Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12DOC-1 is a growth suppressor isolated from normal keratinocytes. We report that p12DOC-1 associates with CDK2. More specifically, p12DOC-1 associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12DOC-1 resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12DOC-1 transfectants (1G1 and2S). The p12DOC-1-mediated decrease of CDK2 was prevented if the p12DOC-1 transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin b-lactone, suggesting that p12DOC-1 may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12DOC-1-mediated, CDK2-associated cell cycle phenotypes. These data support p12DOC-1 as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.

应用重组技术构建野生型及缺失型CDK2基因的真核表达载体，分别使野生型及缺失型CDK2蛋白与增强型绿色荧光蛋白（Enhanced-green Fluorescent Prutein, EGFP）形成融合蛋白。通过脂质体介导的方法将载体转染人宫颈癌细胞系HeLa和中华仓鼠卵巢细胞系CHO，经过细胞周期同步化处理后于荧光显微镜下观察EGFP的亚细胞定位以示踪野生型及缺失型CDK2基因的表达。结果表明，野生型CDK2基因的表达产物定位于细胞核，而两种缺失型CDK2基因分别编码的CDK2蛋白N-端1～201及98～298多肽均主要定位于细胞质。以上结果提示，CDK2蛋白序列中不含有与核定位直接相关的信号，其入核过程可能是由其N-端1～97及202～298多肽范围内的部分氨基酸共同形成高级结构，并依赖此高级结构与其他含有入核信号的蛋白形成复合物，从而被带动进入细胞核的。

Tumor invasion and metastasis are the most common causes of death in gastric carcinoma. Human heparanase influences tumor invasiveness and angiogenesis. Analysis of its expression in gastric carcinoma has been hindered by our inability to procure pure cancer cells from heterogeneous tissue. In the present study, we analyzed heparanase expression in human primary and metastatic gastric carcinoma cells as well as in paired normal gastric epithelial cells by laser capture microdissection coupled with reverse transcriptionpolymerase chain reaction (RT-PCR). Tumor tissues, metastatic lymph nodes, and apparently uninvolved normal gastric tissues were collected from 30 patients who had undergone gastrectomy with radical lymph node dissection for gastric carcinoma without preoperative treatment. Bulk tissues and laser capture microdissected cell groups were separately subjected to RT-PCR analysis with heparanase-specific primers. For bulk tissues, heparanase-specific transcripts were detectable in all primary tumor tissues, metastatic lymph nodes, and almost all matching normal tissues. RT-PCR analysis after laser capture microdissection showed no detectable heparanase expression in matching normal epithelial cell groups. Of the laser capture microdissected primary gastric carcinoma cells, 47% (14/30) were heparanase positive. Expression was closely associated with greater tumor invasiveness, including Borrmann gross type and depth of wall infiltration. For metastatic cell groups dissected from lymph nodes, 95% showed clear heparanase expression. urthermore, the extent of lymphatic spread was directly correlated to heparanase expression at the primary site. In conclusion, laser capture microdissection coupled with RT-PCR is a reliable approach for molecular analysis of heparanase expression in gastric carcinoma. Heparanase may facilitate invasion and metastasis of gastric carcinoma cells.