Mutational changes of EGFR mRNA in oral cancers and functions of a candidate tumor suppressor, doc-1

口腔癌中 EGFR mRNA 的突变变化以及候选肿瘤抑制因子 doc-1 的功能

• 批准号: 10470384
• 负责人: MATSUO Kou
• 金额: $ 8.38万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470384/
• 关键词: EGFR; mutation; truncated EGFR; doc-1; DNA replication; DNA polymerase α; primase; CDK2; RNA edition; base change; DOC-1R; cyclin- dependent kinase; 細胞周期; 口腔癌; point mutation; deletion mutation; 癌抑制遺伝子; primase; deletion murtation

1) We examined the mutations in the coding region of the human Epidermal growth factor receptor (EGFR) gene in normal oral keratinocytes and oral squamous carcinoma cells. First, a tumor-associated silent mutation, which generates a unique BsrI restriction site, was detected at position 2073 only in malignant keratinocytes. The utility of the tumor-associated BsrI site for diagnostic applications is currently being explored. Second, we found that there is a truncated EGFR mRNA (〜1.5 kb) which lacks both transmembrane and kinase domains. The functional analyses of the truncated receptor is in progress.2) We found that pDoc-1 (a protein form of a candidate tumor suppressor, doc-1 gene) associates with DNA polymerase α/primase (pol-α : primase), resulting in suppression of DNA synthesis. Using in vitro DNA replication assay revealed that pDoc-1 suppresses DNA replication, leveling at 〜50 %, by affecting the initiation step rather than elongation phase. The pol- α : primase binding domain in pDoc-1 is mapped to the amino-terminal six amino acids (MSYKPN). On the other hand, we demonstrated that pDoc-1 also associates with cyclin-dependent kinase 2(CDK2). More specifically, pDoc-1 associates with the monomeric nonphosphorylated form of CDK2. Amino acids 109-111 (TER) are necessary for pDoc-l's association with CDK2.

1）我们检测了正常口腔角质形成细胞和口腔鳞状细胞中人表皮生长因子受体（EGFR）基因编码区的突变。首先，仅在恶性角质形成细胞中的 2073 位检测到肿瘤相关的沉默突变，该突变产生独特的 BsrI 限制位点。目前正在探索肿瘤相关 BsrI 位点在诊断应用中的效用。其次，我们发现存在一个截短的 EGFR mRNA（约 1.5 kb），缺乏跨膜结构域和激酶结构域。截短受体的功能分析正在进行中。2）我们发现pDoc-1（候选肿瘤抑制因子doc-1基因的蛋白质形式）与DNA聚合酶α/引物酶（pol-α：引物酶）结合，导致DNA合成受到抑制。使用体外 DNA 复制测定表明，pDoc-1 通过影响起始步骤而不是延伸阶段来抑制 DNA 复制，水平约为 50%。 pDoc-1 中的 pol-α：引物酶结合域被映射到氨基末端的六个氨基酸 (MSYKPN)。另一方面，我们证明 pDoc-1 还与细胞周期蛋白依赖性激酶 2 (CDK2) 相关。更具体地说，pDoc-1 与单体非磷酸化形式的 CDK2 结合。氨基酸 109-111 (TER) 对于 pDoc-1 与 CDK2 的关联是必需的。

项目成果

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Ieyoshi Kobayashi 等：“舌下腺乳头状囊腺癌的免疫组织化学和超微结构研究”J.

Shintani, S, et al.: "pl2^<DOC-1> is a novel CDK2-associated protein"Mol. Cell. Biol.. 20. 6300-6307 (2000)

Shintani，S 等人：“pl2^<DOC-1> 是一种新型 CDK2 相关蛋白”Mol.

Kou Matuo et al.: "p12^<DOC-1>, a growth suppressor, associates with DNA polymerase α/primase."The FASEB Journal. 14. 1318-1324 (2000)

Kou Matuo 等人：“p12^<DOC-1>，一种生长抑制剂，与 DNA 聚合酶 α/引物酶相关。”FASEB 杂志 14. 1318-1324 (2000)

Masayo Morfuji: "Differential expression of cytoskeleton after orthotopic inplamtation of newly established human tongue cancer cell lines of defined metastatic ability."Amerikan Jornal of Pathology. 156・4(in press). (2000)

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K.Matsuo: "p12DOC-1,a growth suppressor,associates with.DNA polymerase α/primase."FASEB J.. (in press). (2000)

K. Matsuo：“p12DOC-1，一种生长抑制剂，与 DNA 聚合酶 α/引物酶相关。”FASEB J.（出版中）。