Molecular pathological study of mechanism in malignant changes of benign odontogenic tumors

本研究旨在探讨口腔良性肿瘤继发性恶变的分子机制。我们主要集中在钙化性囊性牙源性肿瘤(COT)，因为我们已经提出了钙化牙源性囊肿内可能的恶变。为此，我们从一位54岁男性的上颌骨钙化牙源性囊肿中分离出六个细胞系统(命名为COT1至COT6)。COT1-COT6表现出牙源性上皮细胞的特征，呈多边形细胞形态：角蛋白免疫阳性，角蛋白16、釉原蛋白、Tuftelin、BSP、MMP-20和ALP表达不同的mRNA水平。在与成纤维细胞共培养时，COT6细胞生长形成矿化结构，后期出现鬼影细胞和钙化物质。最后，它们在COT6细胞巢内发育成钙化结节。染色体分析表明，该细胞不仅存在…的3n倍体等数量异常平均59条染色体较多，但也有各种结构异常。其中，der(9)t(9；13)(p13.3；q12.3)在所有6个细胞系统中都是共有的。COT细胞在裸鼠体内形成具有鳞状细胞癌样外观的肿瘤。移植瘤灶的角化与鬼细胞分化和钙化密切相关。此外，在COC手术标本中，幽灵细胞表达细胞外基质分子，包括Perlecan和MMP7或β-catenin。这些结果表明COT是一种潜在的肿瘤细胞，这6个COT细胞系统是研究幽灵细胞分化和上皮钙化的分子机制的有用模型。此外，研究结果表明，COT含有癌前肿瘤细胞，这些细胞能够在上述基因改变的情况下转化为恶性肿瘤。本研究是首次在体外证实牙源性良性肿瘤的继发性恶变。较少

This research project was carried out in order to study a possible molecular pathway of secondary malignant transformation from oral benign tumors. We mainly focused on calcifying cystic odontogenic tumor (COT), because we had already proposed a possible of malignant changes within calcifying odontogenic cyst. To this end, we have isolated six cell systems (designated COT1 to COT6) from a calcifying odontogenic cyst arising in the maxilla of a 54 year-old male. COT1-COT6 showed odontogenic epithelial characteristics with polygonal cell shapes: they were immunopositive for keratin and expressed distinct mRNA levels for keratin 16, amelogenin, tuftelin, BSP, MMP-20, and ALP. In co-cultures with fibroblasts, COT6 cells grew to form nestic structures, in which ghost cells and calcified materials appeared in the later stage. Finally, they developed into calcified nodules within the COT6 cell nests. Chromosome analyses showed the cells had not only numeral abnormalities such as 3n ploidies w … More ith average numbers of 59 chromosomes but also various kinds of structural abnormalities. Among them, der(9)t(9; 13)(p13.3;q12.3) was shared by all of the six cell systems. COT cells formed tumors with a squamous cell carcinoma-like appearance in nude mice. Keratinization in transplanted tumor cell foci was closely associated with ghost cell differentiation and calcification. Also in surgical specimens of COC, ghost cells were positive for extracellular matrix (ECM) molecules including perlecan as well as MMP7 or β-catenin. The results indicated that ghost cells were generated by cytoplasmic retention of ECM molecules, which were related to Wnt signaling pathways.These results show that COT is potentially neoplastic, and that the six COT cell systems are useful models for investigating the molecular mechanisms of ghost cell differentiation and epithelial calcification. In addition, the findings suggest that COT contains precancerous tumor cells which are able to transform into malignant with above mentioned gene alterations. The present study is the first in-vitro demonstration of secondary malignant transformation from a benign odontogenic tumor. Less