无关 (P >0 0 5 )。结论 :VEGF在癌前病变中可出现不同程度的表达 ,但在胃癌中有高水平表达 ,与肿瘤的恶性进程和预后有关     

RT—PCR法检测胃癌组织mdr—1基因的表达及临床意义

应用RT－PCR方法检测30例胃腺癌mdr-1基因的表达。结果发现术前化疗的胃腺癌中mdr－1表达阳性率为91％，明显高于术前非化疗组的阳性率（47％）（P＜0．O1）。在术前非化疗组中，中分化胃癌的阳性率高于低分化胃癌的阳性率（P＜0.05）。mdr－l基因的表达与胃癌转移的发生率无明确关系。临床检测胃癌mdr－l基因的表达，有助于选择术后化疗方案。     

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.     

Inactivation of the E-Cadherin Gene in Primary Gastric Carcinomas and Gastric Carcinoma Cell Lines

We investigated the E (epithelial)-cadherin gene for mutations and loss of heterozygosity (LOH) in 24 primary gastric carcinomas (12 differentiated and 12 undifferentiated types, including 3 signet-ring cell carcinomas), as well as 4 gastric carcinoma cell lines of the undifferentiated type (MKN-45, GCIY, HGC-27 and GT3TKB). We utilized PCR-SSCP and RT-PCR followed hy direct sequencing to detect gene mutations and skipped exons, and RT-PCR-SSCP to examine LOH. In primary carcinomas, gene mutations or skipped exons, were detected in 4 of 9 (44%) undifferentiated carcinomas of the scattered type, including 2 signet-ring cell carcinomas, and in none of the 3 undifferentiated carcinomas of the adherent type and 12 differentiated carcinomas. Demonstrated mutations of the E-cadherin gene included an 18 bp deletion (codon 418-423) and a 3 bp deletion (codon 400, calcium-binding domain), both located in exon 9. Skipping of exon 9 with a 1 bp insertion at codon 337, and skipping of exon 8 with a 1 bp deletion at codon 336, also were detected. LOH was confirmed in all of the carcinomas in which gene mutations or skipped exons (3/3 informative cases) were demonstrated. The MKN-45 cell line exhibited an 18 bp deletion at the exon 6-intron 6 boundary with loss of the wild-type allele, and 2 of the remaining 3 cell lines (HGC-27 and GT3TKB) had lost expression without detectable structural alteration of the E-cadherin gene. These data provide support for classic two-hit inactivation of the E-cadherin gene in a high percentage of undifferentiated carcinomas of the scattered type.     

Expression of Thymidine Phosphorylase in Human Gastric Carcinoma

The activity of thymidine phosphorylase (dThdPase) has heen reported to increase in several types of malignant tumors. Experimental evidence has shown that dThdPase is identical to platelet-derived endothelial cell growth factor, and that dThdPase has angiogenic activity. We examined the expression of dThdPase to investigate whether the expression of dThdPase correlates with angiogenesis, clinicopathologic features and the prognosis of patients with human gastric carcinomas. Microvessels were assessed by immnnostaining endothelial cells for factor VIII. We counted microvessels in the tumors of 158 patients whose tumors were completely removed surgically. Microvessels were counted in a × 400 field in the most active areas of neovascularization. We purified a monoclonal antibody (TMA-1) against dThdPase and studied the expression of dThdPase using TMA-1 in the same serial sections as those used for the detection of factor VIII. The correlation between angiogenesis and dThdPase, and the clinicopathological significance of dThdPase, in patients with gastric carcinoma were examined. The positive expression of dThdPase was more frequent (P<0.001) in gastric carcinomas (67/158, 43.4%) than that in normal tissues (12/158, 7.6%). The average microvessel count in dThdPase-positive gastric carcinomas was higher (P<0.001) than that in dThdPase-negative carcinomas. The percentage of gastric carcinoma cells expressing dThdPase was significantly correlated with the microvessel count (P<0.001). Further, the average size of dThdPase-positive carcinomas was significantly larger (P<0.001) than that of negative carcinomas and the mean microvessel count in dThdPase-positive gastric carcinomas was also significantly higher (P<0.001) than that in dThdPase-negative carcinomas. There was a significant correlation between the positive expression of dThdPase and microvessel count (P<0.001) or lymph node metastasis (P=0.013) by multivariate logistic analysis. Further, patients with dThdPase-positive carcinoma showed a significantly worse prognosis than those with dThdPase-negative carcinoma overall and in stage III. These findings indicate that the expression of dThdPase in gastric carcinomas is related to progression and metastasis, and this enzyme affects the prognosis of some patients with the disease.     

血管内皮生长因子C在胃癌中的表达及其与淋巴结转移的关系

目的研究血管内皮生长因子 C(VEGF-C)在胃癌中的表达并探讨其与淋巴结转移的关系。方法应用逆转录-聚合酶链反应(RT-PCR)检测 VEGF-Cm RNA 在5株胃癌细胞株中的表达情况,同时采用免疫组化法,检测63例接受根治性切除手术病例的胃癌组织标本 VEGF-C 蛋白表达。结果 VEGF-C mRNA 表达于胃癌细胞株 MKN45、SGC-7901及 AGS。VEGF-C 蛋白则在52.4%(33/63)的病例中呈阳性表达。在伴淋巴结转移的胃癌中,VEGF-C 表达较无淋巴结转移者更显著(P<0.01)。同时,VEGF-C 表达与淋巴管浸润和 TNM 分期密切相关(P<0.01),但与年龄、性别、肿瘤大小、位置、Lauren 分型、浸润深度和血管浸润均无明显相关。结论 VEGF-C 的表达与胃癌淋巴结转移密切相关,同时,淋巴管生成可能成为治疗胃癌的一个新靶点。     

血管内皮生长因子C在胃癌中的表达及其与淋巴结转移的关系

目的 研究血管内皮生长因子C(VEGF-C)在胃癌中的表达并探讨其与淋巴结转移的关系。 方法应用逆转录-聚合酶链反应(RT-PCR)检测VEGF-Cm RNA在5株胃癌细胞株中的表达情况,同时采用免疫组化法,检测63 例接受根治性切除手术病例的胃癌组织标本VEGF-C蛋白表达。 结果 VEGF-C mRNA表达于胃癌细胞株MKN-45、SGC-7901及AGS。VEGF-C蛋白则在52．4％(33／63)的病例中呈阳性表达。在 伴淋巴结转移的胃癌中,VEGF-C表达较无淋巴结转移者更显著(P<0．01)。同时,VEGF-C表达与淋巴管浸润和TNM分期密切相 关(P<0．01),但与年龄、性别、肿瘤大小、位置、Lauren分型、浸润深度和血管浸润均无明显相关。 结论 VEGF-C的表达与胃癌淋巴结转移密切相关,同时,淋巴管生成可能成为治疗胃癌的一个新靶点。     

TFF1TFF2在胃癌和癌前病变中的表达及意义

目的 :观察三叶因子I(Trefoilfactor1,TFF1)、三叶因子II(Trefoilfactor2,TFF2)在胃癌前病变(不典型增生和肠上皮化生)及胃癌的表达,探讨TFF1、TFF2表达与胃癌发生的关系。 方法 :对正常胃粘膜、胃粘膜肠上皮化生、胃癌组织各30例,采用SP免疫组化方法进行TFF1、TFF2蛋白的定位和半定量检测。 结果 :正常胃粘膜→肠上皮化生→不典型增生→胃癌四种病变中,TFF1、TFF2表达呈逐渐减弱趋势,差异具有显著性(P<0.05)。 结论 :TFF1、TFF2蛋白表达在胃癌发生过程中进行性下调,TFF1、TFF2蛋白表达可作为胃癌生物学行为的客观指标。