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

利用分子生物学技术建立口腔癌恶性分级新系统

• 批准号: 05404069
• 负责人: ENOMOTO Shoji
• 金额: $ 13.12万
• 依托单位: Faculty of Dentistry, Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05404069/
• 关键词: oral squamous cell carcinoma; normal human keratinocytes from oral mucosa; cell line establishment; vascular endoepitheraial protein; PKC eta; p53 tumor suppressor gene; temperature sensitive mutant; molecular diagnosis; Squamous cell carcinoma

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

为了研究口腔鳞状细胞癌（OSCC）原发部位肿瘤细胞和转移肿瘤细胞之间的差异，我们尝试独立于患者使用原发肿瘤和转移肿瘤建立细胞系。此外，还对来自同一患者口腔粘膜的正常人角质形成细胞（NHK）进行细胞系建立，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个在无蛋白培养基中同时建立了原发性和转移性肿瘤细胞系以及正常角质细胞系。通过PCR方法，使用多组任意引物来差异显示来自OSCC和NHK细胞的转录本，以寻找在OSCC或NHK细胞中特异性表达的基因。我们报告了一种由其中一种表达的蛋白质 OSCC 细胞系，被发现可抑制淋巴细胞的生长。此外，还制备了一种单克隆抗体，可识别未知的血管内皮细胞蛋白。PKC eta 是已知在上皮细胞中表达的 PKC 的一种亚型。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度表达 PKC eta。结果发现，12-O-十四烷酰佛波醇13-乙酸酯（TPA）治疗可抑制上皮细胞的生长，而在相同条件下成纤维细胞则不受抑制，这表明可能是OSCC的基因治疗。我们之前报道过p53抑癌基因在OSCC中频繁突变，并被认为是导致该肿瘤发生的原因。为了了解突变的作用，通过转染试验检查了 OSCC 中发现的 p53 突变体。发现具有密码子138的Val的突变体是温度敏感突变体。建立利用p53基因突变的分子诊断体系，详细比较该基因的状态、临床过程和组织学恶性程度。尽管突变频率如此之高，但我们未能发现突变的存在与临床或组织学恶性程度之间存在明显的相关性，这表明p53突变在OSCC的发生中起重要作用。较少的

项目成果

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Sekiguchi, T.等人：“TFIID 基底细胞转录因子 CCG1/TAFII250 亚基中的 tsBN462/13 突变诱导 BHK 细胞凋亡”实验细胞研究。

Yamato, K., et al.: "Temperature-sensitive p53 mutant p53Val-138 : Modulation of the cell cycle, viability and expression of p53-responsive genes" Oncogene. 11. 1-6 (1995)

Yamato, K., et al.：“温度敏感的 p53 突变体 p53Val-138：细胞周期、活力和 p53 响应基因表达的调节”Oncogene。

Hirano.Y.,Yamato K,and Tsuchida N: "A temperature sensitive mutant of the human p53,valine 138 Arrests the growth without induced expression of cip.1-wafinsbi" Oncogine. 10. 1879-1885 (1995)

Hirano.Y.、Yamato K 和 Tsuchida N：“人类 p53、缬氨酸 138 的温度敏感突变体可在不诱导 cip.1-wafinsbi 表达的情况下抑制生长”致癌基因。

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Yasumasa Hirano、Koji Matsumura、Hideki Sakai、Nobuo Tsuchida：“口腔癌 - 临床 DNA 诊断” Kanehara Publishing，3-（1995）

Hirano.Y.,Yamatok,and Tsuchida N: "A temperature sensitive mutant of the human P53,Valiue 138 Arrests the growth without induced expression of cip・1-Wafz-sbi" Oncogine. (in perss).

Hirano.Y.、Yamatok 和 Tsuchida N：“人类 P53 的温度敏感突变体，Valiue 138 在不诱导 cip·1-Wafz-sbi 表达的情况下抑制生长”Oncogine（在 perss 中）。