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

1)我们检测了人表皮生长因子受体（EGFR）基因编码区在正常口腔角质形成细胞和口腔鳞癌细胞中的突变。首先，肿瘤相关的沉默突变，它产生一个独特的BsrI限制性位点，仅在恶性角质形成细胞的位置2073检测。目前正在探索肿瘤相关BsrI位点用于诊断应用的效用。其次，我们发现有一个截短的EGFR mRNA（约1.5 kb），它缺乏跨膜和激酶结构域。2）我们发现pDoc-1（候选肿瘤抑制基因doc-1的蛋白形式）与DNA聚合酶α/引物酶（pol-α：primase）结合，导致DNA合成的抑制。使用体外DNA复制测定显示，pDoc-1通过影响起始步骤而不是延伸阶段来抑制DNA复制，在约50%处保持水平。pDoc-1中的pol- α：引发酶结合结构域定位于氨基末端的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（CDK 2）相关。更具体地，pDoc-1与CDK 2的单体非磷酸化形式结合。氨基酸109-111（TER）是pDoc-I与CDK 2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.