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Immunohistochemical Detection of Erythropoietin, Platelet-Derived Growth Factor and Their Receptors in Ameloblastomas

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Immunohistochemical Detection of Erythropoietin, Platelet-Derived Growth Factor and Their Receptors in Ameloblastomas J.Hard Tissue Biology Vol. 18(1):19-26, 2009 Journal of Hard Tissue Biology 18[1] (2009) p19-26 © 2009 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Original Immunohistochemical Detection of Erythropoietin, Platelet-Derived Growth Factor and Their Receptors in Ameloblastomas Xiaodong Yin1), Jiankai Xu2), Jinna Shi1), Kewen Lv1), Eryang Zhao3), Tenglong Hu1), Ryo Tamamura4), Hitoshi Nagatsuka4) and Xiaohui Jiao1) 1) Department of Oral and Maxillofacial Surgery, School of Stomatology, Harbin Medical University, Harbin, Heilongjiang 150001, China 2) Department of Pharmacogenomics, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, Heilongjiang 150081, China 3) Department of Pathology, School of Stomatology, Harbin Medical University, Harbin, Heilongjiang 150001, China 4)Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University,Okayama 700-8525, Japan (Accepted for publication, January 20, 2009) Abstract: Ameloblastoma (AB) is the most common odontogenic epithelium-derived tumor, but has unclear etiology. Various ligand/receptor systems are implicated in ameloblastoma development. In this study, we explored the expression of erythropoietin (EPO), platelet-derived growth factor (PDGF) and their receptors (EPOR, PDGFR) in ABs, and the potential origins of ABs from keratocystic odontogenic tumors (KCOTs) and tooth germs in control groups. Immunostaining was performed using antibodies for EPO, EPOR, PDGF-A and PDGFR-α. Statistical analysis was performed based on immunoreactivity. We found that EPOR expression was significantly higher in ABs than in KCOTs (P<0.05), but was similar to that of tooth germs. EPOR expression was also significantly different between the AB subtypes (P<0.05). Nevertheless, EPO expression was only significantly different between tooth germs and KCOTs (P<0.05). Immunoreactivity for PDGFR-α and PDGF- A was stronger in ABs than in KCOTs and tooth germs. However, significant differences were only found between individual groups or among types or subtypes of ABs (P<0.05 or P<0.01). In addition, the expression of both EPOR and PDGFR-α in the recrudescent ABs was significantly greater than in primary ABs (P<0.01). We conclude that EPO, EPOR, PDGF-A and PDGFR-α are essential for the growth of human teeth as well as for oncogenesis, development, cell differentiation and biological behavior of odontogenic neoplasm. Keywords: Ameloblastoma, Erythropoietin, Keratocystic odontogenic tumor, Platelet-derived growth factor, Tooth germ Introduction multiple angiogenins that are released by tumor and host cells. Ameloblastoma (AB) is an epithelium-derived neoplasm and Erythropoietin (EPO) is well known for its angiogenic role in is associated with the highest mortality of all odontogenic tumors1). treating cancer-related anemia, thus improving life quality5). It has paradoxical clinical phenotypes, as it is a benign tumor However, recent studies revealed that EPO can stimulate tumor with some malignant features, such as high recurrence rate and progression6), while EPO inhibitors can suppress tumor locally aggressive behavior2), and there are multiple types and angiogenesis and progression7). EPO binds to the erythropoietin- subtypes with differing biological characteristics. Varying receptor (EPOR) to activate its biological activities, and recent expression of genes3) and proteins4) in ABs have been described studies have shown that EPO and EPOR participate in invasion in recent studies, but the precise mechanisms for oncogenesis and and progression of various tumors, such as head and neck development of ABs remain poorly understood. squamous cell carcinoma (HNSCC)8). However, the effects of the Angiogenesis provides a critical link between tumor EPO/EPOR system in the development of ABs are still unclear. progression and embryonic development, and is mediated by Platelet-derived growth factors (PDGFs) are regarded as major mitogens for fibroblasts and other mesenchymal-derived cells9). Corespondence to: Xiaohui Jiao, MD, PhD. Department of Oral and Maxillofacial Surgery, School of Stomatology, Harbin Medical University, Four members of PDGF family (PDGF-A, -B, -C and -D) have Harbin, Heilongjiang 150001, China. Tel: +86 451 855 53926, Fax: +86 been characterized. The PDGF isoforms, by forming homo- or 451 536 50087, E-mail: [email protected] 19 Xiaodong Yin et al.: Detection of EPO&PDGF in Ameloblastoma heterodimers, bind and phosphorylate their receptors, PDGFR α buffer (pH 7.5) and heated in an autoclave (121°C, 2 atm) for 2 - and β-receptors (PDGFR-α and -β) to induce physiological role min. The slices were then incubated with primary antibodies at in embryonal development10) and pathological role in 4°C overnight. The primary antibodies were rabbit anti-EPOR oncogenesis11). However, two recent studies have reached opposite polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, conclusions on the expression of the PDGF/PDGFR system in USA; diluted at 1:600), goat anti-EPO polyclonal antibody (Santa the tooth germ and ameloblastomas3, 4), which prompted us to Cruz Biotechnology; 1:400), rabbit anti-PDGF-A polyclonal further explore these findings. antibody (Santa Cruz Biotechnology; 1:50) and rabbit anti- In recent years, many growth factors and their receptors, PDGFR-α polyclonal antibody (Santa Cruz Biotechnology; 1:50). including insulin-like growth factors4), have been detected in The slices were then reacted with peroxidase-conjugated anti- developing tooth germs and ameloblastic tumors, suggesting the rabbit IgG (for EPOR, PDGF-A and PDGFR-α) or anti-goat IgG importance of these factors in the development of these (for EPO) polyclonal antibodies (Santa Cruz Biotechnology) for odontogenic tissues. The aim of this study was to detect the 20 and 15 min, respectively. The reaction products were observed expression of the EPO/EPOR system and the PDGF/PDGFR by immersing the slices in 0.03% diaminobenzidine (DAB) system in normal and neoplastic odontogenic tissues. solution containing 2 mM hydrogen peroxide for 30 s. The slices were stained again with hematoxylin for 10 s. Then slices were Materials and methods dehydrated in graded alcohols, cleared in xylene and sealed using Sample selection neutral gum. As AB originates from odontogenic epithelium tissues, we used Sections of a case of moderately differentiated lingual KCOTs and tooth germs as control groups to further study the squamous cell carcinoma were used as positive control. For pathogenesis of AB by measuring the expression of the EPO/EPOR negative control sections, the primary antibody was omitted during and PDGF/PDGFR systems. immunostaining. The samples used in this study included 9 tooth germs, 10 keratocystic odontogenic tumors(KCOTs), 36 ABs and 1 Evaluation of staining and statistical analysis metastatic AB. All samples were obtained surgically at Department The staining results were observed and evaluated using a Leica of Oral and Maxillofacial Surgery, School of Stomatology, Harbin DM 2500 microscope by two independent senior pathologists. Medical University, during 2004~2007. For orthodontic reasons, We considered the evaluation criteria as the percentage of positive 9 developing tooth germs of the mandibular third molars were cells and the intensity of staining. Cases in which more than 65% obtained from 5 males and 4 females with a mean age of 14 years. of cells were scored as (–) negative, (+) weak to moderate positive, We randomly selected the 10 KCOTs from 4 males and 6 females and (++) strong positive staining. Descriptive statistical analysis with a mean age of 36 years. The 36 ABs were taken from 20 was performed, and the Mann-Whitney U-test and Kruskal-Wallis males and 16 females with a mean age of 32 years. The AB samples test were used to test for statistically significant differences were classified according to their histological patterns and between two groups or among three or more groups, respectively. biological characteristics, as described in Table 1. The single The criterion for statistical significance was P<0.05. All statistical metastatic AB was from a 52-year-old, female and was a analyses were performed using R version 2.4.1 software (www.r- recrudescent case. project.org). Preparation of samples Results All samples were routinely fixed in 10% neutral formalin for EPOR expression 24~48 hrs, dehydrated in graded alcohols, cleared in xylene, and EPOR was expressed mainly at the cytomembrane and in the embedded in paraffin. 3-µm-thick sections were used for histology cytoplasm of the epithelium in tooth germs, KCOTs and ABs (Fig. and immunohistochemistry. Hematoxylin-eosin-stained slides 1). EPOR expression was similar in tooth germs and ABs. were prepared and were reviewed to confirm the original However, EPOR expression was significantly weaker in KCOTs histological diagnoses, using the WHO classification of head and than in tooth germs or ABs (P<0.05). 7/9 of the tooth germs neck tumors2), particularly to confirm the stage of tooth germs strongly expressed EPOR in the epithelium (Table 1); however, and the types of ABs. the tooth follicle only expressed EPOR weakly (Fig. 1a). 8/10 of the KCOTs only weakly expressed EPOR (Table 1). EPOR Immunohistochemistry expression showed differences among the types and subtypes of The waxed tissue slices were melted by baking in an oven AB (Table 1). 35/36 of the ABs expressed EPOR in peripheral (80°C)
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