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

本课题旨在研究口腔良性肿瘤继发性恶性转化的可能分子途径。我们主要关注钙化性囊性牙源性肿瘤（COT），因为我们已经提出钙化性牙源性囊肿内可能发生恶性变化。为此，我们从一名54岁男性上颌骨的钙化牙源性囊肿中分离出6个细胞系统（指定为COT1至COT6）。COT1-COT6表现出多角形细胞形状的牙源性上皮特征：它们对角蛋白免疫阳性，表达不同水平的角蛋白16、淀粉原蛋白、凝灰质蛋白、BSP、MMP-20和ALP。与成纤维细胞共培养时，COT6细胞生长形成巢状结构，后期出现鬼影细胞和钙化物质。最后，它们在COT6细胞巢内发展成钙化结节。染色体分析表明，这些细胞不仅存在3n个倍体（平均59个染色体）等数量异常，而且存在各种结构异常。其中，der(9)t(9; 13)（p13.3;q12.3）为6个细胞系统共有。裸鼠体内COT细胞形成鳞状细胞癌样肿瘤。移植瘤细胞灶角化与鬼影细胞分化和钙化密切相关。同样在COC的手术标本中，鬼细胞对细胞外基质（ECM）分子（包括perlecan以及MMP7或β-catenin）呈阳性。结果表明，鬼细胞是由ECM分子在细胞质内的滞留产生的，与Wnt信号通路有关。这些结果表明COT具有潜在的肿瘤性，并且6种COT细胞系统是研究鬼细胞分化和上皮钙化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，这些细胞能够通过上述基因改变转化为恶性肿瘤。本研究是首次在体外证明良性牙源性肿瘤继发性恶性转化。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