The Scientific World Journal Volume 2012, Article ID 946060, 8 pages The cientificWorldJOURNAL doi:10.1100/2012/946060 Research Article Ki-67 and MCM-2 in Dental Follicle and Odontogenic Cysts: The Effects of Inflammation on Proliferative Markers Nurhan Guler,¨ 1 Nil C¸omunoglu,˘ 2, 3 and Fatih Cabbar1 1 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Yeditepe University, No. 238 Bagdat Cd, 34728 Goztepe, Istanbul, Turkey 2 Pathology Service, Haydarpas¸a Hospital, 34668 Uskudar, Istanbul, Turkey 3 Department of Pathology, Faculty of Medicine, Yeditepe University, 34755 Istanbul, Turkey Correspondence should be addressed to Nurhan Guler,¨ [email protected] Received 25 February 2012; Accepted 12 April 2012 Academic Editors: A. Baldi and S. E. Hiscox Copyright © 2012 Nurhan Guler¨ et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The aim of this study was to investigate whether there is any association between inflammation and the expression of markers of cell cycle entry (Ki-67 and MCM-2) in dental follicle (DF) of asymptomatic impacted teeth and odontogenic cysts. The study consisted of 70 DFs and 20 odontogenic cysts (radicular cyst (RC), dentigerous cyst (DC) and keratocytic odontogenic tumor (KCOT) located at posterior mandibular region. Histological findings of inflammation for all specimen and mucous cell prosoplasia, squamous metaplasia, glandular epithelium for all DFs were stained with hematoxyline and eosin, periodic acid schiff, alcian blue, and mucin. Epithelial cell proliferation was determined by using immunohistochemical labeling for Ki-67 and MCM-2. The histologic examinations showed 16% mucous cell prosoplasia, 54% squamous metaplasia, 20% glandular epithelium, 37% inflammation. Inflammation was detected in all RCs and %62 in DF, %43 in DC and KCOT. Positive correlation was found between the inflammation of DF and odontogenic cysts (P<0.01). The mean Ki-67 and MCM-2 expressions were found 9, 64 ± 5, 99 and 6, 34 ± 3, 81 in DF, 11, 85 ± 9, 01 and 13, 6 ± 9, 94 in odontogenic cysts, respectively. While the mean Ki-67 expressions were statistically significant in DF and KCOT (P<0.01), MCM-2 were significant in RC and KCOT (P<0.01). MCM-2 expresion in RCs were statistically significant than KCOT (P<0.01). The results of this study indicated that the higher MCM-2 expressions in RC than the KCOT might be related to the inflammation and this protein might be more sensitive to inflammation. 1. Introduction is an odontogenic cyst of inflammatory stimulation of the epithelial rests of Malassez [5]. DC presents clinically as an Odontogenic cysts, osteodestructive lesions affecting jaws, aymptomatic unilocular radiolucency enclosing the crown of are arise from epithelial cells of dental follicle (DF) or from an unerupted or impacted tooth [3], and it can transform the remnants of odontogenic epithelium such as reduced into more serious lesions, such as unicystic ameloblastoma enamel epithelium, malessez, hertwig epithelial shield, or [6]. Keratocystic odontogenic tumor (formerly odontogenic rests of serres [1]. The most common cysts originated keratocysts, KOCT) is a unique cyst because of its locally from these odontogenic cells are dentigerous cyst (DCs), aggressive behavior, high recurrence rate, and characteristic keratocytic odontogenic tumor (KCOTs), and calcified odon- histological appearance [4]. RCs, DCs, and KCOTs show togenic tumor [2, 3]. It is well known that the epithelial distinct growth patterns and biological behaviors. Increased linings of both inflammatory and developmental cysts of activity of the epithelium, confirmed by previous study that odontogenic origin are primarily composed of squamous has compared KCOTs with other odontogenic cysts, may epithelium, but various forms of metaplasia and degenera- explain the high recurrence rates of KCOTs [7]. tion are observed in these epithelial linings such as mucous Cell proliferation plays an important role in several cells, ciliated cells, para and/or ortho-hyperkeratinization, biological and pathological events such as oral cysts and and formation of hyaline bodies [4]. Radicular cyst (RC) tumors [5, 7, 8]. The proliferative potential can be assessed by 2 The Scientific World Journal immunohistochemistry using monoclonal antibodies against enlarged tissues surrounding impacted third molars and 20 specific cell cycle associated proteins. The proliferative activ- inflammatory and developmental odontogenic cysts located ity of the epithelial lining of odontogenic cysts more particu- at the posterior mandibular region (of 6 RCs, 7 DCs and 7 larly in KCOTs has been the subject of various investigations KCOTs) were included. Fifty females and 20 males (mean age examining the expressions of p53, proliferating cell nuclear 27 years, range 16–68 years) were included in DFs group and antigen (PCNA), Ki-67, as well as nucleolar organiser regions seven females and 13 males (mean age 39 years, range 22–67 (AgNORs) [5, 7–10]. Among the immunohistochemical cell years) were included in odontogenic cysts group. The study cycle markers, the monoclonal antibody Ki-67 has been the was approved by Yeditepe University clinical investigations most widely used, which labels all active parts of the cell cycle, ethic committee (2006) and informed consent was obtained rises during the second half of the S phase, reaches a peak from all participating patients. in the G2 and M phases, and rapidly degrades after mitosis DFs of impacted teeth were carried out under local with a half life of detectable antigen being an hour or less. It anesthesia by conventional third molar surgery and biopsy can be expressed actively in proliferating cells, in particular, taken from odontogenic cyst. All specimens were placed neoplasms and its immunoreactivity, has been found to immediately in 10% buffered formalin and processed to correlate closely with other variables of cell proliferation paraffin wax. Sections (5 µ thick) were cut from each [7, 11]. block containing the DF and cysts specimens and stained Minichromosome maintenance proteins (MCM) expres- with hematoxylin and eosin (H&E) for routine histologic sion can be used as a new marker in the determination of examination. All slides were stained with periodic acid the proliferating cells. The main function of MCM proteins is Schiff (PAS), Alcian blue, and mucin for the evaluation of cooperation with other factors in molecular mechanisms that mucous cell prosoplasia. Histologic appearance of normal form the replication fork and in regulation of DNA synthesis. DF described by Glosser and Campbell [16]wasused MCM proteins form a ring-shaped complex, which is and any soft tissue specimens with squamous epithelium activated when other factors are bound. MCM 2–7 complex spreading along the surface of the DF were considered as is one of the prereplication factors. Association of MCM 2–7 cystic [9]. Histologically, the epithelial components consisted complex is a crucial moment initiating the replication fork. of mucous cell prosoplasia, basal epithelial cells, squamous MCM proteins play a role in maintaining genome integrity epithelium, and glandular epithelium, while mesenchymal and prevent rereplication once per cell cycle. Proliferating components were inflammation, rests of the odontogenic cells have high levels of MCM, whereas they are not detected epithelium, and calcified follicles. in quiescent, differentiated or senescent cells. Recent results Immunohistochemical method was described in our also indicate that the presence or absence of licensed origins previous study [9]. Briefly, 5 µ thickness sections were has even deeper implications for cell biology, as it might prepared for incubations of Ki-67 (ScyTek, Logan, UT; play a role in determining the proliferative capacity of 7 mL) and MCM-2 (MCM-2; CRCT2.1 [D1.9H5]; 1/100 cells [12–14]. They are also potential useful markers of dilution) antibodies according to manufacture’s instructions. cell proliferation. This protein can be observed both in Dysplastic cervix epithelium was used as a positive control cells coming out of the cell cycle and during the cellular for Ki-67 and tonsillary tissue for MCM-2. Each slide was proliferation in the normal, premalignant, and neoplastic examined under a multihead light microscope and scored cells [14, 15]. Recent studies suggested that MCMs are good by pathologist. For each of the specimens, intensity and markers of proliferation activity degree, because they are extent of Ki-67 and MCM-2 expression were evaluated by highly expressed in a variety of tumors [12]. a comprehensive scoring formula. One hundred nuclei were Under the influence of pathologic changes, however, assessed in each specimen and scored as a percentage of dental follicles that possess reduced epithelium can prolif- positively stained nuclei out of the total number of nuclei erate into stratified squamous epithelium as far as originate reprehensive microscopic fields counted. dental cysts [16]. In this way, the microscopic analysis Histologic and immunohistochemical characteristics of concomitant to immunohistochemical markers is relevant epithelial and mesenchymal components of DF and odon- for understanding the behavior of DF in and odontogenic togenic cysts were recorded and the Kruskal-Wallis test cysts. It appears that assessing the proliferative capacity of was used to compare data between groups. Two-tailed epithelial cells