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 , 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/ 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 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 -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) for 20 min, and then slices were dewaxed using xylene columnar cells and some central polyhedral cells. Most and graded alcohols, and soaked in distilled water with 3% keratinizing cells of the acanthomatous subtype and granular cells hydrogen peroxide. The slices were immersed in 0.01 M citrate of the granular subtype expressed weak or moderate EPOR. EPOR 20 J.Hard Tissue Biology Vol. 18(1):19-26, 2009 Statistical significance: * P< 0.05, ** 0.01. Immunohistochemical reactivity: (–) negative, (+) weak–to-moderate positive and (++) strong positive.Values in parentheses deno ameloblastomas. Table 1: Immunohistochemical expression of EPO, EPOR, PDGF-A and PDGFR- α in tooth germs, keratocystic odontogenic tumors and te percentages.

21 Xiaodong Yin et al.: Detection of EPO&PDGF in Ameloblastoma

Figure 1. Immunohistochemical expression of EPOR (a) Tooth germ showing strong expression of EPOR in epithelial cells and a few stromal cells (×400). (b) Follicular ameloblastoma with moderate EPOR expression in peripheral cells and some central cells, compared with intense immunostaining in vascular endothelial cells (×200). (c) Plexiform ameloblastoma showing strong EPOR expression in most neoplasm cells (×200).

Figure2. Immunohistochemical expression of EPO (a) Keratocystic odontogenic tumor showing moderate expression of EPO in a ripple-shaped epithelial layer (×200). (b) Follicular ameloblastoma showing moderate EPO expression, mainly in the central polyhedral cells (×200). (c) Plexiform ameloblastoma showing strong EPO expression in most neoplastic epithelium cells (×200). was mainly distributed in the peripheral columnar cells of follicular expressed EPO in peripheral cells than in central cells. In contrast, ABs as well as in endothelial cells of stroma blood vessels (Fig. plexiform ABs showed an opposite pattern of EPO expression 1b). However, EPOR expression in plexiform ABs was relatively (Fig. 2c). Extremely weak expression of EPO was found in the stronger in the peripheral and central cells (Fig. 1c). Very weak mesenchymal cells of these odontogenic tissues. EPOR expression was found in the stromal cells of these odontogenic tissues. PDGF-A expression PDGF-A was mainly expressed in the cytoplasm of epithelium, EPO expression and, to a lesser extent, in the cytoplasm of mesenchymocytes in EPO was mainly expressed in the cytoplasm of the epithelium normal and neoplastic odontogenic tissues (Fig. 3). PDGF-A in normal and neoplastic odontogenic tissues (Fig. 2). EPO expression was slightly stronger in the epithelium of ABs than in expression was similar in tooth germs and ABs; however, EPO tooth germs or KCOTs (P>0.05). Some mesenchymocytes in expression in KCOTs was significantly weaker than in tooth germs dental papillae and dental follicles also showed weak-to-moderate (P<0.05). EPO expression was strong in the epithelium of 6/9 PDGF-A expression (Fig. 3a). PDGF-A expression in follicular tooth germs (Table 1), but the tooth follicle only showed sporadic ABs was significantly different to that in plexiform ABs (P<0.05). and weak expression. In 7/10 KCOTs, EPO expression was PDGF-A expression was slightly higher in the non-cellular negative or weak (Table 1). Most ABs expressed EPO in peripheral variation subtype than the cellular variation subtypes (P>0.05). columnar cells and central polyhedral cells (Fig. 2c), while some Over half of the keratinizing cells in acanthomatous ABs and most ABs expressed EPO mainly in the central cells (Fig. 2b). More granular cells in granular cell ABs showed very weak PDGF-A than half of the keratinizing cells in the acanthomatous subtype expression (Table 1). and granular cells in the granular subtype expressed weak-to- moderate EPO (Table 1). Most follicular ABs more strongly PDGFR-α expression 22 J.Hard Tissue Biology Vol. 18(1):19-26, 2009

Figure 3. Immunohistochemical expression of PDGF-A (a) Tooth germ showing moderate expression of PDGF-A in the cytoplasm of epithelial cells and some stromal cells (×400). (b) Acanthomatous ameloblastoma with obvious cell bridges showing weak expression of PDGF-A, mainly in the cytoplasm of keratinizing cells (×400). (c) Granular cell ameloblastoma showing weak PDGF-A expression principally in cytoplasm of bigger granular cells. (×400)

Figure4. Immunohistochemical expression of PDGFR-α (a) Keratocystic odontogenic tumor showing moderate expression of PDGFR-α in the epithelial layer (×400). (b) Follicular ameloblastoma showing moderate PDGFR-α expression at the cytomembrane of neoplastic cells (×400). (c) Plexiform ameloblastoma showing strong PDGFR-α expression at the cytomembrane and in the cytoplasm of most peripheral cells and some central cells (×400).

PDGFR-α was mainly found at the cytomembrane and Expression profile of recrudescent ameloblastomas cytoplasm of epithelium and mesenchymocytes in odontogenic The immunostaining profiles of these four factors in tissues (Fig. 4). PDGFR-α immunostaining was slightly higher recrudescent ABs were stronger than in primary ABs, and the in ABs than in KCOTs or tooth germs (Table 1). PDGFR-α expression of EPOR and PDGFR-α differed significantly (P<0.01) expression was significantly weaker in KCOTs than in tooth germs between recrudescent and primary ABs. (P<0.05). In tooth germs, PDGFR-α expression was weaker in dental papillae and dental follicles than in the epithelial layer. Discussion PDGF-α expression differed significantly between the follicular Angiogenesis is a critical link in numerous physiological events and plexiform ABs (P<0.01). Some follicular ABs expressed such as embryonic development and pathological events such as PDGFR-α only at the cytomembrane of neoplastic cells (Fig. 4b). tumor growth12), and is mediated by angiogenins including EPO Non-cellular variation ABs showed significantly greater PDGF- and PDGFs4). The EPO/EPOR system is important for α expression than acanthomatous ABs (P<0.05). erythropoiesis during embryonic development. Knockout of the encoding either EPO or EPOR in mice resulted in embryonic Expression profile of metastatic ameloblastomas death owing to deficient liver erythropoiesis13). EPO or EPOR- Although our study did not include any cases of ameloblastic knockout embryos also showed defective angiogenesis and heart carcinoma, we did include one case of metastatic AB. We observed formation owing to enhanced in the endocardium and no morphological difference between the metastatic and non- myocardium14). The PDGF/PDGFR system is also crucial to metastatic ABs. Metastatic AB expressed moderate development. Lack of the PDGF-A affects lung growth immunostaining of all four factors, and the expression of these leading to death of young mice15). PDGFR-α is essential for factors was stronger in the central cells than in the peripheral mesenchymocyte proliferation in the early development of many cells. organs16). PDGFR-α null embryos also develop cleft face and vascular defects leading to death17). Therefore, these data show 23 Xiaodong Yin et al.: Detection of EPO&PDGF in Ameloblastoma that both EPO and PDGFs are very important for the proliferation malignancies including gliomas29). Disrupting the PDGFR-α- and differentiation of tissues and organs. In this study, the mediated signal significantly inhibited the growth of lung expression of EPO, PDGF-A and their receptors was greater in carcinoma30). These data show a high correlation between the the epithelium and mesenchymocyte of developing human tooth PDGF/PDGFR system and neoplasm. In this study, we observed germs compared with oral mucosa. This suggests that these two higher expression of this system in ABs than in tooth germs and systems may participate in the development of human teeth KCOTs, and only the expression of PDGFR-á was significantly through autocrine and paracrine mechanisms. In addition, the tooth stronger in tooth germs than in KCOTs, indicating the potential germ epithelium and the neighboring neoplastic epithelium islands role of this system in the tumorigenesis of odontogenic epithelium expressed these four factors, indicating the tumorigenesis of AB. as well as in the development of teeth. This result is partly EPO has been extensively used since the 1990s to treat cancer- consistent with a previous study4), but inconsistent with another3). related anemia to improve quality of life5). However, unexpected The expression of PDGF-A and PDGFR-á differed significantly adverse outcomes were observed in two recent trials in which between the follicular and plexiform types of AB, hinting that cancer-related anemia was treated with EPO18, 19); concerns have this system may be involved in the development of the tissue gradually been raised for this practice because EPO might promote structure of ABs. Among the AB subtypes, the expression of tumor growth. Studies have hinted that the EPO/EPOR system is PDGF-A and PDGFR-á in the non-cellular variation subtype was involved in ABs. First, the angiogenic potential of EPO was stronger than in the cellular variation subtype. Moreover, PDGFR- considered to be similar to that of vascular endothelial growth á expression differed significantly between the non-cellular variant factor (VEGF)20). Second, VEGF is a major angiogenin for AB21). and the acanthomatous subtype, suggesting that PDGFR-á is Third, HNSCC, which shares the same epithelial origin with AB, involved in cell differentiation of AB. We also found that is invaded by an EPO signal8). Thus it seems likely that EPO is keratinizing cells and granular cells showed similar expressed in AB. EPO works only by binding to and immunoreactivity for PDGF/PDGFR system to that for the EPO/ phosphorylating EPOR22), to block apoptosis and promote survival. EPOR system. Moreover, PDGFR-á expression seemed to be Since EPOR was first found in lung cancer23), it was subsequently stronger than that of PDGF-A, suggesting that PDGFR-á is more identified in many tumors including HNSCC8). In this study, we important in the development of normal and neoplastic detected stronger expression of EPO and EPOR in ABs and tooth odontogenic tissues, which agrees with previous studies16, 17). germs than in KCOTs. EPO expression was also significantly The expression of these four factors in recrudescent ABs was different between tooth germs and KCOTs. These findings suggest stronger than in primary ABs, and the expression of EPOR and that the EPO/EPOR system may have an important part in PDGFR-α, in particular, was significantly difference between oncogenesis of odontogenic epithelium as well as in the recrudescent and primary ABs. This suggests that these factors development of teeth. EPO expression also differed among the may be involved in the high recurrence rate of AB. The EPO signal AB subtypes, suggesting that EPO may, to some extent, be in the progression and invasion of malignancy8) and the PDGF involved in the tissue structure of AB. In addition, EPOR signal in the interaction between neoplasm and bone expression was significantly stronger in the non-cellular variation microenvironment4) indicate that these two systems might be subtype than in the cellular variation subtype, suggesting that involved in the local bone invasiveness of ABs. Thus we believe EPOR is related to cell differentiation of AB. Keratinizing cells that the differing expression of these two systems in the types or of the acanthomatous subtype and granular cells of the granular subtypes of ABs might contribute to the specific biological subtype showed weaker staining of the EPO/EPOR system than characteristics of AB. the other neoplastic cells, suggesting lower immunoreactivity for To our knowledge, we are the first to examine the expression EPO and EPOR in these cell types. Moreover, EPOR expression of the EPO/EPOR and the PDGF/PDGFR systems in ABs, KCOTs seemed to be stronger than that of EPO, suggesting that EPOR and tooth germs using immunohistochemistry. Our work provides may be a more suitable marker for odontogenic tumors. at least some explanation for the angiogenesis, oncogenesis and PDGF/PDGFR systems can transduce mitogenic signals24). progression of odontogenic tumors. Moreover, it contributes to PDGF was first reported as a serum factor that promoted the the understanding of the pathogenesis of AB. Although this study proliferation of arterial smooth muscle cells25). After that, PDGF represents a preliminary investigation of angiogenins and their expression was found not only in other normal cells including receptors in AB, our data lay a foundation to further explore the fibroblasts, vascular endothelial cells and platelets26), but also in relationship between these systems and their mechanisms of multiple tumor cells such as acute megakaryoblastic leukemia27). action. PDGFRs are expressed on erythroid and myeloid precursors in marrow and also in various normal cells comprising fibroblasts26) Acknowledgments and diverse tumor cells28). 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