Identification of tumor suppressor genes by means of a genomic clone-transfer

胰腺癌细胞在1p、6q、9p、12q、17p和18q等位基因发生杂合性丢失的频率较高。在这些缺失的等位基因中，可能存在肿瘤抑制基因。本研究的目的是通过基因组克隆转移的方法寻找胰腺癌缺失区域中的抑癌基因。我们将重点放在6q、12q和18q上，因为尽管LOG频率很高，但还没有发现相关的基因。为了寻找基因组克隆，我们筛选了人酵母和细菌人工染色体(YAC，BAC)文库。鉴定的克隆被构建在重叠群中以覆盖缺失的区域。对于6q21的缺失区域，构建了跨越1-Mb的完整的BAC重叠群。我们在该地区鉴定了42个EST。重叠群可在http：//www-alis.tokyo.jst.go.jp/hgs/plmap.pl？PLID=14。对于12q21，构建了一个由21个BAC克隆组成的重叠群，其跨度为3-Mb。共鉴定出40个新的STSS和10个EST。我们在缺失的区域发现了几个新的基因，包括hGnT-IV-H、hHDC和TUB1。为了了解待转移胰腺癌细胞的遗传状态，我们分析了KRAS、p16、P53和Smad4等肿瘤相关基因。为了了解18q的状态，我们分析和鉴定了导管内乳头状黏液性肿瘤中18q的高频率杂合性缺失，这被认为是传统胰腺癌的早期病变，Smad4突变的频率很低。由于18q基因缺失的范围很广，因此选择了18号染色体的转移来补偿其在胰腺癌细胞中的功能。转移克隆在体外生长受限，在体内有致瘤性。

The pancreatic cancer cells are in high frequency of loss of heterozygosity in some chromosomal allels including 1p, 6q, 9p, 12q, 17P, and, 18q. In these deleted alllels, tumor suppressor genes may exist. Aim of this study was to identify tumor suppressor genes in the deleted regions in the pancreatic cancer by means of a genomic clone-transfer. We focused on 6q, 12q and 18q because no responsible genes have been identified yet despite high frequency of LOH.To find genomic clones, human yeast and bacterial artificial chromosome (YAC, BAC) libraries were screened. Identified clones were constructed in contig to cover the deleted regions. For the deleted region at 6q21, a complete BAC contig to span 1-Mb was constructed. We identified 42 ESTs in the region. The contig was available at http : //www-alis.tokyo.jst.go.jp/HGS/plmap.pl? PlID=14.For 12q21, a contig comprised of 21 BAC clones which could span 3-Mb was constructed. Forty novel STSs and 10 ESTs were identified. We identified several novel genes in the deleted regions including hGnT-IV-H, hHDC, and TUB1. Information and references for these genes were available in Genbank/EMBL/DDBJ.To know genetic status of pancreatic cancer cells to be transferred, we analyzed cancer-related genes including KRAS, p16, p53 and SMAD4. To know a status of 18q, we analyzed and identified high frequency of LOH of 18q in intraductal papillary mucinous tumors, which was thought to be an early lesion of conventional pancreatic cancer, with low frequency of mutation of SMAD4. Since loss of 18q was found to span broad area, transfer of chromosome 18 was chosen and carried out to compensate its function in the pancreatic cancer cells. The transferred clone revealed limited growth in vitro and tumorigenesity in vivo.