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Changes of Tgfb1 and Tgfbrii Expression in Esophageal Precancerous and Cancerous Lesions: a Study of a High-Risk Population in Henan, Northern China

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Changes of Tgfb1 and Tgfbrii Expression in Esophageal Precancerous and Cancerous Lesions: a Study of a High-Risk Population in Henan, Northern China Diseases of the Esophagus (2002) 15, 74–79 Ó 2002 ISDE/Blackwell Publishing Asia http://www.paper.edu.cn Original article Changes of TGFb1 and TGFbRII expression in esophageal precancerous and cancerous lesions: a study of a high-risk population in Henan, northern China Q. Zhou,1 L. Dong Wang,1,2 F. Du,1 Y. Zhou,3 Y. Rui Zhang,4 B. Liu,5 C. Wei Feng,6 S. Shan Gao,1 Z. Min Fan,1 C. S. Yang,7 S. Zheng2 1Laboratory for Cancer Research, College of Medicine, Zhengzhou University, Zhengzhou, Henan; 2Cancer Institute, Zhejing University, Hangzhou, China; 3Department of Oncology, The First Affiliated Hospital of Zhengzhou University and 4Department of Gastroenterology, Henan Province Hospital, Zhengzhou, Henan; 5Department of Gastroenterology, Tong Ren Hospital, Capital Medical University, Beijing; 6Department of Gastroenterology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China; 7Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ, USA SUMMARY. The level of transforming growth factor b1 (TGFb1) and transforming growth factor bII receptor (TGFbRII) was determined immunohistochemically in normal tissues and tissues with different severities of lesions (basal cell hyperplasia, BCH; dysplasia, DYS; carcinoma in situ, CIS; and squamous cell carcinoma, SCC) from surgically resected human esophagi and esophageal biopsies of symptom-free subjects. The samples were from an area with high esophageal cancer incidence in northern China (Linzhou, formerly Linxian, and nearby county Huixian in Henan Province). Peroxidase immunostain (ABC) and conventional hematoxylin and eosin stain were used. The tissue sections were incubated with antibodies of TGFb1 and TGFbRII overnight. The immunoreactivity was observed in cytoplasm of the esophageal specimen. From normal to BCH to DYS to CIS and to SCC, the positive immunostaining rates for TGFb1 increased significantly (P < 0.05). A linear correlation between the positive immunostaining rates of TGFb1 and the different lesions was observed (P < 0.05). From well- to moderately- and poorly differentiated SCC, the positive immunostaining rates for TGFb1 decreased gradually, but the difference was not significant (P > 0.05). In contrast, with the lesions progressing from normal to BCH to DYS to CIS and to SCC, the positive immunostaining rates for TGFbRII decreased significantly (P < 0.05). From well- to moderately- and poorly differentiated SCC, the positive immunostaining rates for TGFbRII decreased significantly (P < 0.05). There was a linear correlation between the positive rates of TGFbRII and different lesions and SCC differentiation (P < 0.05). The present results indicated that the alterations of TGFb1 and TGFbRII is a frequent event in esophageal multistage carcinogenesis, the absent or lower expression of TGFbRII may lead to the loss of cell proliferation control by TGFb1 and the overexpression of TGFb1 may be a negative feedback response caused by the lower expression of TGFbRII protein. INTRODUCTION EC incidence between the high- and low-incidence area could be ashigh as500:1. 1–4 Linzhou city Esophageal carcinoma (EC) is one of the most (formerly Linxian) and nearby county Huixian in common malignant diseases in the world with Henan province have been well recognized ashigh- remarkable geographic distribution; the ratio for incidence area for EC; the average incidence rates for men and women are 161 and 103 per 100 000, respectively. EC remains the leading cause of cancer- related death in these areas.5 Esophageal carcino- Address correspondence to: Dr Li Dong Wang, Laboratory for genesis is considered as a multistage progressive Cancer Research, College of Medicine, Zhengzhou University, Zhengzhou, Henan, 450052, People’sRepublic of China. process. The early indicator for the subject predis- Tel. ⁄ Fax: (+86) 371 6970165; E-mail: [email protected] posed to EC is abnormal hyperproliferation of 74 中国科技论文在线 http://www.paper.edu.cn esophageal epithelial cells, morphologically manifes- China. No selection process was involved. Out of the ted as basal cell hyperplasia (BCH) and dysplasia 32 subjects examined, there were 20 men (28–70 years (DYS), which have been considered as precancerous of age with a mean ± SD of 50 ± 7) and 12 women lesions of EC.5–9 However, the information for the (25–69 yearsof age with a mean ± SD of 46 ± 17). mechanism of esophageal epithelial cell proliferation The biopsies were taken each from the middle-third regulation isvery limited. of the esophagus (30–32 cm from the incisor teeth) Recent studies indicate that some polypeptides from each subject. All the tissues were fixed with 85% may be involved in epithelial cell proliferation and alcohol, embedded with paraffin, and serially sec- differentiation, which may play an important role in tioned at 5 lm. The sections were mounted onto the malignant transformation of the epithelial cells. histostick-coated slides. Four or five adjacent ribbons Transforming growth factor b1 (TGFb1) issucha were collected for histopathologic analysis (hematox- polypeptide, inhibiting cell proliferation and inducing ylin and eosin stain) and for immunohistochemical cell differentiation.10–14 TGFb1 could induce the staining. formation of tissue matrix. It has been frequently observed that many different kinds of human cancer Histopathologic analysis cells lost their response to TGFb1, which hasbeen considered as one of the vital stages in carcinogenesis. Histopathologic diagnosis for esophageal epithelia The inhibition effect of TGFb1 on cell proliferation wasmade according to cellular morphologic changes dependson a compound of two related serine ⁄threon- and tissue architecture using previously established ine kinase trans-membrane proteinscalled receptor I criteria.26 In brief, the normal esophageal epithelium (TGFbRI) and II (TGFbRII). The inactivation of contained one to three proliferating basal cell layers; each receptor will lead to TGFb1 resistance.15–25 So the papillae were confined to the lower half of the far, the information for the alterationsof TGF b1 and epithelium. In BCH, the thickness of the proliferating TGFbRII expression in multistage progress of eso- basal zone surpassed 15% of the total epithelial phageal carcinogenesis is very limited. thickness. DYS was characterized by nuclear atypia To characterize the changesof TGF b1and (enlargement, pleomorphism, and hyperchromasia), TGFbRII expression in human esophageal carcino- loss of normal cell polarity, and abnormal tissue genesis, the present study was undertaken to deter- maturation. SCC wascharacterized by confluent and mine the protein level of TGFb1 and TGFbRII in invasive sheets of cohesive, polymorphous cells with normal tissues and tissues with different severities of hyperchromatic nuclei. lesions of BCH, DYS, carcinoma in situ (CIS), and squamous cell carcinoma (SCC) from surgically Immunohistochemical staining resected human esophagi and esophageal biopsies of symptom-free subjects. The subjects were from Linz- Anti-TGFb1 antibody isa polyclonal rabbit antiserum hou and nearby county Huixian in Henan province, against the C-terminal domain of TGFb1 of human high-incidence areasfor EC in northern China. origin (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-TGFbRII antibody isa polyclonal rabbit anti- serum against the C-terminal domain of TGFbRII of MATERIALS AND METHODS human origin (Santa Cruz Biotechnology, Santa Cruz, CA). The avidin–biotin–peroxidase complex method Tissue collection and processing was used for the immunostaining of TGFb1 and A total of 74 surgically resected primary EC speci- TGFbRII. In brief, after de-waxing, inactivating menswere collected from the patientsin Linzhou and endogenous peroxidase activity and blocking cross- Huixian. Out of the 74 specimens examined, 44 were reactivity with normal serum (Vectastain Elite Kit; from men (40–74 yearsof age with a mean ± SD of Vector, Burlingame, CA), the sections were incubated 56 ± 9) and 30 were from women (37–69 yearsof overnight at 4°C with a diluted solution of the primary age with a mean ± SD of 54 ± 7). All the patients antibodies(1:100 for TGF b1 and TGFbRII). Location had not received either chemotherapy or radiother- of the primary antibodies was achieved by subsequent apy before surgery. The resected esophageal speci- application of a biotinylated antiprimary antibody, an menswere cut longitudinally and flattened. Iodine avidin–biotin complex conjugated to horseradish per- staining with Lugo’s solution was applied to the oxidase, and diaminobenzidine (Vectastain Elite Kit). whole mucosa. Based on the iodine stain results, all Normal serum blocking and omission of the primary the unstained mucosa and part of the stained mucosa antibodies were used as negative controls. Clear were cut and recorded. A total of 175 samples were cytoplasm and cell membrane staining was the cri- collected from the esophageal mucosa-adjacent can- teria for a positive reaction. Several terms were used cer. Esophageal biopsies were taken from 32 symp- to describe immunostaining patterns as previously tom-free subjects who volunteered to participate in a established, including ÔdiffuseÕ, ÔfocalÕ and Ôscat- routine endoscopic screening for EC in Huixian, teredÕ.8,9 中国科技论文在线 http://www.paper.edu.cn Table 1. Immunoreactivity of TGFb1 and TGFbRII in esophageal normal epithelia and the epithelia with different severities of lesions TGFbRII TGFb1 No. of positive No. of positive Histologic No. of samples stains stains types examined (n,%) (n,%) Normal 43 16 (37)* 42 (98)* BCH
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