INVESTIGATION OF THE TUMOR SUPPRESSOR GENES INVOLVED IN THE CARCINOGENESIS OF ORAL SQUAMOUS CELL CARCINOMA BASED ON THE GENETIC COMPLEMENT.

基于遗传互补的口腔鳞状细胞癌癌变抑癌基因研究。

• 批准号: 11671980
• 负责人: NEGISHI Akihide
• 金额: $ 2.24万
• 依托单位: GUNMA UNIVERSITY (2000)Tokyo Medical and Dental University (1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671980/
• 关键词: ORAL CANCER; SQUAMOUS CELL CARCINOMA; GENETICS; CHROMOSOME; MICROCELL FUSION; TUMOR SUPPRESSOR GENE; 細胞

1)Construction of A9 cells containing subchromosomal transferable fragments(STFs)derived from the normal human chromosome 3.A9(neo3)cells, containing normal human chromosome 3 tagged with pSV2neo, were irradiated with doses of 2-12 Gy of X-rays. Irradiated microcells were fused to A9 cells, and G418-resistant A9(STF)clones were selected in DMEM supplemented with 10% CS and 0.8mg/ml of G418. We have constructed 16 A9(STF)clones containing partial fragment derived from normal human chromosome 3. To identify the human sequence in the A9 clones containing STFs, we performed Q-banding analysis and chromosome painting with the human chromosome 3 specific probe. To localize the isolated STFs, DNA samples prepared from all 16 A9(STF)clones were assayed using 54 microsatellite markers on chromosome 3.2)Tumorigenicity of the parental cell line and A9(STF)clones.The tumorigenicity of the parental A9 cells and 6 different A9(STF)clones was examined in nude mice. The parental A9 cells formed progressively growing tumor in all the sites of nude mice within 3 to 5 weeks after subcutaneous inoculation. On the other hand, only one microcell hybrid ; A9(neo3B)cells failed to form tumors even more than 6 month after subcutaneous injection. Another five A9(STF)clones produced tumor in nude mice like the parental A9 cells. These data suggested that the critical tumor suppressor gene(s)which may contribute to the development of tumor in nude mice on human chromosome 3 was expressed only in the A9(neo3B) cells.3)Identification of the critical tumor suppressor gene(s)on human chromosome 3 using suppression subtractive hybridization(SSH).We are now trying to identify the critical tumor suppressor gene(s), which was expressed in the suppressed clone, not in the unsuppressed clones, on human chromosome 3 using SSH method.

1)含正常人3号染色体亚染色体可转移片段(STF)的A9细胞的构建。将辐照后的微细胞与A9细胞融合，在添加10%CS和0.8 mg/mlG418的DMEM中筛选出G418抗性的A9(STF)克隆。我们构建了16个含有正常人类3号染色体部分片段的A9(STF)克隆。为了鉴定含有STF的A9克隆中的人类序列，我们用人类3号染色体特异性探针进行了Q-显带分析和染色体涂染。用染色体上的54个微卫星标记检测亲本细胞系和A9(STF)克隆的致瘤性，并检测亲本A9细胞和6个不同的A9(STF)克隆在裸鼠体内的致瘤性。裸鼠皮下接种后3~5周内，亲代A9细胞在裸鼠各部位形成进行性生长的肿瘤。另一方面，只有一种微细胞杂交体A9(Ne3B)细胞在皮下注射6个月后仍未形成肿瘤。另外五个A9(STF)克隆与亲代A9细胞一样，在裸鼠体内产生了肿瘤。3)利用抑制性消减杂交技术鉴定人3号染色体上的关键抑癌基因(S)，该基因在人3号染色体上的表达存在于抑制克隆中，而不是在未抑制克隆中。

项目成果

Narikazu Uzawa et al.: "Loss of heterozygosity on the short arm of chromosome 3 in oral squamous cell carcinomas -Relationship between loss of heterozygosity on 3p25-pter region and clinical and histological features-."Jpn.J.Oral Maxillofac.Surg.. 46(5).

Narikazu Uzawa 等人：“口腔鳞状细胞癌中 3 号染色体短臂杂合性丢失 - 3p25-pter 区域杂合性丢失与临床和组织学特征之间的关系 -”Jpn.J.Oral Maxillofac.Surg。

鵜澤 成一: "口腔扁平上皮癌における第3染色体短腕上のヘテロ接合性消失について-3p25-pter領域の欠失と臨床的・病理組織学的特性との関連-"日本口腔外科学会雑誌. 46. 455-461 (2000)

Seiichi Uzawa：“关于口腔鳞状细胞癌中 3 号染色体短臂杂合性的丧失 - 3p25-pter 区域缺失与临床和组织病理学特征之间的关系 -”日本口腔颌面外科杂志 46。 455-461 (2000)

Teruo Amagasa et al.: "Clinical characteristics of precancerous lesions and early squamous cell carcinoma in the oral cavity."J.Jpn.Soc.Oral Tumor. 11(4). 357-363 (1999)

Teruo Amagasa 等人：“口腔癌前病变和早期鳞状细胞癌的临床特征。”J.Jpn.Soc.Oral Tumor。

天笠 光雄: "口腔前癌性病変と早期扁平上皮癌の臨床"日本口腔腫瘍学会雑誌. 11. 357-363 (1999)

Mitsuo Amagasa：“口腔癌前病变和早期鳞状细胞癌的临床实践”日本口腔肿瘤学会杂志 11. 357-363 (1999)。

Narikazu Uzawa et al.: "Homozygous deletions on the short arm of chromosome 3 in human oral squamous cell carcinomas."Oral Oncology. (in press).

Narikazu Uzawa 等人：“人类口腔鳞状细胞癌 3 号染色体短臂上的纯合缺失。”口腔肿瘤学。