Identification of a novel tumor suppressor gene on chromosome arm 18q in human pancreatic caner

18q缺失在人类胰腺癌中非常常见。肿瘤抑制基因SMAD 4存在于该区域中，但该基因的引入仅通过ETS抑制血管生成而在体内抑制肿瘤细胞生长。然而，18号染色体的引入在体外和体内均抑制。此外，在大多数胰腺肿瘤发生的早期病变中，18q丢失，但SMAD 4功能正常。等位基因型分析18 q确定了一个常见的丢失区域之间的D18S451和D18S462。数据库检索表明，共有164个蛋白质编码基因。其中一个基因PMAIP1被微阵列分析挑出来。这种基因在胰腺癌中经常被抑制。该基因的上调导致胰腺癌细胞中细胞生长的抑制。相反，siRNA介导的PMAIP1敲低刺激细胞生长。因此，PMAIP 1基因是胰腺癌的候选抑癌基因之一。我们进一步使用siRNA对常见缺失区域的所有蛋白编码基因进行了系统的敲除研究。监测细胞生长的恢复，我们总共挑选了13个候选基因。在这些研究的过程中，我们开发了一种改良的连接缓冲系统。使用该系统，我们可以以廉价，高效和快速的方式进行连接。总共100个连接反应可以在10分钟内完成，100个反应仅花费1美元。因此，我们将这种方法命名为“咖啡中断结扎技术”。"

Loss of 18q is highly frequent in human pancreatic cancer. A tumor suppressor gene, SMAD4, resides in this region, but introduction of this gene inhibit tumor cell growth only in vivo by means of angiogenesis inhibition through ETS. Introduction of chromosome 18, however, inhibit both in vitro and in vivo. Moreover, in most of the early lesions of the pancreatic tumorigeneses, 18q is lost but SMAD4 functions normally. Allelotype analyses on 18q identified a commonly lost region between D18S451 and D18S462. Database search indicated a total of 164 protein coding genes. One of such genes, PMAIP1, was picked up by the microarray analyses. This gene is frequently suppressed in pancreatic cancer. Upregulation of this gene caused suppression of the cell growth in pancreatic cancer cells. On the contrary, siRNA-mediated knockdown of the PMAIP1 stimulated cell growth. Thus, the PMAIP1 gene is one of the candidate tumor suppressor genes for pancreatic cancer.We further performed systematic knockdown studies using siRNAs for all the protein-coding genes in the commonly deleted region. Recovery of the cell growth was monitored and we picked up a total of 13 candidate genes. Further detailed studies are necessary.In the course of these studies, we developed a modified buffer system for ligation. Using this system, we can perform ligations in a cheap, efficient, and rapid manner. A total of 100 ligation reaction can be done within a 10 minutes only spending one dollar for 100 reactions. Thus, we named this method as "Coffee break ligation technique."