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

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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.