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性肿瘤细胞的差异，我们尝试用原发和转移性肿瘤独立于患者建立细胞系。此外，还建立了来自同一患者口腔黏膜的正常人角质形成细胞（NHK）细胞系，作为理想的对照细胞。1993-1996年间成功建立的5个细胞系中，有2个细胞系在无蛋白培养基中同时建立了原发、转移的肿瘤细胞系和正常的kratinocyte细胞系。采用PCR方法对OSCC和NHK细胞的转录本进行差异展示，利用任意引物组寻找OSCC或NHK细胞特异性表达的基因。我们报道了一种由一种OSCC细胞系表达的蛋白，发现它可以抑制淋巴细胞的生长。此外，制备了一种单克隆抗体，可识别未知的血管内皮蛋白。PKC eta是PKC的一个亚型，已知在上皮细胞中表达。为了了解细胞生物学行为，使用腺病毒载体在上皮细胞中过表达PKC eta。结果发现，12- o -十四烷酰磷13-乙酸酯（TPA）抑制了上皮细胞的生长，而成纤维细胞在相同条件下未受到抑制，提示可能存在OSCC的基因治疗。我们之前报道了p53肿瘤抑制基因在OSCC中经常发生突变，并被认为是OSCC发生的原因。为了了解突变的作用，用转染法检测了OSCC中发现的p53突变体。一个密码子138位Val的突变体是一个温度敏感突变体。为了建立p53基因突变的分子诊断体系，详细比较了该基因的现状、临床过程和组织学恶性分级。虽然突变频率如此之高，但我们并未发现突变的存在与临床或组织学恶性分级之间存在明显的相关性，提示p53突变在OSCC的发生中起重要作用。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