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

1)我们检测了正常口腔角质形成细胞和口腔鳞癌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限制性位点。目前正在探索肿瘤相关BsrI位点的诊断应用。其次，我们发现有一个截断的EGFR mRNA（约1.5 kb），缺乏跨膜结构域和激酶结构域。截断受体的功能分析正在进行中。2)我们发现pDoc-1（候选肿瘤抑制基因doc-1的一种蛋白形式）与DNA聚合酶α/引物酶（pol-α：引物酶）相关，导致DNA合成受到抑制。体外DNA复制实验表明，pDoc-1通过影响起始阶段而非延伸阶段抑制DNA复制，抑制水平在50%左右。pDoc-1的pol- α：引物酶结合域被定位到氨基末端6个氨基酸（MSYKPN）。另一方面，我们证明pDoc-1也与细胞周期蛋白依赖性激酶2（CDK2）相关。更具体地说，pDoc-1与CDK2的单体非磷酸化形式相关。氨基酸109-111 （TER）是pdoc -1与CDK2结合所必需的。

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.