Establishment of A Novel System for Malignancy Grading of Oral Cancer Using Molecular Biological Technique

为了研究口腔鳞状细胞癌(OSCC)原发部位的肿瘤细胞和转移的肿瘤细胞的差异，我们尝试建立了独立于患者的原发和转移的肿瘤细胞系。此外，取自同一患者口腔黏膜的正常人角质形成细胞(NHK)也进行了细胞系的建立，作为理想的对照细胞。在1993-1996年间成功建立的5个细胞系中，有2个在无蛋白介质中同时建立了原代和转移的肿瘤细胞系和正常的Krat细胞系。利用随机引物的聚合酶链式反应方法对口腔鳞癌和非小细胞肺癌细胞的转录产物进行差异显示，以寻找在口腔鳞癌和非小细胞肺癌细胞中特异表达的基因。我们报道了一个在口腔鳞状细胞癌细胞系中表达的蛋白质，发现它对淋巴细胞的生长有抑制作用。此外，还发现了一种识别未知血管内皮细胞…的单抗PKC ETA是已知在上皮细胞中表达的PKC的一个亚型。为了了解细胞的生物学行为，利用腺病毒载体在上皮细胞中过表达PKC ETA。研究发现，12-O-十四酰佛波醇13-乙酸酯(TPA)可抑制口腔鳞状细胞癌上皮细胞的生长，但对成纤维细胞无抑制作用，提示口腔鳞癌可能是一种基因治疗方法。我们曾报道P53抑癌基因在口腔鳞癌中经常发生突变，并被认为与口腔鳞癌的发生发展有关。为了了解突变的作用，对口腔鳞状细胞癌中发现的p53突变进行了检测。发现一个密码子为138的Val突变体是一个温度敏感突变体。为了建立利用P53基因突变的分子诊断体系，对P53基因突变的状态、临床过程和组织学恶性程度进行了详细的比较。虽然突变率很高，但突变的存在与口腔鳞癌的临床和组织学恶性程度没有明显的相关性，提示P53突变在口腔鳞癌的发生发展中起重要作用。较少

To investigate the difference between tumor cells of the primary site and metastasized tumor cells of oral squamous cell carcinoma (OSCC), we tried to establish cell lines using primary and metastasized tumors independently from a patient. In addition, normal human keratinocytes (NHK) from oral mucosa of the same patient was also subjected to cell line establishment, which served as ideal control cells. In 2 out of 5 cell lines, which were successfully established between 1993-1996, the primary and metastasized tumor cell lines and normal kratinocyte cell lines were simultaneously established in the protein free media.Transcripts from OSCC and NHK cells were differentially displayd by PCR method using sets of arbitrary primers to search for genes specifically expressed in OSCC or NHK cells.We reported a protein which was expressed by one of the OSCC cell line and was found to suppress the growth of lymphocytes.In addition, a monoclonal antibody, which recognize an unknown vascular endo … More epitherial protein, were prepared.PKC eta is a subtype of the PKC is known to be expressed in the epitherial cells. To understand the cell biological behavior, PKC eta was overexpressed in the epithelial cells using an adenovirul vector. It was found that the treatment by the 12-O-tetradecanoylphorbol 13-acetate (TPA) suppressed the growth of epithelial cells, while fibroblasts were not suppressed under the same condition, suggesting a possible gene therapy of OSCC.We previously reported that p53 tumor suppressor gene is frequently mutated in OSCC,and is considered to be responsible for the development of this tumor. To know the role of the mutation, the p53 mutants found in OSCC were examined by transfection assays. A mutant with Val of codon 138 were found to be a temperature sensitive mutant. To establish a system of molecular diagnosis using the p53 gene mutations, the status of the gene, clinical process and histological malignancy grade are compared in detail. Although the mutation frequency was so high, we could not found an obvious correlation between the existence of mutation and clinical or histological malignancy grade, suggesting that the p53 mutation is important in the initiation of OSCC. Less